Determination of suvorexant in human plasma using 96-well liquid-liquid extraction and HPLC with tandem mass spectrometric detection.

A method, using liquid chromatography with tandem mass spectrometric detection (LC-MS/MS), was developed for the determination of suvorexant (MK-4305, Belsomra(®)), a selective dual orexin receptor antagonist for the treatment insomnia, in human plasma over the concentration range of 1-1000ng/mL. Stable isotope labeled (13)C(2)H3-suvorexant was used as an internal standard. The sample preparation procedure utilized liquid-liquid extraction, in the 96-well format, of a 100µL plasma sample with methyl t-butyl ether. The compounds were chromatographed under isocratic conditions on a Waters dC18 (50×2.1mm, 3µm) column with a mobile phase consisting of 30/70 (v/v %) 10mM ammonium formate, pH3/acetonitrile at a flow rate of 0.3mL/min. Multiple reaction monitoring of the precursor-to-product ion pairs for suvorexant (m/z 451→186) and (13)C(2)H3-suvorexant (m/z 455→190) on an Applied Biosystems API 4000 tandem mass spectrometer was used for quantitation. Intraday assay precision, assessed in six different lots of control plasma, was within 10% CV at all concentrations, while assay accuracy ranged from 95.6 to 105.0% of nominal. Quality control (QC) samples in plasma were stored at -20°C. Initial within day analysis of QCs after one freeze-thaw cycle showed accuracy within 9.5% of nominal with precision (CV) of 6.7% or less. The plasma QC samples were demonstrated to be stable for up to 25 months at -20°C. The method described has been used to support clinical studies during Phase I through III of clinical development.

Effect of disease state on ionization during bioanalysis of MK-7009, a selective HCV NS3/NS4 protease inhibitor, in human plasma and human Tween-treated urine by high-performance liquid chromatography with tandem mass spectrometric detection.

HPLC-MS/MS methods for the determination of a Hepatitis C NS3/NS4 protease inhibitor (MK-7009) in human plasma and Tween-treated urine were developed and validated over the concentration range 1-1000 ng/mL and 0.2-100 microg/mL respectively. A stable isotope labeled internal standard (ISTD), D(4)-MK-7009, was employed. Analytes were chromatographed by reversed phase HPLC and quantified by an MS/MS system. Electrospray ionization in the positive mode was employed. Multiple reaction monitoring of the precursor to product ion pairs m/z 758.6-->637.4 MK-7009 and m/z 762.5-->637.4 ISTD was used for quantitation. Analyte and internal standard were extracted from 250 microL of plasma using an automated 96-well liquid-liquid extraction. Plasma pH adjustment prior to extraction minimized ionization suppression in plasma samples from patients with Hepatitis C. The urine method involved direct dilution in the 96-well format of 0.020 mL Tween-treated urine. These methods have supported several clinical studies. Incurred plasma sample reanalysis demonstrated adequate assay reproducibility and ruggedness.

Determination of the HIV integrase inhibitor, MK-0518 (raltegravir), in human plasma using 96-well liquid-liquid extraction and HPLC-MS/MS.

An HPLC-MS/MS method was developed for the determination of MK-0518 (raltegravir), an HIV integrase inhibitor, in human plasma over the concentration range of 2-1000 ng/mL. Stable isotope labeled (13)C(6)-MK-0518 was used as an internal standard. The sample preparation procedure utilized liquid-liquid extraction with hexane:methylene chloride in the 96-well format with a 200 microL plasma sample size. The compounds were chromatographed on an Ace C(18) (50 x 3.0 mm, 3 microm, titanium frits) column with 42.5/57.5 (v/v %) 0.1mM EDTA in 0.1% formic acid/methanol mobile phase at a flow rate of 0.5 mL/min. Multiple reaction monitoring of the precursor-to-product ion pairs for MK-0518 (m/z 445-->109) and (13)C(6)-MK-0518 (m/z 451-->367) on an Applied Biosystem API 4000 HPLC-MS/MS was used for quantitation. Intraday precision of standard curve concentrations in five different lots of control plasma was within 3.2%, while accuracy ranged from 94.8 to 106.8%. The mean extraction recovery of spiked plasma samples was 87%. Quality control (QC) samples were stored at -20 degrees C. Initial within day analysis showed QC accuracy within 7.5% of nominal with precision of 3.1% or less. The plasma QC samples were demonstrated to be stable for up to 23 months at -20 degrees C. The method described has been used to support over 18 clinical studies during Phase I through III of clinical development.

