Jim X. Shen

A high-throughput LC–MS/MS method for the quantitation of posaconazole in human plasma: Implementing fused core silica liquid chromatography

A rapid and robust liquid chromatographic tandem mass spectrometric (LC-MS/MS) method for the determination of posaconazole concentrations in human plasma was validated. Posaconazole was extracted from human plasma using mixed-mode cation exchange solid phase extraction in a 96-well plate format followed by gradient separation on a fused-core Halo C18 column. The analyte and its corresponding internal standard were detected using a Sciex API 4000 triple quadrupole LC-MS/MS system equipped with a TurboIonSpray ionization source operated in the positive ion mode. The calibration range of the method was 5.00-5000ng/mL using a 50microL aliquot of plasma. The assay inter-run accuracy and precision were-4.6-2.8% and 2.3-8.7%, respectively (n=18). The results from method validation indicate the method to be sensitive, selective, accurate, and reproducible. The method was successfully applied to the routine analysis of clinical samples with the fused-core silica columns providing excellent reproducibility for greater than 1000 injections per column.

Fit-for-purpose bioanalytical cross-validation for LC–MS/MS assays in clinical studies

The paradigm shift of globalized research and conducting clinical studies at different geographic locations worldwide to access broader patient populations has resulted in increased need of correlating bioanalytical results generated in multiple laboratories, often across national borders. Cross-validations of bioanalytical methods are often implemented to assure the equivalency of the bioanalytical results is demonstrated. Regulatory agencies, such as the US FDA and European Medicines Agency, have included the requirement of cross-validations in their respective bioanalytical validation guidance and guidelines. While those documents provide high-level expectations, the detailed implementation is at the discretion of each individual organization. At Bristol-Myers Squibb, we practice a fit-for-purpose approach for conducting cross-validations for small-molecule bioanalytical methods using LC-MS/MS. A step-by-step proposal on the overall strategy, procedures and technical details for conducting a successful cross-validation is presented herein. A case study utilizing the proposed cross-validation approach to rule out method variability as the potential cause for high variance observed in PK studies is also presented.

Overcoming bioanalytical challenges in an Onglyza® intravenous [14C]microdose absolute bioavailability study with accelerator MS

BACKGROUND An absolute bioavailability study that utilized an intravenous [(14)C]microdose was conducted for saxagliptin (Onglyza(®)), a marketed drug product for the treatment of Type 2 diabetes mellitus. Concentrations of [(14)C]saxagliptin were determined by accelerator MS (AMS) after protein precipitation, chromatographic separation by UPLC and analyte fraction collection. A series of investigative experiments were conducted to maximize the release of the drug from high-affinity receptors and nonspecific adsorption, and to determine a suitable quantitation range. RESULTS A technique-appropriate validation demonstrated the accuracy, precision, specificity, stability and recovery of the AMS methodology across the concentration range of 0.025 to 15.0 dpm/ml (disintegration per minute per milliliter), the equivalent of 1.91-1144 pg/ml. Based on the study sample analysis, the mean absolute bioavailability of saxagliptin was 50% in the eight subjects with a CV of 6.6%. Incurred sample reanalysis data fell well within acceptable limits. CONCLUSION This study demonstrated that the optimized sample pretreatment and chromatographic separation procedures were critical for the successful implementation of an UPLC plus AMS method for [(14)C]saxagliptin. The use of multiple-point standards are useful, particularly during method development and validation, to evaluate and correct for concentration-dependent recovery, if observed, and to monitor and control process loss and operational variations.

Stereoselective quantitation of a serine protease inhibitor using LC-MS/MS at elevated column temperature

A LC-MS/MS method using a LC column packed with sub-2 micron particles and elevated column temperatures was validated for the quantitation of SCH 503034 diastereomers (SCH 534128 and SCH 534129) in human plasma. The method was validated over the concentration range of 2.5 to 1250 ng/mL. Inter-assay precision, based on percent relative deviation for n = 18 replicate quality controls, was 4.5% for SCH 534128 and 4.9% for SCH 534129. Inter-assay accuracy based on n = 18 replicate quality controls was +/- 7.8% for both SCH 534128 and SCH 534129. The method involved the novel application of ion pairing reagents to increase the stereoselectivity of the separation. Temperature, types of ion pairing reagent, and concentration of ion pairing reagent were all found to play significant roles in the resolution of the SCH 534128 and SCH 534129 diastereomers on a LC column packed with sub-2 micron particles. Specifically, a sensitivity increase of five-fold was demonstrated by increasing the column temperature. Without sacrificing resolution, the run time was significantly shortened when the column temperature was elevated to 100 degrees C.

Reasons for calibration standard curve slope variation in LC–MS assays and how to address it

A consistent calibration curve slope is a positive indication of assay performance in a validated bioanalytical method using LC–MS/MS. It is one of the quality indicators utilized by bioanalytical scientists during the data review process. However, it is not uncommon that a calibration curve slope varies significantly across different analytical runs during sample analysis. The causes for such variation and their potential impacts on the assay ruggedness have been discussed on multiple occasions [1,2]. Although there is no specific requirements for the acceptability of calibration curve slope in regulatory guidelines, a scientific understanding of what is behind the observed deviation is valuable during method development, validation and sample analysis. Large variance in curve slope often indicates potential issues associated with a method. We advocate always assessing and understanding root cause for such variance and this paper tries to illustrate possible causes for the calibration curve slope variation (including the linearity change) and assess their impacts on the analysis results.