Pharmacokinetic Study In Healthy Subjects Research Articles

Hybrid solid-phase extraction to overcome interference due to phospholipids for determination of neratinib in human plasma using UPLC-MS/MS.

A reliable and robust bioanalytical method was developed to quantify neratinib, a tyrosine kinase inhibitor in human plasma, using UPLC-MS/MS. The extraction of neratinib and its deuterated internal standard, neratinib-d6, was successfully performed on hybrid solid-phase extraction ultra-cartridges to remove the interference of phospholipids and proteins. Chromatographic analysis was performed on a UPLC BEH C18 (50× 2.1 mm, 1.7 μm) column using 0.1% formic acid and acetonitrile under gradient conditions. The total analysis time was 1.5min. Neratinib was quantified using electrospray ionization source operated in the positive-ion multiple reaction monitoring mode. The mass transitions of neratinib and neratinib-d6 were m/z 557.3/112.1 and m/z 563.1/118.2, respectively. The linear concentration range for neratinib was 0.5-500 ng/mL, which adequately covers concentration levels expected in real subject samples. The assay was extensively validated for various validation parameters following standard guidelines for a bioanalytical assay. The intra- and inter-batch precision was ≤4.6%, and neratinib was found to be stable under various stability conditions. The mean internal standard-normalized matrix factor and recovery were 0.997 and 95.4%, respectively. The validated method was successfully applied to a pharmacokinetic study in healthy subjects with different doses.

Bridging Efficacy of Tofacitinib Immediate-Release to Extended-Release Formulations for Treatment of Ulcerative Colitis: Application of a Model-Informed Drug Development Approach.

Tofacitinib is an oral, small molecule Janus kinase inhibitor for the treatment of ulcerative colitis (UC). We report a model‐informed drug development approach for bridging efficacy from immediate‐release (IR) to extended‐release (XR) tofacitinib formulations in patients with UC. IR‐XR efficacy bridging was supported by exposure‐response analysis of phase 3 induction/maintenance studies of the IR formulation in UC to identify exposure metrics relevant for efficacy. Pharmacokinetic studies in healthy subjects were used to confirm similarity of relevant exposure metrics of tofacitinib IR 5 mg twice daily to XR 11 mg once daily, and tofacitinib IR 10 mg twice daily to XR 22 mg once daily, thereby bridging efficacy between IR and XR formulations. Food effect was evaluated at both XR formulation dose levels. Exposure‐response analysis demonstrated that area under the plasma concentration–time curve (average plasma concentration) was a relevant predictor of efficacy. Pharmacokinetic studies demonstrated that area under the plasma concentration–time curve was equivalent between formulations under single‐dose and steady‐state conditions, and other exposure metrics were also similar. These results also supported bridging of safety data for IR‐XR formulations. Food had no impact on tofacitinib XR exposure. These data support efficacy/safety bridging of IR‐XR formulations in patients with UC.

Selective quantification of lacosamide in human plasma using UPLC-MS/MS: Application to pharmacokinetic study in healthy subjects with different doses.

A practical, sensitive, and robust UPLC-MS/MS method was developed and validated to quantify lacosamide in human plasma. A simple one-step protein precipitation was used to extract lacosamide and labeled lacosamide-13C, D3 as an internal standard (IS) from 150-μL plasma. The extracts were analyzed on an Eclipse Plus C18 column (50 × 2.1 mm, 1.8 μm) using 0.1% formic acid in water and methanol:acetonitrile (50:50, v/v) under gradient conditions. The extracts were quantified on LCMS-8040 using electrospray ionization source operated in positive ionization and multiple reaction monitoring modes. The method showed good linearity from 0.02 to 20 μg/mL, which was adequate to cover lacosamide concentration assayed in formulations with different strengths. The bioanalytical assay was fully validated as per current regulatory guidelines. The intra-batch and inter-batch precision values of lacosamide were less than 4.6%. Lacosamide was found to be stable at different storage conditions. The extraction recoveries and IS-normalized matrix factors for lacosamide ranged from 97.17 to 99.68% and from 0.973 to 1.012, respectively. The validated method was successfully applied to a pharmacokinetic study with three lacosamide formulations (50, 100, and 200 mg) in 36 healthy subjects. The assay reliability was determined by reanalysis of 81 subject samples.

