A sensitive and high‐throughput LC‐MS/MS method for inhibition assay of seven major cytochrome P450s in human liver microsomes using an in vitro cocktail of probe substrates

A sensitive and high‐throughput LC‐MS/MS method was established and validated for the simultaneous quantification of seven probe substrate‐derived metabolites (cocktail assay) for assessing the in vitro inhibition of cytochrome P450 (CYP) enzymes in pooled human liver microsomes. The metabolites acetaminophen (CYP1A2), hydroxy‐bupropion (CYP2B6), n‐desethyl‐amodiaquine (CYP2C8), 4′‐hydroxy‐diclofenac (CYP2C9), 4′‐hydroxy‐mephenytoin (CYP2C19), dextrorphan (CYP2D6) and 1′‐hydroxy‐midazolam (CYP3A4/5), together with the internal standard verapamil, were eluted on an Agilent 1200 series liquid chromatograph in <7 min. All metabolites were detected by an Agilent 6410B tandem mass spectrometer. The concentration of each probe substrate was selected by substrate inhibition assay that reduced potential substrate interactions. CYP inhibition of seven well‐known inhibitors was confirmed by comparing a single probe substrate assay with cocktail assay. The IC₅₀ values of these inhibitors determined on this cocktail assay were highly correlated (R² > 0.99 for each individual probe substrate) with those on single assay. The method was selective and showed good accuracy (85.89–113.35%) and between‐day (RSD <13.95%) and within‐day (RSD <9.90%) precision. The sample incubation extracts were stable at 25 °C for 48 h and after three freeze–thaw cycles. This seven‐CYP inhibition cocktail assay significantly increased the efficiency of accurately assessing compounds’ potential inhibition of the seven major CYPs in drug development settings. Copyright © 2014 John Wiley & Sons, Ltd.     

A UPLC-MS/MS assay of the "Pittsburgh cocktail": six CYP probe-drug/metabolites from human plasma and urine using stable isotope dilution

The efficiency of drug metabolism by a single enzyme can be measured as the fractional metabolic clearance which can be used as a measure of whole body activity for that enzyme. Measurement of activity of multiple enzymes simultaneously is feasible using a cocktail approach, however, analytical approach using different assays for drug probes can be cumbersome. A quantitative ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) based method for the rapid measurement of six cytochrome P450 (CYP) probe drugs and their relevant metabolites is described. The six specific probe substrates/metabolites are caffeine/paraxanthine (CYP1A2), flurbiprofen/4'-hydroxyflurbiprofen (CYP2C9), mephenytoin/4'-hydroxymephenytoin (CYP2C19), debrisoquine/4-hydroxydebrisoquine (CYP2D6), chlorzoxazone/6'-hydroxychlorzoxazone (CYP2E1) and dapsone/N-monoacetyldapsone (NAT2). These probes were quantified by stable isotope dilution from plasma and urine. The present workflow provides a robust, fast and sensitive assay for the "Pittsburgh cocktail", and has been successfully applied to a clinical phenotyping study of liver disease. A representative group of 17 controls and patients with chronic liver disease were administered orally caffeine (100 mg), chlorzoxazone (250 mg), debrisoquine (10 mg), mephenytoin (100 mg), flurbiprofen (50 mg) and dapsone (100 mg). Urine (0 through 8 h) and plasma (4 and 8 h) samples were analyzed for drug/metabolite amounts by stable isotope dilution UPLC-MS/MS. The phenotypic activity of drug metabolizing enzymes was investigated with 17 patient samples. Selected reaction monitoring (SRM) was optimized for each drug and metabolite. In the method developed, analytes were resolved by reversed-phase by development of a gradient using a water/methanol solvent system. SRM of each analyte was performed in duplicate on a triple quadrupole mass spectrometer utilizing an 8 min analytical method each, one with the source operating in the positive mode and one in the negative mode, using the same solvent system. This method enabled quantification of each drug (caffeine, chlorzoxazone, debrisoquine, mephenytoin, flurbiprofen, and dapsone) and its resulting primary metabolite in urine or plasma in patient samples. The method developed and the data herein demonstrate a robust quantitative assay to examine changes in CYP enzymes both independently or as part of a cocktail. The clinical use of a combination of probe drugs with UPLC-MS/MS is a highly efficient tool for the assessment of CYP enzyme activity in liver disease.     

