Sensitive and selective methods based on high performance liquid chromatography (HPLC) with tandem mass spectrometric (MS/MS) detection were developed for the determination of vitamin D3 in human serum. Derivatization of vitamin D3 and its stable isotope labeled internal standard provided highly sensitive quantification and selective detection from endogenous compounds. Samples were prepared using the in-tube liquid–liquid extraction (LLE), 96-well plate LLE, and in-tip solid phase micro-extraction (SPME) in 96-well format. In all methods, the MS/MS detection was performed using Applied Biosystems-Sciex API 3000 tandem mass spectrometers interfaced with a heated nebulizer probe and operated in the positive ionization mode. Both tube and plate LLE methods achieved a lower limit of quantitation (LLOQ) of 0.5 ng/mL when 1.0 and 0.4 mL of human serum was processed, respectively, and were validated in the concentration range of 0.5–25 ng/mL; while for the in-tip SPME method, LLOQ was 5 ng/mL with only 0.1 mL of human serum required. Comparisons were made among three different methods, including precision and accuracy, sample throughput, recovery and matrix effects.

Two sensitive and selective methods based on solid phase microextraction (SPME) and liquid–liquid extraction (LLE) in 96-well format, in combination with high performance liquid chromatography (HPLC) with tandem mass spectrometric (MS/MS) detection have been developed to determine a model drug compound in human plasma. Both assays were performed on an Applied Biosystems-Sciex API 4000 tandem mass spectrometer interfaced with a turbo ion-spray probe and operated in the negative ionization mode. A lower limit of quantitation (LLOQ) of 1ng/mL achieved when 0.25mL of human plasma was processed. In both methods, a stable isotope labeled internal standard was utilized. The methods were validated in the concentration range of 1–500ng/mL. The intraday precision (%C.V.) of the method using LLE was 0.8% at LLOQ, and was equal to or lower than 3.3% at all other concentrations, while the intraday precision (%C.V.) of the method using SPME was 6.9% at LLOQ, and was equal to or lower than 5.7% at all other concentrations. Based on the direct comparison of the two methods and their successful applications in clinical sample analysis, it may be concluded that SPME may be considered and used as an alternative approach for quantitative determination of drugs in pharmacokinetic studies.

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.

An automated 96-well solid phase extraction method was developed for the isolation of a selective neuropeptide Y5 receptor antagonist and internal standard from human plasma and urine. Following isolation, the analyte was selectively quantified using a reverse phase HPLC system coupled with atmospheric pressure chemical ionization (APCI) mass spectrometry operated in the positive ionization MRM mode. Based upon the peak area ratio (analyte: internal standard) vs standard concentrations, the analyte was quantified over a concentration range of 2-1000 ng/mL. The absolute and relative matrix effects from different sources of sample matrices on the ionization efficiency were examined and the absence of these effects was confirmed. The extraction efficiency was found to be approximately 90% and reproducible in both matrices. Assay validation results, including intra-day precision and accuracy values, are presented. The validated assay has been used to support several pharmacokinetic studies, and some representative data are presented.

A method for the determination of a prostaglandin D2 receptor antagonist (I, a compound being evaluated for the prevention of niacin induced flushing) and its acyl glucuronide metabolite (II) in human plasma is presented. The method utilized high performance liquid chromatography (HPLC) with tandem mass spectrometric (MS/MS) detection using an atmospheric pressure chemical ionization (APCI) interface operated in the positive ionization mode. The product ion was a radical cation generated via a homolytic bond cleavage. A chemical analog of the drug was used as internal standard (III). The acyl glucuronide metabolite (II) was detected using the same precursor-to-product ion transition used for the parent compound after chromatographic separation of I and II. Drug and metabolite were extracted using semi-automated, 96-well format solid phase extraction (SPE), and chromatography was performed using a reverse phase analytical column with an isocratic mobile phase. The chromatographic retention factor (k′) of II was found to be highly sensitive to mobile phase formic acid concentration. An adjustment in mobile phase formic acid concentration improved the chromatographic separation between II and a mono-hydroxylated metabolite after an unexpected lack of MS/MS selectivity between the two molecules was observed. The dependence of retention factor on formic acid concentration (k′ increased as formic acid concentration decreased) was thought to indicate polar interactions between II and the stationary phase. The stability of II in spiked human plasma was determined. The rate of hydrolysis back to parent compound was relatively low (approximately 0.1 and 0.5% per hour at room temperature and 4°C, respectively) indicating that significant changes in analyte concentrations did not occur during sample processing. The concentration range of the assay was 10–2500ng/mL for both drug and glucuronide metabolite.

