Comparison of urinary 6beta-hydroxycortisol/cortisol ratio between neonates and their mothers

AIMS: To assess CYP3A enzyme activity in human neonates by measuring the urinary 6beta-hydroxycortisol/cortisol (6beta-OHF/C) ratio. METHODS: Fifty-six mature male neonates with normal delivery, seventeen of their mothers and twenty-four healthy non-pregnant young women participated in this study. Urinary 6beta-OHF/C ratio was determined on the day of birth in neonates and their mothers. In addition, changes in the ratio after birth were determined in neonates. RESULTS: On the day of birth, the urinary 6beta-OHF/C ratio of neonates was significantly higher than that of their mothers (20.5 vs 6.9). In contrast, no significant difference was observed in the mean ratio of urinary 6beta-OHF/C between women with and without pregnancy (6.9 vs 9.0). The urinary 6beta-OHF/C ratio after birth was decreased day by day in neonates. CONCLUSION: These results indicate that the high urinary 6beta-OHF/C ratio in mature neonates on the day of birth is independent of the activity of CYP3A enzyme in their mothers.     

DEET (N,N-diethyl-m-toluamide) alone and in combination with permethrin increased urinary excretion of 6beta-hydroxycortisol in rats, a marker of hepatic CYP3A induction.

In this study, the ratio of 6beta-hydroxycortisol (6beta-OHF) to free cortisol (F) was determined in urine following a single dermal dose of 400 mg/kg of DEET (N,N-diethyl-m-toluamide), and 1.3 mg/kg of permethrin, alone and in combination, in rats. Urine samples were collected at 2, 4, 8, 16, 24, 48, and 72 h after application. Recoveries of 6beta-OHF and cortisol (F) from control urine samples were between 75 and 85%, with limits of detection at 30 and 10 ng/ml for cortisol and 6beta-OHF, respectively. A single dermal dose of DEET alone and in combination with permethrin significantly increased urinary excretion of 6beta-hydroxycortisol 24 h after dosing. Permethrin did not significantly alter the urinary excretion of 6beta-hydroxycortisol. These results indicate that DEET, alone and in combination with permethrin, increased urinary excretion of 6beta-OHF in rats following a single dermal dose application.     

An high performance liquid chromatographic method for the quantification of cotinine in the urine of preschool children

Tobacco smoke exposure is an important and preventable cause of morbidity among children. Enviromental tobacco smoke (ETS) increases respiratory symptoms and disease and also decreases lung function in children who live in a household with at least one smoker. We have developed a simple and reliable HPLC method with diode array dedection to determine the urine concentrations of cotinine in children aged 3 to 6 years, exposed to ETS. The assay involved a liquid-liquid extraction with chloroform. The HPLC method utilized a Chromasil C18 column (150 mm x 4.6 mm i.d.) and an isocratic mobile phase of phosphate buffer: acetonitrile (83:17 v/v, 0.02 M containing 0.1% triethylamine, adjusted to pH 6.72 with orthophosphoric acid), at a flow rate of 0.7 ml min(-1). The detection was performed at 260 nm and the total analysis time of analysis was less than 15 min. Linearity ranged from 0 to 80 microg L(-1); correlation coefficients (r2) for calibration curves were greater than 0.99. With 2 mL of urine for extraction, the limit of detection was 0.1 microg L(-1). The mean extraction ratio of cotinine was 88.78%. This analytical method is suitable for the determination of cotinine levels in a large number of urine samples.     

Determination of gabapentin in human plasma and urine by high-performance liquid chromatography with UV-vis detection

A simple and reliable high-performance liquid chromatographic (HPLC) method with UV–vis detection has been developed and validated for the determination of gabapentin (GBP) in human plasma and urine. The clean up of the sample was carried out by solid-phase extraction with C18-cartridge. After the clean up procedure, the samples were pre-column derivatizated with 1,2-naphthoquinone-4-sulphonic acid sodium salt (NQS). A chromatographic separation was achieved on a C18 column with a mobile phase consisting of acetonitrile and 10 mM orthophosphoric acid (pH 2.5) with isocratic elution (35:65). Baclofen was used as an internal standard (I.S.). The method developed for GBP was linear over the concentration range of 0.05–5.0 μg/ml and 0.1–10.0 μg/ml for plasma and urine, respectively. The method is precise (relative standard deviation, R.S.D. <4.05%) and accurate (relative mean error, RME <0.15%); mean absolute recoveries were 72.21% for plasma and 72.73% for urine.     