A high‐throughput inhibition screening of major human cytochrome P450 enzymes using an in vitro cocktail and liquid chromatography–tandem mass spectrometry

A sensitive and high‐throughput inhibition screening liquid chromatography–mass spectrometry (LC‐MS/MS) method was developed and validated for the simultaneous quantification of five probe metabolites (7‐hydroxycoumarin, CYP2A6; 4‐hydroxytolbutamide, CYP2C9; 4′‐hydroxymephenytoin, CYP2C19; α‐hydroxymetoprolol, CYP2D6; and 1‐hydroxymidazolam, CYP3A4) for in vitro cytochrome P450 activity determination in human liver microsome and recombinant. All the metabolites and the internal standard, tramadol, were separated on a Waters 2695 series liquid chromatograph with a Phenomenex Luna C₁₈ column (150 × 2.0 mm, 5 µm). Quality control samples and a positive control CYP inhibitor were included in the method. The IC₅₀ values determined for typical CYP inhibitors were reproducible and in agreement with the literature. The method was selective and showed good accuracy (99.13–103.37%), and inter‐day (RSD < 6.20%) and intra‐day (RSD < 6.13%) precision. Also, the incubation extracts of the sample were stable at room temperature (20 °C) for 48 h and for 96 h in the autosampler (4 °C). The presented method is the first HPLC‐MS/MS method of this combination for simultaneous detection of the five metabolites 7‐hydroxycoumarin, 4‐hydroxytolbutamide, 4′‐hydroxymephenytoin, α‐hydroxymetoprolol and 1‐hydroxymidazolam in a single‐run process. It is possible that the high‐quality and ‐throughput cocktail provides suitable information in drug discovery and screening for new drug entities. Copyright © 2013 John Wiley & Sons, Ltd.     

A validated assay by liquid chromatography–tandem mass spectrometry for the simultaneous quantification of elvitegravir and rilpivirine in HIV positive patients

Because of the large variability in the pharmacokinetics of anti‐HIV drugs, therapeutic drug monitoring in patients may contribute to optimize the overall efficacy and safety of antiretroviral therapy. An LC–MS/MS method for the simultaneous assay in plasma of the novel antiretroviral agents rilpivirine (RPV) and elvitegravir (EVG) has been developed to that endeavor. Plasma samples (100 μL) extraction is performed by protein precipitation with acetonitrile, and the supernatant is subsequently diluted 1:1 with 20‐mM ammonium acetate/MeOH 50:50. After reverse‐phase chromatography, quantification of RPV and EVG, using matrix‐matched calibration samples, is performed by electrospray ionization–triple quadrupole mass spectrometry by selected reaction monitoring detection using the positive mode. The stable isotopic‐labeled compounds RPV‐¹³C6 and EVG‐D6 were used as internal standards. The method was validated according to FDA recommendations, including assessment of extraction yield, matrix effects variability (<6.4%), as well as EVG and RPV short and long‐term stability in plasma. Calibration curves were validated over the clinically relevant concentrations ranging from 5 to 2500 ng/ml for RPV and from 50 to 5000 ng/ml for EVG. The method is precise (inter‐day CV%: 3–6.3%) and accurate (3.8–7.2%). Plasma samples were found to be stable (<15%) in all considered conditions (RT/48 h, +4°C/48 h, ?20°C/3 months and 60°C/1 h). Selected metabolite profiles analysis in patients' samples revealed the presence of EVG glucuronide, that was well separated from parent EVG, allowing to exclude potential interferences through the in‐source dissociation of glucuronide to parent drug. This new, rapid and robust LCMS/MS assay for the simultaneous quantification of plasma concentrations of these two major new anti‐HIV drugs EVG and RPV offers an efficient analytical tool for clinical pharmacokinetics studies and routine therapeutic drug monitoring service. Copyright © 2013 John Wiley & Sons, Ltd.     

