Podium Presentations for Tox / TDM / Endocrine

Evaluation of Analytical Techniques for Drug Checking John Halifax (Presenter) UCSF

To be presented in Track 3 (Steinbeck 3) on Wednesday at 14:00

Introduction:

Drug overdose mortality has risen dramatically in North America over the last two decades. The overdose crisis is now considered to be in its “Fourth Wave,” characterized by high mortality driven by synthetic opioids replacing traditional illicit drugs and augmented by increasing stimulant-involved deaths. In 2020 and 2021 in San Francisco County, cumulative deaths from overdose were almost double those from COVID-19, and an increase in fentanyl-involvement in deaths among individuals without prior evidence of opioid use in San Francisco suggests increased unintentional fentanyl exposure in the city.

Drug checking, the use of chemistry techniques to identify components present in a drug sample, has gained traction as one harm reduction intervention to mitigate overdose. Drug checking in the US has so far been limited to FTIR spectroscopy and fentanyl immunoassay test strip technologies to provide onsite service. San Francisco launched its own FTIR and test strip service in June 2022, with the service augmented with offsite research laboratory confirmation of all drug samples analysis performed by untargeted Liquid Chromatography-Quadrupole Time of Flight-Mass Spectrometry (LC-QTOF-MS). This work demonstrates the advantages of this onsite-offsite hybrid model of drug checking, which provides all the benefits of a point-of-service test for people who use drugs (PWUD), while gaining superior chemical analysis to better comprehend the composition of the illicit substance supply. Improved knowledge of the local illicit substance supply can be utilized to inform public health planning and toxicological case response.

Objectives:

To demonstrate the effectiveness of utilizing a clinically validated LC-QTOF-MS comprehensive drug screen method to analyze street drug products to evaluate, confirm, and augment point of service drug checking efforts.

Methods:

All drug samples submitted for point-of-service drug checking were delivered to the clinical lab dissolved in 1 mL MeOH at approximately 1 mg/mL to circumvent transportation of consumable drug material. All samples were diluted to approximately 10 mcg/mL in mobile phase and analyzed using a method previously validated for clinical testing. Briefly, chromatographic separation was performed using a C-18 column with a 10-minute gradient from 2%-100% organic. Data was collected on a SCIEX TripleTOF®5600 operating in positive-ion mode using a TOF-MS survey scan with IDA-triggered collection of high-resolution product ion spectra (20 dependent scans). Data was analyzed using an in-house library containing >5000 small molecules including >150 fentanyl analogs. Calibrator mixes were made for 25 compounds of interest to enable “semi-quantitation” in the drug products. Accurate quantitation is limited by weighing errors at the point-of-service and issues with drug product dissolution. Results were available to point-of-service drug checking staff within three days of sample intake for the purposes of informing delivery of service and to communicate confirmation results to returning service participants.

Results:

From June to November 2022, 188 drug products were evaluated at the point-of-service using FTIR spectroscopy and immunoassay lateral flow test strips for fentanyl and benzodiazepines followed by analysis using the LC-QTOF-MS method. The LC-QTOF-MS detected an additional 36 compounds in the samples that were missed by point-of-service technologies. Using the LC-QTOF-MS method as the “gold standard,” the sensitivity and specificity of the FTIR was 36.18% and 98.84% respectively for all analytes. Sensitivity and specificity of the FTIR for fentanyl only was 58.70% and 100.00% respectively, improving to 93.48% and 98.59% respectively when fentanyl test strip results were considered. Notably, the FTIR missed all benzodiazepine compounds detected by LC-QTOF-MS (n=4).

Confirmation by LC-QTOF-MS was originally planned for every sample only during the pilot period of the point-of-service program, but due to the overwhelming advantages provided by confirmation, confirmation for all samples has been extended indefinitely.

As determined by LC-QTOF-MS, samples expected to be methamphetamine (n=72) rarely contained other drug classes (n=2, 4.17%). Samples expected to be cocaine (n=42) contained unexpected drug classes more often, (n=10, 23.81%), but 9 of these 10 samples contained only lidocaine or levamisole, established cocaine adulterants. Samples expected to be fentanyl (n=37) were frequently identified as complex mixtures of compounds from a variety of drug classes (n=24, 64.86%), with semi-quantitation identifying widely varied and unpredictable potency of fentanyl in the samples ranging from 0.0092% to 46.37%. Samples expected to be heroin (n=10) also showed frequent contamination with fentanyl (n=3, 30.00%), conflicting with established thought that fentanyl is unlikely to be mixed with West Coast tar heroin. Detection of xylazine, deschloro-etizolam, flubromazolam, bromazolam, and butyryl fentanyl by confirmation but not by point-of-service demonstrate the added benefit of utilizing mass spectrometry to improve public health surveillance efforts to identify drug supply changes to inform overdose crisis response efforts.

Conclusions:

Confirmation of point-of-service drug checking by LC-QTOF-MS provides deeper comprehension of the illicit substance supply in a geographic region, aiding both harm reduction and traditional clinical practice. This information facilitates improved confidence in the capability and limitations of point-of-service drug checking, improves drug checking as public health surveillance intervention, and provides crucial context to interpret comprehensive drug screen results for clinical cases.

Multiplexed Quantification of Venlafaxine and Four Metabolites in Human Plasma Claire Knezevic (Presenter) Lurie Childrens Hospital

To be presented in Track 3 (Steinbeck 3) on Wednesday at 14:20

INTRODUCTION

Venlafaxine (VEN) and its O-demethylated metabolite, O-desmethylvenlafaxine (ODV), are commonly prescribed serotonin-norepinephrine reuptake inhibitors, approved for the treatment of depression and anxiety. Both VEN and ODV have similar antidepressant activity and their three metabolites formed by further demethylation (N-desmethylvenlafaxine (NDV), N,O-didesmethylvenlafaxine (NODDV), and N,N-didesmethylvenlafaxine (NNDDV)) are inactive. This metabolism is accomplished by CYP450 enzymes, namely CYP2D6, CYP3A4, and CYP2C19, all of which are polymorphic and can be inhibited or activated by foods and other drugs. At standard dosing levels, approximately 40% of patients have VEN and ODV concentrations outside the therapeutic range, demonstrating the potential clinical value of quantifying these drugs.

OBJECTIVES

The objectives of this study were to develop and validate a quantitative tandem mass spectrometry method for venlafaxine, desvenlafaxine, and their three inactive metabolites. A secondary objective was to quantify these molecules in remnant patient plasma and observe the relationship between administered drug and metabolites.

METHODS

K2EDTA plasma was spiked with VEN, ODV, NDV, NODDV, and NNDDV. Remnant whole blood from patients prescribed venlafaxine was obtained and plasma was obtained by centrifugation. Collection of remnant whole blood was approved by the IRB. Following the addition of isotopically-labeled internal standards (VEN-d6 and ODV-d6) to plasma and sample extraction via protein precipitation, samples were subjected to analysis by liquid chromatography-tandem mass spectrometry. Chromatographic separation was performed using a ZORBAX Eclipse Plus C18 column and detected on a SCIEX API 4500 mass analyzer operated in positive ionization mode.

RESULTS

The analytical measuring range for VEN and all four metabolites was 5 – 800 ng/mL. Standard curves were generated via weighted quadratic (NNDDV) or linear (VEN, ODV, NDV, NODDV) regression of calibrators. Inter-assay imprecision was less than 10% for all levels of all analytes. No carryover was observed after repeated injections of sample at upper limit of measuring range and no cross-talk was observed between any analyte or internal standard. Only minor matrix effects were observed and both recovery efficiency and process efficiency were >96% for all analytes. Twenty-one remnant plasma specimens were obtained from patients with current venlafaxine prescriptions (prescribed doses of 37.5 – 450 mg/day) and analyzed with this method. VEN and ODV were quantified in all samples, with concentrations ranging from 7-1200 ng/mL. NODDV was quantified in all but one sample and ranged from 10.2 to 430 ng/mL. NDV and NNDDV were quantified in 14 and 6 samples, respectively, at concentrations up to 1060 and 168 ng/mL, respectively. The ratio of total active to total inactive analytes ranged from 0.74 to 14.5, with a median of 6.39.

CONCLUSION

While previous studies have focused on quantifying VEN and ODV, fewer studies have evaluated the contribution of metabolism to the inactive metabolites to the overall pharmacokinetics of these antidepressants. This effort developed an efficient and accurate method for the quantification of VEN, ODV, and all three inactive metabolites in human plasma and demonstrated this capacity in remnant human samples. This method’s performance fulfills the criteria for an FDA Bioanalytical Method and may be used to study the pharmacokinetics of these drugs and metabolites. Additionally, these validation studies demonstrate that this method is suitable for analysis of patient samples. Although the time of dosing relative to the blood collection was not captured, all patients had been prescribed daily doses of venlafaxine for more than 10 days, during which time a daily dose would reach a steady state concentration. In agreement with published reports, ODV was the major metabolite present in most samples (18 of 21). Evaluation of the ratios of active analytes VEN and ODV to the inactive metabolites demonstrates wide inter-individual variation in metabolism. Importantly, this method enables further studies combining drug and metabolite quantification with pharmacogenetic evaluation of metabolizing genes. Future clinical studies can be performed to probe the impact of utilizing VEN and metabolite monitoring on therapeutic efficacy.

Optimized UPLC-MS/MS Assay for Therapeutic Drug Monitoring in Patients with Adenine Phosphoribosyltransferase Deficiency Margret Thorsteinsdottir (Presenter) University of Iceland

To be presented in Track 3 (Steinbeck 3) on Wednesday at 14:40

INTRODUCTION: Adenine phosphoribosyltransferase (APRT) deficiency is an inborn error of purine metabolism characterized by urinary excretion of poorly soluble 2,8-dihydroxyadenine (DHA), causing kidney stones and chronic kidney disease. Treatment with the xanthine oxidoreductase (XOR) inhibitors allopurinol or febuxostat, reduces DHA excretion and ameliorate disease manifestations. However, some patients cannot tolerate currently available therapies while others continue to form stones and progress to end-stages kidney disease despite treatment. Therefore, a more reliable method for therapeutic drug monitoring (TDM) in patients with APRT deficiency is needed.

OBJECTIVES: The aim of this study was to optimize a UPLC-MS/MS assay for simultaneous quantification of DHA, adenine, allopurinol, oxypurinol and febuxostat in plasma, utilizing design of experiments (DoE) for monitoring of pharmacotherapy in patients with APRT deficiency.

METHODS: The chemometric software MODDE Pro 13 was used for optimization of the UPLC-MS/MS plasma assay. A fractional factorial (FF) design was used for experimental screening to reveal the most influential experimental factors. Significant factors were studied via central composite design and related to sensitivity, resolution and retention time utilizing partial least square (PLS)-regression. Absolute quantification of DHA, adenine, allopurinol, oxypurinol and febuxostat was performed in plasma samples from untreated and treated APRT deficient patients and healthy controls.

RESULTS: The DoE approach was efficiently used for optimization of the UPLC-MS/MS assay. For all analytes, accuracy and precision were within the acceptable range of ± 15%. Preliminary data revealed a median (range) plasma concentration of 248 (224-395) ng/mL for DHA and 194 (159-284) ng/mL for adenine, in the untreated patients, and below 50 ng/mL for DHA and 533 (339-1034) ng/mL for adenine, in those on treatment. DHA was not detected in the plasma samples from healthy controls. In patients receiving XOR inhibitor therapy, the median plasma concentration for allopurinol, oxypurinol and febuxostat was 687 (103-2901), 7945 (2199-10943) and 1628 ng/mL, respectively.

CONCLUSION: The UPLC-MS/MS assay has been implemented to monitor the efficacy of pharmacotherapy and treatment adherence among patients with APRT deficiency.

Rapid and Sensitive Protein Quantitation in Biofluids by Paper Spray Mass Spectrometry: Single Instrument Albumin/Creatinine Ratio Measurements Chris Gill (Presenter) Vancouver Island University

To be presented in Track 1 (Steinbeck 1) on Wednesday at 16:10

INTRODUCTION: Direct mass spectrometry approaches such as paper spray mass spectrometry (PS-MS) are presenting new alternatives as candidate methods for clinical workflows. PS-MS, in particular, offers a facile strategy for chemical measurements in complex samples such as biofluids. Small aliquots (i.e., ≤10µL) of sample are deposited on pointed paper strips with co-deposited internal standards. The strips are moistened with a suitable solvent, and upon the application of high voltage, ions are generated in a manner akin to electrospray, allowing direct analyte quantitation via tandem mass spectrometry. The strips are inexpensive and disposed for each measurement, eliminating carryover, and can be used to conduct ‘on-paper’ derivatization reactions as well as replace the extraction/preconcentration steps necessary in other analytical workflows.

OBJECTIVES: To demonstrate the use and effectiveness of chemically modified PS-MS paper substrates for the rapid, sensitive, and quantitative measurement of proteins in biofluids, including the rapid ‘one instrument’ measurement of albumin/creatinine ratios in urine.

METHODS: All measurements were performed by paper spray tandem mass spectrometry with a high-throughput paper spray ion source (Thermo Scientific™ TSQ Altis™ triple quadrupole mass spectrometer with a VeriSpray™ source). PS-MS paper substrates were modified by grafting polyamidoamine (PAMAM) dendrimers to their surface, increasing protein affinity. The extraction / preconcentration of target proteins from urine and serum was accomplished by vortexing the strips in 150 µL biofluid samples, followed by a short wash. Measurement by PS-MS was accomplished using barcode traceable VeriSpray™ PS-MS sample plates, allowing multiplexed measurements of 24 strips per plate, and the unattended measurement of up to 240 samples. Quantitative calibrations were achieved for both proteins and creatinine utilizing co-deposited internal standards.

RESULTS: The PAMAM functionalized PS-MS paper substrates were characterized for direct protein measurements in human urine and serum sample matrices. This included optimizing sample extraction times, washing protocols and evaluating the storage stability of the modified paper substrate. A 30 second protein vortex extraction followed by a 5 second acetonitrile wash was optimal. Once prepared, PAMAM functionalized PS-MS substrate exhibited no deterioration in performance when stored at ambient conditions over a one month period. As examples, the direct measurement of hemoglobin and albumin in urine and lysozyme and insulin in serum is presented, achieving up to 11 fold sensitivity improvements, dependent upon the charge state of the protein. The analytical performance of the functionalized paper substrate was evaluated for the analysis of albumin in urine, achieving linearity with R2 > 0.99, LOD of 1.1 µg mL-1, LOQ of 3.8 µg mL-1, precision <10%, and recoveries of 70-83%. The direct, simultaneous measurement of urinary albumin and creatinine ratios for anonymous patient samples by PS-MS demonstrates excellent comparison with validated clinical results obtained utilizing immunoturbimetry for albumin and the spectrophotometric Jaffe method for creatinine.

CONCLUSION: The use of PAMAM functionalized PS-MS paper substrate enhances the sensitivity of direct protein quantitation from biofluids. The ‘one instrument’ quantitative measurement of urinary albumin/creatinine ratios by PS-MS shows significant promise as a new candidate method for clinical analyses.

PFAS Dark Matter and Slippery Cannabis: Disparate Problems with a Similar Path to a Solution Frederick Strathmann (Presenter) MOBILion Systems

To be presented in Track 1 (Steinbeck 1) on Thursday at 10:20

INTRODUCTION:
PFAS compounds have a long and diverse history of applications yet only recently has sufficient attention been focused on the environmental and toxicological impacts of their use. The number of PFAS compounds has expanded rapidly and it is estimated that 5,000 to 10,000 PFAS compounds exist. Despite the tremendous numbers of PFAS compounds in existence, a relatively small number have been studied in depth and are commercially tested routinely. The term “PFAS Dark Matter” has emerged to signify the recognized gap between Total Organic Fluorine, Total Oxidizable Precursors, and targeted methods using tandem mass spectrometry for PFAS concentration assessment. It is estimated that nearly every individual has at least one PFAS present in their blood, and the toxicological significance of PFAS and the implications of the PFAS Dark Matter are far from being fully appreciated. Similarly multifarious, cannabis has a complex chemical composition that includes terpenes, sugars, hydrocarbons, steroids, flavonoids, amino acids, and other compounds of potential interest. More than 700 natural constituents have been identified and more than 100 are classified as cannabinoids. The toxicological community has been challenged with the appearance of isomers of various cannabinoids causing numerous analytical challenges with limited solutions beyond chromatographic run time extension. Though diverse in the context of the challenges presented, trends in PFAS and cannabinoid production and laboratory-associated detection methods are quickly evolving in a manner reminiscent of Novel Psychoactive Substances with similar complexities involving identification, testing, and interpretation of toxicological data.

OBJECTIVES:
The primary objective of this study was to leverage a relatively novel analytical combination of LC, HRIM and QTOF approaches to unravel the complexity seen with existing separation challenges. A secondary objective of this study was to call attention to the need for clinical laboratories to evolve beyond existing workflows in anticipation of the challenges with emerging analytical needs associated with higher complexity biomarkers on the horizon.

METHODS:
We used the MOBILion HRIM system based on Structures for Lossless Ion Manipulation (SLIM) to assess cannabinoids and PFAS in a variety of matrices. A combination of Flow Injection Analysis or Liquid Chromatography with HRIM was used prior to detection using an Agilent QTOF. Accurate mass, isotope spacing, isotope ratios, and mobility aligned fragmentation were used in various combinations for tentative and absolute identification depending upon available standards. In several cases, CCS values were derived providing a unique, molecular identifier that was leveraged to generate 2 dimensional plots of CCS vs. m/z to elucidate trendlines and characteristic subclasses revealing distinctive relationships within and across compound classes. Lastly, previously established CCS values were used to generate reference plots of CCS vs. m/z as a tool to understand potential impact of interferences with known, endogenous compounds where applicable.

RESULTS:
Herein we report our use of the MOBIE® high-resolution ion mobility system (HRIM) from MOBILion with an Agilent LC-QTOF system to resolve fourteen different cannabinoid species reported in cannabis including positional isomers delta-8 and delta-9 THC, with an approximately 0.4% CCS difference, sufficiently resolved in matrix-free samples in the absence of chromatographic separation. Eight previously identified perfluorooctane sulfonates including the tentative identification of one additional branched form previously unseen, were found with enhancement of existing chromatographic separation by the HRIM system. Lastly, we explored an emerging link between hemp and PFAS in a subset of available extracts as a potential consequence of phytoremediation efforts with implications into as yet unknown toxicological significance.

CONCLUSION:
The LC-HRIM-QTOF system used is a powerful combination that can be implemented to enhance existing LC workflows for deeper sample characterization, reduce LC reliance to boost throughput, and add CCS values to existing compound identification and classification approaches. The examples provided here are relatively simple analytical challenges where existing separation technology has been limited in its utility for long-term, routine use. The clinical laboratory field has been eagerly watching the various “omics” fields with the anticipation of multiomics, diagnostic test availability, yet we have been largely idle in integrating truly novel analytical techniques outside of molecular testing. As the intricacies of each of the omics fields continues to be unraveled, novel technologies being applied to applications residing on the outskirts of existing and established workflows hold promise for meeting the ever-increasing complexity of novel testing expectations to come.

Time to Fly; TOF Mass Spec for Quantitation of Therapeutic Monoclonal Antibodies Paula Ladwig (Presenter) Mayo Clinic

To be presented in Track 1 (Steinbeck 1) on Thursday at 14:00

Introduction:
Therapeutic monoclonal antibodies have drastically improved the treatment of autoimmune disorders, cancers and other rare conditions. As recent as 5 years ago, clinical laboratories were struggling to develop quantitative methods for this class of proteins, based on large molecule drugs, as traditional methods for small molecules were not directly applicable.

Application of research proteomics mass spectrometry methods have increased choices for this class of therapeutics. The first mass spec methods were tryptic peptide and detection of unique peptides in the drug. The peptide methods have been very successful for chimeric therapeutic antibodies, such as infliximab for example (previously published). As therapeutics became more humanized, the peptide method loses some of its specificity due to overlapping peptides within the human proteome. Intact methods were then developed for these more humanized therapeutics such as eculizumab and vedolizumab for example (previously published). These methods use an enrichment method followed by Orbitrap detection.

Recent commercial advances in both hardware and software has allowed for TOF mass spectrometers to enter the arena as both a screening and quantitation tool for therapeutic monoclonal antibodies. The beauty of the TOF platform is the ability to screen or quantitate multiple analytes from the same injection without the loss in sensitivity found when utilizing an Orbitrap; sensitivity and resolution having an inverse relationship. This allows for more versatility in types of testing along with advantages for multiplexing panels.

Objectives:
To provide proof of concept TOF mass spectrometry methods for the quantitation of therapeutic monoclonal antibodies

Methods:
Samples were enriched utilizing Life Technologies CaptureSelect&trade; Affinity Matrix. In a 96-well PVDF plate, 100 mcL of resin was added and washed twice with 200 mcL 1xPBS utilizing positive pressure to move waste to a reservoir. 30 mcL of sample, along with 30 mcL of a stable isotope labeled or surrogate internal standard, were added and incubated shaking for 1 hour. The enrichment was washed 3 times with 150 mcL water. 200 mcL of 5% acetic acid was added and plate was incubated shaking for 15 minutes. Enriched proteins were eluted by positive pressure into a 2mL 96-deep well collection plate. Enrichment was reduced with 100 mcL 100 mM DTT in 1M ammonium bicarbonate; at 55&deg;C for at least 30 minutes.

