Identification of the human cytochrome P450s responsible for the in vitro metabolism of a leukotriene B4 receptor antagonist, CP-195,543.

1. The major human cytochrome P450 (CYP) form(s) responsible for the metabolism of CP-195,543, a potent leukotriene B4 antagonist, were investigated. 2. Incubation of CP-195,543 with human liver microsomes resulted in the formation of three major metabolites, M1-3. M1 and M2 were diastereoisomers and formed by oxidation on the benzylic position. M3 was formed by aromatic oxidation of the benzyl group attached to the 3-position of the benzopyran ring. 3. The results from experiments with recombinant CYPs, correlation studies and inhibition studies with form-selective inhibitors and a CYP3A antibody strongly suggest that the CYP3A4 plays a major role in the metabolism of CP-195,543. Recombinant CYP3A5 did not metabolize CP-195,543. 4. The apparent K(m) and V(max) for the formation of M1-3 in human liver microsomes were determined as 36 microM and 4.1 pmol min(-1) pmol(-1) P450, 44 microM and 10 pmol min(-1) pmol(-1) P450, and 34 microM and 2.0 pmol min(-1) pmol(-1) P450, respectively. The average in vitro intrinsic clearance for M2 was the highest both in human liver microsomes and recombinant CYP3A4 compared with M1 and M3. Intrinsic clearance for M2 in human liver microsomes and recombinant CYP3A4 was 0.231 and 0.736 ml min(-1) pmol(-1) P450, respectively. The intrinsic clearances for M1 and M3 in human liver microsomes and CYP3A4 were 0.114 and 0.060 and 0.197 and 0.088 ml min(-1) pmol(-1) P450, respectively. This suggests that benzylic oxidation is the predominant phase I metabolic pathway of CP-195,543 in man.

A high-capacity LC/MS system for the bioanalysis of samples generated from plate-based metabolic screening.

HPLC/MS is a linear technique characterized by serial injection and analysis of individual samples. Parallel-format high-throughput screens for druglike properties present a significant analytical challenge. Analysis speed and system ruggedness are key requirements for bioanalysis of thousands of samples per day. The tasks involved in LC/MS analysis are readily divided into three areas, sample preparation/liquid handling, LC/MS method building/sample analysis, and data processing. Several automation and multitasking strategies were developed and implemented to minimize plating and liquid handling errors, reduce dead times within the analysis cycle, and allow for comprehensive review of data. Delivering multiple samples to multiple injectors allows the autosampler time to complete its wash cycles and aspirate the next set of samples while the previous set is being analyzed. A dual-column chromatography system provides column cycling and peak stacking and allows rapid throughput using conventional LC equipment. Collecting all data for a compound into a single file greatly reduces the number of data files collected, increases the speed of data collection, allows rugged and complete review of all data, and provides facile data management. The described systems have analyzed over 40 000 samples per month for two years and have the capacity for over 2000 samples per instrument per day.

High-throughput method development approaches for bioanalytical mass spectrometry.

A rational approach to the development and optimization of solid-phase extraction (SPE) methods is described. The semiautomated scheme allows for the simultaneous testing of multiple chemistries using a custom multiple-sorbent 96-well method development plate. Optimized extraction conditions for up to five analytes are determined in a single 2.5-h experiment. The experiment can be tailored to determine SPE conditions (including wash protocols) for related analytes. Data obtained by liquid chromatography-atmospheric pressure ionization-mass spectrometry allows the quantitation of absolute recovery and selection of the best extraction conditions for approximately 100 analytes of diverse structure. Optimized extraction protocols yielding at least 80% recovery are determined for 81% of the analytes. For 96% of the analytes screened, extraction conditions resulting in recoveries of > or = 60% are determined. The most generic set of SPE conditions consist of either C8 or C18 sorbent with an eluent composition of acetonitrile with 5mM nitric acid added.