Characterization of the solubility of a poorly soluble hydroxylated metabolite in human urine and its implications for potential renal toxicity.

The solubility, in human urine, of the major hydroxylated metabolite (M1) of an experimental cognition enhancer was characterized through a series of in vitro experiments in an effort to estimate the probability of crystalluria occurring following oral administration of the parent compound. The aim of these experiments was to determine if a safety margin existed between clinically observed urine concentrations and the solubility of M1. The mean urine concentrations of M1 in young and elderly subjects following oral administration of the parent compound at the highest doses tested, were 4865 +/- 2368 ng/mL and 2764 +/- 791 ng/mL, respectively. In vitro solubility experiments with M1 were conducted in drug-free human urine (37 degrees C) from four male and four female healthy subjects under conditions of high and low urine osmolality. Mean concentrations (n = 16) of M1 in human urine to which solid M1 was added, were 3656 +/- 621 ng/mL, 4678 +/- 1169 ng/mL and 5378 +/- 2474 ng/mL after stirring for 24, 48 and 72 h, respectively, indicating that the ex vivo mean solubility of M1 in human urine is no greater then approximately 5 microg/mL. Addition of solid M1 to urine from human subjects dosed with the parent compound resulted in mean urine M1 concentrations 23.5% greater than those observed in vivo. The results from both experiments indicated a significant overlap between urine concentrations of M1 in vivo following the highest oral administration of the parent drug and M1 solubility measured in vitro, suggesting a high potential for in vivo saturation of urine with M1 with subsequent precipitation, crystalluria, and nephrotoxicity. Consequently, the results of these studies have placed restrictions on the dose that could be administered during clinical development of this compound.

Determination of an investigational HIV integrase inhibitor in human plasma using high performance liquid chromatography with tandem mass spectrometric detection.

An HPLC-MS/MS assay for the determination of an HIV integrase inhibitor, 5-(1,1-dioxido-1,2-thiazinan-2-yl)-N-(4-fluorobenzyl)-8-hydroxy-1,6-naphthyridine-7-carboxamide (I) in human plasma has been developed and validated. Compound I and a stable isotope labeled internal standard (II) were isolated from 0.5 mL plasma samples by solid phase extraction using an Ansys SPEC C-8 96-well plate. Extracts were separated on a Hypersil BDS C-18 HPLC column (3.0 mmx50 mm, 3 microm) with a mobile phase consisting of 25 mM ammonium formate pH 3.0:acetonitrile (60:40) vol%/vol% pumped at 0.5 mL/min. A Sciex API 365 mass spectrometer equipped with an atmospheric pressure chemical ionization source was operated in selected reaction monitoring (SRM) mode with the precursor-to-product ion transitions m/z 431-->109 (I) and m/z 437-->115 (II) used for quantitation. The assay was validated over the concentration range of 10-5000 ng/mL and was found to have acceptable accuracy, precision, linearity, and selectivity. The mean extraction recovery from spiked plasma samples was 69%. The intra-day accuracy of the assay was within 4% of nominal and intra-day precision was better than 4% C.V. Following a 200 mg dose of the compound administered to human subjects, concentrations of I ranged from 21.1 to 1500 ng/mL in plasma samples collected up to 12 h after dosing. Inter-day accuracy and precision results for quality control samples run over a 3-month period alongside clinical samples showed mean accuracies of within 6% of nominal and precision better than 3.5% C.V.

Assessment of the in-vivo stereochemical integrity of aprepitant based on the analysis of human plasma samples via high-performance liquid chromatography with mass spectrometric detection.