The enriched and reduced samples were analyzed using an Agilent 1260 Infinity II HPLC System connected to an AB Sciex ZenoTOF&trade; 7600 mass spectrometer. A volume of 10 mcL was injected onto a Poroshell 300SB C3, 2.1x75 mm, 5-micron column heated at 60 &deg;C. Mobile phase A was water with 1% formic acid and mobile phase B was 10% isopropanol 90% acetonitrile with 0.1% formic acid. The therapeutics eluted during an 8 min gradient from 25%B to 33%B. The diverter valve was used to direct 6.5 minutes of the gradient into the mass spec; otherwise, the LC was diverted to waste. The mass spec using positive ESI was run using intact protein workflow; CUR 30, CAD 7 GAS1 35, GAS2 30, and temperature 500 &deg;C. TOF MS data from collected from 1000 to 2500 m/z; DP 175 and CE 10.

Sciex OS was used for instrument control, data acquisition, along with qualitative and quantitative data viewing and processing of data. Sciex OS Analytics was used for batch quantitation. The +11, +12, and +13 charge states (&plusmn;0.2 m/z) for the light chain mass of the therapeutic and internal standard were used for data extraction; combined to give the extracted ion chromatograph which is integrated and utilized for quantitation. A quadratic curve was drawn from the standards and unknowns back calculated from the curve.

Results:
Extracted trays (N=7) for the current clinical eculizumab quantitation LDT test method on our ThermoFisher Scientific QExactive Plus platform were re-injected on the Agilent 1260 Infinity II HPLC System connected to an AB Sciex ZenoTOF&trade; 7600 mass spectrometer and QC (N=12) and patient results (N=56) where compared. Inter day precision for the QE and TOF (respectively) compared; QCLow 19% and 16%, QCMed 11% and 10% and QCHigh 7% and 5%. Patient comparison gave a Passing-Bablok fit y=1.04x+(-4.476) for those within the AMR.

We also performed N=5 proof of concept runs for a test method for pembrolizumab and nivolumab from 5 to 500mcg/mL. 7 levels of standards were spiked with both pembrolizumab and nivolumab. 4 levels of QC were used to determine the accuracy and precision of the method. Precision was within 20% and accuracy within &plusmn;10% of expected.

Conclusion:
We demonstrate the ability to utilize the Agilent 1260 Infinity II HPLC System connected to an AB Sciex ZenoTOF&trade; 7600 mass spectrometer for quantitation of therapeutic monoclonal antibodies.

Lack of Unique Tryptic Peptides in Fully-Humanized Adalimumab Impedes Mass Spectrometry-based Quantification Jocelyn V. Abonamah (Presenter) Cleveland Clinic

To be presented in Track 1 (Steinbeck 1) on Thursday at 14:20

Introduction:
Bottom-up protein quantification requires digestion of the target protein into component peptides. The abundance of selected peptides is accepted to be a surrogate for the abundance of the protein. However, this method requires that proteotypic peptides be unique and not found endogenously in the target matrix. Adalimumab (Humira) is a monoclonal antibody (mAb) tumor necrosis factor (TNF) inhibitor that is widely used to treat a variety of inflammatory conditions, such as rheumatoid arthritis, Crohn’s disease, and ulcerative colitis. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) assays for mAb quantification in complex matrices are desirable because they allow for measurement of total drug rather than free, are generally more accurate and specific than antibody-based methods, and allow for assay multiplexing. The use of LC-MS/MS internal standards (ISTDs) improves analytical variation by correcting bias and variation. Immunoanalysis methods do not use ISTDs and therefore are prone to variability due to reagents, biologic interferences, etc. Despite literature claiming adalimumab is quantifiable using a simple bottom-up proteomics approach, due to the possibility of endogenous peptides it is likely not possible without utilizing immunoenrichment to select for adalimumab. Adalimumab is a fully-humanized mAb, which reduces its immunogenicity, increases its half-life, and allows for human effector functions to take place at Fc receptors. However, unlike chimeric mAbs which may contain multiple species-specific peptides unlikely to be found in humans, adalimumab did not contain any unique peptide sequences.

Methods:
The adalimumab protein sequence was subjected to in silico tryptic digestion using PeptideMass. Using the peptide sequences generated in silico and with agreement from literature, the peptides APYTFGQGTK (light chain) and NYLAWYQQKPGK (light chain) were selected for evaluation. Standard peptides were purchased from GenScript. Sample preparation consisted of protein enrichment with saturated ammonium sulfate, resuspension of the pellet in tris buffer, denaturation with trifluoroethanol, reduction with dithiothreitol, and alkylation with iodoacetamide. The protein was then digested with trypsin overnight and analyzed using a reversed-phase chromatography method with a Zorbax RR Eclipse XDB-C8 trapping cartridge (2.1 mm x 15 mm, 3.5 µM), an Acquity UPLC Peptide CSH C18 VanGuard Pre-column (2.1 mm x 5 mm 1.7 µM, 130 Å), and an Acquity UPLC Peptide CSH C18 Column (2.1 mm x 100 mm, 1.7 µM, 130 Å) on a Thermo Fisher Scientific Altis MS/MS coupled to a Thermo Fisher Scientific UltiMate3000 Dionex LC.

Results:
When ten naïve patient serum samples were screened for adalimumab, peaks were observed for APYT in eight samples and NYLA in nine samples. The peaks were all above the limit of quantitation of 1 µg/mL. The interferences were likely due to endogenous peptides as the interference signals increased with increased sample volume. Additionally, the interferences were not observed in various blank samples/solvents. Upon further research using UniProt, evidence of both APYT and NYLA was found at the transcript level in human Ig protein.

Conclusion:
Adalimumab was not able to be quantified via LC-MS/MS using the desired sample preparation technique due to the lack of multiple identifiable unique tryptic peptides. Despite the typically high specificity of LC-MS/MS methods, in this case antibody-based quantification methods are more specific and alternative methodologies must be explored. If LC-MS/MS analysis was required, a more complicated sample preparation could remediate the problem, e.g. affinity resin. However, at higher cost and number of sample preparation steps.

Characterization of Adrenocortical Function in Patients with Severe Infections Using Corticotropin-stimulated Serum Steroid Profiles Michael Vogeser (Presenter) University Hospital, LMU Munich

To be presented in Track 3 (Steinbeck 3) on Thursday at 14:40

INTRODUCTION:
Relative adrenal insufficiency in severe sepsis can be corrected by hydrocortisone treatment. However, it is not yet fully understood which patients will benefit from this or similar measures. Therefore, diagnostic tests are needed to characterize the functional status of the adrenal gland in patients with life-threatening infections.

OBJECTIVES:
Exploring a new diagnostic approach to describe adrenocortical function.

METHODS:
Corticotropin stimulation was performed in patients with sepsis and healthy control subjects. Baseline and stimulated samples were analyzed using an isotope dilution-based LC-MS/MS method for multi-steroid profiling.

RESULTS:
The dynamics of the steroids studied after corticotropin stimulation in healthy and severely ill subjects were complex. A cortisol-to-corticotropin ratio of >32 was associated with an unfavorable outcome.

CONCLUSION:
Serum steroid profiling by LC-MS/MS in samples obtained after corticotropin stimulation allows a differentiated description of adrenocortical function as exemplified in patients with sepsis.

Evaluation and Application of Automated Non-contact Reflectance-based Hematocrit Prediction of Dried Blood Spots Liesl Heughebaert (Presenter) Ghent University

To be presented in Track 1 (Steinbeck 1) on Thursday at 15:15

Introduction: Dried blood spot(s) (DBS) microsampling has increasingly attracted interest as a patient-centric alternative to a conventional blood draw. Due to the advances in liquid-chromatography tandem mass spectrometry (LC-MS/MS) equipment, shorter runtimes are being achieved in combination with increased sensitivity. This enables the quantification of lower drug concentrations in samples with limited amount of blood, as low as a few microliters (1). Despite the advances in the field and the many advantages associated with DBS sampling, its widespread use into clinical practice is still hampered, which is mainly caused by the hematocrit (Hct) effect. An important application of DBS lies in the field of therapeutic drug monitoring (TDM), where patient follow-up can be established trough home-sampling for drugs as immunosuppressants (e.g. tacrolimus, sirolimus, everolimus and cyclosporin A). It has previously been demonstrated that fully automated DBS analysis for immunosuppressant drug monitoring suffers from this Hct effect, mainly due to analysis of a partial DBS punch and extractability differences imposed by blood with different Hcts (2). Fortunately, different approaches to cope with this issue have been developed - amongst which the Hct prediction of DBS using ultraviolet-visible (UV-Vis) spectroscopy - which allow Hct correction based on the DBS-predicted Hct (3).

Objectives: Recently, a UV-Vis-based Hct prediction module has been incorporated into the automated CAMAG® DBS-MS 500 HCT system. However, besides a proof-of-principle, no formal in-depth evaluation of this module, or demonstration of its applicability, has been performed. Hence, the aim of this study was twofold. On the one hand, we performed an in-depth evaluation of this module to establish to what extent automated Hct prediction of DBS via this module can universally be applied and generates acceptable results (4). On the other hand, the validated methodology was used to predict the Hct of a relevant set of venous DBS obtained in the framework of TDM of immunosuppressants. More specifically, to demonstrate applicability, the resulting predicted Hct was used to correct for the Hct effect observed in the automated DBS analysis for these compounds and corrected DBS results were compared with paired whole blood concentrations (5).

Methods: Using calibrators (n = 95) and quality control samples (n = 42) generated from authentic patient samples we set up and validated a calibration model using the automated UV-Vis-based hematocrit predicton module. Additionally, we evaluated whether the validated calibration model could serve as a ‘generic’ model for different, independent Hct prediction modules as this would substantially increase user-friendliness and implementation potential. Finally, 48, 47, 58 and 48 paired venous whole blood and venous DBS patient samples were collected for tacrolimus, sirolimus, everolimus, and cyclosporin A quantification, respectively, and analyzed using an automated DBS-MS 500 HCT extraction unit coupled to an LC-MS/MS. Additionally, for all 201 samples the Hct of the DBS was predicted using the automated UV-Vis based Hct prediction module.

Results: A quadratic calibration curve with 1/x² weighting was established. The bias, intra-day and total precision for 8 different cohorts reflecting Hct sub-ranges from 0.157 to 0.537 L/L were below 0.025 L/L, 2.2% and 2.7%, respectively. A lab-lab comparison of the performance of the Hct module of two independently operated instruments demonstrated that the validated model can be used as a generic calibration model. For tacrolimus and cyclosporin A, UV-Vis-based Hct prediction allowed for adequate correction of the Hct effect. Also for sirolimus and everolimus the results greatly improved after Hct correction, although for both a Hct bias remained. Overall, clinical acceptance limits (i.e. ≥ 80% of the samples within 20% difference compared to whole blood) were met for all analytes, demonstrating the feasibility of fully automated DBS extraction (coupled to LC-MS/MS) in combination with UV-Vis-based Hct prediction from DBS for application in clinical practice.

Conclusion: Automated UV-Vis-based Hct prediction of DBS can universally and reliably generate acceptable results. Moreover, by combining Hct prediction with fully automated DBS extraction and LC-MS/MS analysis, we showed that immunosuppressant concentrations can be adequately corrected, yielding a good agreement with whole blood.

REFERENCES:
1. Deprez S, Heughebaert L, Verougstraete N, Stove V, Verstraete A, Stove C. Automation in microsampling: at your fingertips? In: Siple J, Ehrenfeld E, Lee M, Spooner N, editors. Patient Centric Blood Sampling and Quantitative Bioanalysis. In revision: John Wiley & Sons; 2022.
2. Deprez S, Stove C. Application of a Fully Automated Dried Blood Spot Method for Therapeutic Drug Monitoring of Immunosuppressants. Archives of pathology & laboratory medicine. 2022. DOI: 10.5858/arpa.2021-0533-OA.
3. Capiau S, Wilk LS, De Kesel PMM, Aalders MCG, Stove CP. Correction for the Hematocrit Bias in Dried Blood Spot Analysis Using a Nondestructive, Single-Wavelength Reflectance-Based Hematocrit Prediction Method. Analytical chemistry. 2018;90(3):1795-804.
4. Boffel L, Heughebaert L, Lambrecht S, Luginbuhl M, Stove CP. In-depth evaluation of automated non-contact reflectance-based hematocrit prediction of dried blood spots. The Analyst. 2022;147(23):5445-54.
5. Deprez S, Heughebaert L, Boffel L, Stove CP. Application of non-contact hematocrit prediction technologies to overcome hematocrit effects on immunosuppressants quantification from dried blood spots. Talanta. 2022. DOI: 10.1016/j.talanta.2022.124111.

Feasibility of Routine Therapeutic Drug Monitoring for 5-Fluorouracil. Why Aren’t We Doing It? Peter Galettis (Presenter) University of Newcastle

To be presented in Track 2 (Steinbeck 2) on Thursday at 15:15

Introduction:
In 2019 the International Association of Therapeutic Drug Monitoring and Clinical Toxicology (IATDMCT) produced recommendations for the monitoring of 5-Fluorouracil with a target AUC range of 20-30 mg/L*h. However, the monitoring of 5-Fluorouracil is not practiced routinely in Australia, even though approximately two thirds of patients are dosed incorrectly.

Aims:
We set out to determine the feasibility of providing a routine TDM service for 5-Fluorouracil.

Methods:
We conducted a feasibility study of patients receiving infusions of 5-Fluorouracil greater than 24 hours. Plasma samples were sent to the Clinical Pharmacology Laboratory at the University of Newcastle where they were analysed for 5-Fluorouracil using a validated LC-MSMS. The results of the 5-Fluorouracil analysis were returned to the referring oncologist who then decided whether to adjust the dose of 5-Fluorouracil given to the patient.

Results:
The study recruited 38 patients receiving 5-Fluorouracil infusions, which ranged in age from 33 to 77 years. There were 23 males and 15 females. Of the 38 patients 36 received a 46 hour infusion while 2 received a 96 hour infusion. Only 36% (13/36) of patients were within the 5-Fluorouracil therapeutic range consistent with previous studies. Only 1 patient had an AUC greater than 30, while 61% (22/36) of patients had an AUC less than 20. On a second round of monitoring 4/12 achieved an AUC between 20-30, a further 2/4 achieved the target after 3 rounds of TDM and 2 reached the target after 4 and 5 rounds of TDM. Median time to provide results of the clinician was 5 days from collection of the sample with a range of 2 to 9 days.

Discussion:
Only 36% of patients achieved the therapeutic 5-Fluorouracil AUC of 20-30 with standard treatment, this was increased to 58% after clinicians decided to increase the dose of 5-Fluorouracil after performing TDM. We have shown that routine TDM of 5-Fluorouracil is feasible with a sample turnaround time of under a week providing oncologist with sufficient time to adjust 5-Fluorouracil dose if desired.

Poster Presentations for Tox / TDM / Endocrine

Development of a Sensitive Method for Determination of Vitamin D Metabolites in Urine by LC-MS/MS Kentaro Abe (Presenter) JEOL Ltd

Poster #1a View Map

This poster will be attended on Thursday at 11:00 for 1 hour in the Exhibit Hall.

Background:
Recent epidemiological studies have reported that vitamin D deficiency poses a risk of developing not only bone diseases but also various diseases such as cancer and autoimmune diseases. Serum concentration of 25(OH)D is used as a marker to reflect vitamin D sufficiency. Vitamin D metabolites are excreted in the urine in the form of glucuronidation. The relationship between urinary and blood vitamin D metabolites is not yet well understood.　Quantitative analysis of urinary vitamin D metabolites is important for understanding vitamin D metabolism. Vitamin D metabolites in urine are very low concentration, requiring sensitive measurements. In this study, we aimed to develop LC-MS/MS based method to accurately measure vitamin D metabolites in human urine.

Methods:
The JeoQuantTM Kit for LC-MS/MS analysis of vitamin D metabolites (JEOL, Japan) was slightly modified and used for the quantitative measurement of urinary vitamin D metabolites. Deglucronidation was done using β-Glucuronidase. Urine samples were collected from healthy volunteer subjects (22 subjects, collected on 4 separate days, total n= 88) and stored at −80 °C until their use. A urine sample (300 µL) was added with 50µL β-glucuronidase (from E. coli, Type IX-A, 5000 units) in water, then incubated at 37 °C for 1 h. After incubation, 350 μL of sample was mixed with 50 μL of IS solution, followed by solid liquid extraction (ISOLUTE SLE+: Biotage, Sweden) and derivatized as manufacturers instruction. The pretreated samples were analyzed using an AQUITY UPLC I-Class system coupled to a Xevo TQ-XS mass spectrometer (Waters, USA).

Results:
Glucuronic acid conjugated vitamin D metabolites in 24-hour urine collections were treated with β-glucuronidase and released under optimized reaction conditions. The lower limits of quantification (LOQ) is as follows; 25(OH)D3: 3.4pg/mL, 3-epi-25(OH)D3: 5.9pg/mL, 25(OH)D2: 1.4pg/mL, 24,25(OH)2D3: 5.9pg/mL. In 24-hour urine collections, 25 (OH) D3 and 24,25(OH)2D3 could be quantified, but 3-epi-25(OH) D3 and 25(OH)D2 were less than LOQ. The intra-assay CVs were 10.0% and 8.4% for the low level samples and 3.6% and 4.9% for the high level samples for 25(OH)D3 and 24,25(OH)2D3, respectively. Plasma 25(OH)D3 concentrations correlated very well with urinary 24,25(OH)2D3 concentrations (r= 0.63, n= 88).

Conclusions:
We developed the LC-MS/MS based method using JeoQuantTM for the simultaneous quantification of vitamin D metabolites in urine by the treatment with β-Glucuronidase. This method can be expected to be useful for understanding the urinary excretion of vitamin D metabolism and for assessing the clinical conditions caused by vitamin D deficiency.

Can We Transfer Insulin-like Growth Factor 1 Reference Interval between Mass Spectrometry Assays with Different Traceability? Sally Ezra (Presenter) Alberta Precision Laboratories and University of Calgary

Poster #3a View Map

This poster will be attended on Thursday at 11:00 for 1 hour in the Exhibit Hall.

INTRODUCTION: Insulin-Like Growth factor 1(IGF-1) is a key mediator of growth hormone actions. Accurate assessment of IGF-1 is crucial for diagnosis and monitoring of Growth hormone deficiency and acromegaly. Current immunoassays for IGF-1 are subject to a variety of interferences and pre-analytical sample handling issues, as well as having poor agreement between different platforms. LC-MS/MS assays provide an alternative platform not prone to most analytical interferences associated with immunoassays.

OBJECTIVES: The main objective of this study was to determine the feasibility of transferring IGF-1 reference intervals between two liquid chromatography mass spectrometry assays with traceability distinct reference materials.

METHODS: We developed and validated a quantitative IGF-1 assay traceable to NIST reference standard (2926). As clinical interpretation of IGF-1 results are highly reliant on age-specific reference intervals (RIs), a reference interval transference study was conducted against the liquid chromatography-high resolution mass spectrometry (LC-MS/HRMS) IGF-1 assay that is traceable to WHO (02/254) reference standard. The analytical agreement between the assays was evaluated using the linear model and the appropriateness of the linear model for RIs transference was assessed using Deming regression, correlation coefficients, Q-Q plot, and difference plot and studentized residues for the LC-M/MS against IGF-1 immunoassay and the LC-MS/HRMS IGF-1 assay.

RESULTS: Our study showed a strong correlation (R2>0.93) and agreement (slope=1.006, intercept =negligible) between LC-MS/MS and LC-MS/HRMS regardless of the difference in their traceability and all statistical criteria were met per CLSI guidelines. Although, the LC-MS/MS and the immunoassay showed a strong correlation (R2>0.97, slope=1.055), they failed to meet all statistical criteria for RIs transference due to the bias (-44.91) and non-normal distribution of the residues. Finally, the verification of the transferred RIs showed that 95% of reference samples fell within the transferred RIs, meeting CLSI C28-A3 guidelines.

CONCLUSION: Our method employs calibrators traceable to the recently available NIST reference standard (2926) in comparison to the reference laboratory, which was traceable to the WHO reference material (02/254). Therefore, we provide the first known example of an LC-MS method traceable to the NIST standard and demonstrate sufficient agreement between these methods to allow RI transference.

Naturally Occurring Metabolites Can Inhibit β-Glucuronidases Hydrolysis and Result in False Negatives for Urine Drug Testing Anusha Chaparala (Presenter) Integrated Micro-Chromatography Systems Inc

Poster #5a View Map

This poster will be attended on Thursday at 11:00 for 1 hour in the Exhibit Hall.

Introduction
Urine drug testing is one of the most common practices for monitoring the use of prescribed opioid medications. Testing is typically performed by a preliminary screening assay, such as immunoassay, followed by a confirmatory assay such as liquid chromatography coupled with mass spectrometry (LC-MS/MS). Screening and confirmatory assays benefit from the use of enzymes that hydrolyze, or deconjugate, glucuronidated analytes.
New generation of beta-glucuronidases can now effectively cleave glucuronides in urine at room temperature. However, during our studies, we have identified additional challenges in urine across several patient samples. Endogenous compounds in clinical samples can inhibit these enzymes and compromise hydrolysis. This can make analyzing some patient samples more difficult than others and might lead to false-negatives.

Objective
Present data to show that endogenous metabolites in clinical samples can reduce hydrolysis, and commercially available recombinant enzymes are not inhibited equally.

