Translating Pre-Clinical Research to Clinical Patient Care™
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Plenary Presentations
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Metabolomics Enabling Personalized Medicine
Metabolism is at the core of physiology and therefore metabolomics is ideally suited to assess someone’s health state. In this presentation strategies are discussed how personalized medicine can be realized by using metabolomics and by integrating it with other omics data. Examples are shown how disease pathology can be studied using metabolomics in clinical studies, and how mechanistic insights can be obtained using advanced in-vitro models and translational metabolomics. Pharmacometabolomics can help to study how pharmacology modulates disease pathology, and to predict efficacy and adverse effects of pharmacological interventions . An outlook will be given how metabolomics will impact clinical research and ultimately clinical decision support.
What We Can Learn from a Drop of Urine – Metabolomics at its Earliest: Discoveries of Bile Acid Synthesis Disorders, a New Category of Fatal Metabolic Liver Disease and Development of a Treatment
This presentation will highlight how mass spectrometry was successfully applied to define new genetic defects in the cholesterol-bile acid biosynthetic pathway as a specific class of metabolic liver disease. Bile acid synthesis disorders due to single enzyme defects generally present in infancy or early childhood with a progressive cholestatic hepatitis that, unchecked, lead to cirrhosis, liver failure, and death. Prior to the seminal work of Setchell and colleagues in identifying 6 genetic diseases as discrete entities, and conceiving of an effective therapy, children with these autosomal recessive diseases either underwent liver transplantation, or more commonly, were given supportive care until they died of liver failure of unknown origin. To be described are the combined use an untargeted and targeted approach with FAB-MS, GC-MS and ESI-LC-MS/MS that led to the elucidation of the biochemical basis of these diseases, the development of an international screening program, and the evaluation of the therapeutic responses that served to ultimately gain regulatory approval from the FDA for a life-saving therapy based on oral administration of cholic acid. This application of mass spectrometry to clinical chemistry has been a game-changer that has led to a radical change in the evaluation and treatment of patients with idiopathic progressive familial intrahepatic cholestasis syndromes.1
Molecular Tissue-Typing in Clinical Translational Research: Towards Precision Medicine
A multimodal approach for molecular imaging for clinical studies is trending the field of imaging mass spectrometry. More and more researchers realize that a single technology provides only a subset of the molecular information needed to obtain an in depth understanding of a clinical problem. Multimodal approaches enable the study of clinical samples at a variety of molecular and spatial scales. The molecular complexity on the genome, proteome and metabolome level all needs to be taken into account. The distribution of several hundreds of molecules on the surface of complex (biological) surfaces can be determined directly in complementary imaging MS experiment with different desorption and ionization strategies.
Frontiers of Orbitrap Mass Spectrometry
The talk provides an overview of a short but eventful history of Orbitrap mass spectrometry, from laying down the first principles of the technology to its current status in mainstream mass spectrometry as the leading technique for high-resolution, high mass accuracy quantitative analysis. While describing new possibilities arising from the recent extensions of two latest families of instruments, Fusion and (Q) Exactive, a special emphasis is placed on technical solutions that enhance quantitative analysis in these instruments. Future trends and perspectives of Orbitrap mass spectrometry are discussed, particularly in relation to high-throughput clinical analysis.
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Podium Presentations
Poster Presentations
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Pharmacotherapy Monitoring of Patients with APRT Deficiency on Allopurinol Treatment Utilizing UPLC-QqQ-MS/MS Assay
Adenine phosphoribosyltransferase (APRT) deficiency results in excessive urinary excretion of poorly soluble 2,8-dihydroxyadenine (DHA), causing nephrolithiasis and chronic kidney disease. Treatment with allopurinol, which is metabolized to the active metabolite oxypurinol, effectively reduces DHA excretion and prevents urinary stone formation. However, a reliable method for therapeutic monitoring of patients with APRT deficiency is lacking. An UPLC-QqQ-MS/MS assay for quantitative measurement of oxypurinol in patient’s plasma samples was successfully developed and optimized utilizing design of experiments. APRT enzyme assay was used to confirm diagnosis of patients with APRT deficiency.
High-Throughput LC-MS/MS Measurement of Pregnenolone in Human Blood Serum for Research Purposes
Pregnenolone is a biosynthetic precursor to other steroids such as corticosteroids, androgens, and estrogens. We developed a sensitive, robust, high-throughput quantitation assay for pregnenolone which can measure 10 to 500 ng/dL (0.3 to 1.5 nmol/L) in blood serum. The LC-MS/MS method can be multi-channeled with other HESI-MS/MS methods with a total runtime of 4.5 minutes. Derivatization with hydroxyl amine was necessary to reliably achieve the desired analytical range. Selective-reaction monitoring (SRM) within a 1-minute data window produced quantitation and conformation chromatographic peaks.
Resolution and Reproducibility of the Pentafluorophenyl Column is Superior to that of the Octadecylsilane Column in the High-Throughput Profiling of Biofluid
In high-throughput metabolomic profiling, chromatographic separation is crucial because well-performed chromatographic separation may reduce signal suppression from the complex biological matrices and improve the discoverability of low-abundance metabolites. We compared the performance of pentafluorophenyl (PFP)- and octadecylsilane (ODS)-based columns in profiling biological fluids. Total and extracted ion chromatograms demonstrated that the PFP column achieved better analyte separations than did the ODS column. Application of PFP column in metabolomes profiling extracted from urine and serum samples. There were 26 identified lipid species were significantly perturbed in overweight participants. Overall, choline-containing lipids were the most abundant perturbed lipidome, followed by sphingolipids and various phospholipids.
Metabolomic Profiling of Amino Acids and Related Metabolites in Patients with Aortic Stenosis; Application to Identify New Markers of Valvular Calcification
To evaluate the potential relationship between aortic valve stenosis and amino acid (AA) metabolome, a new LC–MS/MS method has been developed and validated for simultaneous determination of AA in human plasma. The optimized method enabled to comprehensively quantify 43 target metabolites using an off-line sample pre-treatment method, followed by reversed-phase ion-pair liquid chromatography (Surveyor, Thermo Scientific) coupled on-line to triple quadrupole mass spectrometry (TSQ Vantage, Thermo) via a positive electrospray ionization source. We found several changes in AA concentrations and their ratios such as ornithine : arginine and Fischer ratio in AS group compared with those in the control.
Detection, Identification and Confidence Rating of Unknown and Unexpected Compounds Found in Plasma Samples by LC-HRMS Analysis
With the capability of high-resolution (HR)-MS instruments to perform sensitive and reliable quantifications in HR-full scan mode, the detection (from the same acquisition) of unexpected and/or possible diagnostic compounds in human plasma samples, is becoming realistic.
After 1) a rapid overview of data treatment for the discovery of unexpected compounds found in test/patient plasma samples, we will review how to 2) identify compounds and 3) rate the confidence of their identification. For this last part, we will base our analysis on 2 key documents: 1) Commission decision 2002/657/EC and 2) Sumner LW et al; Metabolomics, 2007, which are dedicated for screening and metabolomics labs, respectively.
Research into Lysosomal Storage Metabolism Using Plasma Lipid Characterization by LC-Differential Mobility Spectrometry-MS/MS
Research into plasma sphingolipids and determining the concentrations of such is of growing importance in the clinical Research into plasma sphingolipids is of growing importance in the clinical research laboratory, particularly within groups researching Lysosomal Storage Metabolism. Current methods of analysis involve either enzyme activity procedures or derivatization of compounds prior to analysis. Direct analysis of these groups can be complex due to extensive structural homogeneity between individual compounds. Differential Ion Mobility Spectrometry offers an additional level of separation based on differences in molecular size, shape, charge state and chemical interactions, rather than mass alone. This allows for simplification of chromatographic and extraction methodologies, enabling the creation of rapid and facile analytical methods for inherently similar analytes.
Changes in Lipid Constituent of Cervical Tissue During Neoplasia Processes
Changes in lipid constituent of cervical tissue during neoplasia processes.
Development of complex genetic and lipidomic approach for the prediction of the risk and progression of cervical intraepithelial neoplasia in patients with human papillomavirus associated diseases of the cervix uteri. The lipid profiles of the cervical tissues with neoplastic changes and the surrounding healthy tissue from 5 patients with low-grade and 5 - with high-grade squamous intraepithelial lesion were obtained. A group of possible biomarkers for the prediction of evolving neoplastic transformation was proposed. The comprehensive analysis of tissue lipid constituent with molecular genetic markers significantly increases the diagnostic potential of cervical cancer risk assessment.
Simultaneous Determination of Antiepileptic Drugs in Blood by Tandem Mass Spectrometry
Determination of drugs in the blood for the therapeutic treatment of epilepsy has an important diagnostic role . We developed quantitative method for detecting antiepileptic drugs by HPLC / MS. Analytical equipment - liquid chromatograph and a mass spectrometer LCMS-8060 (Shimadzu, Japan). Sample preparation - a protein precipitating from plasma/serum. This method is designed for the monitoring of the antiepileptic drugs phenobarbital, phenytoin, carbamazepine, lamotrigine in serum/plasma. The limits of detection are in the range of from 100-500 ng/ml depending on the analyte. The recovery rate was 93 - 98%. The CV of each quality control sample concentration does not exceed 5%. Our method are validated and suitable for routine analysis.
Keeping the Flow – Equilibrium Analysis of the Renin-Angiotensin-System and Applications in Diagnosis and Treatment of Hypertension
Biochemical analysis of the Renin-Angiotensin-System is technically challenging, since angiotensins undergo rapid turnover during blood sampling, introducing analytical artefacts. Unique features of the circulating RAS allow for a novel diagnostic approach, overcoming these pre-analytical issues. RAS-equilibrium analysis can be applied to standard heparin plasma or serum samples and is based on the LC-MS/MS based quantification of up to 10 angiotensin metabolites (RAS-Fingerprint) providing a comprehensive qualitative and quantitative picture of soluble RAS components. The AA2-Ratio, a RAS-Fingerprint derived diagnostic value, has shown to possess a huge potential as a robust and drug tolerant screening test for primary aldosteronism.
Metabolomic Profiling by UPLC-MS Analysis of Urine Samples of Children Affected by Type 1 Diabetes
Type 1 diabetes (T1D) is a crucial pathology, that influences the life of patients, since from the childhood.
In our work, we studied the T1D in infancy, through the metabolomic approach for a whole assessment of biologic profile of T1D children in insulin therapy and in good glycemic control (mean HbA1c % < 8), and of healthy children comparable for age, sex and puberty.
Applying the high-definition mass spectrometry to the urine samples, we were able, after processing data with specific statistical multivariate and univariate analysis, to clearly separate the peers in two groups and to reveal important alteration in different metabolic pathways in diabetic subjects, even if they are not far from the onset of the diseases, like adults, and they are all in good glycemic control.
Application of Desorption Electrospray Ionisation Mass Spectrometry in Analysing Skin Secretions: A Non-invasive Diagnostic Tool?
Sweat contains many chemicals which may provide valuable clinical information. Desorption electrospray ionisation mass spectrometry is a powerful tool for lipid analysis which can detect endogenous lipids in fingermarks. We developed a methodology for the analysis of sweat and examined its utility to assess differences in humans. Principal component analysis demonstrated that lipid related features allowed the differentiation of subjects, gender and diet. The method will be applied to clinical samples in order to examine its potential as a quick and non-invasive diagnostic tool.