Direct plasma injection for high-performance liquid chromatographic-mass spectrometric quantitation of the anxiolytic agent CP-93 393.

A direct plasma injection method has been developed for the rapid analysis of drugs in biological fluids. A new generation restricted access media column specifically designed to accommodate direct injection of plasma and other fluids is utilized for on-line HPLC-ESI-MS analysis. For rapid analysis the on-line extraction column is linked to a HPLC-ESI-MS system. Good results are obtained for the quantitation of CP-93 393 and deuterated internal standard over the range of 10-1000 ng/ml. The lower limit of detection for the assay was 58 pg injected on column. Accuracy and precision values are 9.0% or better over the entire range of the assay. In addition, more than 200 injections (100 microl) were performed per column with unattended, automated analysis.

Tissue distribution and biotransformation of zopolrestat, an aldose reductase inhibitor, in rats.

Zopolrestat (Alond) is a new drug that is being evaluated as an aldose reductase inhibitor for the treatment of diabetic complications. 14C-labeled zopolrestat was orally administered to rats for a tissue distribution study and a bile duct cannulation metabolism study. Tissue samples from the distribution study were analyzed by complete oxidation and liquid scintillation counting. Urine and bile samples from the bile duct cannulation study were analyzed by microbore HPLC, with simultaneous radioactivity monitoring and atmospheric pressure ionization tandem mass spectrometry. The mass balance in the distribution study demonstrated that the greatest exposure (AUC0-infinity) occurred in the liver, followed by the ileum and large intestine. The time of maximal plasma concentrations for nearly all tissues was 4 hr after the dose, and the half-life of radioactivity in most tissues (8-10 hr) was similar to the half-life in plasma. For the bile duct-cannulated rat study, most of the radioactivity was recovered in the bile, indicating that biliary excretion is a major route of elimination of zopolrestat and its metabolites in rats. Numerous oxidative metabolites, as well as phase II conjugates, were identified in the bile and urine samples. Acyl glucuronides of zopolrestat and unchanged drug accounted for >85% of biliary radioactivity, whereas unchanged drug and degradation products of glutathione conjugates were identified as the major urinary metabolites.

Disposition and metabolism of tenidap in the rat.

Tenidap is a new antirheumatic drug currently undergoing clinical evaluation. It inhibits production and activity of cytokines in vivo and causes significant reductions in plasma markers of disease activity in rheumatoid arthritis. After the oral administration of C-14 labeled tenidap, bile, urine and plasma were examined by HPLC and atmospheric pressure tandem mass spectrometry. Label is excreted primarily in bile/feces and the remainder in urine, with good recoveries. Numerous metabolites were identified and the structures of most were confirmed by comparison with authentic synthetic samples. Hydroxylation in several positions on both the oxindole and thienyl rings of tenidap represents the major routes of metabolism; most of these metabolites are subsequently conjugated. The glucuronide of 5'-hydroxytenidap, excreted primarily in bile, is the major metabolite, constituting about one third of the oral dose recovered. Other pathways include dihydroxylation and methoxylation on the thienyl ring. An unusual reduction of hydroxytenidap took place, resulting in the formation of a novel thiolactone analog. Anaerobic incubation with rat cecal contents generated the thiolactone metabolite, suggesting the involvement of gut microflora.

Development of a high-performance liquid chromatographic-atmospheric pressure chemical ionization-tandem mass spectrometric assay for beta-tigogenin cellobioside in human serum.

A specific high-performance liquid chromatographic-atmospheric pressure chemical ionization tandem mass spectrometric assay for the quantitative determination of beta-tigogenin cellobioside in human serum is described. Serum cleanup and acetylation of the analyte were required to achieve the desired lower limit of quantification, 10 ng/ml. The precision of the assay was better than 13% over a serum concentration range of 10-500 ng/ml.

Development and validation of a high-sensitivity assay for an antipsychotic agent, CP-88,059, with solid-phase extraction and narrow-bore high-performance liquid chromatography.