Achiral and chiral liquid chromatographic methods utilizing mass spectrometric detection were developed to investigate the possibility of inversion of configuration at any or all of the chiral centers of the neurokinin-1 (NK-1) receptor antagonist, aprepitant (5-[[2(R)-[1(R)-(3,5-bistrifluoromethyl phenyl)ethoxy]-3(S)-(4-fluorophenyl)morpholin-4-yl]methyl]-2,4-dihydro-[1,2,4]triazol-3-one), in-vivo, following administration of the compound to man. A structure such as aprepitant, that contains three chiral centers, may exist in eight stereochemical forms or, more specifically, as four diastereoisomeric pairs of enantiomers. The four diastereoisomers were separated from each other using a ProntoSil C18 AQ HPLC column (4.6 x 100 mm, 3 microm particles) with a mobile phase composed of acetonitrile--water (47:53, v/v%). Detection was via a single quadrupole mass spectrometer that was connected to the HPLC system via an APCI interface. Analysis of post-dose plasma samples under these conditions indicated that only aprepitant and or its enantiomer were present following oral administration of the drug. Aprepitant and its enantiomer were separated using a Chiralcel OD-H HPLC column with a mobile phase composed of hexane-isopropanol (80:20, v/v%); tandem mass spectrometric detection using an APCI interface was employed. Post-dose plasma samples analyzed using the Chiracel column were found to contain only aprepitant. The results of these experiments confirm that the products of inversion of configuration at any or all of the three chiral centers of aprepitant are not detectable in human plasma samples obtained following the administration of the drug.

Determination of dextromethorphan and its metabolite dextrorphan in human urine using high performance liquid chromatography with atmospheric pressure chemical ionization tandem mass spectrometry: a study of selectivity of a tandem mass spectrometric assay.

Analytical method for the simultaneous determination of dextromethorphan (1) and dextrorphan (2) in urine, based on solid-phase extraction of drug from acidified hydrolyzed biological matrix, were developed. The analytes (1 and 2) and the internal standard (levallorphan, 3, IS) were detected by high-performance liquid chromatography-mass spectrometry (HPLC-MS/MS) in positive ionization mode using a heated nebulizer (HN) probe and monitoring their precursor-->product ion combinations of m/z 272-->215, 258-->201, and 284-->201 for 1, 2, and 3, respectively, in multiple reaction monitoring mode. The analytes and IS were chromatographed on a Keystone Prism reverse phase (50 mm x 2.0 mm) 5 microm column using a mobile phases consisting of a 35/65 or 27/73 mixtures of methanol/water containing 0.1% TFA adjusted to pH 3 with ammonium hydroxide pumped at 0.4 ml/min for 1 and 2, respectively. The limits of reliable quantification of 1 and 2 were 2 and 250 ng/ml, respectively, when 1 ml of urine was processed. The absence of matrix effect was demonstrated by analysis of neat standards and standards spiked into urine extracts originating from five different sources. The linear ranges of the assay were 2-200 and 250-20,000 ng/ml for 1 and 2, respectively. Assay selectivity was evaluated by monitoring the "cross-talk" effects from other metabolites into the MS/MS channels used for monitoring 1, 2, and 3. In addition, an interfering peak originating from an unknown metabolite of 1 into the quantification of dextromethorphan was detected, requiring an effective chromatographic separation of analytes from other metabolites of 1. The need for careful assessment of selectivity of the HPLC-MS/MS assay in the presence of metabolites, and the assessment of matrix effect, are emphasized.

Elimination of autosampler carryover in a bioanalytical HPLC-MS/MS method: a case study.

A case study in identifying and eliminating the source of autosampler carryover in a bioanalytical HPLC-MS/MS assay is described. Through a series of systematic experiments, the carryover was traced to the injection valve and was eliminated by switching from a partial loop to a full loop injection, which provided more effective flushing of the sample flow path. The susceptibility of the HPLC system to carryover was demonstrated to depend on the absolute sensitivity of the detection method and the mass of analyte injected at the assay lower limit of quantitation (LLOQ).

Determination of fexofenadine in human plasma using 96-well solid phase extraction and HPLC with tandem mass spectrometric detection.

