An UHPLC-MS/MS Method with Protein Precipitation to Quantify Fourteen Antihypertensive Drugs and Two Metabolites in Human Plasma: Update for Adherence Testing Amedeo De Nicolò (Presenter) University of Turin Presenter Bio: Dr. Amedeo De Nicolò was graduated at the University of Turin in Molecular and Cell Biology and got a PhD in Medicine and Experimental therapy in 2017 at the same University. Major interest is in the application of quantitative LC/MS for Therapeutic Drug Monitoring purposes.

Authors: Amedeo De Nicolò1, Valeria Avataneo1, Franco Rabbia2, Elisa Perlo2, Jessica Cusato1, Fabio Favata1, Giovanna Fatiguso1, Debora Pensi1, Sarah Allegra1, Chiara Fulcheri2, Elena Berra2, Paolo Mulatero2, Giovanni Di Perri1, Franco Veglio2 and Antonio D’Avolio1.
1.Laboratory of Clinical Pharmacology and Pharmacogenetics #, University of Turin, Department of Medical Sciences, Amedeo di Savoia Hospital, Turin, Italy. 2.Division of Internal Medicine and Hypertension Unit, University of Turin, Department of Medical Sciences, AOU “Città della Salute e della Scienza”, Turin, Italy.

One of the main problems in the management of resistant hypertension (RH) is the discrimination of real cases of RH from poor therapeutic adherence. In this work our group validated a method for the quantification of fourteen antihypertensive drugs and two metabolites in human plasma within the same run with high sensitivity performance. Considering the high usefulness and reliability of TDM for adherence testing, this method is expected to be widely applied in the near future.

Introduction

To date, the management of resistant hypertension (RH), consisting in high arterial pressure (BP >140/90 mmHg) despite the administration of 3 or more antihypertensive drugs (including a diuretic) [1] is still difficult. One of the major issues is the differentiation of true RH from poor therapeutic adherence (TA): also defined as “pseudo-resistance” [2, 3]. Therefore, the adoption of Therapeutic Drug Monitoring (TDM) of antihypertensive drugs could represent a useful tool for direct testing of TA, especially before proceeding with surgical alternatives to pharmacological treatment (e.g. renal denervation or carotid baroreceptors stimulation). Recently, our group published a UHPLC-MS/MS method for adherence testing in suspected RH patients, capable of testing 10 different drugs [4]. Nevertheless, several other antihypertensive drugs are largely used in this context, so a wider multiplexed method was needed. For these reasons, we updated that method for the quantification of 4 more drugs and 2 active metabolites in plasma samples, suitable for an adherence screening before surgery and for evaluation of drug-drug interactions.

Methods

Validation was performed according to FDA and EMA guidelines. A volume of 200μL of plasma sample, calibration standard and quality control was added with 40μL of internal standard working solution (IS, 6,7-dimethyl-2,3-di(2-pyridyl)quinoxaline + 2H6-Atenolol, 13C-2H2-Telmisartan and 2H6-Nifedipine) and 1mL of pure acetonitrile in amber PTFE tubes (to limit nifedipine photodegradation), and then, after vortex mixing for 5 sec, all samples were centrifuged at 20000 x g for 10 minutes. After drying all the supernatants (about 1.5h), samples were reconstituted with 200 µL of water:acetonitrile 90:10(vol:vol): 5 µL were injected in a Perkin-Elmer UHPLC system, coupled with a Q-Sight 220 tandem mass detector. Chromatographic separation was performed on a Acquity ® HSS T3 1.8μm 2.1x150 mm column (Waters, Milan, Italy), with a gradient of two mobile phases: phase A (water + formic acid 0.05%) and phase B (acetonitrile + formic acid 0.05%), from 92:8 to 5:95 for 7 min and reconditioned again for 3 min (total duration 10 min). The instrument was settled in positive electrospray ionization (ESI+) for all drugs, except for hydrochlorothiazide and chlortalidone, which were detected in negative mode (ESI-). Two different MRM transitions were monitored for each compound, one used as a quantifier and the other one as a qualifier. In order to achieve a strong signal while maintaining the source clean for longer time, the mobile phases flow was diverted (50% ca.) to waste before reaching the detector. Six validation sessions were performed and 6 different plasma lots were used for the preparation of standards and for the evaluation of recovery, matrix effect and IS-normalized matrix effect (IS-nME). Accuracy, intraday and interday precision were evaluated performing the quantification in 5 replicates of 3 different QCs samples. Recovery was evaluated during all validation sessions comparing the areas of the peaks from extracted QCs (pre-spike) and from a blank extract added with the same concentrations of analytes (post-spike). The matrix effect was evaluated comparing the post-spike signal with the one from the direct injection of the same concentration of analytes, without matrix; the IS-nME was evaluated comparing the analytes response/IS response ratios in matrix versus neat solvents at the same concentrations of analytes and IS.

The validated method was tested on real samples from patients with apparent RH, enrolled in the TDM-TO study, all giving informed consent.

Cross-talk between different analytes was evaluated by analyzing plasma samples spiked with high concentrations of each single drug and checking for other analytes signal. Carry-over was checked by injecting blank samples after the highest standard.

Results

All drugs were successfully separated, except for ramipril, amlodipine and nebivolol. No interfering peaks were detected in our conditions.

Accuracy and precision fitted the limits indicated by FDA and EMA guidelines. The evaluation of the reproducibility of IS-nME among different plasma lots was used to identify the analytes needing the adoption of stable isotope labeled IS (SIL-IS), as previously described [5]. Therefore, recoveries, matrix effects and IS-nME resulted consistent and contained, respectively, and reproducible. Calibration curves resulted linear for the drugs with the lowest calibration range and quadratic for the drugs with the highest range.

Sensitivity was high enough to successfully quantifying expected trough concentration of all the considered drugs and metabolites (up to 19.7 pg/mL for nebivolol).

Cross-talk was absent and carry-over was present for nifedipine and valsartan, but 9-8 times lower than the LLOQ, respectively.

This validated method is now allowing to widen the adoption of TDM for TA testing in the context of TDM-TO study and clinical routine. Moreover, compared with TDM on urine samples, this method is more useful to test adherence for nifedipine (less photodegradation and SIL-IS normalization), for Telmisartan (it is not eliminated in urine), Ramipril and Sacubitril, since their major metabolites (sacubitrilat and ramiprilat) are monitored simultaneously.

Conclusions & Discussion

The wide panel of molecules simultaneously tested in this method makes it eligible for a high throughput screening of adherence of the vast majority of suspected RH patients in a really reliable way. Flow divertion and low injection volume , together with the combination of Heated Surface Induced Desolvation [HSID] and laminar flow ion guide, resulted in constant performance of the mass spectrometer for over 5000 injections, without need of advanced maintenance (only monthly cleaning of courtain cap). Actual limit of this approach is the need for blood withdrawal, which could be impossible for “critical” patients, and for centrifugation to obtain plasma. These fallbacks suggest the need for translation of the same application also to urine testing.

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