Adherence monitoring of antihypertensive drugs in finger prick blood microsamples – Development and validation of an analytical procedure

Abstract

Volumetric absorptive microsampling (VAMS) is an emerging technique and has already been established as a tool for adherence monitoring amongst others. This study aimed to add further compounds to the analytical panel for adherence monitoring of antihypertensive drugs (AHD). Based on a previous procedure (Jacobs CM. Anal Chim Acta 2021;1187:339137), sample preparation was performed by hydration of the VAMS-tip and subsequent precipitation with methanol:acetonitrile (30:70, v/v). The supernatant was injected onto a Dionex UltiMate 3000 LC coupled to a ThermoFisher (TF) Q-Exactive. Chromatographic separation was performed on a TF Accucore Phenyl-Hexyl column (100 mm × 2.1 mm, 2.6 μm particle size) within a total runtime of 10 min. The mass spectrometer was operated in switching parallel reaction monitoring mode. The procedure should allow analysis of hydrochlorothiazide, furosemide, lisinopril, and torasemide, as well as the metabolites canrenone, enalaprilat, and ramiprilat. It was validated based on the EMA guideline on bioanalytical method validation and a guideline of the international association of therapeutic drug monitoring and clinical toxicology (IATDMCT) for development and validation of dried blood spot-based methods for therapeutic drug monitoring. Furthermore, as a proof of concept, adherence was assessed in 18 patient samples using individually calculated dose-related cut-off concentrations and determined concentration in VAMS-tips were compared to those in matching plasma samples. VAMS enabled a fast and easy sample preparation. Carry over, selectivity, within and between day accuracy and precision met recommended criteria for all AHD. Matrix effects and recovery, determined at hematocrit (HT) 20%, HT 40%, and HT 60%, were sufficient even at low or high HT values in most cases. Dilution integrity achieved good results, except for lisinopril, using extracted blank matrix to dilute the extract. However, the therapeutic range of lisinopril was already covered by the calibration range. Autosampler stability for 48 h and stability of AHD in VAMS-tips at room temperature (24 °C) in the dark for 2 weeks was given for all analytes. Concentration of ACE-inhibitors and diuretics determined in authentic samples differed between VAMS and matching plasma in most cases. A preexisting method for adherence monitoring of 10 AHD could successfully be supplemented by three ACE-inhibitors and four diuretics, making the method suitable to assess adherence of the most frequently prescribed classes of AHD without changing the analytical setup. Adherence was assessed by calculating individual dose-related cut-off concentrations, a practical tool to estimate drug concentrations at a given point in time. Thus, determined concentrations in VAMS-tips (whole blood) differed from those determined in plasma, as already observed for other antihypertensives (Jacobs CM. Anal Chim Acta 2021;1187:339137). Therefore, specific cut-off concentrations for the adherence assessment need to be evaluated. Although the analytical validation showed satisfying results, further clinical validation is needed. A quantitative method for adherence monitoring of AHD in finger prick blood was successfully developed for three ACE-inhibitors and four diuretics. Specific reference ranges for adherence assessment in whole blood still need to be established.