Methods
Recombinant β-glucuronidases were from IMCS and Kura Biotech. Drug standards were from Cerilliant. Chemicals were purchased from MilliporeSigma and Fisher Scientific. Opioid-positive urine specimens were obtained from a national testing laboratory. Drug free human urine control was from UTAK. Drug free human urine control was fortified with glucuronidated drugs of abuse. Control and patient specimens were buffered and hydrolyzed with two commercially available β-glucuronidases for 15-minutes at room temperature. The two β-glucuronidases were compared by two methods. First, by using an equivalent amount of β-glucuronidases, measured by Bradford Assay, in patient sample hydrolysis. Second, by hydrolyzing patient samples the with a range of β-glucuronidase concentrations.
Hydrolyzed urine samples were cleaned by eluting samples through β-Gone plus plates from Phenomenex and diluted with water. 10 µL of sample was injected on a Thermo Scientific™ Vanquish™ UHPLC system coupled with a Thermo Scientific™ Endura™ Triple Quadrupole Mass Spectrometer using a Phenomenex Kinetex® 2.6 μm Biphenyl 100 Å, 50 x 4.6 mm column. Mobile phase A and B were 0.1% formic acid in water and 0.1% formic acid in acetonitrile, respectively.

Results
Ninety-six patient samples that tested positive for opioids were hydrolyzed with equivalent protein amounts by two different β-glucuronidases. Samples were labeled “positive” if drug free recovery was ≥ 25 ng/mL. One β-glucuronidase consistently reported more positives than the other β-glucuronidase, indicating possible false negatives for the latter.
To determine if enzyme inhibition was involved, four patient samples and a control sample were hydrolyzed with a range of β-glucuronidase amounts (0-100 µg of one β-glucuronidase and 0-200 µg of the second). Hydrolysis was considered complete when < 20% of drug glucuronide was remaining. Several patient samples required more enzyme to complete hydrolysis than the control sample. This indicates that some patient samples may have endogenous inhibitors that can reduce hydrolysis of drug glucuronides.

Conclusion
Some patient samples may contain endogenous urine inhibitors that affect enzyme hydrolysis relative to control samples. Additionally, not all enzymes are inhibited equally by these endogenous molecules.

Direct Quantitation of 77 Therapeutic and Clinical Toxicology Drugs in Dried Blood Spots Using the Fully Automated Transcend DSX-1 System Jingshu Guo (Presenter) Thermo Fisher Scientific

Poster #5b View Map

This poster will be attended on Thursday at 12:30 for 1 hour in the Exhibit Hall.

Introduction:
The dried blood spot (DBS) sampling technique is advantageous over the traditional liquid blood collection due to its minimal invasiveness, smaller sample volume, improved analyte stability, and ease of storage and transportation, resulting in its increasing usage in therapeutic drug monitoring and clinical toxicology. Here, we describe a fully automated workflow to rapidly extract and quantify a wide range of therapeutic drugs and drugs of abuse in DBS using the Thermo Scientific™ Transcend™ DSX-1 UHPLC system, fulfilling routine drug monitoring needs in clinical laboratories.

Method:
Calibrators in blood are spotted on the DBS cards; analytes are extracted from the cards in an automated fashion using innovative flow-through desorption (FTD™) technology, and their corresponding internal standards (IS) are introduced via an automated IS addition module in the dried spot autosampler. Two-dimensional TurboFlow technology allows the removal of interferences from the extracted samples before analytical separation. An integrated software controls every step of the sample desorption and separation. Analyte quantitation is performed on the Thermo Scientific™ TSQ Altis™ Plus triple quadrupole mass spectrometer, and the data is analyzed in Thermo Scientific™ TraceFinder™ software.

Results:
A total of 77 therapeutic and clinical toxicology drugs from 11 compound classes, including anticonvulsants, antidepressants, antihistamines, antipsychotics, benzodiazepines, cocaine, dissociatives, opioids, and stimulants, are quantified in a single injection from DBS cards using a rapid automated method on a Transcend DSX-1 system. The Transcend DSX-1 system combines a dried spot module for direct analyte extraction with a UHPLC for online sample separation using TurboFlow technology. The method only takes 4.3 minutes from analyte extraction to MS detection. Good calibration curves with R2 > 0.98 are achieved using a weighting factor of 1/x, and the limit of quantification (LOQ) values are established with % RSD and % CV < 15, │% Diff│ < 20, and relative ion ratio < %20. The LOQ values are all in the low ng/mL levels, which largely meet the screening and confirmation sensitivity needs of analytical methodologies in routine clinical laboratories.

Conclusion:
Transcend DSX-1 combines a dried spot autosampler and TurboFlow LC-MS/MS and provides a complete workflow for fast and robust quantification of 77 therapeutical drugs and drug-of-abuse analytes in dried blood spots.

Therapeutic Drug Monitoring in Breast Cancer Therapy – Quantification of CDK4/6 Inhibitors by LC-MS/MS Katharina Habler (Presenter) University Hospital, LMU Munich

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Poster #12a View Map

This poster will be attended on Wednesday at 11:00 for 1 hour in the Exhibit Hall.

Introduction
With 1.7 million new cases per year, breast cancer is the third most common cancer worldwide. The cyclin-dependent kinase 4 and 6 (CDK4/6) inhibitors palbociclib, ribociclib, and abemaciclib were approved in recent years by the U.S. Food and Drug Administration (FDA) and European Medicine Agency (EMA) for the treatment of breast cancer. Generally, a fixed dosing regimen of oral tumor therapeutics (OTT) is used. However, several described side effects and pharmacokinetic interactions (CYP3A4 inducers and inhibitors) may affect appropriate drug levels and therapeutic success. OTT extend the options for cancer therapy in the home environment, but also pose challenges for physicians and patients.

Objectives
The aim of our work was the development of a robust liquid-chromatography tandem mass spectrometry (LC-MS/MS) method for all previously approved CDK4/6 inhibitors and to verify adequate serum levels of OTT in routine diagnostics.

Methods
We developed a semi-automated LC-MS/MS method for the simultaneous quantification of abemaciclib, its active metabolites abemaciclib M20 and M2, palbociclib, and ribociclib in human serum. Detuning and measurement of a natural isotopologue of ribociclib enabled measurement in the respective relevant concentration ranges. For chromatographic separation a biphenyl column and acidified mobile phases were used. The method was validated according to the guidance of the FDA and applied to authentic samples.

Results
The LC-MS/MS method was successfully validated according to FDA and showed inaccuracies ≤10.7% and imprecisions ≤8.51%. To demonstrate the applicability of the method, authentic samples were also analyzed of a single individual treated with abemaciclib under a compassionate use authorization. We also assessed pharmacokinetic data of abemaciclib in human serum.

Conclusion
This semi-automated LC-MS/MS method covers all previously approved CDK4/6 inhibitors as well as the similarly pharmacologically active metabolites in human serum simultaneously and was developed for potential future use in routine clinical testing.

Application of LC-MS/MS to Assess the Chemosensitivity of Breast Cancer Spheroids for an in vitro to in vivo Extrapolation Analysis Ramisa Fariha (Presenter) Brown University

Poster #15a View Map

This poster will be attended on Thursday at 11:00 for 1 hour in the Exhibit Hall.

Introduction:
While 2D in vitro cell culture models have been used over the years, 3D cell culture technique is becoming increasingly popular due to its ability to replicate the in vivo tumor environment better. Out of the existing 3D cell culture techniques, MicrotissuesTM has garnered attention due to its scaffold-free nature and high-throughput ability. However, traditional cell culture assessment techniques are unable to fully characterize this 3D platform due to limitations of quantifying trace amounts of absorption taking place within these molds.

Objectives:
Our study expands the use of LC-MS/MS to quantify the absorption of Paclitaxel, a chemotherapeutic, by the 3D MicrotissuesTM molds, and its subsequent impact on MCF7 breast cancer tissue viability.

Methods:
MicrotissuesTM molds were made using 2% agarose and MCF7 cells were allowed to self-assemble to tissue spheroids over DIV= 3. Drug adhesion was initially monitored for the empty wells and molds. Following the initial observation trend and drug absorption, the spheroids were treated with the actual concentration (measured after absorption by mold) versus the concentration it should have been receiving for 12 and 24-hours intervals to assess the impact of the minute change on cellular viability.

Results:
By optimizing the sample preparation technique, our strategy utilizes cell culture media to accurately quantify the drug adhesion to the MicrotissuesTM molds, with LoD= 0.03 μM, optimized for cellular microenvironment with relevance for extrapolation to patient application. The overall assay exhibits linearity of R2>0.99 inclusive of both MRMs, and a percent coefficient of variation that is less than or equal to 10%. We have tested and validated the findings against a traditional live-dead assay for the spheroids. In addition to manual plating with cell culture supernatant testing, we have exhibited the adaptability of our protocol on the JANUS G3 liquid handling workstation for rapid sample preparation, in addition to the fast three minutes total run time per sample analysis. This makes our protocol adaptable for clinical usage, expanding its application to personalized cancer therapies.

Conclusion:
Overall, our work plays an important role in the in vitro to in vivo extrapolation study of chemotherapeutics for breast cancer and how LC-MS/MS can provide a viable and highly sensitive and quantitative method for analysis.

Quantitation of Clinical Research Steroid Analytes from Blood Serum Utilizing Solid Phase Extraction Paired with LC-MS/MS Mackenzie Freige (Presenter) Phenomenex

Poster #15b View Map

This poster will be attended on Thursday at 12:30 for 1 hour in the Exhibit Hall.

Quantitation of Clinical Research Steroid Analytes From Blood Serum Utilizing Solid Phase Extraction Paired With LC-MS/MS

INTRODUCTION: Steroid analysis for clinical research can require very low limits of detection which demand high recoveries from solid phase extraction (SPE) and very clean extracted samples. Labs desire high-throughput methods which consolidate analytes into one panel with fast chromatographic run times. Accurate quantitation makes it necessary to chromatographically separate steroids with the same m/z. Meeting these criteria can be challenging in a single LC-MS/MS method.

OBJECTIVES: Here, we present an effective sample cleanup method and LC-MS/MS analysis method for the quantitation of an 18-analyte steroid panel from serum.

METHODS: The (SPE) technique utilized a reversed phase Strata-X 30mg 96-well plate (Phenomenex, Torrance CA). A Core-Shell 2.6µm, 50x3 mm Kinetex C18 column (Phenomenex) was used for fast chromatographic separation. Detection employed a 7500 Triple Quadrupole LC-MS/MS equipped with an ESI source (Sciex, Framingham, MA).

RESULTS: Linearity is demonstrated for all analytes with an R squared value >0.992. The calibration curves prepared in steroid-free blood serum range from 10 pg/mL to 500 ng/mL, subject to unique cutoff requirements of the analytes of interest. LLOQ values range from 10 pg/mL to 5 ng/mL. The QC samples for 3 replicate extractions at 3 different levels showed relative standard deviation (RSD) below 15% and accuracy between 80-120%. The LC method with the use of a C18 column achieves chromatographic separation of all isomers with an 8-minute method.

CONCLUSION: The proposed sample prep method utilizing a 96-well plate SPE extraction and fast LC method resulted in a simple, rapid cleanup for identification and quantitation of the tested steroid panel analytes from blood serum. The SPE extraction method was robust, reproducible, and can be automated. This makes it a reliable method that combines accurate quantitation with high throughput.

LC-MS Assay for Antibiotics in Intensive Care Unit Clinical Setting Dennis Lee (Presenter) Segal Cancer Proteomics Centre, Lady Davis Institute for Medical Research, Jewish General Hospital, Mcgill University, Montreal, Quebec, Canada

Poster #20b View Map

This poster will be attended on Wednesday at 12:30 for 1 hour in the Exhibit Hall.

INTRODUCTION: Adequate antibiotic dosage is a prerequisite for the successful treatment of bacterial infections. The pharmacokinetics of antibacterial drugs in critically ill patients can be dramatically altered by multiple factors such as large volume infusions, organ dysfunction, attenuated renal elimination, hypoproteinemia, and drug interactions. Therapeutic drug monitoring (TDM) of antibacterials can be used to optimize antimicrobial therapy regimens and improve clinical outcome. β-lactam antibiotics are the most commonly used treatments for bacterial infections in intensive care units (ICUs). Thus, there is an interest in determining the plasma concentrations of meropenem and piperacillin/tazobactam to assess the potential added value of using TDM in an ICU setting.

OBJECTIVES: Here, our goal was to develop and validate a clinical liquid chromatography mass spectrometry (LC-MS) assay for the quantitation of meropenem and piperacillin/tazobactam which are commonly used in ICUs.

METHODS: Patient plasma samples were mixed with isotopically labeled internal standards in acetonitrile, centrifuged, and an aliquot of the supernatant was diluted with deionized water and used for injection. LC-multiple reaction monitoring (MRM)-MS analysis was performed on a Shimadzu Nexera XR UHPLC system coupled to the Sciex QTrap 6500+ mass spectrometer operating in the positive ion mode. The column oven temperature was maintained at 40°C. Chromatographic separation was done using a Zorbax Rapid Resolution High Definition Eclipse Plus C18 reversed phase column (2.1 x 50 mm, 1.8-Micron, Agilent). Mobile phases were 0.1% formic acid in water and 0.1% formic acid in acetonitrile. A gradient of 5-95% B over 3 minutes (with a total run time of 6 minutes) was used. The quantitation was performed based on the chromatographic peak area ratios between the analytes and the corresponding internal standards, using Analyst software (Sciex).

RESULTS: A multiplexed assay was developed, capable of simultaneously measuring meropenem, piperacillin, and tazobactam β-lactams in human plasma samples. The method is currently undergoing validation based on the CLSI guidelines, including accuracy/trueness, imprecision, sensitivity, specificity, carryover, stability, and linearity. Preliminary validation results already indicate that the method will successfully meet the required criteria. Several batches of 10-20 ICU patient samples have been analyzed. The method allows the measurement of the plasma concentrations of these antibiotics with a short turnaround time. Overall analysis time from receiving samples to the reporting values was about 1.5 hours for a batch of 10 samples. The turnaround time obtained is satisfactory for the timely adjustment of ICU-patient treatment.

CONCLUSION: LC-MS method for the determination of meropenem and piperacillin/tazobactam antibiotics was established and used for the analysis of ICU patient samples. The analysis has a short turnaround time and is applicable for clinical testing in ICU settings.

Development and Validation of an LC-MS/MS Assay for Quantification of Glecapravir/Pibrentasvir in Human Plasma Kyana Garza (Presenter) Johns Hopkins University School of Medicine

Poster #21a View Map

This poster will be attended on Thursday at 11:00 for 1 hour in the Exhibit Hall.

Introduction:
The global prevalence of chronic hepatitis C virus (HCV) infection is 58 million. Untreated chronic HCV infections can lead to serious health complications, including liver cirrhosis, damage, and disease. Direct acting antiviral (DAA) therapies for HCV infections are effective in the treatment and management of chronic infections. Glecapravir (GLE) and pibrentasvir (PIB) are DAAs that may be delivered alone or as a fixed-dose oral formulation to treat adolescent and adult patients with chronic HCV infections (genotypes 1-6) with or without cirrhosis. Here, we describe a liquid chromatographic-tandem mass spectrometric (LC-MS/MS) assay for the multiplexed quantification of GLE and PIB in human plasma, with future applications in clinical trials to evaluate the pharmacokinetics of intramuscularly delivered GLE/PIB.

Methods:
Calibrators were prepared in human K2EDTA plasma at final concentrations ranging from 0.25-2,000 ng/mL GLE and 0.25-1,000 ng/mL PIB. Samples at the lower limit of quantification (LLOQ), low, mid, high, and dilution quality control (QC) levels were prepared at 0.25, 0.75, 800, 1800, and 20,000 ng/mL for GLE and 0.25, 0.75, 400, 800 ng/mL, and 10,000 ng/mL for PIB, respectively.

Plasma samples containing GLE and PIB were combined with isotopically labeled internal standards and subjected to protein precipitation. Samples were evaporated to dryness, reconstituted in 50:50 mobile phase A (MPA, 0.1% formic acid in water): mobile phase B (MPB, 0.1% formic acid in 80:20 acetonitrile:water) and subjected to LC-MS/MS analysis.

Chromatographic separation and analyte quantification occurred on an API 6500+ (Sciex, Foster City, CA) interfaced with an LC-30 (Shimadzu, Kyoto, Japan) operated in positive ionization and selective reaction monitoring modes. Chromatographic separation occurred using a Zorbax Eclipse C18 column under a gradient elution from MPA to MPB. Transitions monitored for analytes of interest were as follows: GLE (839.4 -> 684.3 m/z), PIB (557.5 -> 871.4 m/z), and their internal standards (GLE -IS 843.5 -> 684.3 m/z; PIB-IS 561.5 -> 629.3 m/z).

The LC-MS/MS assay was validated in accordance with Food and Drug Administration Bioanalytical Method Validation Guidance for Industry recommendations.

Results:
Inter-assay precision and accuracy ranged from 4.34% to 15.6% and -4.90% to 10.6% for GLE and 3.20% to 9.80% and -7.48% to -1.46% for PIB, respectively. GLE and PIB in plasma are stable following six freeze thaw cycles at <-70°C, with a percent difference (%DIF) ≤±10.8% for GLE and ≤±12.4 for PIB. Additionally, GLE and PIB are stable at room temperature for up to 67 hours and demonstrated re-injection stability at 4°C for up to 101 hours (GLE: %DIF ≤±4.1%; PIB %DIF: ≤±7.8%). Matrix effects were assessed quantitatively; negligible percent matrix effects were observed for PIB (106%) and PIB-IS (111%) across the measuring range of the assay. Significant ion suppression was observed for GLE (59.9%) and GLE-IS (63.9%); however, relative matrix effects were <5% between drug and internal standard and deemed acceptable. Other assay validation assessments in alternative matrices were also deemed acceptable.

Conclusions:
An LC-MS/MS method for GLE and PIB quantification in plasma has been developed and validated and may be used in downstream applications to characterize DAA pharmacology for HCV treatment.

Think Fast: Intelligent Reflex Fast Screening for Alcohol Metabolites in Urine Jennifer Hitchcock (Presenter) Agilent

Poster #26b View Map

This poster will be attended on Wednesday at 12:30 for 1 hour in the Exhibit Hall.

Introduction:
Drug testing labs typically utilize a screening method to look for presumptive positives prior to quantitative confirmation; this process can take hours and will often require multiple aliquots and preparations of the sample. However, using a mass spec-based fast screening method followed by a longer, chromatographic separation for quantitation minimizes bottlenecks from sample prep, while the intelligence built into the mass spectrometer allows for more automated and streamlined runs, as only the samples marked as presumptive positives are analyzed using the longer confirmation method. This data-dependent workflow aims to shorten batch analysis when positive hits are considered infrequent, as is the case with many of the negative mode drug compounds.

Objectives:
The primary objective of this work was to demonstrate a novel workflow for the screening and confirmation of alcohol metabolites and barbiturates in urine using intelligent reflexive logic.

Methods:
Synthetic urine was spiked with a standard mix containing various concentrations of barbiturates, ethyl glucuronide (EtG), and ethyl sulfate (EtS). Both the screening and confirmation methods were performed using an Agilent Poroshell 120 CS-C18 column (2.1x50 mm, 2.7 µm) with a water:methanol mixture containing 0.01% formic acid. The fast screening method was performed in under two minutes, while the confirmation method ran a slower gradient to achieve chromatographic separation in under five minutes. LC-MS/MS analyses was performed in negative ionization mode and multiple reaction monitoring (MRM) of two transitions per analyte. Data was acquired using an Ultivo LC/TQ with a 1290 Infinity II LC stack, and the Intelligent Reflex Fast Screening workflow was activated in MassHunter 12.

Results:
The fast screening workflow consists of two methods: “Tier 1 Method”—a fast semi-chromatographic method for quick screening and “Tier 2 Method”—a comprehensive chromatographic method for confirmation of presumptive positives and quantitation. The screening method was less than two minutes and monitored all transitions for the compounds of interest. It was intended to mark a positive hit as a presumptive positive for further analysis. The confirmation method made use of a shallower, slower gradient to achieve chromatographic separation with baseline resolution for quantitative analysis. Each acquisition method has a quantitative method tied to it for automated processing. The quantitative method for screening sets the minimum detection threshold to trigger a presumptive positive and reflexive confirmation. Upon the marking of a presumptive positive, the sample vial was automatically appended to the bottom of the worklist so that all screened positive samples were reinjected using the longer confirmation method.

Conclusion:
Two simple analytical methods were developed for the screening and confirmation of alcohol metabolites (EtG and EtS) and barbiturates in urine. The screening method achieved results in under two minutes, while the confirmation method demonstrated excellent separation and baseline resolution of the compounds of interest. These methods were paired and, with the intelligence built into the mass spectrometer, used to automate a reflexive workflow for the screening and confirmation of these analytes in urine with no user intervention.

∆8-THC by GC-MS: Unresolved Ion Ratio Failure Investigation Mark Girton (Presenter) University of Virginia

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Poster #32b View Map

This poster will be presented and discussed on Thursday at 12:00 for 15 minutes in De Anza 2 (right outside Exhibit Hall doors across from Ionpath in Booth 36).