Rapid determination of five probe drugs and their metabolites in human plasma and urine by liquid chromatography/tandem mass spectrometry: application to cytochrome P450 phenotyping studies

A liquid chromatography/mass spectrometry method, for rapid determination of five cytochrome P450 (CYP) probe drugs and their relevant metabolites in human plasma and urine, is described. The five specific probe substrates/metabolites, caffeine/paraxanthine (CYP1A2), tolbutamide/4-hydroxytolbutamide/carboxytolbutamide (CYP2C9), omeprazole/5-hydroxyomeprazole (CYP2C19), debrisoquine/5-hydroxydebrisoquine (CYP2D6) and midazolam/1'-hydroxymidazolam (CYP3A), together with the internal standards (phenacetin and paracetamol), in plasma and urine, were extracted using solid-phase extraction. The chromatography was performed using a C18 column with an isocratic mobile phase consisting of acetonitrile and 0.1% formic acid in water (70:30). The triple-quadrupole mass spectrometer was operated in both positive and negative modes, and multiple reaction monitoring was used for quantification. The method was validated over the concentration ranges 0.05-5 microg/mL for caffeine and paraxanthine, 0.02-2 microg/mL for tolbutamide, 0.1-20 microg/mL for 4-hydroxytolbutamide, carboxytolbutamide, debrisoquine and 5-hydroxydebrisoquine, 5-2500 ng/mL for omeprazole and 5-hydroxyomeprazole, and 1-100 ng/mL for midazolam and 1'-hydroxymidazolam. The intra- and inter-day precision were 0.3-13.7% and 1.9-14.3%, respectively, and the accuracy ranged from 93.5-107.2%. The lower limit of quantification varied between 1 and 100 ng/mL. The present method provides a robust, fast and sensitive analytical tool for the five-probe drug cocktail, and has been successfully applied to a clinical phenotyping study in 16 subjects.     

Development and validation of a liquid-chromatography high-resolution tandem mass spectrometry approach for quantification of nine cytochrome P450 (CYP) model substrate metabolites in an in vitro CYP inhibition cocktail

Knowledge about the cytochrome P450 (CYP) inhibition potential of new drug candidates is important for drug development because of its risk of interactions. For novel psychoactive substances (NPS), corresponding data are not available. For developing a general drug inhibition cocktail assay, a liquid-chromatography high-resolution tandem mass spectrometry multi-analyte approach was developed and validated for quantifying low concentrations of O-diethyl phenacetin for CYP 1A2, 7-hydroxy coumarin for CYP 2A6, 4-hydroxy bupropion for CYP 2B6, N-diethyl amodiaquine for CYP 2C8, 4-hydroxy diclofenac for CYP 2C9, 5-hydroxy omeprazole for CYP 2C19, O-dimethyl dextromethorphan for CYP 2D6, 6-hydroxy chlorzoxazone for CYP 2E1, and 6-beta-hydroxy testosterone for CYP 3A in the incubation mixture in the presence of substrates and inhibitors. The tested matrix effects ranged from 63 to 141 % and the recoveries from 95 to 110 %. Time-saving one-point calibration allowed sufficient quantification, although some of the validation results for 7-hydroxy coumarin, 4-hydroxy bupropion, 4-hydroxy diclofenac, and 6-beta-hydroxy testosterone were outside the acceptance criteria (AC) but without influence of the IC₅₀ calculation. Validation showed also that the approach was sensitive and selective using mass spectral multiplexing. In conclusion, the presented assay was suitable for the quantification of the model substrate metabolites and could be used for the development of a CYP inhibition assay for testing most CYPs and a wide range of drugs of abuse.     