Evidence for the validity of cortisol 6 beta-hydroxylation clearance as a new index for in vivo cytochrome P450 3A phenotyping in humans.

This study uses stable isotope methodology to evaluate the validity of 6beta-hydroxylation clearance of endogenous cortisol as a new index for in vivo CYP3A phenotyping in humans. Important factors contradictory to the use of a conventional index of urinary ratio of 6beta-hydroxycortisol to cortisol (6beta-OHF/F) to evaluate in vivo CYP3A activity are also discussed. Stable isotopically labeled cortisol (3-5 mg) was orally administered to three healthy adult subjects to accurately determine the fractional metabolic clearance specific for the 6beta-hydroxylation of cortisol. Plasma concentrations of labeled cortisol and urinary excreted amounts of labeled cortisol and 6beta-OHF were analyzed by gas chromatography-mass spectrometry simultaneously with their endogenous counterparts. There was a good correlation between endogenous and exogenous 6beta-hydroxylation clearances in the three subjects tested (r = 0.7733, 0.9112, and 0.9534 for 2-, 4-, and 6- to 8-h urine collection periods, respectively). This strongly suggests that the endogenous 6beta-hydroxylation clearance can be used as an appropriate index for phenotyping the in vivo CYP3A activity. Furthermore, observed intra- (2.1- to 4.6-fold) and interindividual variabilities (ca. 5-fold) in the labeled cortisol renal clearance suggest that the urinary ratio 6beta-OHF/F, a function of 6beta-hydroxylation clearance and renal clearance of cortisol, does not always reflect the in vivo CYP3A activity. When a macrolide antibiotic, clarithromycin, was administered to a healthy volunteer in a dose of 200 mg every 12 h for 6 days, the inhibitory effects of clarithromycin on the in vivo CYP3A activity were clearly seen by the 6beta-hydroxylation clearance of endogenous cortisol but not by the urinary ratio 6beta-OHF/F.     

HPLC method for the determination of fluvoxamine in human plasma and urine for application to pharmacokinetic studies

A simple, specific and sensitive high-performance liquid chromatographic (HPLC) method has been developed for the assay of fluvoxamine in human plasma and urine. The method was based on reaction of fluvoxamine with 1,2-naphthoquinone-4-sulphonic acid sodium salt (NQS) forming orange colored product. The fluvoxamine-NQ derivative was separated by isocratic reversed-phase HPLC and detected at 450 nm. The chromatographic conditions were as follows: Phenomenex C(18) (250 mm x 4.6 mm i.d., 5 microm) column, mobile phase consisting of acetonitrile/water (80:20 v/v) at a flow rate of 1 ml/min. Tryptamine was selected as an internal standard. The assay was linear over the concentration range of 5-145 and 2-100 ng/ml for plasma and urine, respectively. The limits of detection (LOD) were 1.4 and 1 ng/ml for plasma and urine estimation at a signal-to-noise (S/N) ratio of 3. The limits of quantification (LOQ) were 5 and 2 ng/ml for plasma and urine, respectively. The extraction recoveries were found to be 96.66+/-0.69 and 96.73+/-2.17% for plasma and urine, respectively. The intra-day and inter-day standard deviations (S.D.) were less than 1. The method indicated good performance in terms of specificity, linearity, detection and quantification limits, precision and accuracy. This assay was demonstrated to be applicable for clinical pharmacokinetic studies.     

Simultaneous analysis of naproxen, nabumetone and its major metabolite 6-methoxy-2-naphthylacetic acid in pharmaceuticals and human urine by high-performance liquid chromatography

A high-performance liquid chromatographic (HPLC) method for simultaneous determination of naproxen (NAP), nabumetone (NAB) and its major metabolite, 6-methoxy-2-naphthylacetic acid (6-MNA), was developed for the application to pharmaceuticals and human urine. Isocratic reversed-phase HPLC was employed for quantitative analysis using triethylamine and 1-heptanesulfonic acid sodium salt (HSA) as ion-pair reagents. Urine samples were purified by solid-phase extraction using Bond-Elut Certify II cartridges containing reversed-phase and anion exchange functionalities. The HPLC assay was carried out using a Wakosil ODS 5C18 column (5 microm, 150 x 4.6 mm, i.d.). The mobile phase consisted of 0.5 g of HSA dissolved in 1,000 ml of a mixture of acetonitrile, water and triethylamine (500:500:1, v/v) adjusted with phosphoric acid to pH 3. The calibration curves of NAP and NAB showed good linearity in the concentration range 32-160 microg/ml with UV detection (270 nm) for pharmaceuticals. In the low concentration ranges (8-96 ng of NAP per ml, 24-288 ng of NAB per ml and 5.6-67.2 ng of 6-MNA per ml), the calibration curves were also obtained with fluorimetric detection (excitation 280 nm, emission 350 nm) for biological fluids. The correlation coefficients were better than 0.999 in all cases. The lower limits of detection (defined as a signal-to-noise ratio of about 3) were approximately 0.3 ng for NAP, 1.5 ng for NAB and 0.2 ng for 6-MNA. The procedure described here is rapid, simple, selective, and is suitable for routine analysis of pharmaceuticals and pharmacokinetic studies in human urine samples.     