Oral Cannabidiol Does Not Convert to Δ8-THC or Δ9-THC in Humans: A Pharmacokinetic Study in Healthy Subjects.

Introduction: Recent studies have suggested that cannabidiol (CBD) could interconvert into Delta-8- and Delta-9- tetrahydrocannabinol. Materials and Methods: Thus, we tested the plasma samples of 120 healthy human subjects (60 male and 60 female), 60 in fasting and the other 60 under normal feeding conditions after acute administration of an oral solution containing CBD 300 mg. To do this, we developed a bioanalytical method to determine CBD and the presence of THC in plasma samples by Ultra-High Performance Liquid Chromatography Coupled to Tandem Mass Spectrometry. Results: The results showed that THC was not detected in plasma after the administration of CBD, and those study participants did not present psychotomimetic effects. Conclusions: The findings presented here are consistent with previous evidence suggesting that the oral administration of CBD in a corn oil formulation is a safe route for the administration of the active substance without bioconversion to THC in humans.

Optimization of chromatography to overcome matrix effect for reliable estimation of four small molecular drugs from biological fluids using LC-MS/MS.

The article describes a systematic study to overcome the matrix effect during chromatographic analysis of gemfibrozil, rivastigmine, telmisartan and tacrolimus from biological fluids using LC-ESI-MS/MS. All four methods were thoroughly developed by the appropriate choice of analytical column, elution mode and pH of mobile phase for improved chromatography and overall method performance. Matrix effect was assessed by post-column analyte infusion, slope of calibration line approach and post-extraction spiking. The best chromatographic conditions established were: Acquity BEH C18 (50 × 2.1 mm, 1.7 μm) column with 5.0 mm ammonium acetate, pH 6.0-methanol as the mobile phase under gradient program for gemfibrozil; Luna CN (50 × 2.0 mm, 3 μm) column with a mobile phase consisting of acetonitrile-10 mm ammonium acetate, pH 7.0 (90:10, v/v) for rivastigmine; Inertsustain C18 (100 × 2.0 mm, 5 μm) column using methanol-2.0 mm ammonium formate, pH 5.5 (80: 20, v/v) as the mobile phase for isocratic elution of telmisartan; and Acquity BEH C18 (50 × 2.1 mm, 1.7 μm) with methanol-10 mm ammonium acetate, pH 6.0 (95:5, v/v) as mobile phase for tacrolimus. The methods were thoroughly validated as per European Medicines Agency and US Food and Drug Administration guidance and were successfully applied for pharmacokinetic studies in healthy subjects.

Quantification of fenoprofen in human plasma using UHPLC-tandem mass spectrometry for pharmacokinetic study in healthy subjects.

A rapid, simple and sensitive ultra-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method has been developed to quantify fenoprofen, a nonsteroidal anti-inflammatory drug in human plasma for a pharmacokinetic study in healthy subjects. Owing to high levels of protein binding, protein precipitation followed by solid-phase extraction was employed for the extraction of fenoprofen and fenoprofen-d3 (used as internal standard) from 200 μL human plasma. Separation was performed on a BEH C18 (50 × 2.1 mm, 1.7 μm) column using methanol-0.2% acetic acid in water (75:25, v/v) under isocratic elution. Electrospray ionization was operated in the negative mode for sample ionization. Ion transitions used for quantification in the selected reaction monitoring mode were m/z241/197 and m/z244/200 for fenoprofen and fenoprofen-d3, respectively. Under the optimized conditions, fenoprofen showed excellent linearity in the concentration range 0.02-20 μg/mL (r2 ≥ 0.9996), adequate sensitivity, favorable accuracy (96.4-103.7%) and precision (percentage coefficient of variation ≤4.3) with negligible matrix effect. The validated method was successfully applied to a pharmacokinetic study of fenoprofen in healthy subjects. The significant features of the method include higher sensitivity, small plasma volume for processing and a short analysis time.