A fast and sensitive HPLC-MS/MS method, utilizing atmospheric pressure chemical ionization, for the determination of fexofenadine in human plasma is described. A deuterated analog, d6-fexofenadine is used as the internal standard (IS). Plasma samples are prepared using 96-well solid phase extraction with plates containing Waters Oasis HLB sorbent. The analytes are chromatographed on a Restek Ultra IBD column (3.2 mm x 50 mm, 3 microm) using a mobile phase consisting of a mixture of 90% acetonitrile and 10% 10 mM ammonium acetate buffer and 0.1% formic acid. Quantitation of the analyte is based on the response from the multiple reaction monitoring of the precursor to product ion pairs for fexofenadine (m/z 502 --> 466) and d6-fexofenadine (m/z 508 --> 472). The assay has been validated over the concentration range of 1-200 ng/ml based on the analysis of 0.5 ml aliquots of plasma. Within-day assay accuracy was between 97 and 102% of nominal, while within-day precision was better than 3.5% CV at all points on the standard curve. Analyte extraction recovery was better than 70% over the range of the standard curve. The method was found to be suitable for the analysis of human plasma samples obtained 24 h following the administration of a single 60 mg dose of fexofenadine.

Isolation and structural characterization of the photolysis products of etoricoxib.

Etoricoxib is a potent and novel selective inhibitor of cyclooxygenase-2 (COX-2) which has been developed for the treatment of osteoarthritis, rheumatoid arthritis and several other inflammatory conditions. To support clinical pharmacokinetics studies, a method for the determination of etoricoxib in human plasma was developed. During the development of the method it was found that highly fluorescent products were formed when etoricoxib was exposed to UV light (254 nm). The formation of highly fluorescent products was the basis for the development of a highly sensitive HPLC/fluorescent assay for the indirect determination of etoricoxib in human plasma; the limit of quantification (LOQ) was 1 ng/mL. To unequivocally determine the chemical structures of the photolysis products of etoricoxib, a series of studies was conducted. When etoricoxib was irradiated online in a photochemical reactor, three products were detected in an HPLC-UV system. These products were characterized by HPLC-UV-fluorescence and HPLC-MS/MS. Possible structures of these products were proposed based on these data. The major photolysis products of etoricoxib were further isolated and their structures were elucidated using NMR and HPLC-NMR. The results of these experiments indicate that etoricoxib undergoes a photocyclization reaction when irradiated with UV light (254 nm), leading to the formation of two major isomeric photocyclization products.

Determination of risedronate in human urine by column-switching ion-pair high-performance liquid chromatography with ultraviolet detection.

An HPLC assay for the determination of risedronate in human urine was developed and validated. Risedronate and the internal standard were isolated from 5-ml urine samples in a two-part procedure. First, the analytes were precipitated from urine along with endogenous phosphates as calcium salts by the addition of CaCl(2) at alkaline pH. The precipitate was then dissolved in 0.05 M ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid and subjected to ion-pair solid-phase extraction using a Waters HLB cartridge (1 ml, 30 mg) with 1-octyltriethylammonium phosphate as the ion-pair reagent. Following extraction, the analytes were initially separated from the majority of co-extracted endogenous components on a Waters X-Terra RP18 (4.6 x 50 mm, 3.5 microm) column. The effluent from the X-Terra was "heart-cut" onto a Phenomenex Synergi Polar RP (4.6 x 150 mm, 4 microm) column for final separation. UV detection (lambda=262 nm) was used to quantitate risedronate in the concentration range of 7.5-250 ng/ml. Mean recovery was 83.3% for risedronate and 86.5% for the internal standard. The intra-day precision of the assay, as assessed by replicate (n=5) standard curves, was better than 6% RSD for all points on the standard curve. Within-day accuracy for the standards ranged from 96.3 to 106.1% of nominal. Inter-day precision for quality controls assayed over a 3-week period was better than 5%, while inter-day accuracy was within 90% of nominal. The assay was employed to analyze samples collected during a clinical pharmacokinetics study.

Monolithic silica liquid chromatography columns for the determination of cyclooxygenase II inhibitors in human plasma.