An analytical method has been developed and validated for the quantitation of CP-88,059 in human serum. The compound and internal standard were extracted from serum by solid-phase extraction with a weak cation-exchange phase. The analytes were resolved from endogenous interferences using narrow-bore (2.1 mm I.D.) C18 reversed-phase HPLC. Column effluent was monitored by UV absorbance detection at 215 nm. The standard curve range was 1 to 250 ng/ml. The accuracy and precision values for the method were within +/- 10% and +/- 15%, respectively. A four-fold detectability enhancement was achieved using a 2.1 mm I.D. HPLC column relative to the more common 4.6 mm I.D. column. A performance comparison was made between the 2.1 mm I.D. column used for validation and a 4.6 mm I.D. column with the same stationary phase.

Quantitative determination of the antibiotic azithromycin in human serum by high-performance liquid chromatography (HPLC)-atmospheric pressure chemical ionization mass spectrometry: correlation with a standard HPLC-electrochemical method.

A specific assay for the quantitative determination of the new antibiotic azithromycin in a low volume of human serum is described. The assay uses on-line high-performance liquid chromatography (HPLC) and atmospheric pressure chemical ionization mass spectrometry (HPLC-APCI). Deuterium-labeled azithromycin was synthesized and used as the internal standard of the assay. The drug and the internal standard are extracted from 50 microliters of serum, and aliquots are injected onto a standard reverse-phase HPLC column. The effluent from the HPLC column at 1 ml/min is introduced into the atmospheric pressure source of a SCIEX API III mass spectrometer. Azithromycin concentrations in serum are determined by the selected monitoring of the protonated molecular ions of the drug and the internal standard. Our assay yields accurate and precise results over the range 10 ng/ml to 250 ng/ml. The correlation between the assay and a standard HPLC-electrochemical method, requiring a larger volume of serum, has been determined. The two methods showed excellent agreement. Because of its low volume requirement, our HPLC-APCI assay can be substituted for the standard assay for the investigation of azithromycin pharmacokinetics in children.

Single and multiple dose pharmacokinetics of tenidap sodium in healthy subjects.

1. The absorption, protein binding, clearance and absolute bioavailability of tenidap sodium were studied after single and multiple dosing. 2. Thirteen healthy male volunteers received a single 120 mg oral dose of tenidap sodium and a 20 mg intravenous infusion of deuterated tenidap ([D3]-tenidap) on day 1. This was followed by a 6-day washout period (days 2-7) and then further daily doses of oral tenidap sodium 120 mg for 21 consecutive days (days 8-28) with an additional 20 mg intravenous infusion of [D3]-tenidap on day 28. Twelve subjects were eligible for pharmacokinetic evaluation. 3. Following multiple oral doses, the half-life of tenidap is approximately 23 h. 4. Following single and multiple dose administration, the absolute bioavailability is 85%. 5. Systemic clearance of [D3]-tenidap was 29% greater on day 28 than on day 1 indicating a significant increase in intrinsic clearance (CLint) of tenidap since protein binding of tenidap in plasma did not change during the study. Consistent with the increase in systemic clearance, the half-life of [D3]-tenidap decreased and the ratio of AUC(0,24h) day 28/AUC day 1 following oral dosing was less than one. Tenidap is subject to extensive hepatic metabolism, so the increase in CLint may indicate that tenidap induces its own metabolism. 6. Steady-state was achieved by the eleventh day of dosing. Since numerous studies in patients with rheumatoid arthritis have shown that multiple dosing with tenidap is clinically efficacious, this suggests that the pharmacokinetic differences observed between the first and twenty-first day of multiple tenidap dosing do not influence the clinical response.

Metabolic studies on haloperidol and its tetrahydropyridine analog in C57BL/6 mice.