Pharmacokinetic study of a new angiotensin-AT1 antagonist by HPLC

Recently an innovative novel class angiotensin-AT(1) antagonist has been developed by Rottapharm. In this study, we present a validated method for detecting CR 3834 in biological matrices using high-performance liquid chromatography (HPLC) with diode array detection. After oral administration (30mg/kg) to Wistar rats, the plasma and urine concentrations of CR 3834 and its potential metabolic products were determined. Moreover, the plasmatic time course in rats has been determined after intravenous (IV) administration of CR 3834 (5mg/kg). Biological samples (0.5ml of plasma and 1ml of urine) were purified using solid-phase extraction (SPE) of analytes and the internal standard Idebenone, 2,3-dimethoxy-5-methyl-6-(10-hydroxydecyl)-1-4-benzoquinone. A chromatographic separation was performed on an Adsorboshere C18 at 25 degrees C, with a pre-column of the same matrix; the eluent was made up of acetonitrile/acidified water with CF(3)COOH (pH 2.01) in ratio of 75:25 (v/v); the flow rate was 1.0ml/min and a 100mul loop. The lower limit of detection (LOD) was taken as 25ng/ml in plasma and 50ng/ml in urine samples. The lower limit of quantification (LOQ) was taken as 0.1 and 0.2mug/ml in plasma and urine samples, respectively. The procedures were validated according to international standards with a good reproducibility and linear response (r=0.9916 in plasma; r=0.9997 in urine). The coefficients of variation inter assay ranged between 2.579 and 4.951% in plasma, and between 0.813 and 2.460% in urine. Mean recovery for CR 3834 was 79% in plasma and 97% in urine samples. The experiments performed demonstrated that the method presented was suitable for determining this new angiotensin-AT(1) antagonist in rat plasma and urine.     

Assessment of induced CYP3A activity in pregnant women using 4β-hydroxycholesterol: Cholesterol ratio as an appropriate metabolic marker

AimsThis study was aimed at evaluating changes in CYP3A activity following and during pregnancy by analyzing metabolic markers for CYP3A activity, which can help avoid unnecessary drug exposure and invasive sampling.MethodsForty-eight pregnant women and 25 non-pregnant women were enrolled in this study. Plasma and urine samples were collected from the pregnant women during each trimester and from the non-pregnant women for evaluation of metabolic markers for CYP3A activity. Metabolic markers for CYP3A activity were measured using GC-MS.ResultsAn increased 4β-hydroxycholesterol/cholesterol ratio, consistent with high CYP3A activity, was observed in pregnant women compared with that in non-pregnant women; however, no differences were observed among trimesters. No significant differences were observed in urinary markers.ConclusionsWe observed an increase in the activity of CYP3A following but not during pregnancy when measured using the 4β-hydroxycholesterol/cholesterol ratio. In addition, based on our results, we suggest that the plasma 4β-hydroxycholesterol/cholesterol ratio be used to measure CYP3A activity in pregnant women.     

Determination and assay validation of luteolin and apigenin in human urine after oral administration of tablet of Chrysanthemum morifolium extract by HPLC

A simple, selective, precise, and accurate RP-HPLC assay for simultaneous analysis of luteolin and apigenin in human urine was developed and validated. Prior to HPLC analysis, urine samples were incubated with beta-glucuronidase/sulfatase. Separation and quantification were achieved on an Agilent C18 column under isocratic conditions using a mobile phase (methanol:0.2% phosphoric acid aqueous solution 55:45, v/v) maintained at 1.0 ml/min at 30 degrees C. The standard curves were linear over the range of 0.0975-7.800 and 0.1744-13.95 microg/ml for luteolin and apigenin, respectively (r > 0.999). The assay recoveries for luteolin and apigenin were above 85.7%. The intra-day and inter-day precision (R.S.D.) for luteolin were below 2.2 and 4.0%, respectively, and for apigenin were less than 2.8 and 5.4%, respectively. Stability studies showed three concentration of luteolin and apigenin in urine quality control samples were stable undergoing three freeze-thaw cycles, storage at room temperature for 4 h, and at -20 degrees C for 3 days. The limit of quantitation was 39.20 ng/ml (n = 5) for luteolin and 31.45 ng/ml (n = 5) for apigenin in human urine. The method developed was employed successfully to determine luteolin and apigenin in urine samples obtained from eight healthy volunteers following oral administration of tablet of Chrysanthemum morifolium extract (CME).     