Simultaneous Determination of Five Cytochrome P450 Probe Substrates and Their Metabolites and Organic Anion Transporting Polypeptide Probe Substrate in Human Plasma Using Liquid Chromatography-Tandem Mass Spectrometry

A rapid and selective liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the simultaneous determination of organic anion transporting polypeptide 1B1 (OATP1B1) and cytochrome P450 (P450) probe substrates and their phase I metabolites in human plasma was developed. The OATP1B1 (pitavastatin) and five P450 probe substrates, caffeine (CYP1A2), losartan (CYP2C9), omeprazole (CYP2C19), dextromethorphan (CYP2D6), and midazolam (CYP3A) and their metabolites were extracted from human plasma (50 µL) using methanol. Analytes were separated on a C18 column followed by selected reaction monitoring detection using MS/MS. All analytes were separated simultaneously within a 9 min run time. The developed method was fully validated over the expected clinical concentration range for all analytes tested. The intra- and inter-day precisions for all analytes were lower than 11.3% and 8.82%, respectively, and accuracy was 88.5–117.3% and 96.1–109.2%, respectively. The lower limit of quantitation was 0.05 ng/mL for dextromethorphan, dextrorphan, midazolam, and 1′-hydroxymidazolam; 0.5 ng/mL for losartan, EXP-3174, omeprazole, 5′-hydroxyomeprazole, and pitavastatin; and 5 ng/mL for caffeine and paraxanthine. The method was successfully used in a pharmacokinetic study in healthy subjects after oral doses of five P450 and OATP1B1 probes. This analytical method provides a simple, sensitive, and accurate tool for the determination of OATP1B1 and five major P450 activities in vivo drug interaction studies.

The Effect of Food on Doravirine Bioavailability: Results from Two Pharmacokinetic Studies in Healthy Subjects.

Doravirine is a novel non-nucleoside reverse transcriptase inhibitor being developed for the treatment of HIV-1. In two open-label, single-dose, randomized, two-period, crossover trials, the bioavailability of doravirine administered alone or in a fixed-dose combination (FDC) was determined under fed and fasted conditions. Doravirine 100mg alone or with lamivudine and tenofovir disoproxil fumarate (each 300mg) were administered to healthy subjects fasted or 30min after a high-fat, high-calorie breakfast. Twenty-eight subjects, aged 26-55years, enrolled (doravirine, n=14; FDC, n=14). The sequence of fed/fasted treatment was randomized (1:1). Pharmacokinetic data were analyzed as geometric mean ratios (GMRs) with 90% confidence intervals. Doravirine area under the plasma concentration-time curve (AUC) from time zero to infinity (fed/fasted GMRs: alone 1.16 [1.06-1.26]; FDC 1.10 [1.02-1.20]), AUC from time zero to the last measurement (GMRs: alone 1.18 [1.08-1.29]; FDC 1.10 [1.01-1.20]), and plasma concentration 24h after administration (GMRs: alone 1.36 [1.19-1.55]; FDC 1.26 [1.13-1.41]) values increased in the fed versus fasted state when administered alone or as the FDC; the magnitude was not clinically meaningful. Doravirine maximum achieved concentration was similar after fed or fasted administration for both doravirine alone and FDC (GMRs: alone 1.03 [0.89-1.19]; FDC 0.95 [0.80-1.12]). The pharmacokinetics of tenofovir and lamivudine in the FDC were also slightly altered by administration with food; the changes were not clinically meaningful. All treatments were generally well tolerated. Food had no clinically meaningful effect on doravirine 100mg alone or as part of an FDC.