A simple experimental approach for studying and identifying the relative matrix effect (for example “plasma-to-plasma” and/or “urine-to-urine”) in quantitative analyses by HPLC–MS/MS is described. Using as a database a large number of examples of methods developed in recent years in our laboratories, the relationship between the precision of standard line slopes constructed in five different lots of a biofluid (for example plasma) and the reliability of determination of concentration of an analyte in a particular plasma lot (or subject) was examined. In addition, the precision of standard line slopes was compared when stable isotope-labeled analytes versus analogs were used as internal standards (IS). Also, in some cases, a direct comparison of standard line slopes was made when different HPLC–MS interfaces (APCI versus ESI) were used for the assay of the same compound, using the same IS and the same sample preparation and chromatographic separation conditions. In selected cases, the precision of standard line slopes in five different lots of a biofluid was compared with precision values determined five times in a single lot. The results of these studies indicated that the variability of standard line slopes in different lots of a biofluid [precision of standard line slopes expressed as coefficient of variation, CV (%)] may serve as a good indicator of a relative matrix effect and, it is suggested, this precision value should not exceed 3–4% for the method to be considered reliable and free from the relative matrix effect liability. Based on the results presented, in order to assess the relative matrix effect in bioanalytical methods, it is recommended to perform assay precision and accuracy determination in five different lots of a biofluid, instead of repeat (n=5) analysis in the same, single biofluid lot, calculate standard line slopes and precision of these slopes, and to use <3–4% slope precision value as a guide for method applicability to support clinical studies. It was also demonstrated that when stable isotope-labeled analytes were used as internal standards, the precision of standard line slopes in five different lots of a biofluid was ≤2.4% irrespective of the HPLC–MS interface utilized. This clearly indicated that, in all cases studied, the use of stable isotope-labeled IS eliminated relative matrix effect. Also, the utilization of the APCI interface instead of ESI led to the elimination of the relative matrix effect in all cases studied. When the precision of standard line slope values exceeds the 3–4% limit, the method may require improvements (a more efficient chromatography, a more selective extraction, a stable isotope-labeled IS instead of an analog as an IS, and/or a change in the HPLC–MS interface) to eliminate the relative matrix effect and to improve assay selectivity.

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).

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.

Clinical studies have been performed to investigate the pharmacokinetics and pharmacodynamics of alendronate, an inhibitor of bone resorption for the treatment of osteoporosis. Alendronate is one of the most potent bisphosphonates currently undergoing clinical investigation (> 100-fold more potent than etidronate in vivo). The pharmacokinetics of alendronate are similar to those of other bisphosphonates. After a 2-h intravenous infusion, plasma concentrations of alendronate decline rapidly to approximately 5% of initial values within 6 h. About 50% of a systemic dose is excreted unchanged in the urine in the 72 h following administration. By analogy to its behavior in animals the remainder is assumed to be taken up by the skeleton. After sequestration into bone, the elimination of alendronate is very prolonged. The terminal half-life was estimated to be greater than 10 years. Despite prolonged skeletal residence, the biological effects of alendronate begin to diminish post-treatment, since the duration of effect reflects factors besides dose and cumulative drug exposure. When taken after an overnight fast, 2 h before breakfast, the oral bioavailability of alendronate averages approximately 0.75% of dose with substantial variability (coefficient of variation 55%-75%) both between and within subjects. Reducing the wait before food from 2 h to 1 h, or even 30 min, produces a mean reduction in absorption of 40%. Since the clinical efficacy of alendronate is indistinguishable whether it is given 30 min, 1h, or 3 h before a meal, the observed variability in bioavailability within this range is of little consequence. Dosing up to at least 2 h after a meal dramatically reduces absorption (80%-90%).

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.

Sensitive and selective methods for the determination of hydrochlorothiazide (HCTZ) in human plasma and urine were developed. Both methods were based on high performance liquid chromatography (HPLC) with tandem mass Spectrometric (MS/MS) detection. HCTZ and hydroflumethiazide (HFTZ), used as an internal standard, were isolated from human plasma and urine by liquid-liquid extraction in the 96-well plate format using a Tomtec Quadra 96 workstation. The MS/MS detection was per-formed on an Applied Biosystems-Sciex API 3000 tandem mass spectrometer interfaced with a heated nebulizer probe and operated in the negative ionization mode. The developed methods were validated in the concentration ranges of 1-100 ng/mL and 0.05-20 mu g/mL in human plasma and urine, respectively. The precision of both methods, as expressed by the coefficients of variation (C.V.), was less than 5.6% at all concentrations within the standard curves with acceptable accuracy. The plasma and urine methods provided sufficient sensitivity and high sample throughput to map out the pharmacokinetics of HCTZ in human subjects following an oral dose of HCTZ/Losartan, in support of a human clinical trial designed to evaluate the bioequivalence of HCTZ between two formulations.