HPLC法测定人肝微粒体6β-羟基睾酮和1-羟基咪达唑仑浓度

目的建立和验证人肝微粒体中CYP3A4探药代谢物6β-羟基睾酮和1-羟基咪达唑仑的测定方法,以准确体外评价CYP3A4的活性。方法在人肝微粒体温孵系统中加入睾酮37℃温孵生成6β-羟基睾酮,经过碱化后用乙酸乙酯提取微粒体样本,采用Phenomenex Gemina C18分离,流动相由甲醇和水组成,梯度洗脱,流速1．0mL·min-1,检测波长245nm,测定微粒体中6β-羟基睾酮浓度;或人肝微粒体温孵系统中加入咪达唑仑,37℃温孵生成1-羟基咪达唑仑,经过乙腈处理人肝微粒体样本,采用DiamosilC18分离,20mmoL·L-1醋酸铵-乙腈（60：40,V／V）流动相等度洗脱,流速1．0mL·min-1,检测波长254nm,测定1-羟基咪达唑仑浓度。结果6β-羟基睾酮线性范围为0．111～8．90mg·L-1,1-羟基咪达唑仑线性范围为20-1000mg·L-1,两代谢物的批内、批间差异〈15％,准确度为85％～115％。结论建立的测定微粒体中6β-羟基睾酮和1-羟基咪达唑仑浓度的方法符合生物样品测定的要求,能准确测定微粒体中目标物浓度,可用于评价CYP3A4的活性。     

A Sensitive and Specific Procedure for Quantitation of ADR-529 in Biological Fluids by High-Performance Liquid Chromatography (HPLC) with Column Switching and Amperometric Detection

An HPLC method using electrochemical detection (ED) has been validated for the determination of ADR-529 in plasma and urine using ICRF-192 as an internal standard (IS). Prior to storage and quantitation, both plasma and urine samples require acid stabilization. Acidified plasma samples were prepared for HPLC using a two column solid-phase extraction (SPE). An aliquot of buffered plasma (i.e., pH 6-7) was first deproteinated and desalted on a C-18 SPE column. The analytes were then eluted onto a C-8 SPE column where retention and selective cleanup were achieved in the cation-exchange mode via silanol interactions. Acidified urine samples were diluted in acetonitrile prior to injection. The HPLC system for plasma and urine samples employed two narrow-bore silica columns used in the weak cation-exchange mode and separated by a switching valve. To prohibit late-eluting peaks from passivating the glassy carbon working electrode, a heart-cut containing ADR-529 and the IS was vented from the first silica column to the second using an automated switching valve. Amperometric detection at an oxidation potential of +1050 mV vs a Ag/AgNO₃ reference electrode was used. Linearity was validated between 5 and 500 µg/ml in plasma and between 2 and 100 µg/ml in urine. Imprecision and percentage bias were typically <10% for both plasma and urine controls throughout their respective dynamic ranges. The absolute recoveries for ADR-529 and the IS from plasma were >95%. This method is being successfully applied to the pharmacokinetic/dynamic evaluation of ADR-529 in animals and humans.     