Phenotyping of CYP450 in human liver microsomes using the cocktail approach

The cocktail approach is an advantageous strategy used to monitor the activities of several cytochromes P450 (CYPs) in a single test to increase the throughput of in vitro phenotyping studies. In this study, a cocktail mixture was developed with eight CYP-specific probe substrates to simultaneously evaluate the activity of the most important CYPs, namely, CYP1A2, CYP2A6, CYP2B6, CYP2C9, CYP2C19, CYP2D6, CYP2E1, and the CYP3A subfamily. After cocktail incubation in the presence of human liver microsomes (HLMs), the eight selected substrates and their specific metabolites were analyzed by ultra-high-pressure liquid chromatography and electrospray ionization quadrupole time-of-flight mass spectrometry. Qualitative and quantitative data were simultaneously acquired to produce an overview of the extended phase I biotransformation routes for each probe substrate in the HLMs and to generate phenotypic profiles of various HLMs. A comparison of the cocktail strategy with an individual substrate assay for each CYP produced similar results. Moreover, the cocktail was tested on HLMs with different allelic variants and/or in the presence of selective inhibitors. The results were in agreement with the genetic polymorphisms of the CYPs and the expected effect of the alterations. All of these experiments confirmed the reliability of this cocktail assay for phenotyping of the microsomal CYPs.     

Simultaneous determination of bupropion,metroprolol, midazolam,phenacetin, omeprazole and tolbutamide in rat plasma by UPLC‐MS/MS and its application to cytochrome P450 activity study in rats

A specific ultra‐performance liquid chromatography tandem mass spectrometry method is described for the simultaneous determination of bupropion, metroprolol, midazolam, phenacetin, omeprazole and tolbutamide in rat plasma with diazepam as internal standard, which are the six probe drugs of the six cytochrome P450 isoforms CYP2B6, CYP2D6, CYP3A4, CYP1A2, CYP2C19 and CYP2C9. Plasma samples were protein precipitated with acetonitrile. The chromatographic separation was achieved using a UPLC® BEH C₁₈ column (2.1 × 100 mm, 1.7 µm). The mobile phase consisted of acetonitrile and water (containing 0.1% formic acid) with gradient elution. The triple quadrupole mass spectrometric detection was operated by multiple reaction monitoring in positive electrospray ionization. The precisions were <13%, and the accuracy ranged from 93.3 to 110.4%. The extraction efficiency was >90.5%, and the matrix effects ranged from 84.3 to 114.2%. The calibration curves in plasma were linear in the range of 2–2000 ng/mL, with correlation coefficient (r²) >0.995. The method was successfully applied to pharmacokinetic studies of the six probe drugs of the six CYP450 isoforms and used to evaluate the effects of erlotinib on the activities of CYP2B6, CYP2D6, CYP3A4, CYP1A2, CYP2C19 and CYP2C9 in rats. Erlotinib may inhibit the activity of CYP2B6 and CYP3A4, and may induce CYP2C9 of rats. Copyright © 2015 John Wiley & Sons, Ltd.     

A rapid ultra‐performance liquid chromatography/tandem mass spectrometric methodology for the in vitro analysis of Pooled and Cocktail cytochrome P450 assays

Drug‐drug interaction evaluations of new pharmaceutical candidates are critical to preventing drug withdrawal and are routinely determined through the use of cytochrome P450 assays. The measurement of the effect of test compounds on the metabolism of known substrates allows for the determination of specific CYP450 isoenzyme inhibition and calculation of IC50 values. A sensitive, high‐throughput ultra‐performance liquid chromatography/tandem mass spectrometric (UPLC/MS/MS) method is presented for the evaluation of CYP450 inhibition. The assay was performed using a cocktail of probe substrates and the results were compared to those obtained with the more time‐consuming methodology utilizing individual substrates. The use of a high‐resolution, sub‐2 µm particle, LC system allowed for a high‐throughput assay of just 1 min. The extra resolution of the UPLC/MS/MS system allowed for the complete resolution of the analytes, with a fast switching MS for comprehensive data collection. The CYP450 inhibition results obtained using the substrate cocktail approach were found to be essentially identical to those obtained using individual substrates. Copyright © 2009 John Wiley & Sons, Ltd.     