ABSTRACT SUMMARY:
In October 2022, our laboratory began GC-MS validation experiments to add the ∆8-THC metabolite 11-nor-9-carboxy-∆8-THC (∆8-THCCOOH) on to our existing ∆9-THC urine confirmation assay (metabolite 11-nor-9-carboxy-∆9-THC (∆9-THCCOOH)). Frequent ion ratio failures for ∆8-THCCOOH have become an obstacle. This was initially suspected to be due to a shared transition peak overlap at 488 in the context of high quantities of ∆9-THCCOOH. We were also seeing this issue more frequently on the Agilent 5975C than on the Agilent 5977A, but the latter instrument has been down for maintenance during the month of January 2023.

PROBLEM:
A clinical validation for urine ∆8-THCCOOH by GC-MS is stalled as there are frequent ion ratio failures. While we initially suspected interference from high ∆9-THCCOOH given the shared qualifier transition, further investigation demonstrated a more widespread issue, including both qualifier ions, that has no known systemic cause at this time.

METHOD INFORMATION:
Base hydrolysis:
&bull; 1.0 mL patient urine 50 &micro;L internal standard undergo alkaline hydrolysis with 200 &micro;L 10 M KOH
&bull; Vortex; incubate at 60&deg;C for 20 minutes; cool 5-10 minutes at ambient temperature
&bull; Add 1.5 mL glacial acetic acid and vortex
Extraction:
&bull; Condition 10 mL UCT THC Clean Screen cartridges on positive pressure manifold
&bull; Pour into column reservoir, slowly increase flow (1-2 mL/min)
&bull; Add 3 mL DI H2O; 2 mL THC Wash Solution (100 mM HCl with 5% acetonitrile); air dry 5 min; 200 uL hexane
&bull; Elute with 2 mL 1:1 hexane and ethyl acetate
&bull; Evaporate under nitrogen gas (LABCONCO RapidVap Vertex Evaporator) at 12 psi and dry bath at 60&deg;C for approximately 30 min
Derivatization:
&bull; Add 100 &mu;L BTSFA with 1% TMCS to the dry residue; cap
&bull; Heat at 70 &deg;C for 30 minutes; cool 5-10 min; transfer to vials with micro inserts, crimp seal
GC-MS Analysis:
&bull; Confirmation analysis is using the Agilent MSD system with THC_ACQ.m program. The selected ion monitoring (SIM) mode analyzes for the following ions (m/z) for ∆9-THCCOOH: 371, 473, 488. For deuterated ∆9-THCCOOH: 374, 476, 491. For ∆8-THCCOOH: 303, 432, 488.
&bull; 1 uL aliquots of derivatized samples are injected onto the column by the autosampler
&bull; Agilent Technologies 15m HP-5MS, 0.25mm i.d., 0.25 &mu;m film thickness fused silica capillary column
&bull; GC oven temp. program: 175&deg;C isothermal for 1 minute, then increase at 25&deg;C /minute to 280&deg;C, held at 280&deg; for 2.5 minutes on the MSD 5975 and 175&deg;C increasing at 25&deg;C/min to 280&deg;C, held at 280&deg;C for 2.5 minutes on the MSD 5977
&bull; Helium is used as the carrier gas
&bull; Mass Spec Conditions: Positive Ion Electron Impact (EI).
&bull; Transfer line temp. = 280&deg;C on MSD 5975 and 310&deg;C on MSD 5977.
&bull; MS Source temp. = 230&deg;C for MSD 5975 and 300&deg;C for MSD 5977
&bull; Tune file= Atune.u on MSD 5975 and etune.u on MSD 5977.
&bull; Electron multiplier settings vary with instrument&rsquo;s condition and design.

TROUBLESHOOTING STEPS:
Data analysis (n=304) is ongoing to identify result and parameter characteristics leading to ion ratio failure. Ion ratios are considered acceptable if within +/-15% of an appropriate reference material ion ratio. Overall, there were 117 specimens positive for ∆8-THCCOOH. 58 (49.6%) had acceptable ion ratios, 30 (25.6%) had failure at the 432 transition only, 14 (12.0%) had failure at the 488 transition only, and 15 (12.8%) failed at both transitions. For 20 specimens negative for ∆9-THCCOOH and positive for ∆8-THCCOOH, 14 (70%) have acceptable ∆8-THCCOOH ion ratios for both qualifiers. There are 70 cases positive for both analytes with 25 (37.1%) having ∆8-THCCOOH ion ratio failures. In some cases, high ∆8-THCCOOH results in ion ratio failure, and this is appreciated on chromatography review as broadened peaks with slightly delayed acquisition time. The majority of ion ratio failures do not demonstrate a clear pattern. Ion ratio failures are seen throughout the analytical measurement range for both ∆9-THCCOOH and ∆8-THCCOOH. Occasional cases have signal from unidentified substances which may cause interference, but this does not appear to account for the large majority of ion ratio failures.

OUTCOME:
The Agilent 5977A GC-MS is now operational as of February 1, 2023. This will allow us to perform instrument comparisons with the Agilent 5975C to attempt to resolve issues with unexplained ∆8-THCCOOH ion ratio failures. We will also continue to perform data analysis and chromatography review. Unfortunately, with an approximate 50% ion ratio failure rate, our validation study has been extended as troubleshooting continues.

The Price of Exclusion: What Gets Lost When We Don’t Hydrolyze Urine and Ignore Sulfatase? Ahmed Najar (Presenter) UCSF

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Poster #35b View Map

This poster will be attended on Thursday at 12:30 for 1 hour in the Exhibit Hall.

Introduction:
Many toxicology LC-MS/MS panels have been streamlined by the “dilute and shoot” method for ease of use and the capacity to see both drugs and their conjugates. Labs that hydrolyze tend to only use β-glucuronidase, however drug metabolites can be present as glucuronides and sulfates.
Objective: To evaluate what drug metabolites are missed when using a dilute-and-shoot method for comprehensive drug testing in the clinical setting.

Methods:
Sixty patient samples that tested positive for drugs by routine toxicology testing in a clinical laboratory were selected for evaluation. After hydrolysis optimization, the samples were run by LC-HRMS unhydrolyzed (Neat) and hydrolyzed separately with β-glucuronidase and aryl-sulfatase (IMCSzyme®). Briefly, a hydrolysis mix containing enzyme and buffer was added to an equal volume of urine and left to hydrolyze for 60 min at room temperature, though 15 min is enough for the glucuronidase. The hydrolysis mix was then mixed in acetonitrile (3:1) to precipitate the enzyme. After centrifugation, an aliquot of the supernatant was then diluted 5x in mobile phase A. Subsequently, samples were analyzed using the comprehensive drug test by LC-HRMS in the clinical laboratory at San Francisco General Hospital. A Phenomenex Kinetix method C18 (50x3 mm, 2.6µm) was used for the separation with a 15 min gradient of 5 mM ammonium Formate, 0.05% formic acid as MPA and a mix of methanol and acetonitrile (1:1) with 0.05% formic acid as MPB. Data was acquired on ABSciex TripleTOF®5600 in positive-ion HRMS full scan mode with IDA triggered acquisition of HRMS product ion spectra was used for mass detection. PeakView® (AB Sciex) were used for targeted data analysis where an in house library of 274 drugs and metabolites.

Results:
Across all patient samples, 65 drugs and their metabolites were identified. Samples differed quantitatively and qualitatively for certain compounds. Notably THC metabolites and buprenorphine were only seen in the β-glucuronidase hydrolyzed samples, whereas MDMA metabolites were exclusive in sulfatase digests. We also saw the patient-to-patient variability characteristic of enzymatic digestion in other compound classes. There were no exclusive compounds in the neat samples.

Conclusion:
A combined hydrolysis could ensure a larger capture of some drug metabolites which could make up for the need of running the non-hydrolyzed sample and seeing both drugs and conjugates.

The Study of Three HPLC Column Chemistries for Optimal Separation of THC Isomers Haley Berkland (Presenter) Restek

Poster #36a View Map

This poster will be attended on Wednesday at 11:00 for 1 hour in the Exhibit Hall.

INTRODUCTION: The emergence of Δ9-tetrahydrocannabinol (Δ9-THC) isomers, particularly Δ8- tetrahydrocannabinol (Δ8-THC), have created analytical challenges as they are often not easily resolved by traditional chromatographic methodologies. When consumed, Δ9-THC forms the metabolite 11-Nor-9-carboxy-Δ9-tetrahydrocannabinol (Δ9-THCCOOH). Similarly, Δ8-THC is metabolized to 11-Nor-9-carboxy-Δ8-tetrahydrocannabinol (Δ8-THCCOOH). Traditional methods for separating Δ8-THC and Δ9-THC do not adequately resolve these metabolites, resulting in quantitation issues and the inability to determine an accurate value for one or both isomers. This issue is especially prevalent in urine samples, where these metabolites may be detected at high concentrations.

OBJECTIVES: The primary objective of this study was to evaluate the capability of different HPLC column chemistries to separate Δ8-THCCOOH and Δ9-THCCOOH.

METHODS: Multiple LC-MS/MS methods were developed and optimized to separate Δ8-THCCOOH and Δ9-THCCOOH using a Raptor Biphenyl (2.7 µm, 100 x 2.1 mm), Raptor C18 (2.7 µm, 100 x 2.1 mm), and a Raptor FluoroPhenyl (2.7 µm, 100 x 2.1 mm). Each method used water and methanol as MPA and MPB respectively, both acidified with 0.1% formic acid.

RESULTS: The Raptor Biphenyl did not provide any separation of these isomers under the conditions tested. Utilizing the Raptor C18 column, partial separation of the isomers was obtained in a 7-minute method but was unable to achieve complete resolution. By extending the method to 19 minutes, greater separation was achieved, but the isomers were still unable to be completely resolved. Using the Raptor FluoroPhenyl column, complete resolution of the isomers was achieved in a 7-minute method cycle time. Results were confirmed by analysis of spiked urine samples.

CONCLUSIONS: Of the three stationary phases tested, the Raptor FluoroPhenyl column chemistry provided optimal separation of Δ8-THCCOOH and Δ9-THCCOOH in the shortest analysis time. The separation completely resolved the isomers, preventing quantitation errors caused by the analytes interfering with each other.

The Development of a Virtual Liquid Chromatography Method Development Tool Melinda Urich (Presenter) Restek Corporation

Poster #38a View Map

This poster will be attended on Wednesday at 11:00 for 1 hour in the Exhibit Hall.

INTRODUCTION: The development and optimization of liquid chromatography (LC) separations can be time consuming and costly, often requiring a number of steps including literature research, column selection, method scouting, method development, and method optimization. In an effort to eliminate these steps, an instrument-free, software modeling tool that gives users the ability to select compounds from a database and instantly model a separation on different column phases was developed. Optimization of the model can be performed while maintaining critical pair separations by adjusting for instrument/system effects (e.g. dwell volume and extra column volume), mobile phase preferences, number of gradient steps, and more. The modeler delivers a fast, no-cost starting point. The initial database consists of a Drugs of Abuse (DoA) library containing approximately 250 compounds with plans to continually expand the utility.

OBJECTIVES: To develop a chromatogram modeling tool that allows users to develop and optimize their LC methods virtually, improving data quality and laboratory efficiency without time-consuming in-lab method development.

METHOD: To build the chromatogram modeler, a DoA library containing approximately 250 compounds was created.

Retention times were first collected using a fast/slow gradient, 30°C/60°C temperature points, and ACN/MeOH mobile phases on a single column dimension. Some additional data points outside of these runs were also collected for the development of a semi-empirical correction factor that was used to improve modeling accuracy.

To assess the accuracy of the modeler, experiments comparing compound retention time values between wet-lab and modeled data were conducted. After the initial DoA library was built, the modeler was evaluated over four increasingly more complex stages of verification. In the final, most complex stage, new compounds not previously part of the initial DoA library were added and then compared by testing two different column dimensions, two different columns lengths, two different mobile phases, two different stationary phases, three different gradients programs, and three different temperatures against modeled retention time values. Because the semi-empirical correction factor was developed using only the original library compounds, this stage assessed the viability of adding future compounds to existing libraries.

RESULTS: An online chromatogram modeling tool was successfully developed that allows users to select columns and compounds for separation. A modeled chromatogram and instrument-ready conditions are automatically generated and can be further optimized by users. During software development, the acceptance criteria for retention time agreement between wet-lab and modeled values was set at +/- 15 seconds. This range was chosen because it represents a typical MRM window. In the most complex portion of the verification, 704 retention time data points were collected in total for the 25 compounds used in the evaluation. Only 13 data points exceeded the +/- 15 second window with no compounds missing acceptance criteria by more than five seconds, giving an overall pass rate of 98.2%.

CONCLUSION: For LC method developers, novice and expert, who either lack the expertise, or the time, to develop separations quickly and accurately, this free tool can be used to deliver a fast, no-cost starting point for method development and optimization. This novel, virtual method development software can improve turnaround time, increase throughput to existing methods, and offer an on-demand consultative user experience.

Mass Spectrometry Quantitation of Immunosuppressive Drugs in Clinical Specimens Using Accurate-Mass Full Scan-Single Ion Monitoring Priscilla Yeung (Presenter) Stanford University Department of Pathology

Poster #40a View Map

This poster will be attended on Wednesday at 11:00 for 1 hour in the Exhibit Hall.

INTRODUCTION: Therapeutic drug monitoring (TDM) of immunosuppressive drugs including tacrolimus, sirolimus, everolimus, and cyclosporine A is a critical part of optimizing clinical outcomes for solid organ transplant patients. The most commonly used methodologies for TDM are immunoassays and liquid chromatography-mass spectrometry (LC-MS). Although immunoassays have shorter turnaround times and fewer trained personnel requirements, they can suffer from interference from heterophile antibodies and similar compounds such as other drugs from the same class or metabolites. Current mass spectrometry assays based on multiple reaction monitoring (MRM) can be tedious to optimize and are limited in the number of compounds that can be detected in each run.

OBJECTIVES: The purpose of this study was to implement a mass spectrometry-based test for immunosuppressant TDM using accurate-mass full scan-single ion monitoring (FS-SIM) and online solid phase extraction (SPE).

METHODS: LC-MS analysis was performed on a TLX-2 multi-channel HPLC with a Q-Exactive Plus mass spectrometer. Mobile phase A contained 10 mM ammonium formate in water with 0.1% formic acid and mobile phase B was methanol. A TurboFlow online SPE column was used for sample clean up prior to loading onto the analytical C18 column. The accurate-mass MS was set to positive electrospray ionization mode with FS-SIM for quantification of tacrolimus, sirolimus, everolimus, and cyclosporine A. During the LC-MS analysis, Fragmentation of each analyte was performed using high-energy C-trap dissociation (HCD) for compound confirmation.

RESULTS: The method was validated according to the guidelines for laboratory-developed tests. Within-run and between-run imprecision, analytical bias, limit of quantitation, analytical measurement range, and linearity were all within acceptable limits. Quantification of tacrolimus, sirolimus, everolimus, and cyclosporine A correlated closely with the results from Mayo Clinic Laboratories with R2 = 0.90-0.96. Tacrolimus results also had excellent correlation with Roche immunoassay with R2 = 0.97. Several major metabolites of the four drugs were able to be observed within the LC-MS method.

CONCLUSIONS: Accurate-mass FS-SIM can be utilized for immunosuppressant TDM with tight correlation with results generated by standard methods. Turboflow online SPE allows for a simple “protein crash and shoot” sample preparation protocol. Compared with traditional MRM, analyte quantification by FS-SIM has a more streamlined assay optimization process and allows for detection of more drug metabolites within the same run.

Development and Validation of an Ultra-Performance LC-MS/MS Method for Quantitation of Serum C-peptide Sung-Eun Cho (Presenter) GCLabs ESAC

Poster #42b View Map

This poster will be attended on Wednesday at 12:30 for 1 hour in the Exhibit Hall.

INTRODUCTION : Mass spectrometric methods exhibit higher accuracy and lower variability than immunoassays for measuring serum C-peptide. We developed and validated an ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) assay for measuring serurm C-peptide. We analyzed C-peptide in multiple charge [M+3H]3+, because of its large molecular weight, which could not be analyzed by single charge. We also used derivatization step using 6-aminoquinolyl-N-hydroxysuccinimidylcarbamate (AQC, Cayman Chemical, USA) to increase the ionization efficiency.

METHODS : For the pretreatment step, we first performed the solid phase extraction using Sep-Pak tC18 96-well plate, 100 mg solvent per well, 37-55&micro;m particle size (Waters, USA), and then performed the ion exchange solid phase extraction using Oasis MCX 96-well Plate, 30 mg solvent per well, 30&micro;m particle size (Waters, USA), finally we performed the derivatization step using 1 mg/mL AQC (Cayman Chemical, USA).
We used ExionLC UPLC (SCIEX, USA) system. We used Capcell Pak C18 ACR 2.0 mm &times; 10 mm, 3 &mu;m (OSAKA SODA, Japan) as the Guard Cartridge Column, and we used Capcell Pak C18 ACR, 2.0 mm &times; 150 mm, 3 &mu;m (OSAKA SODA, Japan) column. The total run time was 50 minutes and flow rate was 0.20 mL/min. The LC condition is as belows; 0.1% Formic acid in water for Mobile phase A and 0.1% Formic acid in Acetonitrile for Mobile phase B. The gradient extraction with 0 minute of A85/B15 to 35 minutes of A70/B30 was performed.
We used SCIEX Triple Quad 6500+ (SCIEX, USA) MS/MS in electrospray ionization and positive ion modes with multiple reaction monitoring transitions and 40 Psi of Curtain Gas, 11 Psi of Collision Gas, 5000 Ion Spray Voltage, 400&deg;C of Temperature, etc. The MS conditions of C-peptide and Internal standard are as belows; Q1 Mass 1064.262 Da, Q3 Mass 171.2 Da, 300 msec of Dwell Time, 71 volts of Deculstering Potential (DP), 10 volts of Exit Potential (EP), 169 volts of Collision Energy (CE), 12 volts of Collision Cell Exit Potential (CXP), and for Internal standard which is D8-Val7,10-C-Peptide (Bachem, Switzerland), Q1 Mass 1069.915 Da, Q3 Mass 171.2 Da, 300 msec of Dwell Time, 76 volts of DP, 10 volts of EP, 159 volts of CE, 16 volts of CXP, respectively.

We evaluated precision, accuracy using NMIJ CRM 6901-C (Wako, Japan), linearity, limit of detection (LOD), limit of quantitation (LOQ), carryover, ion suppression.

RESULTS : The intra- and inter-run precision CVs were less than 7 %, and the accuracy bias values were less than 7 %, which were all acceptable. The verified linear interval was 0.05-15 ng/mL, the LLOQ was 0.05 ng/mL. No significant carryover and ion suppression were observed.

CONCLUSION: The UPLC-MS/MS assay showed acceptable performance for measuring serum C-peptide. We will compare the data using this method with those from immunoassay. Even though this method would be difficult to be introduced as the actual clinical test method because of its long performance time and complex sample treatment method, this might be able to be used in a limited way for the purpose of accurate measurement of serum C-peptide.

Analytical Method for the Separation of Isobaric Isomers of Delta 8, 9 and 10 THC and Carboxy THC Metabolites Spiked into in Synthetic Urine Utilizing LC-MS TQ Andrzej Szczesniewski (Presenter) Agilent Technologies

Poster #43a View Map

This poster will be attended on Thursday at 11:00 for 1 hour in the Exhibit Hall.

Background:
This work outlines a highly sensitive, specific, and fast LC/MS/MS analytical method that was developed for the separation and quantitation if isobaric isomer of delta 8, 9 and 10 THC and carboxy THC. The described analytical method achieves high analytical sensitivity and is capable of quantitating analytes over a wide dynamic range.

Methods:
An Agilent 6495 triple quadrupole mass spectrometer with Jet Stream technology in positive electrospray mode and an Agilent Infinity II 1290 UHPLC system were utilized for this analysis. Standards spiked into urine were used for the analysis isobaric isomers. Various columns were evaluated and a Poroshell 120 EC-C18 100 x 2.1 mm, 1.9 um with a water:methanol mixture containing 0.01% Formic Acid and 5 mM Ammonium Formate gradient achieved chromatographic separation in less than 8.0 minute gradient. Quantitative analysis was performed using multiple reaction monitoring (MRM) transition pairs for each analyte and internal standards.

Results:
Good linearity and reproducibility were obtained with a concentration range from 100 pg/ml to 1000 ng/ml for all analytes with a coefficient of variation &gt;0.995 for all sample preparation and chromatographic techniques. Excellent reproducibility was observed for all analytes (CV &lt; 10%) for all techniques and configurations.

Conclusion:
A fast, sensitive, simple, specific, and accurate analytical LC/MS/MS method was developed and verified for the simultaneous measurement of delta 8, 9 and 10 THC and Carboxy THC metabolite spiked into synthetic urine. Future work will include testing different matrixes and checking for interferences that may impact the quantitation of any of the compounds in the analytical method.

A Novel LC-MS/MS Method for the Analysis of Antidepressants in Plasma for Clinical Research Benjamin Dugas (Presenter) Waters Corporation

Poster #44a View Map

This poster will be attended on Wednesday at 11:00 for 1 hour in the Exhibit Hall.

Background:
Depression is common globally, with an estimated 3.8% of the population affected. The condition can impact individuals’ ability to function in work, social and family settings. Many antidepressant drugs are currently prescribed, encompassing selective serotonin reuptake inhibitors (SSRIs), serotonin-nonresponsive reuptake inhibitors (SNRIs) and tetracyclic antidepressants (TeCAs). However pharmacokinetic and drug interactions are known, therefore a reliable quantitative clinical research method may play a role in researching the effects of their administration.