Endometriotic Tissues Differentiation by Mass Spectrometry
Development of instant and non-invasive endometriotic tissues differentiation method. For the first time the Direct Mass Spectrometry approach was applied to analyze endometrium and endometriotic lesions of different localization. Our clinical lipidomics approach defines the net outcome of several imbalanced lipids and fatty acids which may be crucial for endometriosis pathophysiology and tissues differentiation. The results of these studies may bring us closer to the understanding of the pathobiology of the disease and make it possible to elaborate new ways of early diagnostics and “intelligent” surgery.
Exploring Changes in Primary Metabolites in Alzheimer’s Disease Using Targeted LC-MS/MS
Here we investigate biochemical changes in metabolic processes in patients with Alzheimer’s disease using a targeted LC-MS/MS method for primary metabolites in human plasma. Human plasma samples from Alzheimer’s patients were compared with healthy age- and sex-matched controls using a targeted LC-MS/MS approach for amino acids, organic acids, nucleotides, nucleosides and co-enzymes. Sample preparation comprised a two-phase extraction with methyl tert-butyl ether (MTBE) and methanol/water with the methanol/water phase analysed directly by LC-MS/MS. Statistical analysis shows differentiation in profiles from disease and control subjects, with significant changes noted in purine metabolism and perturbations in neurotransmitter pathways including serotonin.
Differential Complex Lipid Plasma Profiles Reveal Perturbations in Preeclampsia
Preeclampsia, the onset of hypertension and proteinuria at =20 weeks of gestation, is a leading cause of maternal perinatal morbidity and mortality. Since preeclampsia is known to affect lipid metabolic pathways, we set out to validate and quantitate the plasma lipidome from women with severe preeclampsia (sPE) and preterm labor (PTL). Using the LipidyzerTM Platform (SCIEX) allowed profiling and accurate quantitation of approximately 1100 lipid molecular species from 13 different lipid classes. Lipid profile abnormalities were found in the triacylglycerides (TAGs) and diacylglycerides species (DAGs) as well as some novel markers found to increase in Preeclampsia.
Isotope Dilution for Measuring Metabolites in Cancer Cells
Mass spectrometry (MS) is already a powerful tool for quantification of metabolites in cancer research and is constantly gaining importance. Nevertheless, through unintentional variations in sample preparation and measurement results are often compromised. To tackle these problems we applied uniformly labeled internal standards (IS). These IS derived from U13C isotopically labelled yeast. As eukaryotic organism yeast shares most of the primary mammalian metabolism and thereby it can be exploited as source for IS. We covered molecules of the Krebs cycle, glycolysis as well as nucleotides, nucleosides and nucleobases in a comparative study of a resistant vs. a non-resistant cancer cell line. Additionally the application of these IS extended the working range and offered the opportunity to accomplish targeted and non-targeted metabolomics at the same time when high resolution MS was applied
Changes of Phospholipids in Mouse Model of Parkinson’s Disease by Using MALDI Imaging and UPLC-MS
Parkinson’s disease (PD) is a common neurodegenerative disease whose pathologic substrate is nigrostriatal dopaminergic degeneration due to the neuronal loss in the pars compacta of the substantia nigra (SN). Thus, it is of great importance to determine early neuronal changes that may contribute to disease progression. In this study, we utilized MALDI Imaging and UPLC-ESI-MS/MS to identify phospholipids related with PD within SN of mouse brain tissue. The SN regions of tissues were collected using laser capture microdissection (LCM). The two phospholipid species (PC 34:1 and PC 32:0) were significantly down-regulated in SN region of PD mouse model.
High-throughput Central Carbon Pathway Metabolomics: Analytical Strategy Based on Hydrophilic Interaction LC Coupled to Targeted Mass Spectrometry
Targeted approach was successfully applied to metabolomics although mainly focused on specific classes of compounds. A more diverse and global coverage is sought to determine the affected pathways and better understand metabolic disease mechanisms. In this study a comprehensive HILIC tandem MS platform has been designed to target metabolites from multiple central carbon pathways. A set of 619 metabolites standards was used to build up the library of transition states for MRM. Chromatographic conditions, including stationary phases, mobile phases composition and pH, were evaluated for optimal separation. As a proof of principle, different biological matrices were characterized using this approach.
Real-time Monitoring of Glutamic Acid in Mouse Brain by Direct Coupling of Microdialysis to Tandem MS with the Newly Developed On-line Desalting System
This study demonstrated the real-time monitoring of Glutamic acid in mouse corpus striatum by direct coupling of microdialysis to tandem mass spectrometer with a newly-developed on-line desalting system. The on-line desalting device showed sufficient desalting efficiency for microdialysate, enabling direct coupling of microdialysis to mass spectrometer. Although there was a small time lag due to flow path length, real-time monitoring of glutamic acid in the mouse striatum was achieved. In conclusion, the present method showed future possibility not only for behavioral study of living animals, but also for in vivo real-time multiple-monitoring such as metabolome analysis.
Application of GC/MS and Untargeted Metabolomics for the Diagnosis of Transaldolase (TALDO) Deficiency
Transaldolase (TALDO) deficiency is a rare disorder in pentose phosphate metabolism. Manifestations of TALDO deficiency include hepatomegaly, dysmorphic features and cardiac defects. Since first described in 2001, about a dozen additional have been published. Diagnosis is typically made by detecting urinary accumulation of intermediates in the pentose phosphate pathway, including erythritol, ribitol, arabitol and sedoheptulose. Here, we report diagnoses of two TALDO cases using GCMS for polyol analysis and DNA sequencing. Untargeted plasma metabolomic profiling also identified several pathognomonic biomarkers for the disorder. The results suggest that untargeted metabolomics may be an efficient screening tool for early diagnosis of TALDO deficiency.
Application of Intact Metabolome Analysis by Probe Electrospray Ionization (PESI)/MS/MS to Local Distribution Analysis and in vivo Real-time Monitoring
Probe electrospray ionization (PESI), which is one of the ambient ionization techniques, enables direct analysis of endogenous compounds in biological tissues without sample preparation. In this study, we applied PESI/tandem mass spectrometry (MS/MS) to intact metabolome analysis of biological tissues and investigated the possibility of applying the present method to local distribution analysis of metabolites and real-time monitoring in a living animal. In conclusion, the present method achieved intact endogenous metabolite analysis without sample pretreatment. PESI/MS/MS also demonstrated its applicability not only to local distribution analysis in brain but also to in vivo real-time analysis of a living mouse liver.
Stable Isotope-assisted Metabolomics to Profile Glucose and Amino Acid Metabolism in Humans
Due to their slow glucose release and thus prolonged energy availability, slowly digestible starch containing food products show promising properties in the context of diabetes management. We analysed plasma samples of a nutritional intervention study conducted by Unilever R&D Vlaardingen in the Netherlands to explore the postprandial turnover of starch- and protein-derived metabolites. We compared the metabolic effects of three different flour blends mixed from different amounts of wheat flour, chickpea flour, barley flour and guar gum. 2% of the flour present in the chapati bread was fully 13C-labeled. We applied targeted GC-MS combined with “Mass Isotopomer Distribution (MID)” analysis to reveal the dynamics of starch- and protein hydrolysis.
HRMS- and MRM-based Screening in Clinical Metabolomics - Sensitivity Matters in Distinguishing Diabetic States
Currently, metabolomics studies start with a broad, non-targeted screening for as many metabolic features as possible. Identified metabolites with potential as biomarker are then analyzed by a targeted, quantitative method(s) to confirm the marker sensitivity and specificity. However, clinical metabolomics often has to deal with very heterogeneous samples. This requires large cohorts to achieve sufficient correlation coefficients for comparison of groups in non-targeted screening and to reveal whether metabolites may be significant for e.g. diagnostics prognostics or patient stratification. Sensitive targeted profiling of metabolites may be suitable to lower heterogeneity in the sample groups and to identify outlier. Here we compared a non-targeted with a targeted workflow for initial characterization of a study group related to pathogenesis of diabetes.
Early Detection of Sepsis by the UPLC-MS Detection of Pathogen Markers
Sepsis is associated with a high morbidity and mortality rate, with the latter reaching up to 70%. However, conventional techniques, such as growth cultures from blood samples, can take several days for clinically informative results. Therefore, there is a pressing need for a fast, accurate, and reliable method for the early detection of sepsis and the identification of the causative pathogen. Here, we demonstrate a UPLC-MS method for the early detection of bacteria in a horse blood model, based upon analysis of the microbial lipidome, which is quick, easy, and pathogen specific.
Rapid Evaporative Ionisation Mass Spectrometry (REIMS) as a Novel Approach to Microbial Community Profiling
Mass spectrometry has become an essential component of clinical microbiology diagnostic workflows, increasing productivity and improving clinical outcomes through reduced sample turnaround time. Present commercial systems depend on isolation of a pure microbial culture, followed by additional sample preparation for MALDI-ToF, leaving room for further improvement. Rapid evaporative ionisation mass spectrometry (REIMS) has demonstrated robust species level identification for a variety of clinically significant microorganisms. Species specific features have been identified in mixed eukaryotic/prokaryotic cultures, indicating potential for taxonomic classification in cross-domain samples. Work is in progress to optimise the application of REIMS direct to clinical samples, removing the requirement for preparatory isolation and culturing of microorganisms.
Rapid and Quantitative Detection of Carbapenemase-Producing Enterobacteriaceae Based on MALDI-TOF MS
Carbapenem is the strongest β-lactam antibiotics and acts as inhibitors of the enzymes that catalyze formation of peptidoglycan in the cell wall of bacteria. Recently, the emergence of carbapenem-resistant bacteria seriously threatens this class of lifesaving drugs. Therefore, rapid detection of carbapenemase-producing enterobacteriaceae (CPE) is very important to prevent spread of these strains. Carbapenemase is an important enzyme that are produced by (CPE) and catalyze the hydrolysis of carbapenem. Typically, MALDI-TOF MS is not appropriate for small molecule analysis because organic matrices make a lot of noise at low m/z range. Parylene-matrix chip was developed for reduce matrix noise, and used to quantitate carbapenem successfully. Finally, MALDI-TOF MS based carbapenem susceptibility test was carried out with different 60 isolates using Parylene-matrix chip.
Use of the MALDI BioTyper System with MALDI-TOF MS for Rapid Identification of Microorganisms Causing Bacterial Urinary Tract Infection from Urine Samples
With the increasing number of cats maintained as pets, the opportunities to treat cats with lower urinary tract disease (LUTD) have recently increased in the clinical veterinary field. We performed direct identification of bacteria in urine using pretreatment kits for the direct application of positive blood culture bottles to MALDI-TOF MS, aiming to improve the low identification rates of E. faecalis. The concordance rates of E. faecalis identification by the MALDI-TOF MS method employing the MALDI Sepsityper Kit for pretreatment were 83.3, showing that the rate was improved.