Sensitive and simultaneous quantitation of 6β‐hydroxycortisol and cortisol in human plasma by LC‐MS/MS coupled with stable isotope dilution method

CYP3A phenotyping provides a means for personalized drug therapy. We focused our attention on the plasma 6β‐hydroxycortisol (6β‐OHF) to cortisol ratio as an index for CYP3A phenotyping. In the present study, we developed a sensitive and reliable method for the simultaneous determination of 6β‐OHF and cortisol in human plasma using high‐performance liquid chromatography/tandem mass spectrometry together with picolinylester derivatization or nonderivatization methods and 6β‐[9,11,12,12‐²H₄]hydroxycortisol and [1,2,4,19‐¹³C₄]cortisol as internal standards for in vivo CYP3A phenotyping in humans. The lower limits of quantification were 38.513 pg/mL for 6β‐OHF and 38.100 pg/mL for cortisol. The relative error and relative standard deviation of the lower limits of quantification were <5% for both methods. The intra‐day and inter‐day assay reproducibilities of the determined 6β‐OHF and cortisol concentrations were consistent with the actual amounts added as relative errors and relative standard deviations for both methods, which were <5.4% and <3.9%, respectively. Both methods were applied for the quantification of plasma 6β‐OHF and cortisol concentrations in healthy subjects taking oral contraceptives. The absolute concentrations and time course of 6β‐OHF and cortisol were found to be consistent when measured using the 2 methods. The ratio as an index for in vivo CYP3A activity decreased after 21 days of taking oral contraceptives for both methods. To the best of our knowledge, this is the first study reporting the detailed investigation of accuracy and precision in the simultaneous measurement of 6β‐OHF and cortisol in human plasma using liquid chromatography/tandem mass spectrometry coupled with stable isotope dilution method, which can be applied to CYP3A phenotyping.     

Development and validation of a sensitive and specific LC–MS/MS cocktail assay for CYP450 enzymes: Application to study the effect of catechin on rat hepatic CYP activity

A sensitive and specific liquid chromatography tandem mass spectrometric (LC–MS/MS) method that enables the simultaneous quantification of probe substrates and metabolites of cytochrome P450 (CYP) enzymes was developed and validated. These substrates (metabolites)—coumarin (7-hydroxycoumarin), tolbutamide (4-hydroxytolbutamide), S-mephenytoin (4-hydroxymephenytoin), dextromethorphan (dextrorphan), and testosterone (6β-hydroxytestosterone)—were utilized as markers for the activities of the major human CYP enzymes CYP2A6, CYP2C9, CYP2C19, CYP2D6, and CYP3A4, respectively. Analytes were separated on Kinetex C₁₈ column (2.1 × 50 mm, 5 μm) using a binary gradient mobile phase of 0.1% formic acid in water and 0.1% formic acid in acetonitrile. Metabolites were detected and quantified by MS using multiple reaction monitoring at m/z 163 → 107.2 for 7-hydroxycoumarin, m/z 235 → 150.1 for 4-hydroxymephenytoin, m/z 287 → 171 for 4-hydroxytolbutamide, m/z 258 → 157.1 for dextrorphan, m/z 305 → 269 for 6β-hydroxytestosterone, and m/z 237 → 194 for the internal standard. The assay exhibited good linearity over a range of 10–500 ng/mL with acceptable accuracy and precision criteria. As a proof of concept, the developed cocktail assay was successfully used to examine the potential impact of catechin on the activity of the major rat liver CYP enzymes.     

In vitro interaction cocktail assay for nine major cytochrome P450 enzymes with 13 probe reactions and a single LC/MSMS run: analytical validation and testing with monoclonal anti-CYP antibodies

A sensitive and rugged LC/MSMS method was developed for a comprehensive in vitro metabolic interaction screening assay with N-in-1 approach reported earlier. A cocktail consisting of ten cytochrome P450 (CYP)-selective probe substrates with known kinetic, metabolic and interaction properties in vivo was incubated in a pool of human liver microsomes, and metabolites of melatonin (CYP1A2), coumarin (CYP2A6), bupropion (CYP2B6), amodiaquine (CYP2C8) tolbutamide (CYP2C9), omeprazole (CYP2C19 and CYP3A4), dextromethorphan (CYP2D6), chlorzoxazone (CYP2E1), midazolam (CYP3A4) and testosterone (CYP3A4) were simultaneously analysed with a single LC/MSMS run. Altogether, 13 metabolites and internal standard phenacetin were analysed in multiple reaction mode. Polarity switching mode was utilized to acquire negative ion mode electrospray data for hydroxychlorzoxazone and positive ionization data for the rest of the analytes. Fast gradient elution was applied, giving total injection cycle of 8 min. The method was modified for two different LC/MSMS systems, and was validated for linear range, detection limit, accuracy and precision for each metabolite. In addition, cocktail inhibition system was further tested using monoclonal anti-CYP antibodies as inhibitors for each probe reaction.     

