ABSTRACT

A simple, accurate and precise method was developed using high performance liquid chromatography with electron spray ionizationtandem mass spectrometry (HPLC-ESI-MS) to quantify the concentration of tacrolimus in human blood with K2EDTA anticoagulant was developed and fully validated. Sirolimus was used as an internal standard (ISTD). The sample extraction procedure utilized solid phase extraction method. The chromatographic analysis was conducted on a Thermo scientific Hypurity (50 x 4.6 mm) 2.6µm within 2 min, using methanol with 10mM ammonium acetate (80:20%, v/v) was used as mobile phase at the flow rate of 0.6mL/min under an isocratic condition. The ionization was performed on electron spray ionization interference with positive mode by multiple reaction monitoring (MRM). The mass transitions were 821.60?768.60 m/z for tacrolimus and 931.70?864.70 m/z for ISTD. Method validated as per USFDA guidelines and calibration curve was found to be linear in the range of 0.200-100.176 ng/mL.The results were within the acceptance limits. The extraction efficiency was 82.52% at the three quality control levels. The lower limit of detection (LLOQ) was found to be 0.200 ng/mL.Stability studies demonstrated that tacrolimus was stable in blood during Bench Top (4hr 10min at room temperature), Auto-sampler (27hr 45 min at 4oC), Freeze-Thaw (5cycles) and Long term analyte stability in blood (37days at -20oC). Keywords: Tacrolimus; Human Blood; Stability; Solid Phase Extraction; Validation
Publication date: 01/10/2021

https://ijbpas.com/pdf/2021/October/MS_IJBPAS_2021_OCT_SPCL_1025.pdf

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https://doi.org/10.31032/IJBPAS/2021/10.10.1025