Development of Bevacizumab Quantification Method in Human Plasma Using Immunoglobulin G Purification and In-solution Digestion
Bevacizumab is a humanized immunoglobulin G (IgG) monoclonal antibody (mAb) used for metastatic colon cancer treatment. As LC-MS offers high selectivity and sensitivity, we developed a method on this platform to analyze mAb. The high complexity of human plasma is the main challenge in obtaining accurate quantification results in LC-MS methods. We used the human IgG purification method to isolate the analyte in human plasma. In-solution digestion was applied for protein digestion. Surrogate peptide was selected to quantify the bevacizumab concentration in human plasma. This proposed method was validated and used to quantify the bevacizumab concentration in patient plasma samples.
Proteome Analysis to Discovery of Biomarkers of Kidney Disease
We performed the proteomic of different tissues of ischemia/reperfusion (I/R) swine model to identify new, predominantly renal biomarker candidates for kidney disease. Urine and serum samples were analyzed in pre-ischemia, ischemia (60 minutes) and 4, 11 and 16 hours post-reperfusion and renal cortex after 24hours of reperfusion. Intersecting the set of proteins up- or down-regulated in the ischemic tissue with both serum and urine proteomes, we identified 55 systemic proteins, four of them predominantly renal, candidates for biomarkers of renal disease. We validated one of the identified biomarkers, DPPIV, in a set of patients with diabetic nephropathy.
Cardiac Amyloidosis Protein Typing by Mass Spectrometry
Amyloidosis is a pathological condition characterized by abnormal deposition of serum proteins in organs and tissues. Specific knowledge of the type of protein deposits in hystologically positive biopsies can be of paramount importance in differential diagnosis and personalized medical treatments. Since AL (light chain amyloid) and ATTR (transthyretin-related amyloid) proteins are most frequently involved in heart amyloidosis, we developed an high sensitivity method for specifically identifying these proteins in heart biopsies with a targeted proteomic approach. The assay has been successfully applied to protein typing in heart histological samples.
Uncovering Target Glycoprotein Biosignatures Using a One-Pot Dual Nanoprobe Mass Spectrometry Assay
We designed a one-pot enrichment strategy to achieve sequential target glycoprotein and glycopeptide purification. By employing dual nanoprobes with divergent separation properties and functionality, a multi-step assay is achieved in a single container ("one-pot"). As demonstrated on alpha-fetoprotein (AFP), the assay has high purification specificity with 2-fold more glycopeptides compared to non-one-pot method. By targeted MS analysis (MRM-MS) of the non-glycopeptides, the assay can quantify low abundant AFP expression (0.5 ng) with good correlation with conventional ELISA method (Pearson's r=0.987). Furthermore, we present the first study revealing AFP glycopeptide patterns of individual liver cancer patients, comprised of 59 heterogeneous glycoforms of bi- and triantennary, core and terminal fucosylation, and mono- to tri-sialylation.
Detection of an Unreported Hemoglobin Variant by Mass Spectrometry – Hemoglobin Charlottesville
Hemoglobin Charlottesville, a previously unreported hemoglobin variant, contains a S138I/L substitution. The presence of the variant hemoglobin was detected during manual review of cation exchange HPLC and capillary electrophoresis data. From our dilute and shoot LC-MS approach, we predicted an amino acid mass difference of 26 in the alpha subunit with polarity of the residue going from polar to non-polar. ETD fragmentation confirmed the predictions and narrowed down the substitution position to 131, 133, or 138. Subsequent MS3 by HCD revealed that the substitution occurred at position 138 of the alpha subunit.
An MS-based Integrated Platform for Intact N-linked Glycopeptide Profiling and Targeted Glycopeptide Quantitation of Liver Cancer Biomarker
Alpha-fetoprotein (AFP) has long been used as a diagnostic and prognostic marker for HCC. However, high false negative rate of diagnosis necessitates the more specific biomarkers and the high sensitive method for detection. Here we developed an analytical pipeline including an automated tool for N-glycopeptide identification and the enzyme-assisted targeted quantitation method for understanding the altered core-fucosylation of AFP. We anticipate applying the proposed protocol to understand the correlation between glycosylation and liver diseases for early detection purpose.
From Thousands of Mass Spectrometry Profiles to Biomarkers Within a Day ? Arion 4 Omics, a Novel Solution to Facilitate and Accelerate Omics-based Decisions
In an era where data science, biology and chemistry have all become finely interlinked, where high-throughput instruments generate staggering amounts of omics data for the purpose of disease profiling and biomarker discovery, much has been written about the problems faced with the storing, processing and analysis of.
Here we present an advanced and fully benchmarked, high performance computing solution to directly address key challenges surrounding the manipulation and analysis of large-scale proteomics data. We demonstrate how ‘Arion 4 Omics’ can analyse and discover biomarkers from many thousands of samples with the results delivered in less than a day.
Accelerating Data Independent Acquisition with Microflow Chromatography
Data independent acquisition (DIA) has been used to increase the comprehensiveness of data collection while maintaining high quantitative reproducibility. As this technique increasingly proves to be a solid tool for biomarker research, larger sample sets are being analyzed, driving the need for further improvements for throughput and robustness. Here microflow LC was investigated in combination with SWATH® acquisition on complex matrices, to assess depth of coverage and robustness relative to current nanoflow strategies.
This approach provides additional workflow options to researchers with higher throughput and robustness needs, enabling to quantify 4500-5000 proteins with CV <20% on ~150 proteomes per week.
Functionalized Surfaces for Direct Immuno-Affinity Mass Spectrometry - Detection of Haptoglobin Phenotypes
Antibody-functionalized MALDI surfaces prepared by ambient ion landing allow efficient immuno-affinity enrichment of antigen from complex samples directly on the plate. The effectivity of antibody-functionalized MALDI surfaces to enrich protein antigen was demonstrated on determination of haptoglobin phenotype - an important biomarker in patient survival.
MALDI plates were functionalized by polyclonal human anti-haptoglobin antibody using the lab-made apparatus. One microliter of serum was applied on the anti-haptoglobin spot and incubated for one hour. After several washing steps, samples were in-situ reduced and mixed with matrix. Automated sample analysis was used to acquire spectra of intact proteins.
Evaluation of a Novel Tandem Quadrupole Mass Spectrometer for the Quantitative Analysis of Peptides Using a Multi-Point Internal Standard Calibration Method
Translational and biomarker verification studies are challenged in that they not only require the analysis of large sample cohorts with high-throughput, but also demand high sensitivity, high resolution and selectivity over a large dynamic range. Targeted LC-MS/MS based assays afford analyte quantification with the reproducibility and throughput required in order to rapidly assess biomarker performance. Multiple Reaction Monitoring (MRM), using tandem quadrupole mass spectrometry, is an enabling technology that provides speed and selectivity, whilst miniaturized LC systems offer additional improved sensitivity. Here, the application of micro-fluidics coupled to a novel tandem quadrupole MS/MS system, using a multi-point internal calibration method for the quantitation of peptides and proteins is presented and considered for speed, sensitivity, accuracy/ bias and selectivity.
Plasma Proteome Profiling Reveals the Effects of Sustained Weight Loss on the Apolipoprotein Family and Systemic Inflammation Status
We have developed an automated and robust shotgun proteomics workflow for the streamlined MS-analysis of single drops of blood (´Plasma Proteome Profiling´, Geyer et al. Cell Systems 2016). Here we applied this approach to 1294 separate plasma proteomes to assess dynamic effects due to weight loss. Among the wide-ranging effects of weight loss (93 significantly affected proteins) were individual specific changes in apolipoprotein profiles. Correlation analysis of plasma proteins to patient data defined a panel of ten inflammation proteins and revealed individual specific-longitudinal inflammation profiles. Participants with high or low level inflammation profiles gained from weight loss.
Proteomic Analysis of Acetaminophen Toxicity in Liver Micro Tissues Using Data Independent Acquisition and Spectronaut 9 Software
Comprehensive data-independent acquisition (DIA) and targeted data analysis with retention-time-normalized spectral libraries have become the methods of choice for discovery and quantitative proteomics. We demonstrate here how thousands of proteins are reproducibly quantified from as little as 12`000 liver derived cells using Spectronaut 9.0. In this study, liver micro tissues were used to show that liver toxicity markers are up-regulated below the acetaminophen’s therapeutic dose. In contrast, proteins expected to contribute to the therapeutic effect are only affected at concentrations higher than the therapeutic dose.
iST: Sample Preparation for High Throughput Clinical Proteomics
Sample preparation workflows are a crucial part of routine mass spectrometry (MS) based proteomics measurements. Complex workflows, extensive sample fractionation and proteolytic digestion are highly time consuming and restrict the overall technical reproducibility limiting the overall applicability of MS-based proteomics for clinical applications. The accuracy and robustness of the MS platform is also strongly affected by sample quality reasoning for high quality proteomic samples. Here we present the straightforward, fast, sensitive and robust in-StageTip (iST) method for streamlined sample processing of complete proteomes.
Development of an LC/MRM-MS Assay for Quantification of Therapeutic Monoclonal Antibody Vedolizumab in Human Serum
Monoclonal antibodies have become commonplace biotherapeutics. Lack of response to monoclonal antibodies (mAbs) has been associated with inadequate mAb serum concentrations. Therapeutic drug monitoring (TDM) of mAbs has the potential to guide to more effective dosing in individual patients [1]. Here we show the development of an liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay for the absolute quantitation of Vedolizumab in serum.
A Validated Amino Acid Analysis Assay for Accurate Quantification of Protein Reference Materials: SIL-Thyroglobulin Case Study
With the emergence of mass spectrometry for the clinical measurement of proteins in biological matrices, the development of biological reference materials will continue to grow in importance. CRM's with values assigned by metrologically valid procedures will be critical to minimize and control experimental variations in all steps of the workflow including protein extraction, fractionation, enrichment, proteolysis and analysis. To this end, we have verified sequence fidelity and isotopic incorporation of an SIL full length Thyroglobulin for use as an internal standard in quantitative MS workflows. We have also developed an amino acid analysis (AAA) method traceable to NIST SRM 2389 and validated the method against NIST SRM 927. This AAA method may be used for accurate quantification of proteins to enable development of accuracy-based protein reference reference materials.
Development of a Mass Spectrometry Method for Quantifying Acute Kidney Injury Biomarkers in Urine
Acute kidney injury (AKI) is an important cause of hospital related morbidity and mortality. Recently two promising urine biomarkers were reported that together offer optimal diagnostic performance for early detection of AKI: Tissue inhibitor of metalloproteinase 2 and insulin-like growth factor binding protein 7. We used quantitative proteomics for detection of the proteotypic peptides of both proteins. Candidate peptides were selected after tryptic digestion of recombinant proteins and conditions for MRM-LCMS were optimized. LOQ for peptides of both proteins were below the expected normal protein concentrations and both proteins could be detected in normal urine pool samples.
Integrated Secretome and Transcriptome Analyses for Identification of Functional Secreted Molecules Involved in Diffuse-type Gastric Cancer Progression
The purpose of this study was to identify functional secreted molecules involved in diffuse-type gastric cancer progression. We integrated the secretomics of six gastric cancer cell lines and gene expression analysis of gastric cancer tissues with publicly available microarray data. This study revealed that GDF15 may be a novel functional secreted molecule for diffuse-type gastric cancer progression, possibly having important roles for cancer progression via the affecting fibroblast function, as well as TGF-ƒÀ.