Waters has developed a clinical research method for the following antidepressants in plasma; citalopram, desmethylfluoxetine, duloxetine, fluoxetine, fluvoxamine, O-desmethylvenlafaxine, sertraline and venlafaxine (10-1000 ng/mL); mirtazapine (5-500 ng/mL) and trazadone (30-3000 ng/mL).

Methods:
Matrix matched calibrators and QCs were prepared using in-house stocks and pooled plasma. Samples (50 µL) were treated with internal standard in acetonitrile. A water/methanol/ammonium acetate gradient was used with a Waters XSelect™ Premier HSS T3 Column on a Waters ACQUITY™ UPLC™ I-Class System followed by detection on a Xevo™ TQD Mass Spectrometer in a 5-minute run.

Results:
No system carryover was observed following analysis of plasma samples containing the highest concentration calibrators. Analytical sensitivity investigations indicated precise quantification (≤20% CV, ≤17.6% bias) at concentrations equal to or lower than the lowest concentration calibrator. Total precision and repeatability were assessed (3 pools, 5 replicates, 5 days; n=25) and determined to be ≤10.0% RSD. Linearity experiments determined the method provided first or second order fits over the ranges analyzed; additionally, each run met acceptance criteria (coefficient of correlation ≥ 0.995, determined concentrations of calibrators ±15% of nominal, ±20% in the case of the lowest calibrator). Post-column infusion experiments demonstrated analytes eluted in regions free of major ion suppression or enhancement. Evaluation of matrix effects at low and high concentrations indicated compensation by the internal standard. Addition of high concentrations of several endogenous and exogenous materials did not affect quantification.

Conclusions:
This quantitative method for clinical research demonstrates very good precision with minimal matrix effects and allows for the multiplexing of a panel of antidepressants in plasma in a short run time.

Effect of the Solriamfetol Drug on Amphetamine Immunoassay Gopal Kumar (Presenter) GME at MetroHealth

Poster #45a View Map

This poster will be attended on Thursday at 11:00 for 1 hour in the Exhibit Hall.

Introduction:
Solriamfetol is used to treat excessive daytime sleepiness (EDS) caused by narcolepsy and residual EDS caused by obstructive sleep apnea/hypopnea syndrome (OSA) despite other treatment. As a schedule class IV-controlled substance, care is taken when prescribing solriamfetol due to the potential for abuse. As such, urinary toxicology screens are monitored during treatment to assess for other illicit substance use. Our providers became concerned when a patient prescribed solriamfetol had a urinary toxicology screen that was positive for amphetamines despite patient denial of illicit drug use. This raised a question regarding a false positive amphetamine screen.

Objectives:
Herein, we tested the effect of Solriamfetol drug (150mg/ml) on the amphetamine assay on the Beckman AU 480 and Citrine™ Triple Quad™ MS/MS Systems.

Methods:
We performed spiking studies with Solriamfetol drug on negative urine. The stock solution of Solriamfetol drug (150mg/ml) was prepared in Phosphate-Buffered Saline. The different concentrations of Solriamfetol drug ranged between (2mg/mL, 200ug/mL, 2ug/mL, 200ng/mL respectively). We then tested each concentration for amphetamine immunoassay on the Beckman AU 480 and the LC/MS-MS. The amphetamine immunoassay on the Beckman AU480 is the Emit® II Plus and the amphetamine assay on the LC/MS-MS was performed on SCIEX Citrine™ Triple Quad™ MS/MS Systems is a laboratory developed test.

Results:
Our data suggests that the amphetamine immunoassay on the Beckman yielded positive results for Solriamfetol at concentrations of 2mg/ml and 200ug/ml and negative results at 2ug/ml and 200ng/ml. However, the Citrine™ Triple Quad™ MS/MS Systems was negative at all concentrations.

Conclusion:
The Beckman AU480 Emit® II Plus amphetamine immunoassay gave false positive results for amphetamines due to Solriamfetol drug usage.

Detection of Nitazene Analogs in Chronic Pain and Behavioral Health Populations Ankit Patel (Presenter) Aegis Sciences Corporation

Poster #45b View Map

This poster will be attended on Thursday at 12:30 for 1 hour in the Exhibit Hall.

INTRODUCTION: Nitazene analogs are 2-benzylbenzimidazole derivatives classified as designer opioids, a group of novel psychoactive substances (NPS). This compound class was originally investigated for its analgesic properties; however, none are currently approved for use in the United States. Several derivatives have been identified in toxicological casework. They are generally characterized as more potent than morphine at the mu-opioid receptor. In recent years, the number of nitazene analogs in the illicit drug supply has increased substantially; their prevalence is a concerning part of the continued global health concern surrounding NPS abuse. Clinicians, especially those in chronic pain and behavioral health settings, should be aware of the prevalence of these compounds in their patient populations.

METHODS: This study was IRB approved. The scope of testing for all samples received in the laboratory depends upon physician request; test offerings include routinely monitored healthcare and behavioral health compliance medications and illicit drugs, including NPS. Nitazene analogs were analyzed in the laboratory as part of a larger NPS panel, which includes designer benzodiazepines, designer opioids, synthetic cannabinoids, synthetic stimulants, and other NPS analytes. The following nitazenes were included in the study: isotonitazene, N-desethyl isotonitazene, etonitazene, N-desethyl etonitazene, metonitazene, metodesnitazene, protonitazene, butonitazene, flunitazene, desnitroisotonitazene, N-piperidinyl etonitazene, N-pyrrolidino etonitazene, and 4-hydroxy nitazene.

Urine samples were hydrolyzed and prepared for analysis by liquid chromatography/tandem mass spectrometry (LC-MS/MS). The mass spectrometer was operated in positive electrospray ionization mode for scheduled multireaction monitoring (sMRM) analysis. Chromatographic separation was achieved using a Restek Biphenyl column. Results were reported qualitatively with detection limits ranging from 1-5 ng/mL depending on the analyte.

Results for target compounds were evaluated from samples received in the laboratory from January 2022 – December 2022. During this time, providers requested 408,823 tests for designer opioids. Positive results were summarized for routine demographic information as well as additional relevant positive results. Co-positive analytes were determined by separate validated methods in the laboratory.

RESULTS: Over the period evaluated, 1,147 samples submitted by 342 patients were determined to be positive for at least one nitazene analog. Positive results were reported from clinics across 20 different states from patients ranging in age from 20-73. 4-hydroxy nitazene, a common metabolite to several different nitazene analogs, was detected in 93% of all positive samples. Metonitazene and N-desethyl isotonitazene were the next most frequently detected, in 54% and 18% of positive samples respectively. Protonitazene, flunitazene, isotonitazene, butonitazene, and N-piperidinyl etonitazene were also detected, but at a much lower frequency.

Relevant co-positive analytes observed in nitazene analog samples include routinely prescribed pharmaceuticals, illicit drugs, THC, alcohol, and other NPS compounds (including fentanyl analogs, designer benzodiazepines, synthetic cannabinoids, synthetic stimulants, and xylazine); co-positivity with fentanyl was notably high (>95%) in this patient population. Nitazene analogs were also frequently detected alongside other NPS compound classes.

CONCLUSIONS: Nitazene analogs are analytes of concern for clinicians in chronic pain and behavioral health settings. In this study, they were detected in patients from a variety of age groups across 20 states. 4-hydroxy nitazene was most often identified; this analyte may be a useful urinary marker for detecting intake of this compound class. Of note, target analytes were regularly observed in the presence of other illicit and prescription drugs, particularly fentanyl. This is especially concerning as the combination of these potent opioids with other substances may increase the risk of adverse events to individuals. The availability of comprehensive analytical testing for NPS compounds, including nitazene analogs, provides a valuable tool for clinicians to utilize to support patient treatment plans.

Method Development and Validation of Paper Spray Mass Spectrometry Method for Quantitation of Remdesivir and Active Metabolite, GS-441524, in Plasma Lindsey Kirkpatrick (Presenter) Indiana University School of Medicine

Poster #47a View Map

This poster will be attended on Thursday at 11:00 for 1 hour in the Exhibit Hall.

Introduction:
Remdesivir (GS-5734) is a nucleoside analog prodrug with antiviral activity against several single-stranded RNA viruses, including ebolavirus (EBOV), severe respiratory distress syndrome virus 1 (SARS-CoV-1), and Middle East respiratory syndrome coronavirus (MERS-CoV). In vitro studies of remdesivir and its active metabolite, GS-441524, also showed antiviral activity against the novel severe respiratory distress syndrome virus 2 (SARS-CoV-2), the causative viral pathogen of COVID-19, and it is one of the FDA-approved antiviral agents for the treatment of individuals with COVID-19. However, remdesivir pharmacokinetics and pharmacodynamics (PK/PD) data in humans is limited. It is imperative that precise analytical methods for the quantification of remdesivir and GS-441524 for use in PK/PD studies, therapeutic drug monitoring, and assessment of toxicity are developed.

Methods:
Seven-point calibration curves for remdesivir and its active metabolite, GS-441524, were created utilizing seven plasma-based calibrants of varying concentrations and two isotopic internal standards of set concentrations. Four plasma-based quality controls were prepared in a similar fashion to the calibrants and utilized for validation. No sample preparation was needed. Plasma samples were spotted on a paper substrate in pre-manufactured Verispray plastic cassettes and allowed to dry. The dried plasma spots were analyzed directly utilizing paper spray-mass spectrometry (PS-MS/MS). The method was validated, and the success of the validation was assessed by evaluating linearity, limits of detection (LOD), accuracy (% bias), precision (% CV), carryover, matrix effects, stability, and metabolic interferences. All experiments were performed on a Thermo Scientific Altis triple quadrupole mass spectrometer.

Results:
The calibration ranges were 20 – 5,000 and 100 – 25,000 ng/mL for remdesivir and GS-441524, respectively. The calibration curves for the two antiviral agents showed excellent linearity (average R2 = 0.99-1.00). The inter- and intra-day precision (%CV) across validation runs at four QC levels for both analytes was less than 11.2% and accuracy (%bias) was within ± 15%. Plasma calibrant stability was assessed and degradation for the 4 °C and room temperature samples were seen beginning at Day 7. The plasma calibrants were stable at -20 °C. No interference, matrix effects, or carryover was discovered during the validation process. The final method had an analysis time of 1.2 minutes.

Conclusion:
The development and validation of the first PS-MS/MS method quantitating remdesivir and its active metabolite, GS-441524, is reported. PS-MS/MS represents a useful methodology for rapidly quantifying remdesivir, which will be useful for clinical PK/PD, therapeutic drug monitoring, and toxicity assessment, particularly during future viral outbreaks.

A Novel LC-MS/MS Method for the Analysis of Antibiotics in Plasma for Clinical Research David Ballantyne (Presenter) Waters Corporation

Poster #48b View Map

This poster will be attended on Wednesday at 12:30 for 1 hour in the Exhibit Hall.

Background:
A reliable clinical research method for the analysis of a large number of antibiotics in a single sample may play a role in understanding the pharmacokinetic and pharmacodynamic effects of their administration. Currently such behaviour is not well understood.

An example of such a clinical research method for a large panel of antibiotic drugs in plasma was developed over several analytical ranges; azithromycin (5-500 ng/mL); ciprofloxacin and clindamycin (0.1-10 μg/mL); ampicillin, cefotaxime, chloramphenicol and linezolid (0.5-50 μg/mL); cefazolin, cefepime, ceftazidime, cefuroxime, flucloxacillin, meropenem and sulbactam (1-100 μg/mL); daptomycin and piperacillin (2-200 μg/mL).

Methods:
Matrix matched calibrators and QCs were prepared using in-house stocks and pooled plasma. Samples (50 μL) were treated with internal standard in methanol. A water/methanol/ammonia gradient was used with a Waters™ ACQUITY™ UPLC™ BEH C18 2.1 x 1.7 μm, 100mm column on a Waters ACQUITY UPLC I-Class FTN and Xevo TQD mass spectrometer utilizing polarity switching in a 5-minute run.

Results:
No system carryover was observed following analysis of plasma samples containing the highest concentration calibrators. Analytical sensitivity investigations indicated precise quantification (≤20% CV, ≤15% bias) at concentrations equal to or lower than the lowest concentration calibrator. Total precision and repeatability were assessed (3 pools, 5 replicates, 5 days; n=25) and determined to be ≤12.5% RSD. Linearity experiments determined the method provided first or second order polynomial fits over the ranges analyzed; additionally, each run met acceptance criteria (coefficient of correlation ≥ 0.995, determined concentrations of calibrators ±15% of nominal, ±20% in the case of the lowest calibrator). Post-column infusion experiments demonstrated analytes eluted in regions free of major ion suppression or enhancement. Evaluation of matrix effects at low and high concentrations indicated compensation by the internal standard. Addition of high concentrations of several endogenous and exogenous materials did not affect quantification.

Conclusions:
This quantitative method for clinical research demonstrates very good precision with minimal matrix effects and allows for the multiplexing of a large panel of antibiotics in plasma in a short run time.

For Research Use Only.Not for Use in Diagnostic Procedures.

Simultaneous Determination of Treosulfan and Fludarabine in Plasma by LC-MS/MS Shelby Hutcherson (Presenter) Memorial Sloan Kettering Cancer Center

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Poster #50a View Map

This poster will be attended on Wednesday at 11:00 for 1 hour in the Exhibit Hall.

Introduction:
Prior to hematopoietic stem cell transplantation (HSCT), a conditioning regimen consisting of high doses of one or more myeloablative agents is given to most patients. This serves to both prevent rejection of the transplanted stem cells and reduce the tumor burden in patients with hematological malignancies. However, myeloablative agents can also cause serious and sometimes irreversible side effects. Treosulfan (Treo) is a structural analog of the alkylating agent busulfan which has been shown in clinical trials to have an improved safety profile compared to busulfan while still maintaining comparable myeloablative activity. Unlike busulfan, Treo is a prodrug which undergoes a pH- and temperature-dependent, nonenzymatic, sequential conversion into two epoxide species with alkylating activity. Because plasma concentrations of Treo exhibit significant interindividual variability, therapeutic drug monitoring (TDM) is necessary to ensure dosages are given that maximize efficacy while minimizing toxicity. Treo is currently being considered for approval by the FDA in combination with fludarabine (Flu) as a conditioning regimen prior to allogeneic HSCT and is also currently being evaluated in combination with Flu and/or other agents in several clinical trials in the United States. As the use of Treo increases in clinical trials and eventually in routine clinical practice, it will be important to be able to quantitate it rapidly and accurately in the clinical laboratory.

Objective:
The objective of this study was to develop an accurate liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay capable of simultaneously quantifying Treo and Flu in plasma across a wide range of concentrations in order to facilitate TDM of both compounds.

Methods:
Deuterated Treo (Treo-d4) and Flu-C13N15 were used as the internal standards (IS) for Treo and Flu, respectively. To prevent the degradation of Treo into the epoxide species, 1 M citric acid was added to all plasma used for validation studies (50 &mu;L per 1 mL of plasma). Drugs were extracted from 100 &mu;L of acidified plasma via protein precipitation with methanol containing IS. After centrifugation at 13,000 rpm for 10 min., 10 &mu;L of the supernatant was injected into the liquid chromatography system and subjected to chromatographic separation followed by electrospray ionization (ESI) tandem mass spectrometry. Treo and Flu measurements were performed on a Thermo Scientific TLX-2 HPLC system coupled to a TSQ Quantum Ultra mass spectrometer in positive ion mode, and compounds were detected using multiple reaction monitoring (MRM) mode. MRM transitions were as follows: Treo 296.0 &gt; 183.1 and 296.0 &gt; 87.1 m/z; Flu 286.1 &gt; 154.0 and 286.1 &gt; 134.0 m/z. Drug concentrations were calculated using a six-point calibration curve. Accuracy was evaluated by spiking Treo and Flu into acidified blank plasma at three concentrations spanning each compound&rsquo;s analytical measurement range (AMR) and calculating the percent recovery after performing LC-MS/MS. Inter-day imprecision was assessed at three concentrations spanning the AMRs by running an LC-MS/MS batch daily for 20 days and calculating the coefficient of variation (CV). To test for the presence of matrix effects, five waste patient plasma samples were acidified and extracted with methanol. Five calibration curves were prepared by spiking Treo and Flu into the plasma extract and five calibration curves were prepared by spiking Treo and Flu into methanol. The mean signal obtained for each compound from the calibration curves prepared in plasma extract after LC-MS/MS was then compared to the mean signal obtained for each compound from the calibration curves prepared in methanol. The stability of Treo at three concentrations spanning the AMR was determined by spiking Treo into acidified or non-acidified blank plasma and performing drug extraction and LC-MS/MS at regular intervals over the course of 24 hours.

Results:
Treo was stable at 4&deg;C in acidified plasma for up to 12 hours but degraded rapidly in non-acidified plasma. Calibration curves for both Treo and Flu were linear over each drug&rsquo;s AMR with R2 values exceeding 0.99. Recovery for both compounds ranged from 95-100% at all three concentrations tested. Similarly, inter-day imprecision was &lt;10% for Treo and Flu at each of the three concentrations tested. Imprecision for Treo ranged from 1.5-9.0%, and imprecision for Flu ranged from 3.9-8.3%. The differences observed when comparing Treo and Flu signal in calibration curve samples prepared in plasma extract to signal obtained from calibration curve samples prepared in methanol were negligible, indicating that no matrix effects are present.

Conclusion:
Our LC-MS/MS method is able to simultaneously quantify Treo and Flu across a wide range of concentrations. Although the validation is ongoing, the assay exhibits acceptable accuracy, imprecision, and linearity, and it is not impacted by matrix effects. The relative instability of Treo and the necessity of acidifying plasma samples directly after collection presents important logistical considerations that will need to be taken into account when implementing Treo measurements clinically. This assay will enable simultaneous TDM of Treo and Flu when used in combination for conditioning prior to HSCT in clinical trials and routine clinical practice.

Tip-on-Tip SPE for the Extraction of Hormones in Saliva Nicholas Chestara (Presenter) DPX Technologies

Poster #50b View Map

This poster will be attended on Wednesday at 12:30 for 1 hour in the Exhibit Hall.

Introduction:
The evaluation of hormone levels in blood and serum have been used to diagnose various diseases and even indicate early signs of a health decline. Some of these hormones include testosterone, progesterone, cortisol, and DHEA, but many others are evaluated. Although current processes are accurate, blood sample collection is considered invasive and potentially uncomfortable for patients, leading scientists to look at other possible non- less invasive collections. One avenue is hormone testing in saliva; saliva can be collected in relatively large amounts (mL) without any invasive measures taken, making it the perfect candidate for clinically relevant testing.

In this study, 9 hormones were evaluated in pooled neat saliva. Utilizing a Tip-on-Tip Solid Phase Extraction (ToT SPE, patent pending) technique with SupelTM Swift HLB (MilliporeSigma), we were able to establish LOQ as low as 1 ppt for some steroids and recoveries averaging 85% or more. for all steroids.
Objectives
Using automation, establish a fast, robust, and sensitive extraction method for the analysis of non-polar hormones in saliva.

Methods:
A MICROLAB® NIMBUS96 automated liquid handler (ALH) was utilized for sample preparation. Saliva was aliquoted into a well plate (1000 µL) that contains 10 mg HLB per well. Internal standards was were spiked into the sample (10 µL). The well plate was then loaded onto MICROLAB® NIMBUS96 system for the rest of the automated protocol. The ALH transfers 500 µL of 30% MeOH into sample well plate and mixes 30 times. The sample is allowed to settle for 3 minutes before 1 mL of supernatant is removed and placed in a waste reservoir. A fresh set of tips is used to transfer and mix 500 µL of 30% MeOH with the sample wells. The filtration tips are placed on the DPX vacuum block (on ALH deck). The sample is then loaded onto the filtration tip, collecting the analyte bound HLB sorbent. The ALH then picks up a set of 300 µL tips for wash transfer- wash 1 is 200 µL of 100% H2O and wash 2 is a subsequent 200 µL of 30% MeOH. The DPX vacuum block then dries the tips for 15 minutes. Once the sorbent is dry, the ALH takes a fresh set of tips, aspirates 275 µL of ACN, goes Tip-on-Tip with the HLB loaded DPX Filtration tips, moves to a new well plate location, and dispenses the ACN through the HLB sorbent for elution. The eluent is then dried down and re-constituted in 100 µL of 75% MeOH for a final concentration factor of 10:1 (1000 µL of saliva to 100 µL of 75% MeOH). The entire automated method takes 35 minutes for 96 samples.

The analysis was performed on an Agilent 1290 LC system coupled with a SCIEX 6500+ tandem mass spectrometer. The chromatographic method was performed achieved on an Agilent InfinityLab Poroshell 120 EC-C18, 3.0 x 100 mm, 2.7 µm, solvent saver LC with a 10 µL injection volume. The column was heated to 45 °C, and the mobile phases were [A] 0.1% formic acid in water and [B] 0.1% formic acid in methanol. Mass spectrometer source parameters included a curtain gas of 35 psi, collision gas of 8 psi, and for positive mode, an IonSpray voltage of 4500 was used. The source temperature was set to 600˚C and 60 psi was used for both ion source gases. Matching internal standards were used for each analyte.

