Validation of a quantitative multiplex LC-MS/MS assay of carvedilol, enalaprilat, and perindoprilat in dried blood spots from heart failure patients and its cross validation with a plasma assay

Andre Joubert; Anton Joubert; Marthinus van der Merwe; Jennifer Norman; Sandra Castel; Paolo Denti; Karen Sliwa; Gary Maartens; Phumla Sinxadi; Lubbe Wiesner

doi:10.1016/j.jmsacl.2022.12.003

Validation of a quantitative multiplex LC-MS/MS assay of carvedilol, enalaprilat, and perindoprilat in dried blood spots from heart failure patients and its cross validation with a plasma assay

J Mass Spectrom Adv Clin Lab. 2022 Dec 12:27:7-17. doi: 10.1016/j.jmsacl.2022.12.003. eCollection 2023 Jan.

Authors

Affiliations

¹ Division of Clinical Pharmacology, Department of Medicine, University of Cape Town, Cape Town, South Africa.
² Cape Heart Institute, Department of Medicine, Faculty of Health Sciences, University of Cape Town, Cape Town, South Africa.
³ Division of Cardiology, Department of Medicine, Groote Schuur Hospital, University of Cape Town, Cape Town, South Africa.

Abstract

Introduction: Adherence to medication is an important determinant of outcomes in chronic diseases like heart failure. Drug assays provide objective adherence biomarkers. Dried blood spots (DBS) are appealing samples for drug assays due to less demanding transportation and storage requirements.

Objectives: To analytically validate a LC-MS/MS method for the simultaneous quantification of carvedilol, enalaprilat, and perindoprilat in DBS and evaluate the feasibility of using the method as an adherence determining assay. To validate the assay further clinically by establishing correlation and agreement between plasma and DBS samples from a pharmacokinetic pilot study.

Methods: The method was validated over a concentration range of 1.00-200 ng/mL according to FDA guidelines. Adherence tracking ability of the assay was evaluated using a pharmacokinetic pilot study. Correlation and agreement were evaluated through Deming regression and Bland-Altman analysis, respectively.

Results: Accuracy, precision, selectivity, and sensitivity were proven with complete and reproducible extraction recovery at all concentrations tested. Stability of the analytes in the matrix and throughout sample processing was proven. The full range of concentrations of the pharmacokinetic pilot study could be quantified for enalaprilat, but not for carvedilol and perindoprilat. The difference between the observed and calculated plasma concentrations was less than 20 % of their mean for >67 % of samples for all analytes.

Conclusions: The assay is suitable as a screening tool for carvedilol and perindoprilat, while suitable as an adherence determining assay for enalaprilat. Equivalence between observed and predicted plasma concentrations proves DBS and plasma concentrations can be used interchangeably.

Keywords: ACE-I, Angiotensin-converting enzyme inhibitors; ALQ, Above the Limit of Quantitation; Adherence; BD, Bidaily; BMI, Body mass index; CHF, Chronic Heart Failure; CID, Collision-induced dissociation; CV, Co-efficient of variation; Carvedilol; DBS, Dried Blood Spots; Dried blood spots; EMA, European Medicines Agency; ESI, Electrospray ionization; Enalaprilat; HF, Heart Failure; ISTD, Internal standard; ITP, Initial testing procedure; LC-MS/MS; LC-MS/MS, Liquid Chromatography with tandem mass spectrometry; LLOQ, lower limit of quantitation; LOD, Limit of detection; MRM, Multiple reaction monitoring; NYHA FC, New York Heart Association Functional Classification; OD, Once Daily; Perindoprilat; QC DIL, Quality control dilution; QC LLOQ, Quality control lowest level of quantification; QC, Quality Control; QCH, Quality control high; QCL, Quality control low; QCM, Quality control medium; S/N, signal-to-noise ratio; SOP, Standard operating procedure; ULOQ, upper limit of quantification; VAMS, volumetric absorptive micro sampling.