Determination of Tacrolimus in Rat Whole Blood Utilizing Triple Quadrupole LC/MS

A simple, sensitive and rapid method was developed for quantitation of tacrolimus in rat whole blood utilizing triple Quadrupole LC/MS. An aliquot of 0.1 mL of whole blood sample was extracted with t-butyl methyl ether using a Heidolph vortexer. The chromatographic separation was performed by using chromolith fast gradient HPLC RP 18e (2mmX50mm.i.d) column with a mobile phase of 90% methanol and 10% 2mM ammonium acetate buffer followed by MS/MS detection. The analyte was quantitated in negative ionization mode. Multiple reaction monitoring (MRM) using the transition m/z 802.4→560.2 and m/z 808.4→548.6 was performed to quantify tacrolimus with IS (pimecrolimus), respectively. The method had a total chromatographic run time of 2.5 min; and linear calibration curves over the concentration range of 20.931 -1000.703 ng/mL.The lower limit of quantification (LLOQ) was 20.931 ng/mL. Use of sodium citrate (3.85%) as an anticoagulant in rat whole blood and samples were stable for at least the time required to assay the number of samples that could be placed in the auto sampler which is maintaining temperature of 10°C. The between and within batch precision and accuracy of the method were determined by using 6 quality control samples. The highest %CV 478.908 ng/mL (8.01% within run & 3.07 between run), with other %CV<5%. The recovery ranged 23.92% for tacrolimus over range of 50.285 to 798.179 ng/mL and was 18.52% for pimecrolimus respectively. The validated method was successfully applied to the quantification of tacrolimus concentration in rat whole blood.