The neuroleptic agent haloperidol (HP) is biotransformed in humans to a pyridinium metabolite, HPP+, that displays neurotoxic properties resembling those of the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-derived neurotoxic pyridinium metabolite MPP+. We report here that HP and its tetrahydropyridine dehydration product 4-(4-chlorophenyl)-1-[4-(4-fluorophenyl)-4-oxobutyl]-1,2,3,6- tetrahydropyridine (HPTP) are metabolized in vivo by the MPTP-susceptible C57BL/6 mouse to several pyridinium metabolites including HPP+ and the 4-(4-chlorophenyl)-1-[4-(4-fluorophenyl)-4-hydroxybutyl]pyridinium species RHPP+, the pyridinium species corresponding to reduced haloperidol (RHP), a major circulating metabolite of HP. Atmospheric pressure ion-spray (API) mass spectral data also suggest the formation of fluorophenyl ring-hydroxylated derivatives of these two pyridinium metabolites. Furthermore, HPLC tracings reveal the presence of HPP+, RHPP+, and two phenolic pyridinium metabolites in brain tissue extracts of HPTP, but not HP, treated mice. The neurotoxic potential of MPTP-type pyridinium species suggests that these metabolites may contribute to some of the neurological disorders observed in humans undergoing chronic HP treatment.

Confirmation of danofloxacin residues in chicken and cattle liver by microbore high-performance liquid chromatography electrospray ionization tandem mass spectrometry.

A specific assay is described for the confirmatory identification of danofloxacin residues in edible tissues of cattle and chicken. The assay utilizes on-line microbore high-performance liquid chromatography and pneumatically assisted electrospray tandem mass spectrometry (MS/MS). Collision-induced dissociation of the danofloxacin protonated molecule results in two significant daughter ions. Monitoring both ions provides the specificity required for this confirmatory assay. Optimum electrospray and MS/MS operating conditions permitted the specific monitoring of danofloxacin and the confirmation of its residues in chicken and cattle liver extracts down to 50 ppb. The analysis of control liver or the commercially available antibacterial quinolones enrofloxacin and its metabolite ciprofloxacin gave no response under the assay conditions. The ratios of the two daughter ions were similar for danofloxacin standard solutions, fortified tissues and incurred tissues.

Pharmacokinetics of azithromycin in pediatric patients after oral administration of multiple doses of suspension.

Azithromycin is an azalide antibiotic. On the basis of data in adults, azithromycin appears to have a greater distribution into tissues, a longer elimination half-life, and a lower incidence of adverse effects than the macrolide antibiotic erythromycin. However, little about the pharmacokinetics of azithromycin in children is known. The objective of our study was to characterize the pharmacokinetics of azithromycin after oral administration of multiple doses of suspension to children with streptococcal pharyngitis. Fourteen children (6 to 15 years of age) received a single oral dose of 10 mg of azithromycin per kg of body weight on day 1 followed by single daily doses of 5 mg/kg on days 2 to 5. Each child fasted overnight before receiving the final dose on day 5. Blood samples were collected at 0, 0.5, 1, 2, 4, 6, 8, 12, 24, 48, and 72 h after this last dose. Concentrations of azithromycin in serum were measured by a specific high-performance liquid chromatography-mass spectrometry method. The mean +/- standard deviation for maximum concentration of drug in serum, time to maximum concentration of drug in serum, and area under the curve (0 to 24 h) were 383 +/- 142 ng/ml, 2.4 +/- 1.1 h, and 3,109 +/- 1,033 ng.h/ml, respectively. Concentrations in serum at 0 h (predose) and at 24, 48, and 72 h after the final dose were 67 +/- 31, 64 +/- 24, 41 +/- 17, and 29 +/- 14 ng/ml, respectively. Thus, once-daily administration of azithromycin resulted in sustained systemic exposure to the drug.

Simultaneous determination of tenidap and its stable isotope analog in serum by high-performance liquid chromatography/atmospheric pressure chemical ionization tandem mass spectrometry.