Effect of bronchodilators in healthy individuals receiving lumacaftor/ivacaftor combination therapy

In an open-label, single-center phase 1 pharmacokinetic study in healthy subjects who received lumacaftor (LUM) in combination with ivacaftor (IVA), review of spirometry data showed a transient decline in percent predicted forced expiratory volume in 1s (ppFEV1) within 4h of drug administration. An additional cohort of healthy subjects with normal baseline ppFEV1 values was studied to evaluate the ppFEV1 response to LUM/IVA administration and assess the effect of long-acting bronchodilators (LABDs) and short-acting bronchodilators (SABDs) on ppFEV1 response. The ppFEV1 decline observed at 4h was attenuated following administration of an LABD and reversed following administration of an SABD. Concomitant administration of LUM/IVA with bronchodilators was well tolerated. These data show that a transient decline in ppFEV1 was observed in healthy subjects following administration of LUM/IVA combination therapy, which can be ameliorated with LABDs or SABDs.

Simultaneous Determination of Trantinterol and One of Its Major Metabolites, 1-Carbonyl Trantinterol, in Human Plasma by LC-MS-MS.

A highly selective and sensitive liquid chromatography-tandem mass spectrometry method was developed and validated for the simultaneous determination of trantinterol and one of its major metabolites, 1-carbonyl trantinterol, in human plasma. An Oasis MCX 96-well solid-phase extraction cartridge and a SeQuantTM ZIC(®)-HILIC LC column were used for sample preparation and chromatographic separation, respectively. The analytes were monitored by a QTrap 5500 mass spectrometer with positive electrospray ionization. Multiple reaction monitoring was used for quantification using the precursor to product ion pairs of m/z 311.1 → 237.9 (trantinterol), m/z 325.1 → 251.9 (1-carbonyl trantinterol) and m/z 368.4 → 294.0 (bambuterol as internal standard). The assay had a calibration range from 0.2 to 50 pg/mL and a lower limit of quantification of 0.2 pg/mL for both trantinterol and 1-carbonyl trantinterol. The inter-day and intra-day precisions were <12.0% and the accuracies were within the range of 87.1-111%. The mean recovery ranged from 82.0 to 97.7% and internal standard normalized matrix effect from 0.813 to 0.899. The analytes were stable under all tested conditions. This validated method was successfully applied to a pilot pharmacokinetic study in healthy subjects administered a single 50 μg oral dose.

Single-dose fentanyl sublingual spray for breakthrough cancer pain

Breakthrough cancer pain (BTCP) is defined as a transient exacerbation of pain that arises in patients with otherwise controlled persistent pain. BTCP typically has a rapid onset and relatively short duration, but it causes a significant amount of physical and psychological distress for patients. Several rapid-onset fentanyl formulations have been introduced in the USA to replace traditional oral opioids for the treatment of BTCP: a transmucosal lozenge, a sublingual orally disintegrating tablet, a buccal tablet, a buccal soluble film, a pectin nasal spray and, the newest formulation to enter the market, a sublingual spray. This article reviews the six rapid-onset formulations of fentanyl approved in the USA for the management of BTCP with emphasis on describing the published literature on fentanyl sublingual spray. The different fentanyl formulations vary in pharmacokinetic properties and ease of use, but all have a rapid onset and a relatively short duration of analgesia. Fentanyl sublingual spray has demonstrated absorption within 5 minutes of administration, with fentanyl plasma concentrations increasing over the first 30 minutes and remaining elevated for 60–90 minutes in pharmacokinetic studies in healthy subjects. Fentanyl sublingual spray shows linear dose proportionality, and changes in the temperature or acidity of the oral cavity do not alter its pharmacokinetic properties. In patients with BTCP, statistically significant pain relief is measurable at 5 minutes after administration of fentanyl sublingual spray, when compared with placebo, with significant pain relief lasting at least 60 minutes after administration. Adverse events are typical of opioid treatment and are considered mild to moderate in intensity. In summary, fentanyl sublingual spray provides rapid onset of analgesia and is a tolerable and effective treatment for BTCP.