Results:
The linear dynamic range, extraction recovery and matrix effects were evaluated with a three-day study (SWGTOX). Recoveries for all analytes ranged from 85%-100% with matrix effects suppression of 5%-18% ion suppression at 5 ng/mL concentration. The linearity was calculated using 10 data points run in triplicate (n=3) each day, giving a data set of n=9 for all analytes. The limit of detection for each analyte was verified based on linear calibration calculations from the 3-day study. Correlation coefficients for the linear calibration plots for all 9 analytes ranged from 0.997-1.00, and % C.V. values were less than 10% for all analytes for inter-day and intra-day precision. Patient samples were evaluated for accuracy and precision separately.

Conclusion:
Due to the viscosity of saliva collected from patients, the use of solid phase extraction (SPE) has been difficult to perform by conventional methods. However, ToT SPE allows for even the most difficult matrices to be extracted using SPE. This automated method of extracting and analyzing steroids is reproducible, high throughput, and provides rapid turnaround time for patient samples.

Establishing Metrological Traceability Based on the Example of Cortisol Judith Taibon (Presenter) Roche Diagnostics GmbH

Poster #52a View Map

This poster will be attended on Wednesday at 11:00 for 1 hour in the Exhibit Hall.

INTRODUCTION: Mass spectrometry (MS) in hyphenation with high-resolution separation techniques, most notably liquid chromatography (LC) and gas chromatography (GC), is the gold standard for reference measurement procedures in clinical diagnostics. The utilization of highly characterized, SI-traceable reference materials and stable isotope-labeled internal standards is of paramount importance for an assay to meet the quality requirements of highest order reference measurement procedures.

OBJECTIVES: By the example of cortisol, we will show how clinically relevant measurement services in endocrinology are made safe and reliable by the establishment of mass spectrometry based reference measurement procedures (RMP) and services.

METHODS: An isotope dilution liquid chromatography mass spectrometry (ID-LC-MS/MS) based candidate RMP was developed for the quantification of cortisol in serum and plasma matrices. To ensure traceability to SI units, either certified primary reference materials or in house quantitative nuclear magnetic resonance spectrometry (qNMR)-characterized material was utilized. A supported liquid extraction (SLE) sample preparation protocol was optimized to minimize matrix effects. To avoid co-elution of interferences, a two-dimensional LC (2D-LC) approach using two orthogonal stationary phases was adopted. An extensive measurement protocol over 5 days was applied to determine precision and accuracy. The measurement uncertainty was evaluated according to the "Guide to the Expression of Uncertainty of Measurement" (GUM).

RESULTS: The RMP allowed the quantification of cortisol in serum and plasma without interference from structurally-related compounds and evidence of matrix effects. Linear range was 0.800 ng/mL – 600 ng/mL. Intermediate precision was ≤ 2.6% and repeatability ranged between 0.6% - 1.9% for all analyte concentrations over the entire measuring range. The relative mean bias was between -1.5% - 0.5% for all levels and matrices. Total measurement uncertainties were found to be ≤ 2.8%; expanded uncertainties were ≤ 5.7% (k=2) for all levels.

CONCLUSION: This highly selective 2D-LC-MS/MS candidate reference measurement procedure, provides a traceable and reliable platform for the standardization of routine tests and for the evaluation of cortisol in clinical samples.

Simultaneous Determination of Prednisone and Prednisolone in Serum by TFLC-MS/MS Ryan Schofield (Presenter) MSKCC

Poster #53a View Map

This poster will be attended on Thursday at 11:00 for 1 hour in the Exhibit Hall.

INTRODUCTION: Many cancer patients who have received bone marrow transplantation (BMT) often have complications with graft versus host disease (GVHD). The donor immune cells that attack malignant tumors also attack other body parts which can lead to liver complications, digestive issues, and skin rashes. The first line treatment for acute GVHD includes the glucocorticosteroid, prednisone (PRED). PRED can have serious side effects for BMT patients whose immune system is already compromised from the transplant procedure. These side effects include susceptibility to infection, muscle atrophy and elevated blood sugar levels which can initiate diabetes and hypertension. PRED is potentially dangerous in high does, especially over long periods of time. When patients experience symptoms that can last months to years this can become problematic. PRED dosing for the treatment of acute GVHD has been set on the drug’s use for other medical conditions which may not be optimal for acute GVHD. It is necessary to have a sensitive and specific assay to quantify PRED routinely in the clinical laboratory to ensure optimal dosage is determined and standardized for treatment of acute GVHD.

OBJECTIVES: The objective was to develop a rapid and simple assay for the simultaneous measurement of PRED and prednisolone (PRDL) in serum by LC-MS/MS. This assay will be used to support clinical trials and pharmacokinetic studies to better define optimal dosing to improve patient outcomes with acute GVHD.

METHODS: PRED and PRDL were isolated from serum samples after protein precipitation with methanol containing internal standards (IS). PRED-D8 was the IS for PRED while PRDL-D8 was the IS for PRDL. Following centrifugation, the supernatant was injected into the turbulent flow liquid chromatography system followed by electrospray ionization tandem mass spectrometry (TFLC-MS/MS). Chromatographic separation was performed using a Thermo Scientific TLX-2 Transcend HPLC system interfaced to a SCIEX 6500+ mass spectrometer operated in positive ion ESI mode. MRM transitions were as follows: PRED 359.1>147.1 and 359.1>171.1 m/z and PRDL 361.2>147.1 and 361.2>128.1 m/z. The results were quantified using a six-point calibration curve. Assay accuracy was determined through recovery studies. Within-day imprecision was evaluated by analyzing 10 specimens in a single day, and between-day imprecision was evaluated by analyzing samples in triplicate over 20 days.

RESULTS: The LOQs of PRED & PRDL were 1 ng/mL; the CVs were <20% over 20 days. For both compounds the calibration curves were linear over the analytical measurement range (AMR) with correlation coefficients (R2) ≥0.999. Dilutions were validated providing a clinical reportable range (CRR) of 1 to 10,000 ng/mL. Within-day and between-day imprecision (CVs) at concentrations spanning the AMR were less than 5% for all analytes. PRED and PRDL were sufficiently stable under all relevant analytical conditions. No significant matrix effects were observed during the method validation. Recoveries ranged from 96-102% for three controls (250, 500, and 750 ng/mL) spanning the AMR for both compounds.

CONCLUSION: We have developed an accurate and sensitive TFLC-MS/MS method for the simultaneous quantification of PRED and PRDL in human serum. The method has been fully validated for imprecision, accuracy, linearity, recovery, carryover, specificity, and matrix effects. This is the first reported method to measure both compounds simultaneously using TurboFlow technology. Due to the assay’s performance, it will be used to support clinical trials and pharmacokinetic studies for patients with acute GVHD.

Therapeutic Drug Monitoring of Mycophenolic Acid, Four Azole Antifungals and One Active Metabolite Using LC-MS/MS Zhicheng Jin (Presenter) University of Wisconsin - Madison

Poster #54a View Map

This poster will be attended on Wednesday at 11:00 for 1 hour in the Exhibit Hall.

Introduction:
Mycophenolate mofetil is an immunosuppressant preventing acute rejection after solid organ transplant. Mycophenolate mofetil and active metabolite mycophenolic acid (MPA) inhibit T and B cell proliferation by blocking inosine monophosphate dehydrogenase, which is required for guanosine monophosphate biosynthesis. However, unlike other immunosuppressants, serum and plasma are optimal samples for the quantification of MPA. Another class of drugs that requires therapeutic drug monitoring for post-transplant patients is antifungal drugs. Triazole antifungal drugs are commonly prescribed for the prevention and treatment of invasive fungal infections for immunocompromised patients or immunocompetent patients with comorbid conditions. Our laboratory offers a liquid chromatography assay for mycophenolic acid quantification. We also have an antifungal drugs panel on an LC-MS/MS platform. Because serum/plasma is the optimal sample for MPA and antifungal drugs, monitoring both mycophenolic acid and azole antifungal drugs simultaneously will increase laboratory efficiency while reducing the cost per test. We reported here the method development and validation of a new LC-MS/MS assay for the quantification of mycophenolic acid and antifungal drugs.

Methods:
The internal standards are mycophenolic acid-D3, voriconazole-D3, posaconazole-D4, itraconazole-D4, isavuconazole-D4, and hydroxyitraconazole-D4, which were purchased from Cerilliant (Round Rock, TX). Calibrators and controls were prepared in-house by spiking drug-free human serum with reference material purchased from Cerilliant (Round Rock, TX) or Toronto Research Company (Toronto, Canada). Serum or plasma samples were protein precipitated. After centrifugation, the supernatant was diluted with mobile phase A and was ready for injection. Analytes were separated on an Ultra C18, 50 x 2.1 mm, 3µm, analytical column (Restek, Bellefonte, PA) on a 1260 HPLC system (Agilent, Santa Clara, CA). Mobile phase A was ammonium formate (10 mM, with 0.1% formic acid); mobile phase B was acetonitrile with 0.1% formic acid. The mass analyzer was either API 6500 or API 4000 triple quadrupole mass spectrometer with an electrospray ionization source. Injection volume was optimized for API 6500 and API 4000 instruments individually. A six-point calibrator and three levels of quality control samples were included in every batch. Raw data were processed using MultiQuant software (SCIEX, Framingham, MA).

Results:
Our laboratory currently offers two separate tests for mycophenolic acid and antifungal drugs panel. MPA assay is on a Thermo Finnigan AS3000 liquid chromatography system and antifungals panel on an LC-MS/MS platform. We sought to combine two methods into one LC-MS/MS assay. This would increase laboratory efficiencies by reducing blood draws, decreasing sample volume, and reducing cost per test. In the meantime, after the new assay goes live, we can retire an aging HPLC system. MRM transitions of MPA and antifungals were optimized on API 6500 and API 4000 triple quadrupole mass spectrometers. The quantification transitions are: mycophenolic acid, 321.0>207.1; voriconazole, 350.1>281.0; posaconazole, 701.3>683.3; itraconazole, 705.2>392.2; isavuconazole, 428.1>215.0; and hydroxyitraconazole, 721.2>408.2. LC separation time is 5 min per sample. The intraday and interday assay precisions are within 1/3 of the total allowable error. The analyte measurable ranges (AMR) are: MPA, 0.4 - 48 mcg/mL; posaconazole, 0.1 - 12 mcg/mL; and 0.2 - 24 mcg/mL for voriconazole, isavuconazole, itraconazole, and hydroxyitraconazole. The test performance on API 6500 vs. API 4000 system will be compared and evaluated.

Conclusion:
We developed a sensitive and robust LC-MS/MS method that can quantify MPA, voriconazole, posaconazole, isavuconazole, itraconazole, and hydroxyitraconazole simultaneously. Our laboratory is in the process of validating this method on both API 6500 and API 4000 mass spectrometers.

LC-MS/MS Analysis of Immunosuppressant Drugs in Whole Blood Using the Xevo TQ Absolute with Capitainer B Device Absorptive Microsampling for Clinical Research Peter Harrsch (Presenter) Waters Corporation

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Poster #57a View Map

This poster will be attended on Thursday at 11:00 for 1 hour in the Exhibit Hall.

Introduction:
Traditional laboratory analysis of the immunosuppressant drugs cyclosporine, everolimus, sirolimus and tacrolimus is well-established in clinical research. However there remains a need for individuals to undergo an invasive, time-consuming and disruptive process under the supervision of trained staff in order to collect a sufficient volume of whole blood for laboratory analysis.

A reliable, remote sampling method may find utility in a clinical research setting. Here we describe the use of Capitainer™ B qDBS Devices to obtain analytically sensitive, precise and accurate data for cyclosporine, everolimus, sirolimus and tacrolimus analysis using small sample volumes.

The Waters ACQUITY™ UPLC™ I-Class System with Xevo™ TQ Absolute Mass Spectrometer was used to analyze these samples.

Methods:
An in-house laboratory developed LC-MS/MS method to analyze all four immunosuppressants in a single run was developed. Waters MassTrak™ Immunosuppressant Calibrator and Control Sets (IVD) and whole blood External Quality Assurance samples (LGC, Bury, UK) were used in conjunction with Capitainer B qDBS Devices to assess the performance of the method.

Samples (10µL) were collected using Capitainer® B qDBS devices, and the sample extracted using solvent containing internal standards. A water/methanol/ammonium acetate gradient was used with a Waters C18 HSS SB column on a Waters ACQUITY™ UPLC™ I-Class and Xevo TQ Absolute Mass Spectrometer operating in positive electrospray ionization mode with run time of less than 2 minutes.

Results:
Analytical sensitivity of the lowest calibrator at 1 ng/mL for everolimus, sirolimus and tacrolimus and 25 ng/mL for cyclosporine was demonstrated with S/N (PtP) > 10 across five analytical runs. We successfully demonstrated linearity of cyclosporine from 25-1500 ng/mL and everolimus, sirolimus and tacrolimus from 1-30 ng/mL, with r2>0.995 over five analytical runs. Total precision and repeatability across the four immunosuppressants (2, 8 and 22 ng/mL for everolimus, tacrolimus and tacrolimus; 150, 400 and 900 ng/mL for cyclosporine) with five replicates over five analytical runs (n = 25) was ≤15% CV. External quality assurance samples for all drugs met the scheme acceptance criteria, with mean bias ≤15% CV.

Conclusions:
Using Capitainer B qDBS devices and sample small volumes (10 µL) of whole blood, an in-house laboratory method was used to meet validation goals for analytical sensitivity, linearity, precision and accuracy for cyclosporine, everolimus, sirolimus and tacrolimus. Furthermore, the advantages conferred by volumetric absorptive microsampling, notably removing the requirement for travel and a venous blood draw and facilitating home sampling, render this technique applicable to clinical research.

For Research Use Only. Not for Use in Diagnostic Procedures.
ACQUITY, UPLC, Xevo, and MassTrak are trademarks of Waters Technologies Corporation. Capitainer is a trademark of Capitainer AB.

An Answer to Vedolizumab Quantitation Interferences Alex Barbeln (Presenter) Mayo Clinic

Poster #58a View Map

This poster will be attended on Wednesday at 11:00 for 1 hour in the Exhibit Hall.

Background: Vedolizumab (VEDO) is a humanized IgG1 kappa therapeutic monoclonal antibody (tmAb) targeting the α4β7 heterodimer, expressed on the surface of gut-specific lymphocytes. This tmAb is routinely used to treat inflammatory bowel disease (e.g. ulcerative colitis and Crohn’s disease). Patients with higher trough concentrations have improved outcomes, so therapeutic drug monitoring (TDM) has become standard of care. Our laboratory has pioneered the use of mass spectrometry for tmAbs. The clinical laboratory developed test (LDT) to quantitate VEDO was the first method clinically available with IgG immuno-enrichment and analysis of the tmAb light chain intact mass, without the use of tryptic peptides or an available stable isotopically labeled (SIL) internal standard (IS). Occasionally, the method has issues with interfering peaks or poor peak shape in a subset of extracted ion chromatograms (XICs) making quantitation more subjective in base line drawing of an XIC or result being unable to be released. Multiple experiments to mitigate this included: changes to mobile phases, columns, enrichment techniques, internal standard, and different instrumentation, all with limited success. Here we set out to investigate whether a specific anti-VEDO antibody enrichment would eliminate the XIC interferences.

Methods:
Samples: 23 residual serum samples with a physician ordered VEDO test which required troubleshooting due to XIC peaks with shoulders, split peaks, or unacceptable retention time (RT) shift were available for this study. Routine LDT method utilizes a surrogate protein IS with Melon Gel enrichment. Samples are reduced with DTT and then analyzed using a Thermo Fisher/Cohesive TLX4 Transcend multi-plex HPLC system connected to a Thermo Scientific Q-Exactive Plus Orbitrap mass spectrometer using Tracefinder for quantitation. The quality of XIC and final report were compared both visually and quantitatively, to the method in development described below. Proof of concept was considered successful if it removed at least 75% of the interferences observed.

Antibody coupling: Approximately 20mg Dynabeads M-280 Tosylactivated (ThermoFisher Scientific) were added to LoBind tubes and placed in a magnetic holder. Beads were washed with 1.5mL borate buffer for 1 minute in an Eppendorf ThermoMixer at room temperature (RT) at 1500RPM. Coupling mixture consisted of 500mcL borate buffer, 100mcg VEDO bridging antibody (Bio-Rad HCA293 or HCA294) and 500mcL ammonium sulfate; mixed overnight for 16-18 hours at 37°C and 1500RPM. The beads were washed with 1.5mL 1xPBS + 5mg/mL BSA for 1 hour at RT and 1500RPM. The beads were washed three times with 1mL of 1xPBS + 0.1% Tween. The beads were resuspended in 500mcL 1xPBS + 0.1% Tween to give a final concentration of 200mcg/mL coupled antibody.

Sample enrichment: Samples were prepared by adding 30mcL unknown/standard/QC to a LoBind tube with 25mcL bead slurry, 50mcL 1xPBS, and 30mcL of 25mcg/mL SIL-VEDO (Sigma-Aldrich). Samples were mixed at RT for two hours at 1500RPM. The beads were washed twice with 1xPBS and twice with water. Elution was done with 50mcL 5% acetic acid and mixing at RT for 10 minutes at 1500RPM. The eluent was then transferred to a PCR plate and reduced with 25mcL 100mM DTT in 1M ammonium bicarbonate. The plate was mixed at 56°C for 45 minutes at 800RPM.

LC-MS: The reduced samples were analyzed using an Agilent 1260 infinity II LC connected to an AB Sciex ZenoTOF 7600 mass spectrometer. A volume of 20mcL was injected onto an Agilent Poroshell 300SB C3, 2.1x75mm, 5 micron column heated at 60°C, running an 8-minute gradient from 25 to 34%B at a flow rate of 300mcL/min. Mobile phases consisted of 1% formic acid in water (A) and 90% acetonitrile, 10% isopropyl alcohol, and 0.1% formic acid (B). Sciex OS was used for data analysis. The +11, +12, and +13 charge states of the VEDO light chain were combined to give an XIC. A calibration curve from 2 to 125mcg/mL was obtained from standards prepared by spiking VEDO in normal human serum.

Results: Changes to mobile phases, columns, enrichment, and instrumentation gave limited success. Substituting the SIL-VEDO IS into the current clinical Melon Gel method eliminated the interference (peak splitting and shoulders) in the IS XICs seen in 4 of the 23 residuals from the clinical method. The SIL-VEDO also enhanced analyte peak confirmation without using any extra macros to calculate RT differences as is needed currently with the surrogate IS.

The next step was evaluating the anti-VEDO antibody capture using the SIL-VEDO as IS. The proof-of-concept new method gave a linear analytical measuring range from 2 to 125mcg/mL, matching the clinical range. For 4 of 23 residuals that had a significant analyte XIC peak but with a slight RT shift, clinically released as “unknown interfering substance present, unable to obtain result”, the anti-VEDO antibody method gave an analyte peak (with exact RT match to the IS) for 2 with quantitation of 4.9 and 5.0mcg/mL respectively and eliminated the interference in the other 2 with reportable of <2.0mcg/mL. For another set of 3 residuals with quantifiable XIC peaks with a light retention time shift, but clinically resulted as <2.0mcg/mL, the anti-VEDO antibody method confirmed 2 residuals as <2.0mcg/mL and gave a reportable result for the 3rd at 4.7mcg/mL. 2 residuals with shoulders (released as 9.3 and 5.1mcg/mL) where re-evaluated with the new method giving symmetrical XICs and quantitation of 9.1 and 6.0mcg/mL.

Conclusion: The anti-VEDO antibody enrichment using the recently available commercial form of the SIL-VEDO IS has shown potential to eliminate interferences in the XIC (split peaks, shoulders or unacceptable RT deltas) related to quantitating VEDO found with the current method along with providing added specificity. Although in early stages of development, this method could be a viable improvement for the clinical lab that would benefit patient care.

Development of a Dried Blood Spot Lead Test by ICP-MS to Increase Screening Compliance in at Risk Populations Cody Orahoske (Presenter) University of California San Francisco

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Poster #59a View Map

This poster will be attended on Thursday at 11:00 for 1 hour in the Exhibit Hall.

Introduction:
Despite significant progress decreasing blood lead levels among US children 1-5 years of age, demonstrated by a 97.5th percentile of 3.5 µg/dl, lead poisoning remains a public health threat. Lead exposure disproportionately affects Black children, and those below poverty levels, in Medicaid, and living in housing units built before 1978. To improve public safety and testing compliance in children younger than 2 years of age, sample collection can be done by using filter paper and dried blood spots. These cards offer the advantage of easy handling and a less invasive collection method. Lead testing from the dried blood spot is comparable to the gold standard detection of lead from a venous whole blood samples although may be prone to contamination and other interferences. We describe a robust and reproducible test which can be used to measure lead from dried blood spots.

Objective:
The primary objective of this study was to develop a method using dried blood spot cards for lead testing using inductively coupled plasma-mass spectrometry (ICP-MS).

Methods:
This method was developed on a Thermo Fisher iCAP RQ and TQ ICP-MS (Thermo Fisher Scientific, Waltham, MA, USA) in kinetic energy discrimination (KED) mode. The method was calibrated with certified reference material traceable to NIST SRM 3128 (VHG labs, Manchester, NH, USA) in whole blood spotted in Whatman 903 cards at various concentrations and dried for at least two hours. Briefly, 5 ml of extraction buffer was added to a 6mm filter paper disc, followed by a 1-minute vortex at 2,000 RPM, incubation at room temperature for 30 minutes and centrifugation at 4,000 RPM for 5 minutes. Samples were loaded onto an Elemental Scientific Inc. (ESI, Omaha, NE, USA) SC-FSAT sample introduction system and subjected to analysis by the ICP-MS. To assess the performance of the method, the following characteristics were established: linearity, reproducibility, and accuracy. Accuracy and linearity across the measurement range were assessed using a reference standard solution traceable to NIST CRM 3128 (Inorganic Ventures, Christiansburg, VA, USA). Reproducibility and accuracy were assessed using used UTAK controls (Valencia, CA) and 16 patient samples with lead values obtained in a whole blood method.