Cytochrome P450 2C9 mediated metabolism in people with and without cancer

OBJECTIVES: To compare cytochrome P450 activity in people with and without cancer and examine the relationship between CYP2C9 activity and serum cytokine levels. PATIENTS AND METHODS: 10 subjects with cancer who were currently receiving treatment and 10 additional subjects without cancer who were matched to the subjects with cancer based on gender and race were enrolled into the study. Serial blood samples were drawn to measure tolbutamide in the plasma before and after oral tolbutamide 500 mg. Total urine excreted was collected from 0 to 12 h following the dose. Tolbutamide and its metabolites were measured in plasma and urine by HPLC. CYP2C9 genotype was determined by PCR and pyrosequencing and cytokine values were determined by ELISA. RESULTS: The mean apparent oral clearance (cancer, 19.5 +/- 10.5 vs. non-cancer, 15.8 +/- 5.0 ml/min) and the mean urinary metabolic ratio from 0 to 12 h were similar (838 +/- 693 vs. 775 +/- 390). Neither age nor genotype statistically affected the outcomes. Mean interleukin-6 (7.2 +/- 9.4 vs. 1.5 +/- 1.3 pg/ml) and tissue necrosis factor-a (26.2 +/- 71.2 vs. 1.5 +/- 1.3 pg/ml) were 5- to 7-fold higher, respectively, in subjects with cancer. No statistically significant correlation between cytokine values and oral clearance or urinary metabolic ratio was found. CONCLUSIONS: CYP2C9 activity as measured by apparent oral clearance and urinary metabolic ratio following oral tolbutamide appear similar in people with and without cancer. Serum cytokine values appear higher in patients with cancer, although the differences did not reach statistical significance.     

A Stability-indicating High Performance Liquid Chromatographic Assay for the Simultaneous Determination of Atenolol and Lercanidipine Hydrochloride in Tablets

A simple, rapid, precise and accurate isocratic reversed phase stability indicating HPLC method was developed and validated for the simultaneous determination of atenolol and lercanidipine hydrochloride in commercial tablets. The chromatographic separation was achieved on phenomenex Gemini C18 (250×4.6 mm, 5 μm) column using a mobile phase consisting of acetonitrile and buffer (20 mM potassium dihydrogen phosphate pH 3.5) in the ratio of (55:45, v/v) at a flow rate of 1.0 ml/min and UV detection at 235 nm. The linearity of the proposed method was investigated in the range of 40-160 μg/ml (r(2)=0.9995) for atenolol and 8-32 μg/ml (r(2)=0.9993) for lercanidipine. Degradation products produced as a result of stress studies did not interfere with the detection of atenolol and lercanidipine and the assay can thus be considered stability-indicating.     

Determination of furosemide in plasma and urine using monolithic silica rod liquid chromatography

In the present study we developed a fast and reliable HPLC assay for the determination of the loop diuretic furosemide in plasma and urine, using a Chromolith RP 18e (100 mm x 4.6 mm) monolithic silica rod HPLC column. After liquid-liquid extraction with diethylether plasma or urine samples were separated with a gradient consisting of solvent A (20% acetonitrile) and solvent B (80% acetonitrile), both in 0.25% acetic acid. The flow rate was 3.5 ml/min and the effluent was monitored by fluorescence with excitation at 230 nm and emission at 410 nm. The retention times for the internal standard (naproxen) and for furosemide were 2.1 and 3.7 min, respectively, and total run time was 8 min. The calibration curves were linear between 7.8 and 1000 ng/ml, and within-assay and between-assay coefficients of variation were <6.5% and <10%, respectively. The proposed assay for furosemide in plasma and urine using monolithic silica rod chromatography is fast, sensitive, and reliable, and, thus, well suited for pharmacokinetic studies.     

A Chromatographic Determination of Aripiprazole using HPLC and UPLC: A Comparative Validation Study

A simple, precise, and accurate isocratic reversed-phase (RP) stability-indicating HPLC assay method was developed and validated for determination of Aripiprazole in bulk and solid pharmaceutical dosage form. A reversed-phase C8 (250×4.0 mm, 5 μm particle size) column for HPLC and C8 (50×2.1mm, 1.7 μm particle size) for UPLC method in isocratic mode was used. The mobile phase consists of acetonitrile: 20 mM ammonium acetate (90:10, v/v), flow rate was set at 1.0 ml/min and 0.250 ml/min for HPLC and UPLC, respectively and the detection was performed for both methods were at 240 nm. Further the validation of both developed method was performed and subsequently compared to prove its better applicability.     

Development of validated UV spectrophotometric method for <Emphasis Type="Italic">in vitro</Emphasis> analysis of sumatriptan in pharmaceutical preparations in comparison with HPLC

An accurate, simple reproducible and sensitive method for the determination of sumatriptan succinate was developed and validated. According to this, sumatriptan succinate was determined using a UV spectrophotometric (UVS) technique and the results were compared with the data of isocratic HPLC procedure on a Bondapack reversed-phase ODS or C18 Waters column (chemically bonded to Spherisorb⁽particles (5 μm), 4.6 mm × 25 cm,) under conditions of isocratic elution at a flow rate of 1.0 ml/min. The mobile phase composition was acetonitrile – buffer (420 : 580, v/v), pH 7.5. The buffer composition was dibutylamine, 85% phosphoric acid, and doubly distilled water (0.18 : 0.06 : 99.76, v/v), which was adjusted to pH 7.5 with 0.8 N sodium hydroxide solution. Both the proposed UVS procedure and HPLC with UV detection were carried out at 282 nm. The linear range of detection for sumatriptan succinate by means of both UVS and HPLC methods was between 21 and 39 μg/ml of sumatriptan. The UVS method is shown to be linear, reproducible, specific, sensitive and robust.     