Annexin A2 Contributes to Chromosomal Instability by Coilin-mediated Centromere Damage
Most human cancers show chromosomal instability (CIN), but the precise mechanisms remain uncertain. We found that annexin A2 is overexpressed in the nuclei of CIN cells compared to cells with microsatellite instability (MIN). Ectopic annexin A2 expression in MIN cells results in a high level of aneuploidy and induces lagging chromosomes. These results suggest that nuclear accumulation of annexin A2 plays a crucial role in CIN by disrupting centromere function. In addition, we are carrying out a search of chemotherapy sensitive marker of esophageal cancer that involved in chromosomal instability by using the latest proteomic analysis techniques.
Best Practices for Long-term Quality Assurance of Quantitative Clinical Chemistry Proteomics Tests
Mass spectrometry (MS) is a promising technology that experiences an increased interest for its application in quantitative clinical chemistry proteomics (qCCP), due to its high specificity and its potential for multiplexing. However, MS is a complex technology, which requires expanded quality assurance procedures and predefined quality criteria to guarantee accurate and reliable results in the clinical laboratory setting. Here, we review our best practices for quality assurance of MS-based qCCP tests in time and space. We are confident that our findings can be generalized and transferred to other applications and labs, enabling ISO 15189:2012 accreditation of qCCP tests.
Multiplex Plasma Protein Analysis: Quantitation of Dozens of Biomarkers from a Droplet of Blood Using Immunoaffinity Mass Spectrometry
Mass spectrometry-based immunoassays enable the measurement of dozens of target proteins from a minute amount of sample. Biological samples like plasma are enzymatically fragmented and peptides derived from proteins of interest are immunoprecipitated by peptide-specific antibodies immobilized on a solid-surface. Indirect quantification could be achieved by using isotopically labelled reference peptides. Here, we present our results for the accurate quantification of more than 12 target proteins in plasma requiring only 5 µl sample material. This low volume meets the assay requirements to monitor plasma proteins in preterm infants to measure plasma parameter relevant to organ development. We quantified those proteins in more than 100 plasma samples from newborns and preterm infants.
Solid-Phase-Free Immunocomplex Isolation for Mass Spectrometry-Based Immunoassays
A novel method integrating solid-phase free immunocomplex isolation for LC/MS based immunoassay approaches was established. Usually, LC/MS based immunoassays include an immunoprecipitation step capturing the endogenous and isotopic labelled reference peptides with antibodies conjugated to solid surfaces in bead or column format prior to MS analysis. Here, we present a method where the antibody-peptide complex is not precipitated, but isolated from plasma digest peptides by asymmetric field flow fractionation (AFFF). The use of AFFF allowed a solid-phase free immunocomplex enrichment from plasma digest, thereby avoiding contamination caused by high abundant peptides through solid-phase interaction with carrier surfaces and allowing the detection of C-reactive protein.
Evaluation of an MS-based Workflow to Determine Absolute Protein Concentration Using Protein Epitopes Signature Tags (PrESTs) in Plasma Samples
We evaluated the use of Protein Epitopes Signature Tags (PrESTs) (Zeiler et al., 2012) for mass spectrometry-based absolute quantification of clinically relevant proteins in plasma samples. We showed that single or mixtures of PrESTs can be spiked into plasma for high accurat quantification of clinically relevant proteins, such as C-reactive protein (CRP). We think that such applications can be translated into the clinics for high-throughput quantification of biomarkers.
Multiplexed MRM-based Protein Quantitation in Human Plasma Using Two Different Stable Isotope Labeled Peptides for Calibration
Precise and accurate quantitation of the endogenous plasma proteome has tremendous potential for clinical applications. Targeted detection of peptides in a bottom-up strategy is the most common and precise MS-based quantitation approach when combined with the use of stable isotope labeled peptides. However, when measuring protein in plasma, these unknown endogenous levels prevent the implementation of ideal calibration strategies since no blank matrix is available. Consequently, several alternate calibration strategies are employed by different laboratories. In this study, these methods were compared to a new approach using two stable isotope standard (SIS) peptides, differentially labeled, enabling an external calibration curve and quality control samples to both be prepared in pooled human plasma without interference from endogenous peptides.
Accuracy Evaluation of High- and Low-density Lipoprotein Cholesterol Assays in Clinical Laboratories by Comparison with Isotope Dilution Mass Spectrometry
We evaluated the performance of five HDL/LDL cholesterol assays currently used at 12 clinical laboratories in Korea to assess the traceability of current HDL/LDL cholesterol in vitro diagnostic products to commutable frozen serum (CFS) reference materials (Toshiba-Kyowa [n = 2], Hitachi-Sekisui [n = 3], Siemens ADVIA [n = 2], Roche Cobas or Modular [n = 2], and Beckman Coulter AU series [n = 3]). The HDL-C assays tended to be higher than GC-IDMS measurements, and the degree of bias was not acceptable by National Cholesterol Education Program criteria. Whereas, LDL-C assays did not show any tendency to GS-IDMS measurements, but the degree of bias was acceptable except the Beckman Coulter assays by National Cholesterol Education Program criteria.
Rapid Quantification of Total Urinary Cortisol and its Major Metabolites by UPLC Tandem Mass Spectrometry
Clinical assessment of daily cortisol production is conventionally done by measuring urinary free cortisol, thus ignoring the fact that only 1% of total endogenous cortisol production appears in urine. Additional measurements of cortisol metabolites are likely to provide a more reliable estimation of total daily cortisol production. We therefore developed and validated a sensitive, selective, and rapid LC-MS/MS method for simultaneous assessment of total cortisol, cortisone, tetrahydrocortisol (THF), allo-tetrahydrocortisol (a-THF) and tetrahydrocortisone (THE) in urine. Total run-time of our method was 5.5 minutes and intra- and inter-assay coefficients of variation were <10% for all components. Linearity in the calibration range (r2) was consistently >0.99 for each component and all results were in agreement with those obtained from routine LC-MS/MS and GC-MS/MS analyses.
The Challenges and Strategies for LC-MS/MS Method Development of Accurate Assays of Steroids in Human Serum
The challenges for LC-MS/MS method development of accurate assays of steroids in human serum are: 1) low ionization efficiency and low concentrations in the circulation; 2) many isobaric isomers; 3) easily interfered by unknown compounds. Three strategies have been proposed and implemented in a fully validated method of simultaneous quantitation of seven androgen- and estrogen-related compounds at the level of pg/mL in postmenopausal serum, and in a fully validated method for the simultaneous quantitation of 7alpha, 7beta OH DHEA and 7 keto DHEA with a novel derivavtization reagent.
Tandem Mass Spectrometry Analysis of Podocyturia from Urine Sample Collected on Paper Filter
Urinary podocytes are potential biomarkers for several kidney diseases. Current analytical techniques for podocyte evaluation are complex, tedious, and the interpretation of results is operator-dependent. The main objective of the project is to develop and validate a simple and robust tandem mass spectrometry method for the simultaneous analysis of specific proteins related to podocyturia using urine samples dried on filter paper in Fabry disease patients, women with preeclampsia, and patients with chronic kidney diseases. The method is based on the elution of urine from the filter paper and the production of specific tryptic peptides. The goals are to facilitate the diagnosis, provide better management, monitoring and long-term follow up of patients by transferring this method to the clinical field.
Estimation and Correction of the Blood Volume Variations of Dried Blood Spots Using a Postcolumn Infused-internal Standard Strategy with LC-ESI-MS
Dried blood spot (DBS) is considered a promising technique in advancing personalized medicine. Whole-spot extraction is advantageous because it is less affected by HCT-caused bias, but blood volume variation limits the use of this sample processing technique. This study proposed a novel approach using the postcolumn infused-internal standard (PCI-IS) method for correcting the blood volume variation on DBS samples. The PCI-IS method utilized the isotonic property of blood samples, by evaluating the extent of the ion suppression at the first significant ion suppression zone, the blood volume on the DBS cards can be calculated.
Throughput and Sample Preparation Improvements for Endocrine Research by Microflow LC-MS/MS
Simple and rapid analysis of endocrine samples is necessary in a clinical research laboratory. Additionally, as financial and environmental concerns become more prevalent, heavy use of solvents for high flow LC-MS/MS methods, particularly those involving online extraction, can be an issue. Microflow LC offers a solution to these concerns, whilst greatly enhancing on column sensitivity and chromatography performance, reducing sample consumption and improving system robustness and uptime. As the sample injection volume is minimized, robustness and reproducibility of the entire method is
dramatically increased as matrix effects are significantly reduced.
Steroid Analysis in Different Biological Matrices: Solving Problems and Breaking New Grounds with LC-MS3
Finally, LC-MS/MS assays are applicable to quantitative steroid analysis in clinical routine diagnostics substituting immunoassays due to superior selectivity and comparable sensitivity. The broadsword MRM allows massive multiplexing of analytes for maximum flexibility. Matrices like hair, dried blood and urine, however, can outplay said approach generating the need for a higher method specialization, meaning increased specificity and selectivity while sacrificing flexibility. Introducing the scalpel MS3 provides that specialization. It offers superior quantitation in the analysis of hair cortisol and dried blood 17-OHP and can even function as an emergency tool in randomly occurring problems in serum or saliva analysis.
Exploring Fragmentation Pathways of Nucleotides by ESIMS/MS: Towards Improved Identification and Quantification of Therapeutically Relevant Nucleotide Analogues
Tandem mass spectrometry with electrospray ionization enables to the performance of the controlled fragmentation of molecules. We applied this technique to perform a broad study on the fragmentation of nucleotide derivatives. Identification and structural analysis of these compounds are important because of the crucial role of nucleotides in many biochemical processes. General conclusions regarding nucleotide fragmentation patterns could be drawn from the results.
LCMS-based CSF Neurotransmitter Quantification for Following Psycho-pharmacotherapy in Psychiatric Disorders
Psychiatric disorders such as depression and bipolarity are leading causes of disability and have huge socioeconomic consequences. The molecular mechanisms underlying psychiatric diseases still remain elusive. Pharmacotherapy of these conditions aim at modulating the neurotransmitter equilibrium in the synaptic cleft. However, appropriate molecular markers to monitor psycho-pharmacotherapy effects are not available. We developed a SPE-LCMS method for quantification of neurotransmitters in CSF. Neurotransmitter quantification was employed to establish comprehensive CSF profiles in suicidal patients that underwent psychopharmaca treatment. The data aid to improve diagnosis, prognosis of psychiatric conditions as well as monitor treatment effects and provide insight in the underlying molecular pathology.
QuEChERS Sample Preparation Prior to UHPLC-MS/MS Determination of Benzodiazepines
A method for routine analysis of 43 benodiazepines by UHPLC-MS/MS was developed. An original sample preparation method was used and validated to simplify workflow in the laboratory and accelarate result reporting. The method was validated in blood, serum and urine and performances were tested in 50 pateint samples.