Once-daily gastroretentive gabapentin for the management of postherpetic neuralgia: an update for clinicians

Gabapentin was originally developed as an add-on anticonvulsant drug, but has been widely used in the USA for the management of postherpetic neuralgia since its approval for this indication in 2002. Gabapentin has a short elimination half life and limited absorption due to a saturable L-amino acid transport system, which is expressed predominantly in the proximal small intestine. Hence, the original immediate-release gabapentin formulation (gabapentin TID) must usually be taken three times a day for optimal efficacy. Gabapentin TID is also associated with a high incidence of dizziness and somnolence and some patients are unable to tolerate the doses required for maximum pain relief. A once-daily, gastroretentive formulation of gabapentin was recently approved by the US Food and Drug Administration (FDA) for the management of postherpetic neuralgia. This formulation provides gradual release of gabapentin to the optimal site of absorption in the proximal small intestine and reduces the chance of saturating intestinal uptake, thus enabling once-daily dosing of gabapentin. This gradual release and absorption have been demonstrated in pharmacokinetic studies in healthy subjects. The efficacy of once-daily gastroretentive gabapentin for the management of postherpetic neuralgia has been demonstrated in placebo-controlled clinical studies. In addition, data from these studies suggest that the incidence of dizziness and somnolence may be reduced compared with similar studies using gabapentin TID. This article provides an overview of the pharmacokinetics, efficacy, and safety of once-daily gastroretentive gabapentin for the management of postherpetic neuralgia.

Rapid and validated fluorometric HPLC method for determination of gabapentin in human plasma and urine for clinical application

A rapid derivatization and validated HPLC method for gabapentin in human plasma and urine is needed for clinical use. The objective of this study was to establish a rapid and validated analytical method for the determination of gabapentin in human plasma and urine using isocratic fluorometric HPLC for clinical application. This analytical method is based on precolumn fluorescent derivatization using 4-fluoro-7-nitro-benzofurazan. The derivatization was coupled to fast HPLC separation using a 2·3 μm-particle size ODS column (100 × 4·6 mm i.d.). The derivatization of gabapentin was optimized and HPLC separation was achieved over an ODS column with a run time of 3·5 min. Calibration curves in human plasma and urine were linear over the concentration ranges of 0·05-10 and 10-1000 μg/mL, respectively. Intra- and inter-assay precision and accuracy values of plasma were within 8·0% and 101-109% and within 8·3% and 94-108%, respectively. Those of urine were within 8·5% and 97-106% and within 9·5% and 97-105%, respectively. This validated method was applied to a pharmacokinetic study in healthy subjects. Interindividual variations in plasma disposition and urinary excretion of gabapentin were observed. A rapid and validated isocratic fluorometric HPLC method for the determination of gabapentin in human plasma and urine for clinical application has been established. This method can be utilized to evaluate the pharmacokinetic disposition of gabapentin in humans.

Simultaneous determination of 18α‐ and 18β‐glycyrrhetic acid in human plasma by LC‐ESI‐MS and its application to pharmacokinetics

A highly sensitive and selective LC-ESI-MS was developed, validated for the simultaneous determination of 18alpha-glycyrrhetic acid (alpha-GA) and 18beta-glycyrrhetic acid (beta-GA) for pharmacokinetic studies in healthy subjects. Sample preparation was performed by liquid-liquid extraction with ethyl acetate and the separations were achieved using a C(18) column with the mobile phase composed of 10 mmol/L ammonium acetate solution-methanol-acetonitrile (40:36:24, v/v/v) at a flow rate of 1 mL/min. The internal standard was honokiol and the epimers were quantified using a single quadrupole mass spectrometer employing ESI in the negative ion mode. The separation factor, alpha, was 1.512 for alpha- and beta-GA. The standard curves were linear for both epimers with coefficients of determination (r >or= 0.9998) over the concentration range of 1-150 ng/mL. The precision and accuracy were <or= 4.33 and <or= 6.57%, respectively. The mean plasma extraction recoveries were 82.23 +/- 1.91 and 84.29 +/- 2.09% for alpha-GA and beta-GA, respectively. The assay was successfully applied to evaluation of the pharmacokinetic properties of alpha-GA and beta-GA from 19 volunteers who had received oral administration of diammonium glycyrrhizinate capsules. The initial data suggest that glycyrrhizin metabolizes to glycyrrhetic acid fairly slowly and the elimination of alpha-GA is slower than that of beta-GA.

