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

Background: An absolute bioavailability study that utilized an intravenous [C-14]microdose was conducted for saxagliptin (Onglyza(R)), a marketed drug product for the treatment of Type 2 diabetes mellitus. Concentrations of [14C]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 [C-14]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.