Methods employing monolithic HPLC columns for the determination of the cyclooxygenase II inhibitors rofecoxib (I) and 3-isopropoxy-4-(4-methanesulfonylphenyl)-5,5'-dimethyl-5H-furan-2-one (DFP, III) in human plasma are described. Each analyte, together with an internal standard was extracted from the plasma matrix using solid-phase extraction in the 96-well format. The analytes were chromatographed on a Chromolith Speed Rod monolithic HPLC column (4.6 x 50 mm). Analyte detection for rofecoxib was via fluorescence following post-column photochemical derivatization. Detection for III was based on the native fluorescence of the compound. The precision, accuracy, and linearity of the methods were found to be comparable to those obtained using methods employing conventional packed HPLC columns. Use of the monolithic column permitted mobile phase flow-rates of up to 6.5 ml/min to be employed in the assays. The use of elevated flow-rates enabled the per sample analysis time to be reduced by up to a factor of 5 compared with assays based on packed HPLC columns. The results of experiments aimed at evaluating the ruggedness and reproducibility of monolithic columns employed in bioanalytical methods are presented.

Determination of efavirenz, a selective non-nucleoside reverse transcriptase inhibitor, in human plasma using HPLC with post-column photochemical derivatization and fluorescence detection.

Methods for the quantitative determination of efavirenz in human plasma and the qualitative assessment of the stereochemical integrity of efavirenz in post-dose human plasma samples are described. After the addition of an internal standard, plasma samples were extracted with hexane-methylene chloride (65/35, v/v%). The extracts were evaporated to dryness and reconstituted in mobile phase. Upon exposure to UV light, the analyte was found to form fluorescent products; the major fluorescent product was isolated and identified as a substituted quinoline. Thus, the plasma extracts were analyzed via HPLC with post-column photochemical derivatization and fluorescence detection. Reverse phase chromatography was used for the quantitative assay, whereas chromatography with a column containing a chiral stationary phase (dinitrobenzoyl leucine) was used for the stereochemical assessment. The quantitative assay has been validated in the concentration range of 50-1000 ng/ml using 0.5 ml samples. Analyte recovery was better than 89% at all points on the standard curve. Intra-day precision was better than 5% C.V., while accuracy was between 95 and 104% of nominal over the range of the assay. The selective detection method reduces the likelihood of interference by co-administered medications or endogenous species. The stereochemical configuration of efavirenz was confirmed to remain intact in post-dose human plasma samples. The quantitative method has been successfully utilized to support a study in which a possible drug interaction between co-administered HIV protease inhibitors and efavirenz was evaluated.

Improved procedure for the the determination of rofecoxib in human plasma involving 96-well solid-phase extraction and fluorescence detection.

An improved assay for the determination of rofecoxib in human plasma samples is described. The analyte and an internal standard were extracted from the plasma matrix using solid-phase extraction in the 96-well format with an Empore C8-SD extraction plate. The analytes are chromatographed on a Waters Symmetry C18 analytical column (3.5 microm, 50x4.6 mm) with a mobile phase consisting of acetonitrile-water (35:65, v/v). Analyte detection was via fluorescence following post-column photochemical derivatization. Eight point calibration curves over the concentration range of 0.5-80 ng/ml yielded a linear response when a 1/y weighted linear regression model was employed. Based on the replicate analyses (n=5) of spiked standards, the within-day assay precision was better than 8% RSD at all points on the calibration curve, within-day accuracy was within 6% of nominal at all standard concentrations. The between-run precision and accuracy of the assay, as calculated from the results of the analysis of quality control samples, was better than 7% RSD and within 5% of nominal. Assay throughput was improved by a factor of three as compared to previously described methods. The method was partially automated using a combination of a Packard Multi-Probe liquid handling system and a TomTec Quadra 96 workstation.

Simultaneous determination of unlabeled and carbon-13-labeled etoricoxib, a new cyclooxygenase-2 inhibitor, in human plasma using HPLC-MS/MS.