Development and validation of liquid chromatography–tandem mass spectrometric method for simultaneous determination of fosinopril and its active metabolite fosinoprilat in human plasma

A highly sensitive and selective liquid chromatography–tandem mass spectrometry (LC–MS/MS) method was developed and validated for simultaneous determination of the prodrug fosinopril and its major active metabolite fosinoprilat for pharmacokinetic studies in healthy subjects. In order to get the lower limit of quantification (LLOQ), especially for analysis of fosinopril, key points of the method have been investigated including chromatographic conditions and selection of LC–MS/MS conditions. The analytes were extracted from plasma samples by liquid–liquid extraction, separated on a reversed-phase C 8 column using gradient procedure, and detected by tandem mass spectrometry with a triple quadrupole ionization interface. The analytes and internal standard zaleplon were detected using positive electrospray ionization (ESI) in the SRM mode. The LLOQ of the method down to 0.1 ng mL −1 for fosinopril and 1.0 ng mL −1 for fosinoprilat were identifiable and reproducible. The standard calibration curves for both fosinopril and fosinoprilat were linear over the ranges of 0.1–15.0 and 1.0–700 ng mL −1 in human plasma, respectively. The within- and between-batch precisions (relative standard deviation (RSD)%) and the accuracy were acceptable. The validated method was successfully applied to reveal the pharmacokinetic properties of fosinopril and fosinoprilat after oral administration.

Distinct absorption characteristics of oral formulations of valproic acid/divalproex available in the United States

Five oral formulations of valproic acid (VPA)/divalproex sodium are approved and commonly used in the US for treatment of epilepsy, mania/bipolar disorder and migraine prophylaxis. These formulations have unique pharmacokinetic and formulation characteristics and are designed to treat distinct patient populations. We compared the absorption characteristics of all five oral VPA/divalproex formulations currently available in the US. Plasma VPA concentration-time profiles, following single oral dose (250mg) administration of five VPA/divalproex formulations under fasting conditions, from three pharmacokinetic studies in healthy subjects (N=9-15 each) were compared. The five VPA/divalproex formulations demonstrated marked absorption differences. The rate of absorption, as characterized by maximum concentration (C(max)) and time to C(max) (T(max)), may be rank-ordered as: VPA syrup (34.2mg/L, 0.9h)>VPA capsule (31.4mg/L, 2.2h)>divalproex sodium sprinkle capsule (20.7mg/L, 4.0h; lag-time congruent with1h) congruent withdivalproex sodium enteric-coated delayed-release tablet (26.0mg/L, 3.4h; lag-time congruent with2h)>divalproex sodium extended-release (divalproex-ER) tablet (11.8mg/L, 19.7h). Divalproex-ER had approximately 11% lower exposure (AUC). The comparable AUC across the five formulations, when corrected for bioavailability differences, demonstrates that formulation primarily affects the drug-release and in vivo absorption of VPA. Only divalproex-ER demonstrated true sustained-release characteristics.

Ginkgo biloba: Evaluation of CYP2C9 Drug Interactions In Vitro and In Vivo

Ginkgo biloba extract is one of the most widely used herbal products in the United States. However, bleeding episodes in patients taking Ginkgo biloba and warfarin have been documented. Therefore, in vitro and in vivo inhibition studies were done to ascertain the influence of ginkgo on CYP2C9, the P-450 isozyme responsible for the metabolism of the most potent warfarin enantiomer, (S)-warfarin. Ginkgo extract inhibited human liver microsomal CYP2C9 with an apparent Ki=14.8 microg/mL, and the inhibition was increased by acid hydrolysis (apparent Ki=9.1 microg/mL). Two open-label, crossover pharmacokinetic studies in healthy subjects were performed using tolbutamide and diclofenac as probe CYP2C9 substrates. In contrast to the in vitro inhibition of CYP2C9, no interactions between Ginkgo biloba extract and CYP2C9 probe substrates were observed in vivo as evidenced by the lack of effect on the steady-state pharmacokinetics of diclofenac or on the urinary metabolic ratio of tolbutamide.

