Development of high performance liquid chromatography tandem mass spectrometric methods for the determination of a novel ascomycin-based immunoregulant in human whole blood and plasma: A case of potential analyte loss during sample collection

Abstract

Methods for the determination of a novel, ascomycin-based macrolide immunosuppressant in human plasma and whole blood are described. Following protein precipitation, the analyte and an internal standard were extracted from each matrix using solid phase extraction on an end-capped cyano column. The analytes were chromatographed on a Zorbax SB-CN analytical column (5.0 μm, 150×4.6 mm) with a mobile phase consisting of 70∶30∶0.1 v/v/v acetonitrile/ammonium acetate (10 mM)/formic acid. A tandem mass spectrometer equipped with an APCI interface was used as the detector. Multiple reaction monitoring using the parent→product ion combinations of m/z 1009→217 and 979→187 was used to detect the analyte and internal standard, respectively. Seven point calibration curves over the concentration range of 0.25–20 ng mL−1 yielded a linear response when a 1/yweighted linear regression model was employed. Based on the replicate analyses (n=5) of spiked standards, the withinday assay precision for both assays was better than 7.5% C.V. at all points on the calibration curves. The within-day accuracy for both assays was within 5% of nominal at all standard concentrations. The between-run precision of each of the assay, as calculated from the results of the analysis of quality control samples, was better than 5%. C.V.A special collection procedure for whole blood, in which samples were stored in the tubes that were used in the initial step of the assay procedure, was developed to eliminate assay error resulting from adsorption of the analyte to the sample storage tube.