A method for the simultaneous determination of etoricoxib and its carbon-13 analog ((13)C(6)-etoricoxib) from human plasma has been developed and used to support bioavailability studies. Plasma samples (0.5 mL) were extracted by using a 3M Empore 96-well plate (C(8)) and the resulting extracts were analyzed by using a PE-Sciex API-3000 HPLC-MS/MS with a heated nebulizer interface (500 degrees C). The method was validated with two different calibration curve ranges, one for etoricoxib (5 to 2500 ng/mL) determined in the presence of lower concentrations of (13)C(6)-etoricoxib (0.5 to 250 ng/mL), and a second curve for the quantitation of similar concentrations of both etoricoxib and (13)C(6)-etoricoxib (0.5 to 250 ng/mL). Extraction recoveries of etoricoxib, (13)C(6)-etoricoxib, and a methylated internal standard were >70% over the range of concentrations included in both calibration curves. Intraday precision and accuracy for the quantitation of etoricoxib were 7.8% relative standard deviation (RSD) or less and within 3.4% respectively over the range of 5 to 2500 ng/mL, and 10.8% RSD or less and within 4 % respectively over the range of 0.5 to 250 ng/mL. Within-batch precision and accuracy for the quantitation of (13)C(6)-etoricoxib over the range of 0.5 to 250 ng/mL were 8.3% RSD or less and within 2.3%, respectively. The validated assay was used in support of human clinical trials.

Dose proportionality of oral etoricoxib, a highly selective cyclooxygenase-2 inhibitor, in healthy volunteers.

To assess dose proportionality of etoricoxib across the anticipated clinical dose range, a single panel of 12 healthy subjects was administered single oral doses of etoricoxib of 5, 10, 20, 40, and 120 mg in an open, two-part, five-period crossover study. Plasma samples were collected aftereach dose and analyzed for etoricoxib concentrations. The pharmacokinetics of etoricoxib appear to be linear over the entire dose range examined, from 5 to 120 mg. Etoricoxib was found to be well tolerated across the 5 to 120 mg dose range.

In-vitro metabolism of celecoxib, a cyclooxygenase-2 inhibitor, by allelic variant forms of human liver microsomal cytochrome P450 2C9: correlation with CYP2C9 genotype and in-vivo pharmacokinetics.

In-vitro studies were conducted to assess the impact of CYP2C9 genotype on the metabolism (methyl hydroxylation) and pharmacokinetics of celecoxib, a novel cyclooxygenase-2 inhibitor and CYP2C9 substrate. When compared to cDNA-expressed wild-type CYP2C9 (CYP2C9*1), the Vmax/Km ratio for celecoxib methyl hydroxylation was reduced by 34% and 90% in the presence of recombinant CYP2C9*2 and CYP2C9*3, respectively. These data indicated that the amino acid substitution at position 359 (Ile to Leu) elicited a more pronounced effect on the metabolism of celecoxib than did a substitution at position 144 (Arg to Cys). The Vmax/Km ratio was also decreased in microsomes of livers genotyped CYP2C9*1/*2 (47% decrease, mean of two livers), or CYP2C9*1/*3 (59% decrease, one liver). In all cases, these changes were largely reflective of a decrease in Vmax, with a minimal change in Km. Based on simulations of the in-vitro data obtained with the recombinant CYP2C9 proteins, it was anticipated that the pharmacokinetics of celecoxib (as a much as a five-fold increase in plasma AUC) would be altered (versus CYP2C9*1/*1 subjects) in subjects genotyped heterozygous or homozygous for the CYP2C9*2 (Cys144) or CYP2C9*3 (Leu359) allele. In a subsequent clinical study, the AUC of celecoxib was increased (versus CYP2C9*1/*1 subjects) approximately 2.2-fold (range, 1.6-3-fold) in two CYP2C9*1/*3 subjects and one CYP2C9*3/*3 subject receiving a single oral dose (200 mg) of the drug. In contrast, there was no significant change in celecoxib AUC in two subjects genotyped CYP2C9*1/*2.

High-throughput, semi-automated determination of a cyclooxygenase II inhibitor in human plasma and urine using solid-phase extraction in the 96-well format and high-performance liquid chromatography with post-column photochemical derivatization-fluorescence detection.