Differences in the urinary excretion of 6-beta-hydroxycortisol/cortisol between Asian and Caucasian women.

The urinary ratio of 6-beta-hydroxycortisol/cortisol has been used as a noninvasive probe for human cytochrome P450 3A4 isoforms (CYP3A4). Ethnic-related differences in the ratio have not been evaluated. The aim of this study was to determine if there are differences in the ratio between Asian and Caucasian women over a menstrual cycle. First-morning urine samples were collected every other day starting from the second day of menstruation for a complete menstrual cycle from 15 Asians and 16 Caucasian women who were 18 to 40 years old, healthy, nonsmoking, and alcohol and drug free, including oral contraceptives. Urine concentrations of 6-beta-hydroxycortisol and cortisol were measured by high-pressure liquid chromatography (HPLC). For statistical analysis, three phases of the menstrual cycle were evaluated: menstruation (days 1-4), follicular or postmenstruation (days 6-10), and the luteal phase (days 21-24) based on the average menstrual cycle (28 days). Statistical analysis was performed by an independent sample t-test using the Bonferroni correction for repeated measures. Large intersubject and intrasubject variations of the 6-beta-hydroxycortisol/cortisol ratios were observed during the menstrual cycles in both ethnic groups. Asian women had a statistically significant lower ratio than Caucasian women did for all three phases of the menstrual cycle: 2.2 +/- 1.1 versus 5.1 +/- 3.5, 2.1 +/- 1.1 versus 6.0 +/- 4.9, and 2.8 +/- 1.6 versus 5.6 +/- 3.0 for the menstruation, follicular, and luteal phases, respectively. The two- to threefold lower 6-beta-hydroxycortisol/cortisol ratios in Asian women suggest that Asian women may have a lower CYP3A activity compared with Caucasian women. Differences in ethnicity may mask potential gender-related effects if ethnic background is not evaluated as a contributing factor.     

Evaluation of accelerator mass spectrometry in a human mass balance and pharmacokinetic study-experience with 14C-labeled (R)-6-[amino(4- chlorophenyl)(1-methyl-1H-imidazol-5-yl)methyl]-4-(3-chlorophenyl)-1- methyl-2(1H)-quinolinone (R115777), a farnesyl transferase inhibitor.

Accelerator mass spectrometry (AMS) has been used in a human mass balance and metabolism study to analyze samples taken from four healthy male adult subjects administered nanoCurie doses of the farnesyl transferase inhibitor 14C-labeled (R)-6-[amino(4-chlorophenyl)(1-methyl-1H-imidazol-5-yl)methyl]-4-(3-chlorophenyl)-1-methyl-2(1H)-quinolinone ([14C]R115777). Plasma, urine, and feces samples were collected at fixed timepoints after oral administration of 50 mg [14C]R115777 (25.4 Bq/mg or 687 pCi/mg i.e., equivalent to 76.257 x 10(3) dpm) per subject. AMS analysis showed that drug-related (14)C was present in the plasma samples with C(max) values ranging from 1.6055 to 2.9074 dpm/ml (1.0525-1.9047 microg/ml) at t(max) = 2 to 3 h. The C(max) values for acetonitrile extracts of plasma samples ranged from 0.3724 to 0.7490 dpm/ml in the four male subjects. Drug-related 14C was eliminated from the body both in the urine and the feces, with a mean total recovery of 79.8 +/- 12.9% in the feces and 13.7 +/- 6.2% in the urine. The majority of drug-related radioactivity in urine and feces was excreted within the first 48 h. High-performance liquid chromatography (HPLC)-AMS profiles were generated from radioactive parent drug plus metabolites from pooled diluted urine, plasma, and methanolic feces extracts and matched to retention times of synthetic reference substances, postulated as metabolites. All HPLC separations used no more than 5 dpm injected on-column. The radioactive metabolite profiles obtained compared well with those obtained using liquid chromatography/tandem mass spectometry. This study demonstrates the use of AMS in a human phase I study in which the administered radioactive dose was at least 1000-fold lower than that used for conventional radioactive studies.