The validation of an LC-MS/MS assay for perhexiline and major hydroxy metabolites, and its application to therapeutic monitoring in patient plasma.

Aim: Perhexiline (PEX), being developed to treat hypertrophic cardiomyopathy, is toxic at levels above the therapeutic range. Plasma level monitoring is therefore essential. The absence of a UV-absorbing chromophore has in the past required quantitative analysis of PEX in plasma using lengthy derivatization methods, followed by HPLC and fluorescence detection.

Use of a carboxylesterase inhibitor of phenylmethanesulfonyl fluoride to stabilize epothilone D in rat plasma for a validated UHPLC-MS/MS assay.

A sensitive, accurate and rugged UHPLC-MS/MS method was developed and validated for the quantitation of Epothilone D (EpoD), a microtubule stabilizer in development for treatment of Alzeimer's disease, in rat plasma. The ester group in EpoD can be hydrolyzed by esterases in blood or plasma, which creates a stability concern for the bioanalysis of EpoD. Species differences in the stability of EpoD in plasma were observed.

Application of a stabilizer cocktail of N-ethylmaleimide and phenylmethanesulfonyl fluoride to concurrently stabilize the disulfide and ester containing compounds in a plasma LC-MS/MS assay.

BMS-753493, a conjugate of the active epothilone moiety, BMS-748285, to folic acid, has been evaluated for the treatment of cancer. The presence of a disulfide bond in BMS-753493 and an ester group in both BMS-753493 and BMS-748285 results in their being unstable in blood or plasma. A stabilization strategy using a cocktail of N-ethylmaleimide and phenylmethanesulfonyl fluoride was developed and applied to stabilize both analytes in human blood during sample collection, processing, and storage.

Automated dried blood spots standard and QC sample preparation using a robotic liquid handler.

Background: A dried blood spot (DBS) bioanalysis assay involves many steps, such as the preparation of standard (STD) and QC samples in blood, the spotting onto DBS cards, and the cutting-out of the spots. These steps are labor intensive and time consuming if done manually, which, therefore, makes automation very desirable in DBS bioanalysis.

Results: A robotic liquid handler was successfully applied to the preparation of STD and QC samples in blood and to spot the blood samples onto DBS cards using buspirone as the model compound.

Systematic evaluation of the root cause of non-linearity in liquid chromatography/tandem mass spectrometry bioanalytical assays and strategy to predict and extend the linear standard curve range.

Rationale: The linear range of a liquid chromatography/tandem mass spectrometry (LC/MS/MS) bioanalytical assay is typically about three orders of magnitude. A broader standard curve range is favored since it can significantly reduce the time, labor and potential errors related to sample dilution - one of the bottlenecks in sample analysis. Using quadratic regression to fit the standard curve can, to a certain degree, extend the dynamic range.

Rapid detection and characterization of in vitro and urinary N-acetyl-L-cysteine conjugates using quadrupole-linear ion trap mass spectrometry and polarity switching.

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.

A sensitive method for the determination of entecavir at picogram per milliliter level in human plasma by solid phase extraction and high-pH LC-MS/MS.

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.

LC-MS/MS-based approach for obtaining exposure estimates of metabolites in early clinical trials using radioactive metabolites as reference standards.

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.

Characterization of the UDP glucuronosyltransferase activity of human liver microsomes genotyped for the UGT1A1*28 polymorphism.

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).

Absence of a pharmacokinetic interaction between entecavir and adefovir.

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.

Entecavir pharmacokinetics, safety, and tolerability after multiple ascending doses in healthy subjects.

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.

Biotransformation of carbon-14-labeled muraglitazar in male mice: interspecies difference in metabolic pathways leading to unique metabolites.

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.