A Brief Summary of Four-years Experience with a Simply, Accurate, Sensitive, Ready to Use Kit for Simultaneous TDM of 16 AEDs in Serum and Plasma by LC-MS/MS
In the past 4 years we tested 56776 samples of patients treated for different kind of epilepsy. 27296 samples were analyzed by chromatography and 29480 by different immunological methods. Approximately one hundred samples of both groups were also retested by LC-MS/MS with a commercial kit (Alifax-Eureka, Italy). The remaining of samples to be tested in chromatography were then tested only by LC-MS/MS with same kit on a Thermo-Fisher TSQ Quantum Access Max triple quadrupole. This kit showed for all the analyzed drugs (carbazepine, oxcarbazepina and their active metabolites, phenytoin, phenobarbital, primidone, topiramate, lamotrigine, levetiracetam, felbamate, zonisamide, rufinamide, lacosamide) consistent and reproducible recovery, sensitivity, selectivity, linearity and high correlation in comparative assays. This kit is now widely used in our TDM routine.
Fast and Sensitive Method for Simultaneous Quantification of 9 Antibiotic Drugs in Human Plasma by LC-MS/MS
Therapeutic drug monitoring (TDM) plays a key role in understanding what is the most effective therapy to be applied to the patient and, at the same time, putting in the foreground his state of health.
This work describes results obtained using Eureka Lab Division ready to use kit. The kit permits to dose in a single chromatographic run and with single extraction procedure, 9 most common antibiotic drugs in human plasma by LC-MS/MS in a sensitive and accurate way.
Its easiness of use and its robustness match perfectly with the needs of the laboratory routine.
Rapid and Sensitive Measurement of Bisphenol A and Bisphenol A Glucuronide in Serum and Urine Using UHPLC-MS/MS
Used in plastics and thermal paper, bisphenol A (BPA) has received much attention due to its endocrine disruption potential and widespread human exposure. BPA can leach into food and beverages from BPA-containing containers, bottles or thermal paper and be consumed or absorbed through the skin. After absorption, BPA is rapidly metabolized to BPA glucuronide and to a small extent, BPA sulfate. Both conjugates are excreted in the urine. The measurement of BPA in biological samples is essential for risk assessment, but such analyses are challenging due to the trace levels of BPA in these samples and the possibility of background contamination. We developed methods based on UHPLC-MS/MS for the rapid quantitative analysis of both BPA and BPA-glucuronide in serum, urine and environmental samples.
Towards a Random Access Workflow for LC-MSMS Testosterone with the Tecan AC Extraction Plate - is It Feasible?
We validated an LC-MSMS testosterone method using the Tecan AC Extraction Plate and observed excellent precision and stability. We then evaluated (1)an historical calibration (from another batch within +/-14 days) or (2)partial calibration (only 2 standards) versus (3)calibration with 5-10 standards in each batch. CVs for between run precision ranged from 2.9-8.6% for 4 QC levels, 6 batches, across 14 days for the 3 calibration schemas. The bias for QC means and 100 patient samples between calibration schemas 1 or 2 versus 3 was <3.4ng/dL or 5.4%. Less intensive calibration schema to enhance productivity appears promising and deserves additional investigation.
Removal of Phospholipids from Plasma Samples with an On-line Zirconium Particle Packed Guard Cartridge Followed by LC/MS/MS Analysis
Protein precipitation is widely used for sample preparation of biological fluid samples such as plasma, serum and whole blood, prior to an LC/MS/MS analysis. While the method is simple and efficient in removal of the proteins, there are present other highly abundant interference's in the biological fluids. In particular, phospholipids have been reported giving rise to severe effects on LC/MS/MS analysis including ion suppression and fouling on chromatographic separation. This poster describes the development and application of an on-line guard cartridge with the zirconia resins to further simplify the sample cleanup workflow.
A LC-MS Assay for Identification and Quantification of Marinobufagenin in Human Plasma: A Novel Approach for Preeclampsia Risk Evaluation
Marinobufagenin (MBG) is a bufadienolide cardiac inotrope implicated in the early diagnosis of volume expansion-mediated hypertensive states such as preeclampsia. The enhanced production of MBG in preeclamptic patients has been described using poor-specific immunoassays. This demonstrates the need for a sensitive analytical method to detect MBG in plasma at low levels. The identification of marinobufagenin in human plasma (men, non-pregnant women and early-pregnant women) using a sensitive and specific LC-MS assay will be presented, leading to a promising perspective concerning the preeclampsia risk assessment. An observational clinical study is currently designed in order to confirm previous results.
Quantification of Catecholamines and Free Metanephrines in Plasma by LC-MS/MS
To enable diagnosis of of pheochromocytomas and paragangliomas, we developed two LC-MS/MS methods: method A for the quantification of catecholamines, and method B for the quantification of free metanephrines. After semi-automated solid phase extraction, chromatography was performed under acidic conditions on a Hypersil Gold C8 column. As mass spectrometer, an AB Sciex QTrap 6500 was used. A sufficient dynamic range could be achieved for all substances. Imprecision was <9.9% for all substances, accuracy between 100-102%. Sufficiently low lower limits of quantification could be achieved, and matrix effects were tested as well. Sufficient sample stability could be shown. All substances tested could be shown as non-interfering. The described methods were successfully introduced into our routine laboratory practice.
Reference Ranges for Free Urine Metanephrines Measured by Tandem Mass Spectrometry
Phaeochromocytoma and paraganglioma (PPGL) are characterised by increased secretion of catecholamines and their metabolites. The biochemical test that is recommended by the Endocrine Society to diagnose and monitor these neuroendocrine tumours is either total metanephrines in urine or free metanephrines in plasma. However, free metanephrines in urine may prove to be the most convenient and best performing test for PPGL diagnosis in the future. In this study, we have used our LC-MSMS method for their measurement in the clinical laboratory to derive gender-specific reference ranges for free metanephrines for both spot urines and 24-hour urine collections.
High Throughput Quantification of Colistin in Dried Blood Spot by Using Ultra-high Pressure Liquid Chromatography-tandem Mass Spectrometry
Colistin, an old lipopeptide antibiotic with known nephrotoxicity and neurotoxicity is now being used to treat Gram-negative multidrug resistant bacterial infections. To ensure the efficacy and safety of colistin, therapeutic drug monitoring is recommended. Dried blood spot (DBS) has been applied to therapeutic drug monitoring. In this study, we developed an analytical method for determination of colistin from DBS by using liquid chromatography-tandem mass spectrometry. The validated method is efficient and high throughput which shows the potential for therapeutic drug monitoring of colistin.
One Platform for Determination of Urinary and Plasma Metabolites Relevant for Diagnostic Assessment of NETs such as Feochromocytoma or Carcinoid
Neuroendocrine tumors, an uncommon group of cancers, represent clinically and etiologically diverse group of disorders. These tumors exhibit secretion of many hormonally active substances which cause various endocrine syndroms. The analysis of catecholamine metabolites, both in plasma and in urine, are of importance for the diagnosis of tumors such as pheochromocytomas or paragangliomas. In case of carcinoid the diagnostic assessment is based on determination of 5-HIAA in urine and serotonin in serum. The aim of this work was to develop methods in such a way to maintain the same configuration of the LC-MS/MS system. Methods for wider group of analytes were developed, as follows: metanephrine and normetanephrine in plasma and in urine; serotonin in serum; vanilmandellic, homovanilic and 5-hydroxyacetic acid in urine. Methods fulfilled validation requirements.
Automated Sample Preparation of Whole Blood for Therapeutic Drug Monitoring and Diagnostics by LC-MS Using a Commercial Autosampler
In this poster the parameters necessary to automatically prepare whole blood samples for on-line LC-MS applications in the field of diagnostics and therapeutic drug monitoring have been investigated. A strategy and the most important parameters are shown for the optimization of a PAL RTC autosampler for the preparation of whole blood.
Evaluation of the Analytical Parameters for Sensitive and Robust Quantitative Analysis of Catecholamines in Human Plasma with LC-MS for Research
We evaluated an analytical method to support analysis of catecholamines (epinephrine, norepinephrine and dopamine) in human plasma for research. The method used SPE for sample preparation, a 9 min gradient chromatographic separation and a triple quadrupole mass spectrometer collecting two SRM for each analyte to calculate ion ratio. The limit of quantitation in donor plasma was 5 pg/mL for dopamine and 25 pg/mL for epinephrine and norepinephrine. Method precision obtained for RECIPETM QC samples was better than 4.6% and accuracy ranged from 86.6-119%. Ionization suppression was observed only for epinephrine and it was corrected by deuterated internal standard.
The Analysis of Emerging Drugs of Abuse: Updating an Existing Method with New Compounds
The determination of psychoactive drugs and their metabolites has become routine in many forensic toxicology laboratories. The optimization of analysis time, resolution between metabolites, method robustness, and the ability to add emerging compounds is of ultimate importance when developing an efficient method for validation. The Raptor™ Biphenyl column combines the speed of superficially porous particles (SPP) with the resolution of highly selective USLC® technology to give the analyst the ability to produce fast dilute and shoot methods while staying current with the ever changing landscape of illegal drugs.
The Analysis of C3-Epimers of 25-Hydroxyvitamin D in Serum by LC-MS/MS
Vitamin D exists in two forms, vitamin D2 and vitamin D3; each undergoes metabolism to form 25-hydroxyvitamin D2 [25(OH)D2] and 25-hydroxyvitamin D3 [25(OH)D3] which are used as the biomarkers for the assessment of vitamin D status. The epimeric forms of 25(OH)D, 3-epi-25-hydroxyvitamin D2 and D3 may contribute to a large portion of the total 25(OH)D concentration. Since these epimers are isobaric, chromatographic separation is necessary for accurate quantitation. In this study, the Raptor™ FluoroPhenyl column was used for chromatographic separation of 25(OH)D and their C3-epimers.
A Method for the Quantification of PEth 16:0/18:1 in Human Blood Based on UHPLC and Orbitrap High Resolution Accurate Mass Spectrometry (HRAM)
Phosphatidylethanol (PEth) is an abnormal phospholipid that is only formed in red blood cells membrane in the presence of ethanol and its usage as a potential alcohol biomarker is being investigated by the Swedish Healthcare system; PEth 16:0/18:1 is the most abundant form. An analytical research method based on liquid chromatography high resolution mass spectrometry for the quantification of PEth 16:0/18:1 in human blood is described. Mass spectrometric detection was performed by monitoring of the intact mass on a Thermo Scientific™ Exactive Plus™ high resolution mass spectrometer using heated electrospray ionization.
Testosterone Serum Calibrator - Quantitation of Serum Testosterone Across the Entire Therapeutic Range Using LC-MS/MS
Development of accuracy-based calibrators in biological matrices for clinical diagnostic applications requires reference measurement calibrators and materials with high accuracy and sensitivity. A method for in vitro diagnostic (IVD) use in LC-MS/MS-based laboratory developed tests (LDTs) for quantitation of testosterone across the entire therapeutic range from 2-2,000 ng/dL in serum by Liquid Chromatography Mass Spectrometry (LC-MS/MS) was developed and validated. A certified reference material (CRM) grade serum calibrant kit, designed to bracket male and female testosterone clinical reference ranges, including 10 levels from 2-2,000 ng/dL along with a blank and 13C-labeled internal standard was developed and validated towards stability under various storage conditions.
