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ABSTRACT: The present study describes a novel methodology for the rapid detection and structural characterization of unknown N-acetyl-L-cysteine (NAC) conjugates using polarity switching of triple quadrupole mass spectrometry. This method utilizes a negative neutral loss (NL) scan of 129 Da or multiple reaction monitoring (MRM) from predicted m/z values to product ions derived from the NL of 129 Da as a survey scan to trigger the acquisition of enhanced product ion (EPI) spectra in the positive ion mode. Thus, selective detection of NAC conjugates and acquisition of fragment-rich MS/MS spectra were accomplished in a single LC/MS run. The utility of this methodology was evaluated through analysis of NAC conjugates of acetaminophen in human urine after an oral dose. The MRM-EPI approach, which showed better sensitivity than the NL-EPI approach in analyzing urine samples, revealed three NAC-acetaminophen conjugates in the human urine, including two minor NAC conjugates that were derived from hydroxyl acetaminophen and methoxy acetaminophen. In addition, the methodology was applied to screening for reactive metabolites of clozapine and diclofenac using NAC as a trapping agent. Results showed reactive metabolite profiles comparable to those obtained from glutathione (GSH) trapping experiments, while MS/MS spectra of NAC conjugates provided more valuable structural information than those of GSH adducts. The study demonstrates that NAC trapping followed by NL-EPI analysis is a useful approach for high-throughput screening of reactive metabolites and that the MRM-EPI method is well-suited for analysis of low levels of NAC conjugates in urine.
Chemical Research in Toxicology 07/2009; 22(7):1246-55. · 3.78 Impact Factor
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ABSTRACT: Entecavir is a guanine nucleoside analogue used in the treatment of hepatitis B virus (HBV) infection. In this paper, we describe an LC-MS/MS method that was developed and validated for the quantitation of entecavir in human EDTA plasma with both high sensitivity (lower limit of quantitation (LLOQ) of 5 pg/mL) and a wide concentration range (5000-fold) intended for low dose ascending clinical studies. High enrichment was achieved by taking advantage of the excellent loading capacity and reproducibility of Oasis HLB 96-well solid phase extraction plate, which allowed 1 mL of plasma samples to be processed in two equal sequential loading steps. Lobucavir, a structural analogue, was used as the internal standard. A filtration step following the reconstitution proved to be vital for the method robustness. The analyte and internal standard were separated on an Xterra MS C18 column with a gradient elution and high-pH mobile phases. Analytes were detected by positive ion electrospray tandem mass spectrometry. The high-pH mobile phase provided both excellent analyte on-column retention and peak shape, leading to the desired sensitivity. Validation results show good intra-assay (12.3%CV) and inter-assay (3.1%CV) precisions, and good assay accuracy (+/-7.6%Dev). Recovery was high (approximately 80%), however, the large volume of plasma used did result in a considerable matrix effect (approximately 0.45) which was well compensated by the analog internal standard. The method was applied to sample analysis of a Phase I clinical study.
Journal of pharmaceutical and biomedical analysis 06/2009; 49(4):1027-33. · 2.45 Impact Factor
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ABSTRACT: An LC-MS/MS-based approach that employs authentic radioactive metabolites as reference standards was developed to estimate metabolite exposures in early drug development studies. This method is useful to estimate metabolite levels in studies done with non-radiolabeled compounds where metabolite standards are not available to allow standard LC-MS/MS assay development. A metabolite mixture obtained from an in vivo source treated with a radiolabeled compound was partially purified, quantified, and spiked into human plasma to provide metabolite standard curves. Metabolites were analyzed by LC-MS/MS using the specific mass transitions and an internal standard. The metabolite concentrations determined by this approach were found to be comparable to those determined by valid LC-MS/MS assays. This approach does not requires synthesis of authentic metabolites or the knowledge of exact structures of metabolites, and therefore should provide a useful method to obtain early estimates of circulating metabolites in early clinical or toxicological studies.
Drug metabolism letters. 01/2008; 1(4):293-8.
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Donglu Zhang, Duxi Zhang,
Dan Cui,
Janice Gambardella,
Li Ma,
Anthony Barros,
Lifei Wang,
Yunlin Fu,
Sandhya Rahematpura,
Julia Nielsen,
Michael Donegan,
Hongjian Zhang,
W Griffith Humphreys
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ABSTRACT: The UGT1A1*28 polymorphism is known to correlate with altered clearance of bilirubin (Gilbert syndrome) and drugs such as 7-ethyl-10-[4-(1-piperidino)-1-piperidino] carbonyloxy camptothecin (CPT-11). Although this polymorphism is clinically relevant and leads to significant drug-related toxicity of CPT-11, in vitro tools to allow prediction of how it will affect the clearance of new chemical entities have not been completely developed. To allow a more complete assessment of whether new chemical entities will be affected by the UGT1A1*28 polymorphism, a panel of microsomes was prepared from 15 donor livers genotyped as UGT1A1*1/*1, UGT1A1*1/*28, and UGT1A1*28/*28 (five donors per genotype). The microsomes were phenotyped by measuring activities of a panel of substrates, both those reported to be conjugated specifically by UGT1A1 or by other UDP glucuronosyltransferase enzymes. Bilirubin, estradiol (3-OH), ethinyl estradiol (3-OH), and 7-ethyl-10-hydroxycamptothecin (SN-38) were found to show significantly lower rates of metabolism in the UGT1A1*28/*28 microsomes with no change in K(m) values. In addition, microsomes genotyped as UGT1A1*1/*28 showed intermediate rates of metabolism. Acetaminophen, 3'-azido-3'-deoxythymidine, muraglitazar, estradiol (17-OH), and ethinyl estradiol (17-OH) were all found to show similar rates of metabolism regardless of UGT1A1 genotype. Interestingly, muraglitazar (UGT1A3 substrate) showed an inverse correlation with glucuronidation of UGT1A1 substrates. These genotyped microsomes should provide a useful tool to allow a more comprehensive prediction of UGT1A1 metabolism of a new drug and gain insight into the effect of the UGT1A1*28 polymorphism.
