In recent years, high-performance liquid chromatography (HPLC) with tandem mass spectrometric (MS/MS) detection has been demonstrated to be a powerful technique for the quantitative determination of drugs and metabolites in biological fluids. However, the common and early perception that utilization of HPLC-MS/MS practically guarantees selectivity is being challenged by a number of reported examples of lack of selectivity due to ion suppression or enhancement caused by the sample matrix and interferences from metabolites. In light of these serious method liabilities, questions about how to develop and validate reliable HPLC-MS/MS methods, especially for supporting long-term human pharmacokinetic studies, are being raised. The central issue is what experiments, in addition to the validation data usually provided for the conventional bioanalytical methods, need to be conducted to confirm HPLC-MS/MS assay selectivity and reliability. The current regulatory requirements include the need for the assessment and elimination of the matrix effect in the bioanalytical methods, but the experimental procedures necessary to assess the matrix effect are not detailed. Practical, experimental approaches for studying, identifying, and eliminating the effect of matrix on the results of quantitative analyses by HPLC-MS/MS are described in this paper. Using as an example a set of validation experiments performed for one of our investigational new drug candidates, the concepts of the quantitative assessment of the "absolute" versus "relative" matrix effect are introduced. In addition, experiments for the determination of, the "true" recovery of analytes using HPLC-MS/MS are described eliminating the uncertainty about the effect of matrix on the determination of this commonly measured method parameter. Determination of the matrix effect allows the assessment of the reliability and selectivity of an existing HPLC-MS/MS method. If the results of these studies are not satisfactory, the parameters determined may provide a guide to what changes in the method need to be made to improve assay selectivity. In addition, a direct comparison of the extent of the matrix effect using two different interfaces (a heated nebulizer, HN, and ion spray, ISP) under otherwise the same sample preparation and chromatographic conditions was made. It was demonstrated that, for the investigational drug under study, the matrix effect was clearly observed when ISP interface was utilized but it was absent when the HN interface was employed.

Contrary to common perceptions, the reliability of quantitative assays for the determination of drugs in biological fluids using high-performance liquid chromatography with tandem mass spectrometric (LC/MS/MS) detection methods and the integrity of resulting pharmacokinetic data may not be absolute. Results may be adversely affected by lack of specificity and selectivity due to ion suppression caused by the sample matrix, interferences from metabolites, and "cross-talk" effects. In this paper, an example of the effect of the sample matrix on the determination of finasteride (I) in human plasma is presented. The ion suppression effect was studied by analyzing standards of I injected directly in mobile phase and comparing the response (peak areas) of I and an internal standard (II) with the peak areas of the same analytes spiked before extraction into five different plasma pools and standards spiked into the plasma extracts after extraction. The LC/MS/MS analyses were performed using a turbo ion spray interface (TISP) under chromatographic conditions, characterized by minimal (total run time of 2 min, capacity factors, k' of 1.50 and 1.75 for I and II, respectively) and high retention of the analytes (total run time 6 min, k' of 3.25 and 13.25 for I and II, respectively). The absolute peak areas for I and II in different plasmas were calculated, and the slopes and peak area ratios at all concentrations within the standard curve ranges were compared. When analyses were performed under conditions of minimal HPLC retention, the slope of the standard line for one set of plasma samples was substantially different (about 50% higher) from that from other plasma sources. The precision of the assay, expressed as coefficient of variation (CV, %) was also inadequate and varied from 15 to 30% at all concentrations within the standard curve range. When the same experiments were repeated using high HPLC retention, the slopes from different plasma sources were practically the same, and the CV was improved to 6-14%. By increasing k' and providing more chromatographic retention of analytes, the "unseen" interferences from plasma matrix were mostly separated from analytes, practically eliminating the ion suppression. In addition, by eliminating from plasma extracts a number of endogenous components through more selective extraction, the ion suppression was also minimized. The detailed data and the design of these experiments are presented. In addition, development of a highly sensitive assay for I in human plasma at low picogram per milliliter concentrations using LC/MS/MS with a heated nebulizer (HN) interface, instead of a TISP interface, is described. In this case, the effects of sample matrixes were not observed.

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A microdose cocktail containing midazolam, dabigatran etexilate, pitavastatin, rosuvastatin, and atorvastatin has been established to allow simultaneous assessment of a perpetrator impact on the most common drug metabolizing enzyme, cytochrome P450 (CYP)3A, and the major transporters organic anion-transporting polypeptides (OATP)1B, breast cancer resistance protein (BCRP), and MDR1 P-glycoprotein (P-gp). The clinical utility of these microdose cocktail probe substrates was qualified by conducting clinical drug interaction studies with three inhibitors with different in vitro inhibitory profiles (rifampin, itraconazole, and clarithromycin). Generally, the pharmacokinetic profiles of the probe substrates, in the absence and presence of the inhibitors, were comparable to their reported corresponding pharmacological doses, and/or in agreement with theoretical expectations. The exception was dabigatran, which resulted in an approximately twofold higher magnitude for microdose compared to conventional dosing, and, thus, can be used to flag a worst-case scenario for P-gp. Broader application of the microdose cocktail will facilitate a more comprehensive understanding of the roles of drug transporters in drug disposition and drug interactions.

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