Abstract A semi‐automated assay for the determination of caspofungin in human plasma is presented. High assay throughput was achieved through the use of a robotic sample processor and 96 well format solid phase extraction (SPE). Drug and internal standard (an isostere) were extracted from plasma using a silica based, C8 stationary phase. The extraction yielded a highly purified extract, as retention was mediated by a combination of reverse phase and secondary ionic interactions. Conditioned SPE plates (50 mg sorbent/well) were loaded with buffered (pH 4.9) plasma containing drug and internal standard. The wells were washed with water and neat methanol prior to elution with a reagent optimized for both recovery and selectivity (0.25 M ammonium hydroxide/0.05% trifluoroacetic acid in methanol). Excess residual water in the SPE wells during the methanol wash was found to cause variable drug recovery and was eliminated by centrifugation of the SPE plate. After evaporation of the SPE eluent, plasma extracts were dissolved in mobile phase and analyzed using a Keystone Betasil C18 analytical column (4.6×50 mm, 3 μm) with fluorescence detection (excitation 220 nm, emission 304 nm). The mobile phase was composed of a 38∶62 (v∶v) mixture of acetonitrile and 0.1% trifluoroacetic acid (adjusted to pH 3 with triethylamine) and was pumped at a flow rate of 1.5 mL/minute. Seven‐point calibration curves over the concentration range 125–10,000 ng/mL yielded a linear response (drug concentration vs drug/internal standard peak height ratio) using a weighed (1/x) linear regression model. Based on the replicate analyses (n=5) of spiked plasma standards, intra‐day assay precision was better than 5.7% coefficient of variation (CV) and intra‐day accuracy was within 1.7% of nominal at all points of the standard curve. Inter‐day precision, as assessed by daily analysis of high, mid, and low concentration quality control samples (n=6), was better than 5.3% CV. Inter‐day accuracy was within 10.7% of nominal value.

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.

A sensitive and selective method for the determination of a compound with activity at the benzodiazepine site of GABA-A receptors (I, 3-(5-methylisoxazol-3-yl)-6-(1-methyl-1H-[1,2,3]triazol-4-yl-methoxy)-[1,2,4]-triazolo-[3,4-A] phthalazine) and its active 5-hydroxymethyl-isoxazole metabolite (II) has been developed. The method is based on high performance liquid chromatography (HPLC) with tandem mass spectrometeric (MS/MS) detection. The analytes (I and II) and internal standard (III) were isolated from human plasma by a 96-well solid phase extraction technique using a Tomtec Quadra 96 system. The MS/MS detection was performed on a PE Sciex API 3000 tandem mass spectrometer interfaced with a heated nebulizer probe. This semi-automated HPLC-MS/MS method was validated in the concentration range of 0.1-25 ng/mL, with the lower limit of quantification (LLOQ) of 0.1 ng/mL for both analytes. The precision of the assay, as expressed by the coefficients of variation (CV), was less than 8% at all concentrations within the standard curve for both I and II, with acceptable. accuracy. This method provided sufficient sensitivity and high sample throughput to completely map out the pharmacokinetic time-course in human subjects following oral doses of I.

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 (3.2mm×50 mm, 3 μm) 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.

A peptide-doxorubicin conjugate (I) is a drug candidate that has been evaluated for the treatment of prostate cancer. During the high-performance liquid chromatographic (HPLC)-fluorescence analysis of clinical samples for compound I and its two known metabolites [doxorubicin (II) and leucine-doxorubicin (III)], additional metabolites are observed in postdose human plasma extracts. Using neutral loss, precursor, and product ion tandem mass spectrometric (MS-MS) experiments, two of these metabolites are identified as doxorubicinol (IV) and leucine-doxorubicinol (V), the active 13-hydroxy metabolites of doxorubicin and leucine-doxorubicin, respectively. A third metabolite, 7-deoxydoxorubicinol aglycone (VI), is detected using single-ion monitoring at m/z 399 in the negative ionization mode. The product ion mass spectrum of this metabolite contains a major fragment at m/z 351, resulting from the loss of water and formaldehyde from the pseudomolecular ion. An HPLC-MS-MS method for simultaneous analysis of II, III, IV, V, and VI is developed utilizing gradient HPLC with a combination of positive/negative ionization MS in the multiple reaction monitoring mode and monitoring the appropriate MS-MS transitions. Using this methodology, rat, dog, and human plasma metabolite profiles are compared and found to be qualitatively similar. Simultaneous fluorescence and MS detection experiments confirm that the peaks observed in the HPLC-fluorescence chromatograms of plasma extracts correspond to each of the five metabolites (II-VI).