Drug Metabolism and Disposition 01/2008; 35(12):2270-80. · 3.73 Impact Factor
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ABSTRACT: This study evaluated the effect of entecavir on the pharmacokinetics of adefovir and the effect of adefovir on the pharmacokinetics of entecavir using a fixed-sequence crossover design in healthy adult subjects. Subjects received 10 mg of adefovir once daily on days 1 to 4, 1 mg of entecavir on days 5 to 14, and 1 mg of entecavir plus 10 mg of adefovir on days 15 to 24. Pharmacokinetic assessments were performed on days 4 and 24 for adefovir and on days 14 and 24 for entecavir. The geometric mean ratios (90% confidence interval) for area under the plasma concentration-time curve in 1 dosing interval, peak plasma concentration, and 24-hour postdose plasma concentration of entecavir when coadministered with adefovir and of adefovir when coadministered with entecavir were within the prespecified 0.80 to 1.25 no-effect range. Entecavir and adefovir were well tolerated when administered in combination. Therefore, the pharmacokinetic data generated in this study indicate that entecavir and adefovir can be coadministered without the need for dosage adjustment.
The Journal of Clinical Pharmacology 11/2007; 47(10):1327-34. · 2.91 Impact Factor
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ABSTRACT: A double-blind, placebo-controlled, multiple oral dose escalation study was conducted to investigate the pharmacokinetics, safety, and tolerability of entecavir in healthy subjects. Eight subjects were assigned to each of the 3 dose panels (0.1 mg, 0.5 mg, and 1 mg or matched placebo once daily for 14 days). Blood and urine samples were collected for pharmacokinetic analyses. Entecavir was rapidly absorbed, with peak plasma concentration occurring within 1 hour of dosing. Steady-state plasma concentrations of entecavir were achieved by 10 days following the initial dose. At steady state, the mean area under the plasma concentration-time curve over 1 dosing interval, increased approximately proportional to dose. Entecavir had a mean terminal half-life ranging from 128 to 149 hours and an effective half-life of approximately 24 hours. Elimination was predominantly through renal excretion, with mean urinary recovery ranging from 62% to 73%. Entecavir was safe and well tolerated when administered at doses ranging from 0.1 mg to 1 mg/d for 14 days.
The Journal of Clinical Pharmacology 12/2006; 46(11):1250-8. · 2.91 Impact Factor
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ABSTRACT: Muraglitazar (Pargluva; Bristol-Myers Squibb), a dual alpha/gamma peroxisome proliferator-activated receptor activator, is under development for treatment of type 2 diabetes. This study describes the biotransformation profile of carbon-14-labeled muraglitazar in plasma, urine, feces, and bile samples from male CD-1 mice [intact and bile duct cannulation (BDC)] after single oral doses of 1 and 40 mg/kg. The major drug-related component circulating in mouse plasma was the parent compound for up to 4 h postdose. Similar to excretion profiles of muraglitazar in humans, monkeys, and rats, urinary excretion was the minor and fecal excretion via the biliary route was the major elimination pathway for muraglitazar in mice. The parent compound was a minor component in urine, bile, and feces, indicating that muraglitazar was extensively metabolized in mice. Major biotransformation pathways of muraglitazar in mice included taurine conjugate formation, acyl glucuronidation, hydroxylation, and O-dealkylation. In addition to those metabolites previously identified in humans, monkeys, and rats (M1-M21), several unique metabolites identified in mice included taurine conjugates (M24, M25, M26a,b,c, and M31), oxazole-ring-opened metabolites (M27 and M28), glutathione conjugates (M29a,b and M30), a dihydroxylated metabolite (M32), hydroxylated metabolites (M33 and M35), and a dehydrogenated metabolite (M34). The taurine conjugate of muraglitazar, M24, was a major metabolite in mice, accounting for 12 to 15% of the total dose in BDC mice or 7 to 12% of the total dose in intact mice. None of these taurine and glutathione conjugates were found in the bile samples of humans, monkeys, or rats.
Drug Metabolism and Disposition 06/2006; 34(5):807-20. · 3.73 Impact Factor
