Assessment of an LC-MS method for plasma quantification of the new immunosuppressant FK778 through comparison with HPLC-UV.
FK778 is a new immunosuppressive agent, derived from the leflunomide-active metabolite A77 1726. It inhibits de novo pyrimidine nucleotide synthesis showing efficacy in the prevention and treatment of rejection in experimental transplant models. The aim of this work was to develop an HPLC-MS method to measure FK778 in plasma for pharmacokinetic studies. The equipment used for mass evaluation was an HLPC coupled to an ion trap analyzer through an electrospray source. After precipitation of plasma proteins with acetonitrile, the supernatant was injected onto an analytical RP-C18 column. Chromatographic separation was performed under isocratic conditions, using a mobile phase consisting of ammonium acetate buffer and acetonitrile (55:45. vol/vol). MS detection was performed in the negative ionization mode by monitoring the molecular ion of FK778 (m/z 307) and IS (m/z 269), using selected ion monitoring for both. However, we observed peaks corresponding to dimers, trimers, and tetramers of FK778 (m/z 637, m/z 945, m/z 1274). The HPLC-MS method was applied to pharmacokinetics in animal models showing comparable results to those obtained by an HPLC-UV assay at 290 nm. Good agreement was observed in the plasma FK778 concentration versus time curves. The rapid preparation of samples and the short run-time make this method attractive for use in clinical practice.
Molinaro
M
M
Department of Pharmacology IRCCS-Policlinico S. Matteo, Pavia, Italy. m.molinaro@smatteo.pv.it
Carazzone
C
C
Barbano
D
D
Abbiati
F
F
Alessiani
M
M
Regazzi
M
M
D000480:Alkynes; D002138:Calibration; D002851:Chromatography, High Pressure Liquid; D006085:Graft Survival / Q000187:drug effects / Q000276:immunology; D016027:Heart Transplantation / Q000276:immunology*; D006801:Humans; D007166:Immunosuppressive Agents / Q000097:blood*; D007555:Isoxazoles / Q000097:blood*; D016030:Kidney Transplantation / Q000276:immunology*; D000077339:Leflunomide; D013058:Mass Spectrometry; D009570:Nitriles; D021241:Spectrometry, Mass, Electrospray Ionization
D000480:Alkynes; D007166:Immunosuppressive Agents; D007555:Isoxazoles; D009570:Nitriles; C115284:2-cyano-3-hydroxy-N-(4-(trifluoromethyl)phenyl)-2-hepten-6-ynamide; D000077339:Leflunomide