Separation of a BMS drug candidate and acyl glucuronide from seven glucuronide positional isomers in rat plasma via high‐performance liquid chromatography with tandem mass spectrometric detection

Abstract

A high-performance liquid chromatography/tandem mass spectrometry (LC/MS/MS) method has been developed and validated for the determination of a BMS drug candidate and its acyl glucuronide (1-O-beta glucuronide) in rat plasma. A 50-microL aliquot of each plasma sample was fortified with acetonitrile containing the internal standard to precipitate proteins and extract the analytes of interest. After mixing and centrifugation, the supernatant from each sample was transferred to a 96-well plate and injected into an LC/MS/MS system. Chromatographic separation was achieved isocratically on a Phenomenex Luna C(18), 3 mm x 150 mm, 3 microm column. The mobile phase contained 0.075% formic acid in 70:30 (v/v) acetonitrile/water. Under the optimized chromatographic conditions, the BMS drug candidate and its acyl glucuronide were separated from its seven glucuronide positional isomers within 10 min. Resolution of the parent from all glucuronides and acyl glucuronide from its positional isomers was critical to avoid their interference with quantitation of parent or acyl glucuronide. Detection was by positive ion electrospray MS/MS on a Sciex API 4000. The standard curve, which ranged from 5 to 5000 ng/mL, was fitted to a 1/x(2) weighted quadratic regression model for both the BMS drug candidate and its acyl glucuronide. Whole blood and plasma stability experiments were conducted to establish the sample collection, storage, and processing conditions. The validation results demonstrated that this method was rugged and repeatable. The same methodology has also been used in mouse and human plasma for the determination of the BMS drug candidate and its acyl glucuronide.

Systematic LC‐MS/MS bioanalytical method development that incorporates plasma phospholipids risk avoidance, usage of incurred sample and well thought‐out chromatography

“…We illustrated this type of interference in our original article on this subject in 1999 and since then there have been a number of publications dealing with the subject matter Kapron et al, 2005;Jemal, 2005;Vanderhoeven et al, 2006;Xue et al, 2006Xue et al, , 2008Schwartz et al, 2006). Drugs producing metabolites that can potentially interfere in this manner include those containing lactone, carboxylic acid and sulfhydryl (thiol) functional groups Jemal, 2005).…”

LC‐MS in Drug Metabolism and Pharmacokinetics: A Pharmaceutical Industry Perspective

“…However, sometimes nonspecifi c binding of enzymatic reaction, redistribution between plasma and blood cells, or a combination of the above factors. A very common example of chemical instability can be seen in acyl glucuronide conjugates [104,105] . A carboxyl group -containing drug may undergo phase II metabolism to form an acyl glucuronide conjugate, which can be unstable and undergo hydrolysis and/or positional rearrangement under neutral or slightly basic pH.…”

Analytical Method Development and Validation in Accordance to the Regulatory Guidelines

“…Direct assays for acyl glucuronides must ensure specificity to the 1-β isomer, as one cannot assume that other migrated isomers yield the same MS response. It is therefore important that the 1-β anomer be resolved from all other forms [54]. One should always consider reducing the pH when collecting samples of an acid-containing drug to avoid the breakdown of any potential acyl glucuronides.…”