Quantification of 25-OH Vitamin D2 and D3 with Chromatographic Resolution of the C3-epimer in Human Plasma or Serum by LC-MS
A clinical research analytical method for the quantification of 25-hydroxyvitamin D2 and D3 in human plasma or serum with chromatographic resolution of 3-epi-25-OH-vitamin D3 is reported. The method involves a simple protein precipitation step followed by online SPE using a Thermo Scientific™ Transcend™ II system; a Thermo Scientific™ TSQ Endura™ triple quadrupole mass spectrometer with atmospheric pressure chemical ionization is used for detection by single reaction monitoring (SRM) using 25-hydroxyvitamin D3-d6 as the internal standard. The method was analytically validated using the MS MS7000 ClinMass® LC-MS/MS Complete Kit 25-OH-Vitamin D2/D3 in Plasma and Serum – On-Line Analysis from RECIPE and limit of quantification, linearity range, accuracy and intra- and inter-assay precision were evaluated for both analytes.
Quantification of Total Homocysteine in Human Plasma or Serum by LC-MS/MS
An analytical method for clinical research for the quantification of total homocysteine in human plasma or serum is reported. The method involves a fast and simple sample preparation followed by injection onto a Thermo Scientific™ Transcend™ II system. Mass spectrometric detection is performed by single reaction monitoring on a Thermo Scientific™ TSQ Endura™ triple quadrupole mass spectrometer using heated electrospray ionization in positive mode. The method was analytically validated using the MS2000 ClinMass® Complete Kit Homocysteine in Plasma / Serum from RECIPE and limit of quantification, linearity range, accuracy and intra- and inter-assay precision were evaluated.
Development of a LC-MS/MS Method for the Simultaneous Measurement of Voriconazole, Posaconazole and Itraconazole
In response to recent clinical guidance advocating the need for antifungal therapeutic drug monitoring and the increased use of antifungal therapies both prophylactically as well as for active treatment has resulted in an increase in requesting. In response to this we have validated a simple and robust method for the determination of voriconazole, posaconazole and itraconazole concentration in serum using LC-MS/MS. This new assay will result in a greatly improved sample turnaround time and will therefore vastly increase the clinical utility of azole antifungal drug monitoring.
Integration of Steroids Analysis in Serum Using LC-MS/MS with Full-automated Sample Preparation
Currently sample preparation for the detection of steroids in serum by LC-MS/MS involves complex offline extraction methods such as solid phase extraction or liquid/liquid extraction, all of which require additional sample concentration and reconstitution in an appropriate solvent. These sample preparation methods are time-consuming, often taking 1 hour or more per sample, and are more vulnerable to variability due to errors in manual preparation. Our approach to offering a high sensitivity steroid detection method and timely, automated analysis of multiple samples is to use the automated sample preparation system coupled to the detection capabilities of a high-sensitivity LC-MS/MS.
Measurement of Vitamin B1 and Vitamin B6 in Whole Blood by LC-MS/MS
An LC-ESI-MS/MS method is described for the quantification of thiamine-pyro-phosphate (TPP) and pyridoxal-5-phosphate (PLP), the biologically active forms of vitamin-B1 and vitamin-B6. A linear gradient of 0.1% formic acid/methanol was used for simultaneous separation on a Symmetry C18 column. The method was linear until 5000 nmol/L for both analytes at an LLOQ of 12 nmol/L for TPP and 6 nmol/L for PLP. Inter-day and intra-day precision were <10.4% (TPP), <5.5% (PLP) and 5.5% (TPP), <3.8% (PLP) respectively. The relative matrix effect was 96.7% for TPP and 93.2% for PLP. Method-comparison of patient samples showed a correlation of y=1.01x-1.6 for PLP and y=1.00x+16.0 for TPP.
A Novel Approach to Urine Catecholamines LC-MS/MS Analysis
The catecholamines, epinephrine (E) and norepinephrine (NE) are very small and polar molecules, causing additional challenges to LC-MS/MS method development, as they are poorly retained on reverse-phase C18 columns. This work presents the development and validation of an LC-MS/MS method for determining catecholamines in urine, based on a new approach to ion-pair chromatography (IPC). Here, the ion-pair reagent 1-Heptane Sulfonic Acid (HSA) is added into the extracted samples instead of the mobile phases (MP) while using conventional LC/MS friendly MP for analysis. This overcomes some of the disadvantages of traditional IPC and the method shows very good performance characteristics.
Optimization of Solid Phase Extraction for the LC-MS/MS Analysis of Vitamin D Metabolites in Clinical Research
Matrix interferences are challenging when analyzing vitamin D metabolites by LC-MS/MS for clinical research. An extraction method using Waters® Oasis® PRiME HLB was developed demonstrating an increase in analytical sensitivity of vitamin D metabolites through the removal of >99% of the targeted lysophosphatidylcholines when compared to protein precipitation. A simple workflow was created; loading the protein precipitate, wash and elute. The extraction method coupled with chromatographic conditions to separate 25OHD2, 25OHD3, 24,25diOHD3 and C3-epi-25OHD3 enabled good precision, analytical sensitivity and accuracy for these vitamin D metabolites using an ACQUITY UPLC Xevo TQ-S micro system.
For Research Use Only, Not for use in diagnostic procedures.
Forensic and Toxicological Drug Screening Using LC-MS/MS with MS/MS Library-based Identification
Forensic and toxicological drug screening has a number of challenges with the need for rapid sample analysis and reporting with quantitative results. Multi Target Screening (MTS) methods were developed using multiple reaction monitoring (MRM) with threshold triggered MS/MS product ion scans at three collision energies to enable spectral based library searching. The MS/MS library was created using certified reference materials and included electrospray spectral data on over 1200 compounds relevant to clinical and forensic toxicology in both positive and negative ion modes. These experiments were applied to reduce false positive and negative reporting using MS/MS spectral library based identification.
Metrological Traceability of a New Waters MassTrak™ Vitamin D Assay
Robust metrological traceability has been incorporated in to the design, development and manufacture of the Waters MassTrak™ Vitamin D kit, to meet the requirements of ISO 17511:2003. The MassTrak™ Vitamin D kit calibrator materials are traceable to NIST SRM2972 via a documented unbroken chain of calibrations. The accuracy of this traceability to NIST SRM2972 has been verified through participation of the Vitamin D Standardisation Certification Program (VDSCP) in which the MassTrak™ Vitamin D Assay achieved a mean % bias of 0.6 % from the VDSCP reference values and an imprecision of 4.9%.
Analysis of Serum Androgens and Corticosteroids for Clinical Research by LC-MS/MS
Here we evaluate an offline automated method for the measurement of serum androgens; testosterone, androstenedione and dehydroepiandrosterone sulfate (DHEAS), and serum corticosteroids: 17-hydroxyprogesterone (17-OHP), cortisol, 11-deoxycortisol and 21-deoxycortisol, enabling steroid profiling for the investigation of metabolic dysfunction biomarkers for clinical research. An LC-MS/MS method was developed using a novel Solid Phase Extraction (SPE) sorbent, reducing sample preparation time and removing more matrix interference in comparison to other sample preparation techniques. Chromatographic resolution between structurally related steroid species was achieved. This offline automated method demonstrates excellent linearity, analytical sensitivity, selectivity, precision and accuracy, while providing high sample throughput capabilities.
MagSiMUS Sample Preparation Technology for LC-MS/MS Analysis
MagnaMedics developed the MagSiMUS sample preparation method for LC-MS/MS analysis. Proteins are precipitated on a magnetic silica based solid support and removed from the sample by magnetic separation. The levels of immunosuppressants, benzodiazepines, vitamin D, and steroids could be accurately determined in clinical samples using this process. MagSiMUS is a reliable sample preparation methods which can be applied flexibly in a manual or medium to high throughput automated work-flow. The absence of a centrifugation step improves the total sample throughput and reduces the preparation time.
Analysis of 25-OH Vitamin D2/D3 in Serum by LC-MS/MS with Full-automated Sample Preparation
Vitamin D measurement has become an important component in clinical assays largely because deficiency is associated with a number of disorders. LC-MS/MS has become essential tool for monitoring the concentration of vitamin D2/D3 in biological samples due to its high level of sensitivity and specificity; however, manual sample preparation often involves several complicated manual steps which can introduce error into the results. In this study, we investigated the ability to analyze for 25-OH Vitamin D2 /D3 by LC-MS/MS using automated sample preparation to process large sample sets.
Fully Automated Platform for Determination of Immunosuppressant Drugs in Whole Blood
Therapeutic drug monitoring of immunosuppressant agents needs to be accomplished by extremely accurate techniques. Liquid chromatography coupled to tandem mass spectrometry (LC–MS/MS) shows higher sensitivity and superior specificity compared to immunoassay-based approaches; however, LC-MS/MS approaches lack in standardization since they require sample preparation procedures that usually involves complex offline extraction methods. These procedures are time-consuming and are more vulnerable to variability due to errors in manual preparation. To increase the data quality, safety, and throughput of LC-MS/MS quantitation of immunosuppressant drugs, a fully automated platform for the quantitation, has been introduced.
High Speed UHPLC-MS/MS Determination of Multiple Steroids in Human Plasma Using the Nexera MX System for Multiplex Analysis
Steroid hormones play an important role as modulators of the autoimmune disease onset/perpetuation. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) is considered to be the method of choice for quantification of steroids as a consequence of the selective detection, precision and high sensitivity. To enhance the scope of steroid research a UHPLC-MS/MS was developed to support higher sample throughput and extended steroid panels.
Analytical Validation of a Multiplex LC-MS/MS Test for Quantifying Serum Steroid Hormones
We developed an in house test for simultaneous quantification of six clinically relevant steroids [11-deoxycortisol, 21-deoxycortisol,delta-4-androstenedione, free dehydroepiandrosterone (DHEA), 17-alpha-hydroxyprogesterone and testosterone] in human serum. After extraction of steroids using µ-elution Oasis MCX SPE sorbent, an UPLC-MSMS test was developed using an Agilent UPLC and 6495 triple quadrupole. Matrix-matched calibrators were used which showed excellent linearity and reproducibility. Total imprecision, measured on pool sera, ranged between 3.6 and 18 % for the six steroids. Accuracy of testosterone was of 103%, checked using NIST human serum reference material. Lowest limits of quantification were <0.5 nmol/L for all steroids, except for DHEA which was 3.0 nmol/L. The calculated total error met the desirable analytical performance criteria required for clinical care.
Flexibility of Multichannel HPLC with a Single Mass Spectrometer to Simplify Workflow Complexity and to Improve Throughput of LC-MS
The recent growing interest of LC-tandem MS in clinical laboratories is mainly because this analytical technique can provide definitive identification and accurate quantitation of target compounds. Today’s clinical laboratories are constantly challenged to increase sample throughput with method flexibility and reduce sample turnaround time. Multichannel HPLC in couple with a tandem-MS allows effective utilization of a single mass spectrometer with method flexibility. Two approaches of multichannel HPLC designs are presented to address different clinical labs’ needs for speed, efficiency and flexibility; One design with TurboFlow and laminar flow for both sample cleanup and separation, while the other design with laminar flow for separating compounds only. For in vitro diagnostic use. Not available in all countries.