High-throughput screening of inhibitory potential of nine cytochrome P450 enzymes in vitro using liquid chromatography/tandem mass spectrometry

The early detection of potential drug-drug interactions is an important issue of drug discovery that has led to the development of high-throughput screening (HTS) methods for potential drug interactions. We developed a HTS method for potential interactions of inhibitory drugs for nine human P450 enzymes using cocktail incubation and tandem mass spectrometry in vitro. This new method involves incubation of two cocktail doses and single cassette analysis. The two cocktail doses in vitro were developed to minimize solvent effects and mutual drug interactions among substrates: cocktail A was composed of phenacetin for CYP1A2, coumarin for CYP2A6, paclitaxel for CYP2C8, S-mephenytoin for CYP2C19, dextromethorphan for CYP2D6, and midazolam for CYP3A4; and cocktail B was composed of three substrates including bupropion for CYP2B6, tolbutamide for CYP2C9, and chlorzoxazone for CYP2E1. In the incubation study of these cocktails, the reaction mixtures were pooled and simultaneously analyzed using liquid chromatography/tandem mass spectrometry employing a fast gradient. The method was validated by comparing the inhibition data obtained from the incubation of each individual probe substrate alone with data from the new method. The IC50 value of each inhibitor in the cocktail agreed well with that of the individual probe drug as well as with values previously reported in the literature. As a HTS method for potential interactions of the inhibition of these nine P450 enzymes, this new method will be useful in the drug discovery process and for the mechanistic understanding of drug interactions.     

Development and validation of a LC‐MS/MS method for the in vitro analysis of 1‐hydroxymidazolam in human liver microsomes: application for determining CYP3A4 inhibition in complex matrix mixtures

Complementary and alternative medicines (CAM) can affect the pharmacokinetics of anticancer drugs by interacting with the metabolizing enzyme cytochrome P450 (CYP) 3A4. To evaluate changes in the activity of CYP3A4 in patients, levels of 1‐hydroxymidazolam in plasma are often determined with liquid chromatography–quadrupole mass spectrometry (LC‐MS/MS). However, validated LC‐MS/MS methods to determine in vitro CYP3A4 inhibition in human liver microsomes are scarce and not optimized for evaluating CYP3A4 inhibition by CAM. The latter is necessary because CAM are often complex mixtures of numerous compounds that can interfere with the selective measurement of 1‐hydroxymidazolam. Therefore, the aim was to validate and optimize an LC‐MS/MS method for the adequate determination of CYP3A4 inhibition by CAM in human liver microsomes. After incubation of human liver microsomes with midazolam, liquid–liquid extraction with tert‐butyl methyl ether was applied and dried samples were reconstituted in 50% methanol. These samples were injected onto a reversed‐phase chromatography consisting of a Zorbax Extend‐C₁₈ column (2.1 × 150 mm, 5.0 µm particle size), connected to a triple quadrupole mass spectrometer with electrospray ionization. The described LC‐MS/MS method was validated over linear range of 1.0–500 nm for 1‐hydroxymidazolam. The results revealed good inter‐assay accuracy (≥85% and ≤115%) and within‐day and between‐day precisions (coefficient of variation ≤ 4.43%). Furthermore, the applicability of this assay for the determination of CYP3A4 inhibition in complex matrix mixtures was successfully demonstrated in an in vitro experiment in which CYP3A4 inhibition by known CAM (β‐carotene, green tea, milk thistle and St. John's wort) was determined. Copyright © 2013 John Wiley & Sons, Ltd.     

