Assessing inter-method agreement of drug-based phenotyping metrics between dried blood spot and plasma sampling

Abstract

Introduction: Pharmacokinetic variability in response to pharmacotherapy contribute to adverse drug reactions, drug-drug interaction and therapeutic failure seen in clinical practice. Poor therapeutic response to medication has been attributed to inter-individual and interethnic variability in cytochrome P450 (CYP450)-dependent metabolism and altered drug absorption via expressed transport channels such as P-glycoprotein (P-gp). An individualised approach in therapeutic management would be beneficial in a South-African population considering the country’s large genetic diversity. A single time point, non-invasive capillary sampling, combined with a low dose probe drug cocktail, to simultaneously quantify in vivo drug and metabolite concentrations, would enhance the feasibility and cost-effectiveness of routine phenotyping in clinical practice and guide personalised prescribing to individual patients. A recent development in dried blood spot sampling is the Mitra™ device, using Volumetric Absorptive Micro Sampling (VAMS™) technology to collect an accurate volume (10-30 µL) of whole blood onto a hydrophilic polymeric tip as an alternative to plasma sampling. Small volume blood sampling however presents bioanalytical challenges in terms of the reproducibility and sensitivity of the quantitative method and the agreement between quantitative measurement from a dried blood spot (DBS) and that from plasma sampling. The physicochemical diversity of the structurally related aromatic probe drugs, used together in a drug cocktail, further require optimised analytical procedures for simultaneous quantification. Phenotyping cocktails are compounded from commercially available dosage forms and introduce challenges with regards to dosage homogeneity, chemical interference or degradation and possible incompatibilities of drugs when used in combination. Aim and objectives: The purpose of this study was to compound the validated “Geneva phenotyping drug cocktail”, from available API sources and develop a validated, targeted, analytical LC-MS/MS method to quantify the seven probe drugs and six respective metabolites in dried blood spots when using the Mitra™ volumetric absorptive micro-sampling device for blood collection. The aim was to assess inter-method agreement of the measured probe drug and metabolite concentrations between the low sample volume, from a dried blood spot, and conventional plasma sampling. Methods: An Agilent binary series LC system coupled to a Sciex 4000 QTRAP triple quadrupole tandem mass spectrometer was used for method optimisation and validation. Targeted LC-MS/MS methods, in both negative and positive ESI mode, were validated according to ICH guidelines for matrix effects, recovery, linearity, limits of quantitation and detection, carry-over, inter and intraday precision and accuracy and analyte stability. The selectivity of the structurally related ionisable analytes was compared between a Kinetex C18 and Kinetex Biphenyl column and the influence of changes in the analytical conditions (involving mobile phase pH and solvent mixture composition as well as the solvent type) studied. An initial assessment of statistical in vitro agreement between plasma and DBS sampling were carried out. USP assays were performed to determine the weight and content uniformity of the compounded phenotyping cocktail containing six of the seven probe drugs. Content uniformity was evaluated with an Acquity UPLC system coupled to a Synapt G2 QTOF mass spectrometer. Results and discussion: A biphenyl stationary phase in combination with methanol as the organic eluent, provided improved resolution and analyte selectivity of the structurally related aromatic compounds. Results from the robustness experiment further confirmed the importance of controlling analytical conditions to ensure reproducibility and reliability of the quantitative method. Separation selectivity and higher throughput were prioritised over optimised ionisation efficiency, although the sensitivity of the analytical method for individual analytes were still within the expected in vivo concentration ranges to infer metabolic and transport phenotypes. This study successfully validated the use of DBS, collected with the volumetrically controlled absorptive microsampling device Mitra™, to measure expected probe drug and metabolite concentrations using the “Geneva phenotyping cocktail”. The validated method met all the required standards accepted in bioanalytical chemistry for specificity, sensitivity, linearity, accuracy, precision, carry-over and stability. From the initial in vitro assessment of agreement, it was concluded that blood cell distribution kinetics are regulated by the blood-to-plasma concentration ratio and time dependent equilibrium between different blood compartments, the physicochemical properties of the analytes, temperature during extraction, analyte concentration and stability. A conclusive confounding factor was the extent to which the extraction procedure liberated bound drug from either plasma proteins or erythrocytes. It was further concluded that the compounded low dose phenotyping cocktail capsules could be used successfully to assess inter-method agreement of drug-based metabolic ratios and drug transport between plasma and DBS collected with the Mitra™ device. Conclusion: To our knowledge, this is the first DBS validation study using the Mitra™ device for the purpose of simultaneous phenotyping of the in vivo P-gp transport and CYP450 metabolic activity of the CYP1A2, -2B6, -2C9, -2C19, -2D6 and -3A4 enzymes and activity.