Results:
The assay is linear across the analytical measurement range of 1 to 100 µg/dl, and recovery of the linearity materials ranged from 92% to 102%, with an average SD of ± 0.8 µg/dl. In 16 patient samples with concentrations ranging from 1.4 to 19.5 µg/dl run in six replicates, the coefficient of variation (CV) range was 0.6% to 21.1%. In these samples, the bias ranged from -4.4 µg/dl to 0.1 µg/dl. In controls recovery ranged from 88% to 100% and CV ranged from 2.6% to 6.0%. Finally, this method successfully quantified five proficiency testing samples from the Dried Blood Spot Lead Proficiency Testing Program (Wisconsin State Laboratory of Hygiene, University of Wisconsin, Madison, WI).

Conclusion:
The dried blood spot method was reproducible and comparable to the routine testing method in whole blood. This dried blood spot lead test meets the performance criteria to be validated as a clinical test in pediatric patients.

Development and Validation of LC-MS/MS Method for Therapeutic Drug Monitoring of Treosulfan and Busulfan Jeayeon Ryu (Presenter) University of Ulsan College of Medicine

Poster #61a View Map

This poster will be attended on Thursday at 11:00 for 1 hour in the Exhibit Hall.

Background:
Hematopoietic stem cell transplantation (HSCT) is a treatment that can cure various malignant and nonmalignant disorders. A conditioning regimen treatment is performed to prevent rejection and reduce tumor burden before HSCT. Treosulfan and busulfan are alkylating agents administered under the conditioning regimen. High busulfan exposure is associated with drug toxicity and low busulfan exposure with rejection or disease recurrence. There is also an association between treosulfan exposure and early toxicity, such as skin toxicity and mucositis. As both busulfan and treosulfan have narrow therapeutic ranges, therapeutic drug monitoring (TDM) is essential. Since the clinical laboratory tests for both drugs are few, a method that can simultaneously perform TDM for both drugs under the same conditions would be helpful in practice. In this study, a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for simultaneous measurement of treosulfan and busulfan was developed and validated. Furthermore, an active metabolite of treosulfan, S,S-EBDM, was also determined by the same method.

Methods:
For preparing calibrators and quality control materials, commercialized reference standards were used for treosulfan, S,S-EBDM, and busulfan. Treosulfan-D4 and busulfan-D8 isotopes were used as internal standards (ISs). The analytes were separated from 25 µL of plasma after protein precipitation with acetonitrile containing ISs. After centrifugation, the supernatant was diluted with 3% formic acid and then injected into the liquid chromatography system followed by tandem mass spectrometry. Chromatographic separation was performed using ACQUITY Ι-Class plus ultraperformance liquid chromatography (UPLC) system coupled with a XEVO TQ-XS mass spectrometer operated in positive ion electrospray ionization mode. The sample was eluted with mobile phases comprising 2-mM ammonium acetate and 0.1 % formic acid in water or methanol. The analytes and ISs were detected in the multiple reaction monitoring (MRM) mode. The limit of detection (LoD), limit of quantification (LoQ), selectivity, linearity, accuracy, precision, carryover, matrix effect, and sample stability were validated according to the Clinical and Laboratory Standards Institute (CLSI) guidelines.

Results:
Retention times for treosulfan and S,S-EBDM and IS for both analytes were 0.44 min, 0.43 min, and 0.44 min, respectively. Retention times for busulfan and IS for busulfan were 1.20 min and 1.18 min, respectively. The mass-to-charge (m/z) transitions used for quantification in the MRM mode were as follows: treosulfan 296.2 > 87.1; S,S-EBDM 200.2 > 87.1; and busulfan 264.1 > 55.1. Total run time was 4 min. The LoDs of treosulfan, S,S-EBDM, and busulfan were 0.21, 0.23, and 0.001 µg/mL, respectively. The linear range of the calibration curves of treosulfan, S,S-EBDM, and busulfan spanned concentrations of 3.13–100, 0.63–5.00, and 0.16–5.00 µg/mL, respectively. Within-run and between-run precision of the developed method fulfilled the analytical criteria, except for S,S-EBDM. The LC-MS/MS method was adequately selective and accurate and showed no carry-over. It provided acceptable matrix effect for treosulfan prodrug and busulfan; however, the matrix effect could not be excluded for S,S-EBDM. Busulfan and treosulfan were stable in plasma for 2 h at room temperature. When the samples were stored at 10°C, busulfan was stable for up to a week and treosulfan was stable for up to 3 days, and both were stable for up to a week when stored frozen at -20°C.

Conclusion:
We developed the LC-MS/MS method to simultaneously measure treosulfan and busulfan. The method met the validation requirements of the CLSI guidelines and showed good performance. Therefore, this method is expected to be helpful in TDM after treosulfan or busulfan treatment in clinical laboratories.

A Combined LC-MS/MS Method for the Analysis of Aldosterone and Plasma Renin Activity for Clinical Research Using the Xevo TQ Absolute Mass Spectrometer Dominic Foley (Presenter) Waters Corporation

Poster #63b View Map

This poster will be attended on Thursday at 12:30 for 1 hour in the Exhibit Hall.

Background:
Primary aldosteronism (PA) is a common cause of hypertension, whereby uncontrollable amounts of aldosterone are produced by a benign tumour or hyperplasia of the adrenal glands. Excess aldosterone results in significant sodium reabsorption in the kidneys, increasing water retention and blood volume, thereby causing hypertension. The renin-angiotensin-aldosterone system (RAAS) regulates the production of aldosterone and in this system, renin and aldosterone should move in synchronicity with each other throughout the day. Therefore, these two components are used to assess the status of the RAAS, particularly in the evaluation of new therapies in clinical research studies.
Historically, the assessments of aldosterone and plasma renin activity (PRA) have been performed using separate methods using immunoassay or more recently liquid chromatography – tandem mass spectrometry (LC-MS/MS) platforms. One of the benefits of using LC-MS/MS for clinical research is the ability to measure multiple analytes across the proteome and metabolome using the same system and even in the same analysis to provide more information in less time and save costs. Here we evaluate a single LC-MS/MS method for the combined measurement of plasma aldosterone and renin activity for clinical research purposes.

Methods:
Aldosterone certified reference material (Merck, UK) and Angiotensin I (Cambridge BioScience, UK) were used to create calibrators in 2% Bovine Serum Albumin (BSA) in Phosphate Buffered Saline (PBS). In-house QC material prepared in both 2% BSA in PBS and K2EDTA plasma (BioIVT, UK), were used to evaluate method precision. Plasma samples were analyzed using the newly developed method and the quantified results were compared to separate independent LC-MS/MS methods for aldosterone and plasma renin activity. Plasma samples were treated with generation buffer (Sodium acetate, EDTA, acetic acid, SBTI and PMSF) and mixed for three hours at 37°C. Samples were precipitated, diluted and centrifuged prior to SPE. Sample supernatant was transferred to a Waters Oasis™ MAX µElution 96 Well Plate, followed by a wash and elution. Using an ACQUITY™ UPLC™ I-Class System, samples were injected onto a Waters XBridge™ C8, 2.5µm, 2.1 x 50 mm Column using a water/methanol/ammonium fluoride gradient elution profile and quantified with a Waters Xevo™ TQ Absolute Mass Spectrometer.

Results:
The method demonstrated no significant carryover or matrix effects and was shown to be linear from 10 – 2500 pg/mL for aldosterone and 0.1 – 25 ng/mL/hr for PRA. Analytical sensitivity investigations indicate the analytical sensitivity of this method would allow precise quantification (<20%) at 10 pg/mL and 0.1 ng/mL/hr, for aldosterone and PRA, respectively. Coefficients of variation (CV) for total precision and repeatability on 5 analytical runs for low, mid and high QCs were all < 10% (n = 25) for aldosterone and PRA. Comparison with samples previously analyzed by an independent LC-MS/MS method demonstrated good agreement for aldosterone and PRA.

Conclusions:
We have successfully quantified aldosterone and renin activity in plasma in a single method using an SPE protocol with LC-MS/MS analysis, for clinical research purposes. The method demonstrates excellent linearity and precision, with minimal matrix effects.

For Research Use Only. Not for Use in Diagnostic Procedures.

Using LC-MS/MS LDT to Determine Fentanyl Prevalence and Evaluate the FEN2 Immunoassay’s Real-World Clinical Performance in Tertiary Care Settings Marlen Menlyadiev (Presenter) UCSD Health

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Poster #66b View Map

This poster will be attended on Wednesday at 12:30 for 1 hour in the Exhibit Hall.

Introduction
LC-MS/MS laboratory-developed tests (LDTs) are the mainstay of modern clinical toxicology testing. While widely used to support the development of FDA-cleared drug immunoassays, their significance in the clinical implementation and evaluation of such assays is less recognized. In this work we report on the use of our LC-MS/MS opiates method for determining the prevalence of fentanyl in urine drug screen (UDS) samples and assessing the real-world clinical performance of the Roche FEN2 fentanyl assay in routine clinical use. This work expands on our previous report of the FEN2 assay implementation.

Methods
Excess specimens from a total of 250 consecutive random UDS clinical samples were collected between 05/04/22 and 05/17/22 under UCSD IRB protocol 181656. These specimens were first screened using the DRI assay (Thermo Fisher Scientific) followed by the analysis by the FEN2 assay (Roche Diagnostics). Each specimen in the study was sent to the clinical toxicology laboratory for quantitative analysis by LC-MS/MS method for fentanyl and norfentanyl. Fentanyl prevalence in the tested population was calculated by examining the extracted ion chromatograms from analyzed samples for fentanyl and norfentanyl peaks (retention times, quantifier-to-qualifier ion, and signal-to-noise (S/N) ratios, etc.). For calculation of the clinical sensitivity and specificity of the FEN2 (and the DRI) immunoassay, sample were classified as true positive if they contained at least 2 ng/mL of fentanyl and/or norfentanyl. The same cutoffs were used when querying EHR to evaluate real-world clinical performance (screening and confirmation positivity rates, rates of false positives and negatives) of the FEN2 and the DRI immunoassays. False positive and negative rates in queried EHR cohorts were determined from samples that screened positive by immunoassay and did not confirm by LC/MS/MS or, conversely, from samples that screened negative, but had fentanyl and/or norfentanyl by LC-MS/MS.

Results
Thirty-eight of 250 study samples were found to contain fentanyl and 49 samples - norfentanyl at ≥ 2ng/mL concentration. Fifty-one samples contained fentanyl, norfentanyl or both analytes at ≥ 2ng/mL. The median fentanyl and norfentanyl concentrations in these 51 samples were 5 and 15.5 ng/mL, respectively, with corresponding inter-quartile ranges (IQRs) of 43 and 85 ng/mL. In addition, in 6 samples from the 250-sample study pool, fentanyl and/or norfentanyl were detected (signal-to-noise ratio >3, acceptable quantifier-to-qualify ion ratios, etc.) but not quantified. These findings corresponded to 22.8% prevalence of fentanyl in our study population.
Of the 51 LC-MS/MS true positive samples in the study, 31 and 50 were classified correctly by the DRI and the FEN2, respectively. Both assays classified 198 of the 199 LC-MS/MS-confirmed true negatives as negative. The clinical sensitivity and specificity calculated from these data were 61% and 99.5% for the DRI and 98% and 99.5% for the FEN2. The real-world screening positivity rate with the DRI and the FEN2 assays during 1 month testing period was, respectively, 13.3% and 17.3%, with the corresponding LC-MS/MS confirmation rate for immunoassay-positive samples of 88.8% and 96.8%. Higher immunoassay positivity rate for FEN2 was likely due to its ability to detect norfentanyl. The false positive rates for the DRI and the FEN2 in queried EHR cohorts (1000+ entries) were, 11.2% and 3.2%, respectively, while false-negativity rates (using smaller subset of total immunoassay screens) were 22% and 5.5% for the DRI and the FEN2 assays.

Conclusion
The use of LC-MS/MS LDT enabled estimation of the prevalence of the fentanyl in the study population (urban tertiary care hospital) and evaluation of the clinical performance of the FEN2 and DRI assay in both the study population and during 1 month of routine clinical use. Our results demonstrate the FEN2 assay has greater clinical sensitivity and is less prone to false positive results as compared with the DRI assay. These findings support the implementation of the FEN2 in a routine clinical practice and underline the broader role of mass spectrometry-based LDTs in clinical toxicology testing.

Are Koreans Overexposed to Perfluoroalkyl Substances? Result of One Referral Laboratory Junhyung Lee (Presenter) GC Labs

Poster #70b View Map

This poster will be attended on Wednesday at 12:30 for 1 hour in the Exhibit Hall.

Introduction:
Perfluoroalkyl substances (PFAS) are a group of chemicals used to prepare fluoropolymer coatings and heat-, oil-, stain-, grease-, and water-resistant products. However, high PFAS levels may affect reproduction, thyroid function, immune system, and injure the liver. This study aimed to analyze the concentrations of perfluorooctanoic acid (PFOA) and perfluorooctane sulfonic acid (PFOS), which are representative PFAS, in the Korean population and compare the results with those of the American population.

Methods:
We analyzed 654 patients referred to our laboratory from March 2020 to May 2022. Two target analytes, PFOA and PFOS, were quantified using high-performance liquid chromatography (HPLC; 1200 series, Agilent, Santa Clara, CA, USA) coupled with a Triple Quad MS/MS system (API 4000, Sciex, Framingham, MA, USA). All analytes were separated on Unison UK C18 column (75 x 3 mm, Imtakt, Japan). Acetonitrile and water were used as mobile phases. PFOA and PFOS concentrations of the American population were obtained from the Fourth National Report on Human Exposure to Environmental Chemicals (survey years: 2015&ndash;2016) as a subset of the National Health and Nutrition Examination Survey (NHANES).

Results:
Geometric mean (95% confidence intervals) of PFOA and PFOS concentrations in the participants were 6.40 (6.05&ndash;6.97) and 6.27 (5.96&ndash;6.60) ng/mL, respectively, while those of the American population were 1.56 (1.47&ndash;1.66) and 4.72 (4.40&ndash;5.07) ng/mL, respectively, and the mean of the two populations were significantly different (P&lt;0.01). Assuming that the cutoff of overexposure was 95 percentile American population, 76.6% (501/654) of our subjects showed high PFOA concentrations corresponding to overexposure and 5.7% (37/654) of our subjects showed high PFOS concentrations.

Discussion and Conclusions:
Serum PFOA concentrations in Koreans were significantly higher than those in Americans. Recently, the Korean government has restricted PFOA use, but this result shows that these efforts are insufficient and additional attention is needed. Since there is a time lag of about 5 years in the data collected between the two population groups, and the number of subjects was limited, it is difficult to make a quick conclusion based on our results. Nevertheless, continuous caution and avoidance of these endocrine-disrupting chemicals is recommended because there is no clear definition of safe concentrations.

Clinical Performance of Mass Spectrometry for 25-OH Vitamin D Measurement Yeonjae Lee (Presenter) Konkuk University Medical Center

Poster #77a View Map

This poster will be attended on Thursday at 11:00 for 1 hour in the Exhibit Hall.

Background:
Accurate measurement of 25-OH vitamin D(25-OH-D) is important for vitamin D status monitoring. Although Isotope dilution liquid chromatography mass spectrometry (ID-LC-MS/MS) is confirmed as reference measurement procedure, many laboratories or researchers still use immunoassays because of the convenience and availability, regardless of standardization. In present study, authors evaluated the clinical performance of mass spectrometry and immunoassay for 25-OH-D measurement.

Method:
The accuracy of MS tests was evaluated by measuring standard reference material produced by National Institute of Standards and Technology (NIST SRM 972a). Then, using the mean values from two laboratory-developed test (LDT) mass spectrometrys (MS) for vitamin D as standard, the qualitative decision concordance and quantitative correlation with another LDT MS test and four commercialized immunoassays; Roche Elecsys Vitamin D Total II, Siemens Atellica IM Vitamin D Total assay, Abbott Alinity 25-OH Vitamin D, and Beckman Coulter Access 25-OH Vitamin D Total, were evaluated for 150 residual serum samples.

Results:
In accuracy, it was confirmed that the mean values obtained from MS tests for standard materials were within the range of assigned value. When the measured 25-OH-D values from each assay were compared to those determined by MS, ranged 5.86-67.75 ng/ml, In contrary to the equivalence between two MS tests with regression slope and intercept those containing 1 and 0 in their 95% confidence interval (CI) and r2 larger than 0.95, none of the immunoassays was equivalent to MS, by showing slope or intercept not containing 1 and 0 in their 95% CI, r2 smaller than 0.95. The regression estimates at 10, 20, and 30 ng/ml as medical decision limit (MDL) were within 5.0% the Vitamin D Standardization-Certification program (VDSCP)-claimed acceptable bias limit, in Beckman test at 30 ng/ml, only. The concordance of qualitative decision with MS ranged 90.0-94.0% in immunoassays.

Conclusion:
It was revealed that the two MS showed good qualitative decision agreement and quantitative correlation. However, despite having been achieved the certification by VDSCP, most immunoassays showed difference over acceptance criteria in the 25-OH-D measurement at MDL levels, which may affect clinical decision-making. Therefore, the superiority of MS test in measuring 25-OH-D compared to immunoassay has confirmed by this study, and attention for results interpretation and efforts for test standardization is required for immunoassays.

Comparison of Whole Blood and Precipitated Blood for the Quantitation of Drugs of Abuse Using Paperspray Katherine Walker (Presenter) Thermo Fisher Scientific

Poster #79b View Map

This poster will be attended on Thursday at 12:30 for 1 hour in the Exhibit Hall.

Introduction:
PaperSpray-MS is a technique for rapidly quantifying analytes in dried matrix spots such as urine or whole blood. Little or no sample preparation is required, and sample analysis times are 2 minutes or less. The new Thermo Scientific™ VeriSpray™ PaperSpray ion source system utilizes PaperSpray technology to make clinical research workflows faster and more efficient by combining ease-of-use and increased automation with the speed that PaperSpray technology provides. Since PaperSpray-MS is a direct analysis technique with no chromatographic separation, the sample matrix can lower the signal and the LLOQ. In complex matrices, such as whole blood, simple precipitation of proteins may improve the quantitation of analytes using PaperSpray.

Objectives:
The primary objective of this study is to compare drug of abuse quantitation in untreated whole blood and whole blood that has been precipitated by zinc sulfate in methanol.

Methods:
Twenty one drugs in the benzodiazepine, opiate, cocaine, stimulant, and sedative classes and their corresponding internal standards were prepared in whole blood and methanol. Spiked whole blood was mixed in a 1:3 ratio with a precipitation solution (2:1 methanol:0.2 ZnSO4), centrifuged down, and the supernatant was removed for analysis. The spiked whole blood, precipitated blood, and methanol were deposited onto VeriSpray sample plates, dried, and then analyzed using a Thermo Scientific VeriSpray PaperSpray ion source coupled to a Thermo Scientific™ TSQ Altis™ MS. The one-minute chronograms were integrated using Thermo Scientific™ TraceFinder™ software and calibration curves were generated.

Results:
For drugs in methanol, all had an LLOQ of 5 ng/mL (lowest concentration tested). In whole blood the analyte signal of calibrators and the background signal decreased due to matrix effects on ionization. Compared to drugs in methanol, the signal-to-noise was worse for drugs in whole blood. For three benzodiazepines, the signal-to-noise and LLOQ was improved by the precipitation method. These compounds either suffer from decomposition or from protein-binding in whole blood. For the remaining 18 drugs of abuse, precipitation does not improve quantitation. Nine analytes had the same LLOQ in whole blood and precipitated blood. Nine analytes had a worse LLOQ in precipitated blood due to the four-fold dilution during precipitation.

Conclusion:
PaperSpray is a sensitive, direct technique for the analysis of drugs in whole blood. For benzodiazepines, precipitation with zinc sulfate is a simple, rapid clean-up method to improve the LLOQ. For other challenging compounds, including protein-binders, this precipitation method should be tested when optimizing sample preparation for PaperSpray.

Best Practices for an Enzymatic Hydrolysis Method of a Drug Comprehensive Panel with Opioids Janet Jones (Presenter) Kura Biotech

Poster #81b View Map

This poster will be attended on Thursday at 12:30 for 1 hour in the Exhibit Hall.

Introduction: Best practices of enzymatic hydrolysis methods are necessary to ensure the robustness of LC-MS/MS assays for clinical drug testing. Many drugs such as opioids, benzodiazepines, cannabinoids, and TCAs go through extensive phase II metabolism to produce conjugated metabolites, as these more polar compounds are easier for the body to excrete. Enzymatic hydrolysis is routinely used in drug testing labs to convert these metabolites back to their parent compounds for analysis by mass spectrometry. Enzymatic hydrolysis is a crucial step in sample prep that requires careful optimization for reliable, accurate results, and it is recommended to follow the suggested protocols set forth by the enzyme manufacturer. B-OneⓇ is an “all-in-one” formula of recombinant beta-glucuronidase and its buffer. It is effective at hydrolyzing conjugated compounds at room temperature in minutes without supplemental buffering, increasing the efficiency of drug testing methods.

