title: Quantitative analysis of the immunosuppressant CP-690,550 in whole blood by column-switching high-performance liquid chromatography and mass spectrometry detection.
abstract: A fast and accurate method to quantify the new immunosuppressive JAK3 inhibitor CP-690,550 in whole blood using a dual-pump liquid chromatography-liquid chromatography-mass spectrometry (LC/LC-MS) system was developed and validated in nonhuman primate blood. Before injection, blood samples were prepared by precipitation with a reagent that included methanol and acetonitrile (30:70, vol/vol) along with the internal standard (CP-istd). Column-switching LC/LC-MS analysis used online extraction followed by separation on a C8 analytic column and MS detection of the [M + H] CP-690,550 (m/z = 313.1) and CP internal standard (m/z = 288.1). Linearity was always better than r = 0.99 (n = 7) for CP-690,550 (range 2.5-750 ng/mL), with a lower limit of quantification (LLOQ) of 2.5 ng/mL. The intrarun accuracy and precision ranged from 103.0% to 105.4% and 2.7% to 4.3%, respectively (n = 5), and the interday precision ranged from 8.7% to 11.1%, and the interday accuracy ranged from 98.1% to 103.8% of nominal values (n = 14). The injection repeatability for the method was 1.3% (n = 7). Except for the LLOQ, the intraday accuracy and precision in human blood were also within 15% (n = 5). The combination of simple sample preparation and short analytic run time of this sensitive procedure makes it effective for monitoring the concentration of CP-690,550 in whole blood in organ-transplant recipients.
authors.affiliation: Transplantation Immunology Laboratory, Department of Cardiothoracic Surgery, Stanford University School of Medicine, Stanford, California 94305, USA.
authors.forename: Ricardo
authors.initials: R
authors.lastname: Paniagua
authors.forename: Andrew
authors.initials: A
authors.lastname: Campbell
authors.forename: Paul S
authors.initials: PS
authors.lastname: Changelian
authors.forename: Bruce A
authors.initials: BA
authors.lastname: Reitz
authors.forename: Chandra
authors.initials: C
authors.lastname: Prakash
authors.forename: Dominic C
authors.initials: DC
authors.lastname: Borie
mesh_terms: D000818:Animals; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D016903:Drug Monitoring; D007166:Immunosuppressive Agents / Q000097:blood*; D053616:Janus Kinase 3; D008252:Macaca fascicularis; D008297:Male; D010880:Piperidines; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors*; D011743:Pyrimidines / Q000097:blood*; D011758:Pyrroles / Q000097:blood*; D012680:Sensitivity and Specificity; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*
chemical_list: D007166:Immunosuppressive Agents; D010880:Piperidines; D011743:Pyrimidines; D011758:Pyrroles; C479163:tofacitinib; D011505:Protein-Tyrosine Kinases; C507933:JAK3 protein, human; D053616:Janus Kinase 3