Simultaneous quantification of mycophenolic acid and its glucuronide metabolites in human plasma by an UPLC–MS/MS assay

The aim of this study was to develop a reliable UPLC–MS/MS assay for accurate quantification of mycophenolic acid (MPA) and its glucuronide conjugates in human plasma. Plasma proteins were precipitated with acetonitrile and the chromatographic separation was achieved on a C₁₈ column with a gradient elution. The detection was performed by a triple quadrupole mass spectrometer in the positive electrospray ionization and multiple reaction monitoring mode. Linearity of the assay was demonstrated over the range of 20–10,000 ng/mL for MPA and MPA glucuronide (MPAG), and 2–1000 ng/mL for acyl MPA glucuronide in human plasma. The assay was precise and accurate with coefficient of variation and bias <15%. MPA and MPAG were stable at 25 °C up to 1 day in both heparin‐ and EDTA‐treated blood. In heparin‐ and EDTA‐plasma, MPA and MPAG were stable for at least 1 week at 25 and 4 °C, and 1 month at ?20 °C. However, 99% acyl MPA glucuronide degraded in both heparin‐ and EDTA‐blood as well as plasma when stored at room temperature for 1 day. All the analytes remained stable for at least 3 months in acidified EDTA‐plasma at ?80 °C. The assay was successfully applied on patients post hematopoietic stem cell transplantation. Copyright © 2016 John Wiley & Sons, Ltd.     

An analytical method for cyclosporine using liquid chromatography–mass spectrometry

A liquid chromatographic mass spectrometric (LC‐MS) assay has been developed for cyclosporine A (CyA) in rat plasma using amiodarone as internal standard (IS). Rat plasma (100 µL) containing drug and IS were extracted using liquid–liquid extraction with 4 mL of 95:5 ether:methanol. After evaporation of the organic layer the residue was reconstituted with 500 µL of water. Then the aqueous layer was transferred to LC‐MS sample vials. A 10 µL volume was injected. The analysis was performed on a C₈ column 3.5 µm (2.1 × 50 mm) heated to 60°C with a mobile phase consisting of acetonitrile:methanol:0.2% NH₄OH (60:20:20) at an isocratic flow‐rate of 0.2 mL/min. The ions used for quantitation of CyA and IS were m/z 1202.8 and 645.9, with retention times of 3.35 and 4.72 min, respectively. Linear relationships (r² > 0.99) were achieved between plasma or blood concentration and peak height ratios (drug:IS) over the concentration range 50–5000 ng/mL. The CV% and mean error were <19%. Based on validation data, the lower limit of quantification for the assay was 50 ng/mL. The reported assay method displayed high measures of linearity, sensitivity, reliability and precision, allowing its applicability in pharmacokinetic studies in rat. Copyright © 2009 John Wiley & Sons, Ltd.     

Simultaneous Determination of Ebastine and Its Active Metabolite (Carebastine) in Human Plasma Using LC–MS–MS

A sensitive and rapid LC–MS–MS method was developed for the simultaneous determination of ebastine and carebastine in human plasma. Solid-phase extraction was used to isolate the compounds from the biological matrix followed by separation on a Symmetry C18 column under isocratic conditions. The mobile phase was 10 mM ammonium formate in water/acetonitrile (40:60, v/v). Detection was carried out using a triple-quadrupole mass spectrometer in positive electrospray ionization and multiple reaction monitoring mode. The method was fully validated over the concentration range of 0.1–10 ng mL^?1 for ebastine and 0.2–200 ng mL^?1 for carebastine in human plasma, respectively. The lower limit of quantification (LLOQ) was 0.1 ng mL^?1 for ebastine and 0.2 ng mL^?1 for carebastine. For ebastine and carebastine inter- and intra-day precision (CV%) and accuracy values were all within ±15% and 85–115%, respectively. The extraction recovery was on average 60.0% for ebastine and 60.3% for carebastine.     

