Rapid and Sensitive Ultra Performance Liquid Chromatography Tandem Mass Spectrometry for Quantitation of Tacrolimus in Human Whole Blood

Therapeutic drug monitoring of immunosuppressive agents in organ transplanted Patient’s is crucial to prevent intoxication or transplant rejection due to inadequate dosage. The commonly used immune assays have been gradually undergoing replacement by mass spectrometry since this physical method offers both a higher Sensitivity and specificity. A simple rapid, novel, sensitive Ultra performance liquid Chromatography (UPLC) coupled with electron spray mass spectrometry method was developed and validated for quantification of tacrolimus (I) in human plasma. The analyte and internal standard (Sacrolimus II) were extracted by liquid-liquid extaction with hexane and four levels of quality control samples of k2 EDTA human whole blood were used. The chromatographic separation was performed on reverse phase thermo hypurity advance column (46 x 50 mm, 5um) with mobile phase of 90% methanol and 10mM ammonium acetate buffer (90:10)low at a flow rate of 0.5ml/min and run time of 2.5 minute .The retention time of both analyte and ISD were 1.37mints.The deprotonate of analyte was quantitated in positive ionization by multiple reaction monitoring (MRM)with mass spectrometry. The mass transitions m/z 821-768.54 and m/z 931.6-864.8 were used to measure I&II in whole blood –I(Q1)and m/z 821.5-768.54 and m/z 931.5884.6for I&II in whole blood –II(Q2) respectively. The method exhibited a linear response in the range of 0.495-99.430ng/ml for tacrolimus in human plasma with co-relation coefficient of greater than 0.998.The lower limit of quantification was 0.5ng/ml with C V % of 5%.The %accuracy for QC samples were 98.33%LQC, 93.03%, GMQC 93.60%MQC and 96.87%HQC.The %CV for QC samples were 5.19%LQC 931.78%GMQC, 3.94%MQC and 0.21%HQC. The % stability by freeze and thaw stability was 97.27%for LQC, 96.42%for HQC and by bench top stability was 100.33% & 98.20% for LQC and HQC respectively. The % accuracy and CV% for dilution integrity in the ratio of 1:5 was 159.248&4.02%. This method can be used for the quantification of tacrolimus in human whole blood in routine and bioequivalence studies.