Objective: Our main objective is to demonstrate under a real study how to apply best practices while evaluating B-One’s performance with multiple glucuronide analytes when exposed to competitive hydrolysis conditions. The goal is to test the enzyme under “realistic” conditions as this is more representative of a comprehensive panel with opioids in a production lab. Quantitative analysis was performed by LC-MS/MS.

Materials and Methods: A comprehensive drug panel that includes the following glucuronide standards; amitriptyline, buprenorphine, carboxy-THC, codeine-6, hydromorphone, morphine-3, naloxone, norbuprenorphine, oxazepam, oxymorphone, and tapentadol were prepared at low (500 ng/mL), medium (5,000 ng/mL), and high (20,000 ng/mL) concentrations in human drug-free urine samples. Acetaminophen-glucuronide (100,000 ng/mL) was added as an enzyme competition substrate to each sample. NGX custom standard mixes (calibration & ISTD) along with glucuronides were used for this study. Then, a hydrolysis method was performed following the enzyme manufacturer`s instructions (B-OneⓇ from Finden by Kura Biotech). Samples were followed by a clean-up protocol (XTR tips 5 mg HLB from DPX technologies). Finally, samples were diluted with DI water and injected into LC-MS/MS for data analysis.

Results: A quantitative method was used to calculate the recovery of free drugs for each analyte in quadruplicates. Results demonstrated great recovery percentages for each analyte regardless of the substrate competitor present for low, medium, and high concentrations. Also, our enzymatic hydrolysis method performed equally with both hard-to-cleave and easy-to-cleave analytes.

Discussion and Conclusion: B-OneⓇ “All-in-One” room temp hydrolysis solution reaches high percentages of parent drug recovery in 15 minutes, and combined with DPX XTR tips and NGX custom standard mixtures, provides a streamlined protocol for ease of use for a drug comprehensive panel with opioids following best practices suggested here. Additionally, B-OneⓇ demonstrated high substrate specificity when acetaminophen was added to the reaction as an enzymatic competitor. We also summarize the best practices that were utilized in this study that include the importance of the organic content, how to properly prepare glucuronide hydrolysis controls, and how to calculate hydrolysis efficiency.

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Method Optimization of Estrone in Serum as a Biomarker at 8 Seconds per Sample Using the LDTD-MS/MS Technique Serge Auger (Presenter) Phytronix

Poster #83a View Map

This poster will be attended on Thursday at 11:00 for 1 hour in the Exhibit Hall.

Introduction
The rapid quantification of estrone in serum can be useful to monitor estrogen levels and help explain abnormal menstrual cycles, infertility, symptoms of menopause, abnormal or heavy bleeding or any other hormonal alterations.

Objectives
For this project, an extraction method is optimized to develop a diagnostic tool to rapidly quantify estrone in serum, which will allow the rapid diagnosis of certain disorders. Laser Diode Thermal Desorption and tandem mass spectrometry (LDTD-MS/MS) is chosen as a fast-analytical technique.

Methods
The extraction is performed as follows: 400µL of serum sample are mixed with 10µL of the internal standard solution (Estrone-d4, 30 ng/mL in methanol). Then, 800µL of Hexane-MTBE (90:10) are added and mixed. After the centrifugation step, 500µL of upper layer is transferred in a new tube and evaporated to dryness. Dry extract is reconstituted with 125µL of methanol:water (1:1) and 6µL of the reconstitute sample is spotted onto LazWell96 plates and evaporated to complete dryness before analysis by LDTD-MS/MS.

The mass spectrometer is operated in negative ionization mode. A flow rate of 5 L/min with air as a carrier gas and a ramp of 3 seconds to 45% laser power with 2 seconds hold are used on the LDTD system.

Result
Human serum has different endogenic levels of estrone. A mixture of bovine serum albumin (BSA), 20 mg/mL diluted in phosphate buffered saline (PBS) buffer was used as a negative matrix. Calibration curves ranging from 10 to 250 pg/mL are prepared in synthetic matrix. A QC set is prepared in synthetic matrix (QC-L, QC-M and QC-H). Replicate extractions are deposited onto a LazWell plate and dried before analysis. The peak area against the internal standard (IS) ratio is used to normalize the signal.

Preliminary data show the regression correlation coefficients (r) obtained are greater than 0.997. For the between-run accuracy, values between 92.3 and 105.2 were obtained and the precision results were lower than 5.9% CV.

For the LDTD-MS/MS analysis, instead of studying the autosampler’s stability, the wet stability (extracted solutions kept at 4°C for 1 week) and dry stability (extracts on LazWell plate for 4 hours at room temperature) is evaluated. After the given stability time, calibration curves are analyzed. The precision obtained for QCs ranges between 2.1 and 8.1%CV and their accuracy ranges between 94.5% and 110.3% of the nominal values.

Serum samples from real patients are tested with this method to correlate with results obtained by traditional LC-MS/MS for a cross validation study. The percentage difference between the values is evaluated. A difference of less than 20% is obtained.

Conclusion
LDTD-MS/MS analysis of Estrone in serum with simple extraction procedure at 8 seconds per samples.

Undetectable Serum Thyroglobulin in Patients with Differentiated Thyroid Cancer: Antibodies, Assay Limitation, or Other? Benjamin Andress (Presenter) Mayo Clinic

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Poster #84a View Map

This poster will be attended on Wednesday at 11:00 for 1 hour in the Exhibit Hall.

Introduction:
Serum thyroglobulin (Tg) measurement is used in the post-operative monitoring for differentiated thyroid cancer (DTC) recurrence. Following total thyroidectomy, serum Tg should remain undetectable, and any measurable Tg indicates residual or recurrent disease. In addition, Tg measurement in fine-needle aspirate biopsy (FNAB) saline washings is used for the evaluation of potential DTC metastases to lymph nodes. Despite the development of highly sensitive Tg assays, serum Tg monitoring does not successfully detect all DTC recurrence. There are several known limitations in the measurement of Tg. Most notably, antibodies to Tg (TgAb), which develop in up to 30% of carcinoma patients, are likely to falsely suppress thyroglobulin results by immunoassay. Interference from heterophile antibodies, which can cause both false positive and false negative results, are also known limitations of immunoassays for Tg. Mass spectrometry (MS) assays were developed to address antibody interference and have been reported as not subject to TgAb or heterophile antibody interference. Analytical sensitivity has historically been a limitation in some Tg-MS assays, but this has been overcome in recent years. The Tg-MS assay at our institution has a lowest reportable concentration of 0.2 ng/mL, versus 0.1 ng/mL for our immunoassay. Despite the availability of sensitive Tg-MS assays, recurrent DTC with unexplained undetectable serum Tg is a persistent occurrence. The objective of this study was to determine whether post-thyroidectomy patients with undetectable serum Tg and cytology-confirmed lymph-node DTC metastases have detectable Tg in FNAB washing and whether the failure to detect serum Tg in this patient population is a result of TgAb interference.

Methods:
FNAB washing (n=30) from patients with cytology-confirmed DTC lymph node metastases but with an undetectable serum Tg by immunoassay were collected and assayed for Tg and TgAb. Serum Tg and TgAb concentrations were obtained within three months (mean: 14 days) prior to FNAB. In a subset of patients (n=8) a residual serum sample was available for additional investigations. Tg was measured on serum and FNAB washing using the Beckman Access Thyroglobulin (Tg2) immunoassay (Tg-IA) on a DxI analyzer, and an in-house developed LC-MS/MS Tg assay (Tg-MS). TgAb in serum was measured using the Beckman Access Thyroglobulin Antibody II assay on a DxI analyzer. Spike-recovery experiments were performed in a subset of samples (n=8) by adding the patient’s Tg-positive FNAB or certified reference material (BCR 457) to matched patient serum. A control serum (TgAb negative, Tg negative) was included for all spiked recovery experiments. Spiked serum was assayed using Tg-IA and percent recovery calculated as (expected – measured)/expected *100. Recovery (percent difference) between immunoassay and MS methods was calculated for FNA samples, and mean recovery for antibody positive versus antibody negative patients was compared by Student’s t-test.

Results:
Of 30 patients with cytology-confirmed DTC lymph node metastases and an undetectable serum Tg, 26 (87%) had detectable Tg in the FNAB washings by Tg-IA, and 25 (83%) were also Tg-positive by Tg-MS. Fifty-eight percent of these patients (15/26) had detectable TgAb in serum, while 42% (11/26) did not. In 8 samples with residual serum (4 TgAb negative and 4 TgAb positive), measurement of serum Tg by a sensitive MS method only detected Tg in one patient (Tg-MS= 0.27 ng/mL). Interestingly, this one discrepant patient was not TgAb positive, and exhibited Tg recovery of >90% in spike-recovery experiments. Spike-recovery experiments performed on the TgAb positive samples showed an interference for the immunoassay as expected, with 4/4 patients exhibiting <90% recovery (range: 27-87%) of Tg-positive FNA or Tg reference material; while TgAb negative serum exhibited no signs of interference (recovery between 94-111% for Tg-positive FNAB or Tg reference material). No evidence of TgAb interference in FNA washings was detected, as there was no significant difference in recovery between antibody positive and antibody negative patients’ Tg in FNA measured by Tg-MS versus Tg-IA.

Conclusions:
This study investigated a subset of post-thyroidectomy patients with DTC lymph-node metastases and undetectable serum Tg by immunoassay and MS methods. Tg was detectable by both MS and immunoassay methods in the majority of FNAB washing samples from patients with cytology-confirmed DTC lymph node metastases and Tg-negative serum. The absence of detectable serum Tg in these patients does not appear to be completely explained by the presence of TgAb, as a subset of patients were TgAb negative and showed >90% recovery of Tg on spike-recovery experiments. These findings imply that in a subset of patients with DTC lymph node metastases, Tg may not be secreted into the circulation, and the lack of measurable serum Tg is not related to the analytical limitations of the assays. Additional studies and testing in a larger number of patients are needed to further prove this concept.

Ion Suppression in Busulfan Monitoring Justine Cole (Presenter) NIH Clinical Center

Poster #84b View Map

This poster will be attended on Wednesday at 12:30 for 1 hour in the Exhibit Hall.

Case Description:
A 55-year-old female known with bone marrow failure, chronic kidney disease and immune dysfunction secondary to deficiency of deaminase 2 (DADA2), presented for administration of a 3-hour busulfan infusion prior to hematopoietic stem cell transplant. As a consequence of multiple red blood cell transfusions, the patient also suffered from hepatic disease due to iron overload, which put her at high risk of drug-induced liver injury. A busulfan concentration-time curve was requested to assess her individual pharmacokinetics during the infusion. Plasma was collected pre-infusion, and during infusion at 0, 1, 2 and 4 hour time-points. Busulfan concentrations were quantified by LC-MS. Between the pre-infusion and during infusion samples, the area under the curve (AUC) of the internal standard decreased by >30%, exceeding the allowable limit of variability for this assay.

Background:
Deficiency of adenosine deaminase 2 is characterized by a spectrum of potential clinical manifestations that span autoinflammation, vasculopathy, immune deficiency and hematological disorders. The mechanism by which DADA2 results in bone marrow failure remains to be fully elucidated. Hematopoeitic stem cell transplant is considered curative of DADA2, particularly in children, but requires preparative treatment that poses a risk of hepatotoxicity. In patients with prior liver disease, myeloablation using two busulfan infusions over two days is recommended, with pharmacokinetic monitoring to reduce toxicity while ensuring adequacy of myeloablation. In-house measurement of busulfan concentration by LC-MS during the first infusion provides timely pharmacokinetic information that is used for adjustment of the second busulfan dose.

MS Method and Results:
Busulfan is measured by HPLC with triple quadrupole mass spectrometry using multiple reaction monitoring in positive mode. Isotope dilution is carried out by addition of busulfan-d8 in acetonitrile as internal standard (IS). The acetonitrile serves to deproteinize the specimens, which are centrifuged before addition to auto sampler glass vials and dilution with Optima LC/MS grade water. The system is flushed with Optima LC/MS grade methanol after the highest calibrator, the highest QC and after every fifth patient sample. In this case, the IS peak AUC decreased from 9.16e5 pre-infusion to between 3.76e5 and 5.40e5 post-infusion, while the patient’s results were unremarkable at 0.024 ng/mL pre-infusion and decreasing from 1331 to 774 ng/mL post-infusion. The patient’s 0-hour sample was rerun neat and in 2x, 5x and 10x dilution to determine whether the IS corrected for the apparent ion suppressing agent within the sample. The IS AUC recovered to 8.46e5 at 10x dilution and the original patient results were deemed accurate.

Discussion and Conclusion:
Internal standards are used in LC-MS to compensate for variability produced by extraction and preparation techniques, sample factors, injection, ionization and flow rates, and detector response. Any fluctuations are expected to affect the IS and the analyte of interest similarly, such that the ratio between the two remains constant. In this case, the post-busulfan infusion IS peak AUC decreased by >30%, signifying the presence of an ion suppressing agent. Busulfan injection contains PEG-400, which co-elutes with and suppresses busulfan signal. Results were briefly withheld while dilution studies were performed to ensure that the IS corrected for the degree of suppression and to validate result accuracy. Going forward, ion suppression is acknowledged in these samples, and a greater degree of variability is accepted in the IS peak AUC.

Concordance of Urine and Meconium Drug Screen Results by Immunoassay and Mass Spectrometry Hannah Brown (Presenter) Washington University School of Medicine in St. Louis

Poster #57b View Map

This poster will be attended on Thursday at 12:30 for 1 hour in the Exhibit Hall.

INTRODUCTION
Newborn urine drug testing faces preanalytical and analytical challenges due to difficult sample collection protocols, low drug concentrations, and unique drug metabolites differing from those targeted by immunoassay (IA) or mass spectrometry (MS)-based methods. For these reasons, paired testing of urine and meconium is often performed . Further, the testing of meconium is also used to assess drug exposure throughout the latter half of gestation and helps with diagnosis of neonatal abstinence syndrome.

OBJECTIVE
The primary objective of this study was to evaluate concordance of drug screening results of newborn urine samples using both IA and MS-based methods with paired testing on meconium.

METHODS
The number of positive drug screen results for newborns with paired urine and meconium samples from January 2021 to October 2022 were tabulated retrospectively for two independent, de-identified datasets (n=881, dataset 1; n=2056, dataset 2). Different drug screening methods were used in the two datasets. In dataset 1, urine was screened with IA and confirmed with MS, while in dataset 2, both the screen and confirmatory testing were performed by MS. All paired meconium samples were sent to an outside reference laboratory where meconium samples were screened by IA and confirmed with MS. Common drug classes tested for in urine and meconium in both datasets include amphetamines (AMP), cocaine (COC), and cannabinoids (THC). The cutoffs for the IA urine screening method (in ng/mL) were: AMP 500, COC 150, OPI 300, THC 50. The cutoffs for the MS urine screening method (in ng/mL) were: AMP 5, COC 1, OPI 25-250 (depending on specific compound), THC 20 (as 11-nor-9-Carboxy-Δ9-tetrahydrocannabinol glucuronide). The cutoffs for meconium screening (in ng/g) were: AMP 100, COC 100, OPI 100, THC 20.

RESULTS
In dataset 1, 66 newborns screened positive for amphetamines in urine and/or meconium. Of these, 3% screened positive in urine alone, 33% were positive in meconium alone, and 64% were positive in both urine and meconium. In comparison, in dataset 2 utilizing MS, 123 newborns tested positive for amphetamines. Of these, 13% were positive in urine alone, 13% were positive in meconium alone, and 74% were positive in both urine and meconium.

For cocaine, 7 newborns screened positive in dataset 1. Of these, 0% screened positive in urine alone, 29% tested positive in meconium alone, and 71% were positive in both urine and meconium. In dataset 2, 65 newborns tested positive for cocaine and of these 15% were positive in urine alone, 14% were positive in meconium alone, and 71% were positive in both meconium and urine.
For cannabinoids, in dataset 1, 435 newborns screened positive. Of these, <1% were positive in urine alone, 66% were positive in meconium alone, and 32% screened positive in urine and meconium. The vast majority of the IA urine screens presumptively positive for cannabinoids did not confirm due to either negative confirmatory results or quantity insufficient to test. In dataset 2, 874 newborns tested positive for cannabinoids. Of these, <1% were positive in urine alone, 97% were positive in meconium alone, and 3% were positive in both meconium and urine.

CONCLUSION
Conventional urine IA screening methods may be inappropriate for urine samples from newborns. Lower cutoffs afforded by the MS approach (dataset 2) increased the sensitivity for detecting both amphetamines and cocaine, which were present in urine only for a significant number of newborns. The higher cutoffs for meconium testing may explain why the observed discordance between urine and meconium drug screen results (i.e., positive in urine and negative in meconium). Even with lower cutoffs afforded by MS, meconium was positive alone in a substantial number of newborns which may reflect drug exposure earlier in gestation or dilute urine. A large number of urine cannabinoid presumptive positive screens did not confirm. Given the poor analytical performance of screening for cannabinoids in urine, meconium only testing may be appropriate for this drug class, in combination with maternal urine drug testing.

Development and Evaluation of an LC-MS/MS Method for Therapeutic Drug Monitoring of Infliximab for Use in the Routine Clinical Laboratory Melissa Sam (Presenter) NSW Health Pathology, Australia UNSW South-Western Sydney Clinical School UNSW School of Chemistry Ingham Institute for Applied Medical Research, Sydney, Australia

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Poster #23a View Map

This poster will be attended on Thursday at 11:00 for 1 hour in the Exhibit Hall.

Introduction:
Therapeutic drug monitoring (TDM) of anti- tumour necrosis factor-alpha drug, infliximab (IFX) is increasingly used in patient management protocols in several autoimmune diseases. Traditionally both drug and anti-drug antibody (ADA) levels are measured using immunoassays, most commonly, ELISA. Measurement of serum drug levels of IFX and ADM and their anti-drug antibodies has been shown to improve patient outcomes over clinical assessment alone. Yet, as discussed by Papamichael et al (2022) in their review, there are significant ‘un-met needs’ in the current modes of testing. Reasons hypothesised by the authors include lack of harmonisation between different assay methods, cost efficiency and long turn-around times between sample collection and result. In addition, most immunoassay methods are not drug tolerant. The presence of the mAb prevents detection of the ADA, and in the same way, the presence of ADAs prevent measurement of the mAb levels. This is due to the formation of complexes between drug (mAb) and ADAs. This produces a gap in information for the clinician in cases of primary or secondary non-response to mAb therapy. Currently, there are several pre-treatment protocols for dissociating drug-antibody complexes in serum and measuring the ADAs on ELISA. However, these are not standardised, nor approved by the therapeutic goods administration, Australia (TGA) and so are not used routinely.

There is limited data on the utility of total drug levels, and along with that, limited ability to detect early development of ADAs that would otherwise be masked by drug interference. The questions is, can patient outcomes be improved if ADAs could be detected earlier? To answer this question, there needs to be further development in assays that can quantify ADAs in the presence of free drug. Recently, assays for the measurement of IFX drug levels have been developed using LC-MS/MS techniques. The benefit of LC-MS/MS assays are that their sensitivity and specificity provide information on drug levels and potential ADA development that is otherwise unavailable. The aims of this study are to develop and validate LC-MS/MS assays for measuring IFX drug levels (both total and free) for routine use and to use these assays to investigate the development of ADAs in patients with undetectable levels on traditional immunoassay.

Methods:
We spiked IFX into buffer at concentrations of 0.5-100 mg/L to produce a calibration curve. Samples were digested using an optimised trypsin digestion protocol. Replicates were analysed to determine intra- and inter-assay precision (%CV). For determination of total IFX spiked into serum, IFX was isolated using ZEBA© and Melon Gel© columns. Free IFX in serum was isolated using an immunoassay capture sample pre-treatment and subsequent on plate tryptic digestion. Samples containing spiked IFX and commercial anti-IFX (ATI) were measured using total IFX method. IFX and ATI ELISAs were performed using a Triturus autoanalyser and commercial ELISA kits.

Results:
We optimised an LC-MS/MS assay for measurement of IFX on the Shimadzu 8050 that has been evaluated for recovery rate, precision, sample stability and carryover. We developed standard curves for the measurement of IFX in serum across the range of 0.5- 100 mg/ml (for total IFX) and 0.5-50mg/ml (for free IFX). Results show good precision for both total IFX assay (R2 = 0.99, %CV < 7) and free IFX assay (R2 = 0.99, %CV < 12). Sample pre- treatment method to isolate total IFX showed good recovery compared to no sample pre-treatment when measured by ELISA. Samples spiked with IFX and commercial ATI were measured on total IFX LC-MS/MS method and compared to ELISA (mean =11.1 mg/L vs <0.49 on ELISA) demonstrating the interference of ATIs in traditional methods. We found that samples spiked with IFX and commercial ATIs performed similarly to samples spiked with IFX alone (Mean =11.1 mg/L vs 12.5 mg/L) on our total IFX, LC-MS/MS assay.

Conclusions:
We have developed an LC-MS/MS assay for the measurement of total IFX and free IFX in serum, which has a good recovery rate, precision and a broader measurement range compared to ELISA. Samples containing IFX-ATI complexes produced similar results to samples containing IFX alone using total IFX assay, showing proof of principle that total IFX can be measured in the presence of ATI. We are currently testing IFX-ATI spiked samples on free IFX assay, and patient samples with suspected ADA development on both total and free IFX assays.