Compound I, 5-chloro-3-(4-methanesulfonylphenyl)-6'-methyl-[2,3']bipyridinyl, has been found to be a specific inhibitor of the enzyme cyclooxygenase II (COX II). The anti-inflammatory properties of this compound are currently being investigated. HPLC assays for the determination of this analyte in human plasma and human urine have been developed. Isolation of I and the internal standard (II) was achieved by solid-phase extraction (SPE) in the 96-well format. A C8 SPE plate was used for the extraction of the drug from human plasma (recovery >90%) while a mixed-mode (C8/Cation) SPE plate was used to isolate the analytes from human urine (recovery approximately 71%). The analyte and internal standard were chromatographed on a Keystone Scientific Prism-RP guard column (20 x 4.6 mm) connected to a Prism-RP analytical column (150 x 4.6 mm), using a mobile phase consisting of 45% acetonitrile in 10 mM acetate buffer (pH = 4); the analytes eluted at retention times of 5.2 and 6.9 min for I and II, respectively. Compounds I and II were found to form highly fluorescent products after exposure to UV light (254 nm). Thus, the analytes were detected by fluorescence (lambda(ex) = 260 nm, lambda(em) =375 nm) following post-column photochemical derivatization. Eight point calibration curves over the concentration range of 5-500 ng/ml for human plasma and human urine yielded a linear response (R2>0.99) when a 1/y weighted linear regression model was employed. Based on the replicate analyses (n = 5) of spiked standards, the within-day precision for both assays was better than 7% C.V. at all points on the calibration curve; within-day accuracy was within 5% of nominal at all standard concentrations. The between-run precision and accuracy of the assays, as calculated from the results of the analysis of quality control samples, was better than 8% C.V. and within 8% of nominal. I was found to be stable in human plasma and urine for at least 8 and 2 months, respectively. In addition, the human plasma assay was semi-automated in order to improve sample throughput by utilizing a Packard liquid handling system and a Tom-Tec Quadra 96 SPE system. The precision and accuracy of the semi-automated procedure were comparable to the manual procedure. Over 5000 clinical samples have been analyzed successfully using these methods.

High-throughput simultaneous determination of the HIV protease inhibitors indinavir and L-756423 in human plasma using semi-automated 96-well solid phase extraction and LC-MS/MS.

A method for the simultaneous determination of the HIV protease inhibitors indinavir and L-756423, in human plasma has been developed. Plasma samples (0.5 ml) were extracted using a 3M Empore 96-well plate in the mixed phase cation exchange (MPC) format. The extraction method was automated through the application of both the Packard 204DT and TOMTEC Quadra 96 work stations, and the resulting extracts were analyzed using a PE-Sciex API-3000 LC-MS/MS with a heated nebulizer interface (500 degrees C). The assay was linear in the concentration range 1-2500 ng/ml for indinavir and 5 2500 ng/ml for L-756423 when 0.5-ml aliquots of plasma were extracted. Recoveries of indinavir and L-756423 were greater than 76 and 80%, respectively, over the calibration curve range when using the described sample preparation method. Within-batch precision and accuracy for the quantitation of indinavir over the range 1-2500 ng/ml were 5.4% R.S.D. or less and within 4.0%, respectively. Within-batch precision and accuracy for the quantitation of L-756423 over the range 5-2500 ng/ml were 5.3% R.S.D. or less and within 3.4%, respectively. Interbatch variability for the analysis of indinavir QC samples at low (3 ng/ml), middle (250 ng/ml) and high (2250 ng/ml) were 3.2, 2.9, and 1.9%, respectively. Interbatch variability for the analysis of L-756423 QC samples at low (15 ng/ml), middle (250 ng/ml) and high (2250 ng/ml) concentration were 2.0, 2.5, and 3.3%, respectively. The validated assay was used in support of human clinical trials.

Determination of celecoxib in human plasma by normal-phase high-performance liquid chromatography with column switching and ultraviolet absorbance detection.

A method is described for the determination of celecoxib in human plasma. Samples were extracted using 3M Empore membrane extraction cartridges and separated under normal-phase HPLC conditions using a Nucleosil-NO2 (150x4.6 mm, 5 microm) column. Detection was accomplished using UV absorbance at 260 nm. The HPLC method included a column switching procedure, in which late eluting compounds were diverted to waste, to reduce run-time to 12 min. The assay was linear in the concentration range of 25-2000 ng/ml when 1-ml aliquots of plasma were extracted. Recoveries of celecoxib were greater than 91% over the calibration curve range. Intraday precision and accuracy for this assay were 5.7% C.V. or better and within 2.3% of nominal, respectively. The assay was used to analyze samples collected during human clinical studies.