A Novel and Cost Effective Method of Removing Excess Albumin from Plasma/Serum Samples and Its Impacts on LC-MS/MS Bioanalysis of Therapeutic Proteins

We have developed an innovative method to remove albumin from plasma/serum samples for the LC-MS/MS quantitation of therapeutic proteins. Different combinations of organic solvents and acids were screened for their ability to remove albumin from plasma and serum samples. Removal efficiency was monitored by two signature peptides (QTA­LVE­LVK and LVN­EVT­EF­AK) from albumin. Isopropanol with 1.0% trichloroacetic acid was found to be the most effective combination to remove albumin while retaining the protein of interest. Our approach was compared with a commercial albumin depletion kit on both efficiency of albumin removal and recovery of target proteins. We have demonstrated that our approach can remove 95% of the total albumin in human plasma samples while retaining close to 100% for two of three therapeutic proteins tested, with the third one at 60–80%. The commercial kit removed 98% of albumin but suffered at least 50% recovery loss for all therapeutic proteins when compared to our approach. Using BMS-C as a probe compound, the incorporation of the albumin removal approach has improved both assay sensitivity and ruggedness, compared to the whole plasma protein digestion approach alone. An LC-MS/MS method was developed and validated based on this new approach for the analysis of BMS-C in monkey serum. This assay was successfully applied to a toxicological study. When the albumin removal method was used in another clinical LC-MS/MS method, the sensitivity improved 10-fold to 50 ng/mL LLOQ comparing to a typical pellet digestion method

Selective Reaction Monitoring of Negative Electrospray Ionization Acetate Adduct Ions for the Bioanalysis of Dapagliflozin in Clinical Studies

Dapagliflozin (Farxiga), alone, or in the fixed dose combination with metformin (Xigduo), is an orally active, highly selective, reversible inhibitor of sodium-glucose cotransporter type 2 (SGLT2) that is marketed in United States, Europe, and many other countries for the treatment of type 2 diabetes mellitus. Here we report a liquid chromatography–tandem mass spectrometry (LC–MS/MS) bioanalytical assay of dapagliflozin in human plasma. A lower limit of quantitation (LLOQ) at 0.2 ng/mL with 50 μL of plasma was obtained, which reflects a 5-fold improvement of the overall assay sensitivity in comparison to the previous most sensitive assay using the same mass spectrometry instrumentation. In this new assay, acetate adduct ions in negative electrospray ionization mode were used as the precursor ions for selective reaction monitoring (SRM) detection. Sample preparation procedures and LC conditions were further developed to enhance the column life span and achieve the separation of dapagliflozin from potential interferences, especially its epimers. The assay also quantifies dapagliflozin’s major systemic circulating glucuronide metabolite, BMS-801576, concentrations in human plasma. The assay was successfully transferred to contract research organizations (CROs), validated, and implemented for the sample analysis of pediatric and other critical clinical studies. This assay can be widely used for bioanalytical support of future clinical studies for the newly approved drug Farxiga or any combination therapy containing dapagliflozin