Liquid chromatography–negative ion electrospray tandem mass spectrometry method for the quantification of tacrolimus in human plasma and its bioanalytical applications

Abstract

A simple, rapid, novel and sensitive liquid chromatography–tandem mass spectrometry method was developed and validated for quantification of tacrolimus (I) in human plasma, a narrow therapeutic index, potent macrolide immunosuppressive drug. The analyte and internal standard (tamsulosin (II)) were extracted by liquid–liquid extraction with t-butylmethylether using a Glas-Col Multi-Pulse Vortexer. The chromatographic separation was performed on reverse phase Xterra ODS column with a mobile phase of 99% methanol and 1% 10 mM ammonium acetate buffer. The deprotonate of analyte was quantitated in negative ionization by multiple reaction monitoring (MRM) with a mass spectrometer. The mass transitions m/ z 802.5→560.3 and m/ z 407.2→151.9 were used to measure I and II, respectively. The assay exhibited a linear dynamic range of 0.05–25 ng/ml for tacrolimus in human plasma. The lower limit of quantitation was 50 pg/ml with a relative standard deviation of less than 20%. Acceptable precision and accuracy were obtained for concentrations over the standard curve ranges. Run time of 2 min for each sample made it possible to analyze a throughput of more than 400 human plasma samples per day. The validated method has been successfully used to analyze human plasma samples for application in comparative bioavailability studies. The tacrolimus plasma concentration profile could be obtained for pharmacokinetic study. The observed maximum plasma concentration ( C max) of tacrolimus (5 mg oral dose) is 440 pg/ml, time to observed maximum plasma concentration ( T max) is 2.5 h and elimination half-life ( T 1/2) is 21 h.