Enatiomer separation of dihydropyridine derivative calcium antagonists by high-performance liquid chromatography using modified Pirkle-type chiral stationary phases and the effect of grapefruit juice on the pharmacokinetics of dihydropyridine enantiomers.

The separation of enantiomers of dihydropyridine derivatives by high-performance liquid chromatography (HPLC) was studied using modified Pirkle-type chiral stationary phases. Enatioseparations of manidipine and nicardipine were achieved by a normal-phase mobile phase utilizing n-hexane-l, 2-dichloroethane-methanol-trifluoroacetic acid on the urea-derivative type chiral statinary phase (SUMICHIRAL OA-4500). The enantioselective HPLC methods of manidipine and nicardipine in human plasma involve a rapid and simple extraction based on a bonded-phase extraction, which were applied to an enatioselective pharmacokinetic study in healthy subjects. Also, the effects of grapefruit juice on the stereoselective dispositions of manidipine and nicardipine in healthy subjects were examined. (+)-Manidipine plasma level was 1.5 fold higher than (-)-manidipine, and both enantiomers plasma level increased to 2.3 fold with grapefruit juice. Additionally, on the study of nicardipine, subjects were received intravenous doses and oral doses. After oral administration, grapefruit juice significantly increased the plasma concentrations of (+)-nicardipine and (-)-nicardipine, but not affect after intravenous administration. It is suggested that grapefruit juice inhibited CYP3A4-mediated presystemic metabolism in the small intestine.

High-performance liquid chromatographic determination of the conjugate metabolites of moxisylyte in human plasma and urine.

Sensitive and specific high-performance liquid chromatographic methods with fluorescence detection are described for the determination of the metabolites of moxisylyte (4-(2-dimethylaminoethoxy)-5-isopropyl-2-methylphenyl acetate) in human plasma and urine. Deacetylmoxisylyte glucuroconjugate (DAM-G) was hydrolysed enzymatically using 1-glucuronidase and quantified as the difference between the DAM concentrations determined after and before hydrolysis. The two sulphate derivatives (deacetylmoxisylyte sulphoconjugate, DAM-S and monomethyldeacetylmoxisylyte sulphoconjugate, MDAM-S), were analysed without prior hydrolysis. Their extraction from plasma and urine, as well as that of DAM from plasma, involved the use of C18 cartridges adapted on a Benchmate workstation. DAM in urine was quantified after liquid-liquid extraction. The two methods were validated for specificity, linearity, intra- and inter-day precision and accuracy. Precision was generally < or = 15% and accuracy < or = 12%. In plasma, the limits of quantification were 2.5 ng/ml for DAM and 2.8 ng/ml for the two sulphates, in urine, they were 40 ng/ml for DAM and 200 ng/ml for the sulphates. These methods were used for pharmacokinetic studies in healthy subjects.

Determination of piroximone in Plasma and urine by high performance liquid chromatography

Abstract A reversed-phase high-performance liquid chromatographic (HPLC) method with UV detection was developed for quantifying piroximone (PI) in plasma and urine. A solid phase extraction of PI simultaneously with an internal standard with an average recovery higher than 86% was needed to avoid endogenous interferences especially in urines from patients with reduced renal functions. The sensitivity limit was 2 ng/ml in plasma and 100 ng/ml in urine. The method was reproducible with intra- and inter-assay coefficients of variations below 7 %. This method was applied to the determination of plasma and urine levels during a pharmacokinetic study in healthy subjects and renally impaired patients. It was found suitable to follow the concentrations until 24 h after a single intravenous infusion of 0.5 mg/kg body weight of piroximone.