A sensitive method for determination of furanodiene in rat plasma using liquid chromatography/tandem mass spectrometry and its application to a pharmacokinetic study

Furanodiene, a sesquiterpene component extracted from the essential oil of the rhizome of Curcuma wenyujin Y.H. Chen et C. Ling (Wen Ezhu), is widely used in traditional Chinese medicine. A sensitive analytical method was established and validated for furanodiene in rat plasma, which was further applied to assess the pharmacokinetics of furanodiene in rats receiving a single dose of furanodiene. Liquid chromatography tandem mass spectrometry (LC/MS/MS) in multiple reaction monitoring mode was used in the method and costundide was used as internal standard. A simple protein precipitation based on methanol was employed. The simple sample cleanup increased the throughput of the method substantially. The method was validated over the range of 1–1000 ng/mL with a correlation coefficient >0.99. The lower limit of quantification was 1 ng/mL for furanodiene in plasma. Intra‐ and inter‐day accuracies for furanodiene were 88–115 and 102–107%, and the inter‐day precision less than 14.4%. After a single oral dose of 10 mg/kg of furanodiene, the mean peak plasma concentration of furanodiene was 66.9 ± 23.4 ng/mL at 1 h, the area under the plasma concentration–time curve (AUC_{0–10 h}) was 220 ± 47.8 h ng/mL, and the elimination half‐life was 1.53 ± 0.06 h. After an intravenous adminstration of furanodiene at a dosage of 5 mg/kg, the area under the plasma concentration–time curve was 225 ± 76.1 h?ng/mL, and the elimination half‐life was 2.40 ± 1.18 h. Based on this result, the oral bioavailability of furanodiene in rats at 10 mg/kg is 49.0%. Copyright © 2011 John Wiley & Sons, Ltd.     

Simultaneous determination of peimine and peiminine in rat plasma by LC‐ESI‐MS employing solid‐phase extraction

A simple and reliable LC‐ESI‐MS method for the determination of peimine and peiminine in rat plasma was developed for the first time. The method was proven to be specific and sensitive by carrying out validation. The analytes were extracted from rat plasma via solid‐phase extraction on Waters Oasis MCX cartridges. Chromatography separation was achieved on a C₁₈ column using 10 mM ammonium acetate (adjusted to pH 3.0 with glacial acetic acid)–acetonitrile (85:15, v/v) as mobile phase. The linear range was 1–100 ng/mL for peimine and peiminine. Intra‐ and inter‐day precisiond were less than 10%. Accuracies were within 85–115% of their nominal concentrations. The limit of quantification was 1 ng/mL for both analytes. The developed assay was successfully applied to pharmacokinetic study of peimine and peiminine in rats orally administered the alkaloids extracts from Bulbus Fritillariae, demonstrating a possible broader spectrum of applications of this method. Copyright © 2009 John Wiley & Sons, Ltd.     

Application of dried blood spot sampling combined with LC-MS/MS for genotyping and phenotyping of CYP450 enzymes in healthy volunteers

An early clinical development study (phase I) was conducted to determine the usefulness of dried blood spot (DBS) sampling as an alternative to venous sampling for phenotyping and genotyping of CYP450 enzymes in healthy volunteers. Midazolam (MDZ) was used as a substrate for phenotyping CYP3A4 activity; the concentrations of MDZ and its main metabolite 1'-hydroxymidazolam (1-OH MDZ) were compared between the DBS method from finger punctures, plasma and whole blood (WB), drawn by venipuncture, whereby several methodological parameters were studied (i.e. punch width, amount of dots analyzed and storage time stability). Genotyping between DBS and venous WB samples was compared for CYP2D6 (*3, *4, *6), CYP2C19 (*2, *3), CYP3A4 (*1B) and CYP3A5 (*3C). In addition, the subject's and phlebotomist's satisfaction with venous blood sampling compared with the DBS method was evaluated using a standardized questionnaire. An LC-MS/MS method for the quantification of the MDZ and 1-OH MDZ concentrations in DBS samples was developed and validated in the range of 0.100-100 ng/mL. No compromises were made for the limits of quantification of the DBS-LC-MS/MS method vs the authentic plasma and WB methods.