A specific high-performance liquid chromatographic/atmospheric pressure chemical ionization tandem mass spectrometric assay for the quantitative determination of tenidap and its D3 analog in human serum is described. The assay exhibited a linear dynamic range of 0.1-25 micrograms ml-1. Its precision for the two analytes over the range was 17% or better. The assay validity and its utility to investigate changes in tenidap bioequivalence and pharmacokinetics are presented.

Identification of potentially neurotoxic pyridinium metabolite in the urine of schizophrenic patients treated with haloperidol.

Evidence that the parkinsonian inducing agent MPTP is biotransformed to a pyridinium species that selectively destroys nigrostriatal neurons in humans and subhuman primates has prompted studies to evaluate the metabolic fate of the structurally related neuroleptic agent haloperidol. With the aid of a highly sophisticated atmospheric pressure ionspray HPLC/MS/MS assay, unambiguous evidence has been obtained for the presence of the haloperidol pyridinium species in extracts of urine obtained from haloperidol-treated patients and in extracts of NADPH-supplemented human liver microsomal incubation mixtures containing haloperidol. The potential significance of the formation of this putative neurotoxic pyridinium species is considered.

Electrospray ionization mass spectrometry of semduramicin and other polyether ionophores.

Pneumatically assisted electrospray mass spectrometry of polyether ionophores yields several molecular ions. A single metal adduct molecular ion can be obtained by the addition of a neutral salt to the HPLC mobile phase. This approach may be useful in structural studies of unknown ionophores and in the development of specific methods for their analysis in complex matrices. Collision-induced dissociation of the molecular ions provides additional structural information and enhanced specificity for trace analysis. HPLC mobile-phase composition and flow rates have been optimized for on-line analysis. Best response and lowest background noise were obtained at the flow rate of 40 microL/min of a mobile phase containing a 20/80 mixture of water and acetonitrile. The development of a specific confirmatory assay for the new ionophore semduramicin in chicken liver demonstrates the usefulness of on-line HPLC pneumatically assisted electrospray mass spectrometry.

Determination of carbadox-related residues in swine liver by gas chromatography/mass spectrometry with ion trap detection.

The ion trap detector (ITD), in combination with a capillary gas chromatograph and under chemical ionization conditions, offers sufficient sensitivity to determine carbadox-related residues as the methyl ester derivative of quinoxaline-2-carboxylic acid at 3 micrograms/kg or higher in porcine liver. A tetradeuterated internal standard of QME effectively compensates for losses incurred during sample preparation. The method produced mean levels of 3.3 (+/- 0.5), 5.5 (+/- 0.8), and 10.1 (+/- 0.9) micrograms/kg for liver fortified at 3, 5, and 10 micrograms/kg. When applied to analysis of samples containing incurred residues of 14C-carbadox at the low microgram/kg level, results were comparable to those obtained by reverse isotope dilution analysis.

Metabolism of a new thiazolidinedione hypoglycemic agent CP-68,722 in rat: metabolite identification by gas chromatography mass spectrometry.

1. After i.v. administration to rat of CP-68,722, a new thiazolidinedione antidiabetic drug, four metabolites were excreted in bile, as glucuronide conjugates. 2. Incubation of the drug with a rat liver microsomal preparation yielded the four in vivo metabolite aglycones and several additional in vitro metabolites. 3. Seven in vivo-generated metabolites were isolated by h.p.l.c. Each metabolite was converted to stable isotope labelled or non-labelled derivatives. Capillary g.l.c.-mass spectrometric analysis of the derivatives indicated that five metabolites result from hydroxylation and one from oxidation to the chromanone. The sites of metabolism were deduced from the electron ionization spectra. 4. Authentic standards for five metabolites were synthesized. Agreements of mass spectra and chromatographic retention times confirmed the five proposed structures. Two metabolites, detected only in vivo, await structure confirmation.