A semi-automated, 96-well solid phase extraction (SPE) method combined with high performance liquid chromatography (HPLC) was developed, validated, and implemented to demonstrate its high sample throughput utility for the determination of a selective gamma aminobutyric acid (GABA)-A receptor agonist in human and rat plasma. A Tomtec Quadra 96 system was utilized to perform the typical 96-well SPE steps, such as plate conditioning and washing, sample loading, drug and internal standard elution, dilution, and liquid transfers. The extracted and diluted eluents were analyzed by HPLC with fluorescence detection with excitation and emission wavelengths at 256 and 447 nm, respectively, Precision and accuracy for standard curve and quality control samples in human and rat plasma were all within acceptable limits. Post-dose plasma samples from three rats were prepared and analyzed using this semi-automated 96-well SPE method. Full details of the analytical methodology, method validation, and the results of analyses of rat plasma samples are presented.

Methods based on high-performance liquid chromatography (HPLC) with atmospheric-pressure chemical ionization (APCI) mass spectrometric (MS) detection using either single (MS) or triple (MS/MS) quadrupole mass spectrometric detection for the determination of (2R)-[1(R)-(3,5-bis-trifluoromethylphenyl)ethoxy]-3(S)-(4-fluoro-phenyl)morpholin-4-ylmethyl]-5-oxo-4,5-dihydro-[1,2,4]triazol)methyl morpholine (Aprepitant, Fig. 1) in human plasma has been developed. Aprepitant (I) and internal standard (II, Fig. 1) were isolated from the plasma matrix buffered to pH 9.8 using a liquid–liquid extraction with methyl-t-butyl ether (MTBE). The analytes were separated on a Keystone Scientific’s Javelin BDS C-8 2mm×4.6 mm 3 μm guard column coupled to BDS C-8 50mm×4.6 mm 3 μm analytical column, utilizing a mobile phase of 50% acetonitrile and 50% water containing 0.1% formic acid and 10 mM ammonium acetate delivered at a flow rate of 1 ml/min. The single quadrupole instrument was operated in a single ion monitoring (SIM) mode analyzing the protonated molecules of Aprepitant and II at m/z 535 and 503, respectively. The triple quadrupole mass spectrometer was operated in multiple reaction monitoring mode (MRM) monitoring the precursor→product ion combinations of m/z 535→277 and 503→259 for Aprepitant and II, respectively. The linear calibration range for both single and triple quadrupole detectors was from 10 to 5000 ng/ml of plasma with coefficients of variation less than 8% at all concentrations. Both single and triple quadrupole instruments yielded similar precision and accuracy results. Matrix effect experiments performed on both instruments demonstrated the absence of any significant change in ionization of the analytes when comparing neat standards to analytes in the presence of plasma matrix. Both instruments were used successfully to support numerous clinical trials of Aprepitant.

A method for the simultaneous determination of Aprepitant, I (5-[2(R)-[1(R)-(3,5-bistrifluoromethylphenyl)ethoxyl-3(S)(4-fluorophenyl) morpholin-4-yl]methyl]-2,4-dihydro-[1,2,4] triazol-3-one) and two active metabolites (II and III) in human plasma has been developed. The method was based on high-performance liquid chromatography (HPLC) with atmospheric pressure chemical ionization tandem mass spectrometric (APCI-MS-MS) detection in positive ionization mode using a heated nebulizer interface. The analytes and internal standard (IV) (Fig. 1) were isolated from basified plasma using liquid-liquid extraction. The organic extracts were dried, reconstituted in mobile phase and injected into the HPLC-MS/MS system.The analytes were chromatographed on a narrow bore (50 mm x 2.0 mm, 3 mum) Keystone Scientific's Prism R.P. analytical column, with mobile phase consisting of acetonitrile (ACN):water containing trifluoroacetic acid with pH adjusted to 3 (40:60, v/v) pumped at a flow rate of 0.5 ml/min. The MS-MS detection was performed on a Sciex API 3000 tandem mass spectrometer operated in selected reaction monitoring mode. The precursor --> product ion combinations of m/z 535 --> 277, 438 --> 180, 452 --> 223 and 503 --> 259 were used to quantify I, II, III, and IV, respectively, after chromatographic separation of the analytes. The assay was validated in the concentration range of 10-5000 ng/ml for I and II and 25-5000 ng/ml for III when 1 ml of plasma was processed. The precision of the assay (expressed as coefficient of variation, CV) was less than 10% at all concentrations within the standard curve range, with adequate assay accuracy. Matrix effect experiments were performed to demonstrate the absence of any significant change in ionization of the analytes when comparing neat standards to analytes in the presence of plasma matrix. This assay was utilized to support a clinical study where multiple oral doses of I were administered to healthy subjects to investigate the pharmacokinetics, safety, and tolerability of Aprepitant. Concentrations of the two most active metabolites, which if present in high concentrations would increase the neurokinin-1 (NK1) receptor occupancy level and therefore potentially contribute to the antiemetic action of Aprepitant, were determined. (C) 2004 Elsevier B.V. All rights reserved.