Simultaneous Extraction of Catecholamine and Metanephrines from Plasma Prior to Analysis Using LC-MS/MS
This poster discusses the impact of optimization of various parts of the method development process to maximise sensitivity while delivering a combined assay for the analysis of plasma catecholamines and metanephrines. Method parameters; precursor ion selection, MRM transitions, chromatography and solid phase extraction protocols were optimized for increased sensitivity. LC-MS/MS analysis was performed using a Shimadzu Nexera UHPLC system coupled to an AB SCIEX 5500 triple quadrupole MS. SPE methods demonstrated recoveries greater than 80 % with RSDs below 10 %. Calibration curves from 20 to 1280 pg/mL demonstrated good linearity and r2 values greater than 0.99 for all analytes.
Anion-exchange LC-MS/MS Analysis of the Most Stable Nerve Agent Biomarker in Urine
Very simple, rapid and direct approach for the determination of the most stable nerve agent biomarker – methylphosphonic acid in urine samples by anion-exchange liquid chromatography tandem mass spectrometry was firstly proposed and developed. Chromatographic separation was performed using anion-exchange column. Application of anion-exchange chromatography allowed completely overcome matrix effect influence of urine substances. Tandem mass spectrometric detection has provided reliable and very sensitive determination of methylphosphonic acid in urine. Achieved limit of detection in urine 4 ng ml-1 were low. Precision of the method was good, within run and between run repeatability were lower than 12%.
LC/MS/MS Analysis for Drugs of Abuse Using Biocompatible Solid Phase Microextraction (BioSPME)
The field of illicit drug testing has recently become a constantly changing environment with the rapid development of unregulated designer and synthetic compounds. This study demonstrates the benefits of Biocompatible Solid Phase Microextraction (BioSPME) over traditional “dilute and shoot” and protein precipitation methods for the enrichment of illicit drugs and drugs of abuse directly from biological matrices. BioSPME methods are shown to produce cleaner extracts and enable lower detection limits from biological samples compared to both protein precipitation and dilution methods.
Biphenyl-based Stationary Phases for Improved Selectivity in Complex Steroid Assays
We developed an UHPLC-MS/MS assay that combines a simple sample preparation with a powerful MS method quantifying a broad steroid panel (cortisol, cortisone, corticosterone, 11-deoxycortisol, 21-deoxycortisol, 17-OH-progesterone, 11-deoxycorticosterone, progesterone, aldosterone, testosterone, dehydroepiandrosterone and dehydroepiandrosterone sulfate) in human serum. After a manual protein precipitation step, the eluates were directly injected into the UHPLC-MS system. Stable isotope-labelled counterparts of the targeted analytes were employed as internal standards. For detection the mass spectrometer operated in the ESI positive mode. Chromatographic separation of all isobaric compounds was achieved employing a Kinetex Biphenyl column. In combination with a mobile phase consisting of 0.2 mM ammonium fluoride and methanol we were able to establish a selective and sensitive method.
Potential and Limitations of Solid Phase Microextraction Coupled to High Sensitive LC-MS/MS System in Analysis of Prohibited Substances from Saliva
One of the directions in the area of monitoring prohibited substances in biological matrices is utilization of alternative specimens i.e oral fluid, sweat, etc. This forces development of new analytical protocols permitting to meet high requirements of data quality to be considered acceptable by international supervising agencies.
Current studies present the use of solid phase microextraction coupled to LC-MS/MS platform for analysis of banned substances in oral fluid. Up-to-date results show the limits of detection at pg/mL levels, thus showing potential of the approach to be used as fast, and sensitive tool for screening of prohibited substances in alternative matrices.
High-Throughput LC-MS/MS Measurement of 17-Hydroxyprogesterone in Human Blood Serum for Research Purposes
Here we present a sensitive, robust, high-throughput quantitation workflow for analysis of 17-Hydroxyprogesterone in serum implemented on 4-channel UHPLC system coupled to a triple-quadrupole mass spectrometer with atmospheric-pressure chemical ionization. Sample preparation involved liquid-liquid extraction. Processed samples were analyzed with a 4-minute water-to-methanol gradient LC method. A two SRM transitions were collected for analyte and for internal standard to calculate ion ratio. The limit of quantitation was 10 ng/dL, method precision was better than 6%RSD, negligible ionization suppression was observed. For method comparison, 40 donor samples were analyzed and results were consistent with those from reference lab.
Investigating the Cisplatin Uptake by Visualizing the Platinum Distribution in the Model Organism Caenorhabditis Elegans
Cisplatin is one of the most important and frequently used cytostatic drugs within treatment of cancer. The cytostatic effect is based on binding the DNA. The associated DNA deformations and the interference with DNA repair mechanisms lead to apoptosis in cancer cells. One important factor of these repair mechanisms is poly(ADP-ribose)-polymerase-1 (PARP-1).
To analyze the bioavailability of Cisplatin in Caenorhabditis elegans, a method laser ablation-inductively coupled plasma-mass spectrometry (LA-ICP-MS) and total reflection x-ray fluorescence (TXRF) were developed. Therefore, L4 stage wildtype worms and poly(ADP-ribose)metabolism enzyme 1 (pme-1) deletion mutants were treated with Cisplatin. Loss of pme-1, the ortholog of human PARP-1, is resulting in a disturbed DNA repair mechanism. The influence of Cisplatin was analyzed for situations, when DNA damage already exists.
Mass Spectrometry Method for the Instant Diagnosis of Endometriosis.
We recruited 80 patients with ovarian cysts and with peritoneal endometriosis who then underwent laparoscopic surgery. The comparison group consisted of 40 patients with uterine fibroids and without endometriosis, which was proved by laparoscopy. Differences in the mass spectrometric profiles of the endometriotic tissues and comparison group were analyzed in combination with the morphological features of endometrioid heterotopy in order to find the specific metabolic biomarkers. Identified biomarkers were verified in the blood and peritoneal fluid of patients with endometriosis. A new possible non-invasive method for endometriosis diagnostic was proposed based on mass-spectrometric analysis of blood plasma.
Desorption Electrospray Ionisation Imaging: A Complementary Tool for Histological Diagnosis of Human Breast Cancer
Breast cancer is a highly heterogeneous disease and one of the most prevalent form of cancers in women worldwide. Accuracy of diagnosis is often compromised as it is dependent on the pathologists’ subjective interpretation. Over the past decade, desorption electrospray ionisation (DESI) has been increasingly used for imaging based cancer studies. DESI mass spectrometry imaging enables spatial visualisation of lipid species across tissue sections allowing direct correlation with morphological features. The technique combined with multivariate statistical analysis serves as an excellent complementary technique for histological diagnosis of breast cancers and to establish key diagnostic lipid species as potential biomarkers.
Mass Spectrometry Imaging of Human Orthotopic Pancreatic Tumor Xenografts for Peptide Profile Characterization
Pancreatic cancer is aggressive and difficult to treat with a 5 year survival rate of just 1-16% respective to stage at diagnosis. Mass spectrometry imaging (MSI) is a powerful analytical tool to investigate pancreatic adenocarcinoma (PDAC) tumors in order to characterize the invasive front in the human orthotopic PDAC tumor models, differentiate stroma reaction, and grades of differentiation of primary PDACs, depending on the model. Based on macroscopic and microscopic annotations of the orthotopic PDAC xenograft tumors, peptide profiles were characterized to compare differences between the tumors, non-tumor tissue, tumor margins, invasive regions and metastatic lesions. This information may provide clinically relevant markers for tumor classification, tumor margin assessment and/or therapeutic response prediction.
Cytological and Histological Workflow Compatible Automated and Semi-Automated Sample Profiling and Analysis Methods
The flowprobe system facilitates atmospheric pressure extractive sampling without additional extensive preparation for spot based targeted profiling as well as arrayed imaging and scanned profiling. Presented here are a selection of emerging applications of the sample introduction technique at the levels of clinical, quantitative, and basic research, biomarker discovery, and drug deposition analysis. The identification and characterization of molecules of interest via direct and continuous in situ microextraction and ionization based surface analysis is shown to be, importantly, possible from histologic and cytological preparations in such a way that the samples are preserved and useful for subsequent orthogonal analysis.
MALDI Imaging Mass Spectrometry Analysis of Lipids Around Cortical and Hippocampal Amyloid-β Plaques of Transgenic Alzheimer’s Disease Mice (tgArcSwe)
A number of biochemical and clinical studies suggest that in addition to peptides, dysregulated lipid metabolism may also linked to the Alzheimers disease (AD) pathogenesis. Here, gangliosides were identified to bind to Amyloid-β peptides and thereby promote fibrillogenesis. Moreover, hypercholesterolemia was found to accelerate the AD pathology. Here, we performed MALDI imaging, to study the lipid microenvironment around cortical and hippocampal Amyloid-β plaques in transgenic AD mice carrying the Arctic and Swedish mutation of amyloid-beta precursor protein (tgArcSwe). The results show distinct localization patterns of ganglioside and other biogenic lipid species to Amyloid-β pathology, suggesting a prominent role of neuronal lipid species in AD pathogenesis.
Structure Specific Immunolabeling and Mass Spectrometric Probing of Amyloid Beta Plaque Pathology in Alzheimer’s Disease
Alzheimer's disease (AD) is a chronic, neurodegenerative disease, of which the underlying pathological mechanism is still not understood. The disease is characterized by accumulation of amyloid-beta (Abeta) peptides into different extracellular plaques. Plaques have also been found in non-demented pathological ageing patients. Therefore, discrimination between structural and molecular plaque architecture are of essential interest to resolve Abeta plaque pathology in AD. Here, hyperspectral imaging paradigm employing the Abeta aggregate binding luminescent conjugated oligothiophenes (LCO) in combination with an in-house software was used to differentiate between different types of plaques. The approach was further shown to be applicable for laser microdissection and offline mass spectrometric analysis, which validated the presence of various C- and N-terminal Aβ in the plaques.
MALDI imaging Mass Spectrometry of Cortical Lipids in Human Alzheimer’s Brain
Alzheimers disease is the most common neurodegenerative disease affecting 1 in 8 over the age of 65. Genetic predisposition with the apolipoprotein E (APOE) e4 allele, a lipid transporter protein, was identified as the major risk factor to develop sporadic AD, implicating a prominent role for lipids in AD pathogenesis. This highlights the need for further investigation of chemical changes in brain lipid species in AD pathology.
Here, MALDI imaging mass spectrometry has been used to determine lipids mass profile around plaques of post mortem human cortical brain from AD patients. In detail, sublimation with 1,5 diaminonaphtalene (1,5 DAN) was used for high resolution imaging of lipid species followed by amyloid staining on the same tissue. The data show distinct lipid localisations, including ganglioside species that were correlated to AD pathology.
Development of a LC-MS/MS Method to Quantify Urinary 18-hydroxycortisol,18-oxocortisol and Tetrahydroaldosterone
Evidence suggests that Primary hyperaldosteronism subtype classification through specific urinary steroid quantification may help guide treatment. This work describes the development and validation of a LC-MS/MS method to quantify urinary 18-hydroxycortisol,18-oxocortisol and tetrahydroxyaldosterone.
Urine samples were prepared using liquid- liquid extraction. Chromatographic separation was achieved over 4.5 min using a phenyl 1.7 µm 2.1x 100 mm column and detected using a Waters TQMS. No interference or ion suppression were observed. Intra- and inter-assay imprecision was within the admissible range. Linearity and the LOQs covered clinically relevant concentrations. This method has been successfully developed and validated for clinical analysis.
Improving Transplant Patient’s Welfare; Standardising and Advancing the Therapeutic Drug Monitoring of the Immunosuppressant Drug Tacrolimus.
The immunosuppressant drug tacrolimus requires Therapeutic Drug Monitoring. External Quality Assurance data for tacrolimus demonstrates that there is currently an unacceptable inter-laboratory variability. LGC has developed a Reference Measurement Procedure (RMP) for tacrolimus in whole blood which has been applied to the characterisation of Certified Reference Materials and assigning target concentrations in laboratory intercomparisons.
This RMP has been adapted for the evaluation of a novel Volumetric Absorptive Microsampling (10 µL) device. Preliminary data using a ‘wet’ device provided fit for purpose data. However use of dried tips resulted in bias which could not be accounted for and requires further investigation.
Assessing Ion Suppression in On-Line Solid Phase Extraction LC-MS/MS
In an online SPE-LC-MS/MS method for the analysis of immunosuppressant drugs in whole blood, we observed differences in response between injections of standards prepared in mobile phase and standards prepared in matrix. We configured the system and method to permit concomitant injection within the same method cycle to both first and second dimension columns. We injected standard solutions to the second dimension while injecting either mobile phase or matrix blanks to the first dimension. This approach has allowed us to directly assess the influence of residual matrix on signal. Having indirectly established that the signal differences were related to matrix and not to recovery in the first dimension of the separation, we evaluated changes to the analytical gradient to correct the problem.
Interference Co-eluting with Aldosterone
After implementing a new LC-MSMS method for aldosterone in serum into routine use, we detected a reoccurring interference in about 3-5 % of the samples. We were unable to identify this interference and ended up making an alternative method for these samples.
What Makes Testosterone Different from Other Steroids in Trouble Shooting?
In our steroid profile (kit Perkin Elmer, LC-MS/MS Waters) we were confronted with a failure of our QC for testosterone in home-made QC samples, while the performance of commercial, stripped serum QC samples is good. We ruled out various errors in the pre-treatment of the samples. After contacting the MS vendor, several parts of the MS were cleaned and/or replaced. The problem couldn’t be solved by the vendor so far. This case is illustrating that commercial QC samples are not useful in detecting sensitivity related problems.
Systematic Troubleshooting of Assay Weaknesses During Method Development
Development of a Multi-analyte psychostimulant panel comprising Fluphenazine, Chlorpromazine, Risperidone, 9-OH Risperidone, Methylphenidate and Haloperidol was undertaken using TFC-LC-MS/MS. The assay development posed an initial challenge related to assay LLOQ (0.1ng/mL for Fluphenazine and 10ng/mL for Chlorpromazine) with a common measurement range (250 fold). Sequential observations were resolved including phospholipid removal (TFC loop injection and LC gradient modulation), transition detuning (and selection for equivalent response ranges), transition summing (least sensitive ionization/transmission efficiency analyte focus), improved imprecision/accuracy (echo transition summing, scheduled MRM and differential transition selection between IS and analyte) and determination of LC eluent stream multiplexing (staggered parallel LC compatibility).
The Center did not Hold – what happened to the peaks in the middle of the run?
It is a common complaint that LC problems occur more frequently than MSMS problems in the clinical mass spectrometry laboratory. Poor chromatography for early eluting analytes, caused by guard and column aging or sample preparation errors, is encountered fairly often. Occasionally bizarre peak shapes or missing peaks only at the end of the run is observed, potentially LC pump or mobile phase related. Acceptable chromatography at both the beginning and end of the run with missing peaks in the middle of the run is a more interesting situation to troubleshoot. We describe the presentation and resolution of such a case.
α-Methyldopa Interference in Urinary Normetanephrine Measurement by LC-MS/MS?
α-Methyldopa is known for analytical interference in HPLC methods for metanephrine measurement, but at present it is unclear whether this also holds for LC-MS/MS. We present a case of analytical interference from α-methyldopa in our LC-MS/MS method for urinary normetanephrine analysis. A closely eluting interfering peak had been mistakenly integrated by the MS instrument software as being normetanephrine and was reported to the clinic, despite notification of an ion ratio failure. The interference could be eliminated by modification of column type and chromatographic conditions. Our finding emphasizes the importance of critical evaluation of each chromatogram and notification of ion ratio failure. Particularly, for compounds with unresolvable mass fragmentation, chromatography remains key to guarantee accurate patient results.
Using a Potassium Ion Complexation Method to Estimate Hematocrit in DBS Samples Using Flow Injection Analysis Electrospray Ionization Mass Spectrometry
Dried blood spot (DBS) is an important sampling technique, but individual variation of hematocrit (HCT) is the major sources of quantification errors for this technique. This study developed a potassium ion complexation (PIC) method to calibrate HCT on DBS samples using flow injection analysis electrospray ionization mass spectrometry. Extraction of potassium ion (K+) followed by complexation with 18-crown-6 aided the measurement of K+ from DBS samples which was used to calibrate HCT. Linearity, precision and accuracy tests were done to validate the method. We anticipate that the developed PIC method can be used to calibrate the HCT from DBS samples.
UHPLC-MS/MS Method with On-line SPE to Quantify Tacrolimus and Everolimus in Peripheral Blood Mononuclear Cells: Application of “IS-normalized” Matrix Effect
Although the use of tacrolimus and everolimus is currently guided through TDM in whole blood, this does not necessarily reflect concentrations in lymphocytes. In this work we describe a method for their dosage in peripheral blood mononuclear cells (PBMCs).
PBMCs were isolated from blood with CPT vacutainers, washed twice and lysed. Drugs were extracted from cell lysates with on-line SPE platform and then separated and detected through reverse-phase UPLC-MS/MS.
Validation parameters successfully fitted FDA and EMA guidelines and samples from pediatric patients resulted within calibration range.
This method results eligible for use on real PBMC samples from pediatric patients.
A UHPLC-MS/MS Method to Quantify Tacrolimus in Tissues: Standardization Based on the Real Volume of Cells
For many drugs, blood or plasma measurements have a poor prediction of their efficacy and toxicity in the site of action. This method is capable of obtain concentration as ng/mL from different kind of tissues: we described the example of tacrolimus. After tissue lysation, done by a MagNA Lyser Instrument (Roche), tacrolimus was extracted with on-line SPE platform and then separated and detected through a UHPLC-MS/MS. This method could be used for evaluating the intra tissue concentrations of different drugs and for understanding the correlation between plasmatic/blood/PBMCs and tissue concentrations, with the same results expression (ng/mL).
The Benefits of Design of Experiments for Optimization of Quantitative LC-MS/MS Clinical Diagnostic Assays
Liquid chromatography-tandem mass spectrometry (LC-MS/MS) is a powerful technique for quantification of biomarkers in various biological matrices for support of clinical diagnosis and therapeutic drug monitoring. Method optimization involves many experimental factors which need to be simultaneously studied to obtain maximum sensitivity and selectivity at minimum retention time. This paper will illustrate that using design of experiments (DoE) for optimization of LC-MS/MS methods is much more efficient with only fraction of experiments that would been required by changing one-factor-at-time (OFAT) approach. Examples will be given to illustrate how DoE works for optimization of LC-MS/MS clinical diagnostic assays.
A Fatal Fall Due to the Multiple Drug Overdose
A 34-year-old woman was found dead in her building basement. Drugs and metabolites concentrations in ug/L measured by LCMS/MS in serum were: diazepam 2039, temazepam 148, sertraline 117, N-desmethylsertraline 271, nitrazepam 25, all in or below therapeutic range and nordiazepam 1447, demoxepam 2420, alprazolam 52, mirtazapine 2201 and fluvoxamine 427 above therapeutic range.
Although literature shows very low acute toxicity of benzodiazepines and antidepressants, demoxepam and mirtazapine concentrations were above toxic range. Also, combination of these drugs have synergistic pharmacological effects. There is a possibility that multiple drugs overdose caused accidental fall and/or contributed to possible suicide attempt.
A High-Throughput Solution for Therapeutic Drug Monitoring (TDM) with LC-MS/MS – 132 Drugs on One Column
For accurate, sensitive and fast Therapeutic Drug Monitoring (TDM) in clinical routine analysis, RECIPE has developed its ClinMass® TDM Kit-systems MS9000 and MS9050. Each system works with just one analytical column and the same solvents for all analytes, so high-throughput with no change in hardware between methods can be realized. The MS9000 Kit-system offers a fast sample preparation technique, the MS9050 Kit-system is working with fully automated sample preparation without the need of a liquid handling system. Both Kit-systems show outstanding validation results for Tricyclic Antidepressants, Antiepileptics, Neuroleptics, Antidepressants, Benzodiazepines and Antimycotics with more drug classes in progress.
Novel Devices for Blood Microsampling
A new device set that enables an easy blood microsampling for an accurate quantitative analysis of drug compounds and endogenous compounds in body is presented. They are for contributing to two scientific and medical aspects. The one is to facilitate 4Rs initiative, namely Replacement, Reduction and Refinement in animal studies on drug R&D by fulfilling our Responsibility for it. The other one is to promote an easy blood sampling from seriously sick patients and neonates/infants where stress and sense of fear upon the sampling can be reduced as well as the sampling size.
Solid Phase Micro Extraction for Determination of Free Testosterone and Metabolites
The extraction mechanism for Biocompatible Solid Phase Micro Extraction (Bio-SPME) enabled for the determination of unbound testosterone and some of its metabolites at low levels by LC/MS/MS. Improved sample preparation techniques allowed for reproducible and accurate quantitation of the unbound/bound analytes in human and animal serum samples. Advantages over current methodologies concerning matrix interference removal and pre-concentration of the analytes to achieve low detection limits will be presented.
HPLC-MS Method Development for Multi-component Determination of Less Polar Ginsenosides in Urine
Ginseng saponins are used in Traditional Chinese Medicine for more than 2000 years. Developed analytical method uses mass spectrometric detection in selected ion monitoring mode for sapogenin fragmentation ions of less polar ginsenosides in urine after ginseng infusion administration. Main advantage of this technique is the ability to detect simultaneously protopanaxatriol and protopanaxatriol derivatives and its deshydroxy analogues. Limits of detection were on the level of 0.01-0.02 mg/L. Good linearity and reproducibility were observed for this method. Developed approach was applied for pharmacokinetics study of ginsenosides less polar metabolites in healthy volunteers after ginseng infusion administration.
Evaluation of the Mitra™ Micro-sampling Device Against Dried Blood Spot Cards for Measurement of 25-dihyroxy Vitamin D3 by LC-MS/MS
The use of dried blood spots (DBS) has been extensively reported as a convenient and less invasive alternative to venepuncture, yet the haematocrit present in whole blood can adversely affect the quality of measurements. Haematocrit affects the viscosity of whole blood thus altering the distribution of the blood droplets on the cards. Mitra™ tips are volumetric absorptive micro-samplers (VAMS) which take up a fixed volume of blood via an absorptive tip. We have developed a LC-MS/MS assay for the quantitation of 25(OH)D3 in whole blood collected using DBS and Mitra™ tips. In this study we compared the two micro-sampling techniques against the plasma assay and investigated the impact of different haematocrit levels on the 25(OH)D3 concentration.
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