Development and validation of a liquid chromatography–mass spectrometric assay for simultaneous determination of tacrolimus and 13-O-desmethyl tacrolimus in rat kidney tissue

Tatian Kirresh, Sony Tuteja, D. Russo, P. D. Brophy, D. J. Murry

Research output: Contribution to journalArticle

2 Scopus citations


A sensitive and robust LC–MS/MS method has been developed and validated to determine the concentrations of tacrolimus and its major metabolite 13-O-desmethyl tacrolimus (13-ODMT) in kidney tissue from rats who received tacrolimus intra-peritoneally at doses of 0.5 mg/kg and 2 mg/kg. The samples were prepared by a liquid-liquid extraction procedure using ethyl ether as the extraction solvent and ascomycin as the internal standard. Chromatographic separation was achieved using Phenomenex Kinetex column (2.6 μm C18 100 Å, 100 × 2.1 mm, Phenomenex, Torrance CA) and a gradient mobile phase of water and methanol-acetonitrile (50:50, v/v) both containing 0.1% formic acid. The limit of quantification was 0.25 ng/ml and the calibration curves covered a concentration range from 0.25 to 50 ng/ml. Intra-and inter-assay precision and accuracy for both tacrolimus and 13-ODMT were all within FDA guidelines for bioanalysis. Extraction efficiency for tacrolimus ranged from 67.00 to 74.90% and from 66.70 to 78.40% for 13-ODMT. Several challenges interfering with the performance of the method such as phospholipid build-up have also been addressed. Kidney tissue samples from six rats receiving either 0.5 or 2 mg/kg dose were analyzed and resulted in a median concentration of 11.54 and 0.72 ng/ml for tacrolimus and 13-ODMT, respectively, for the lower dose level, and a median concentration of 8.89 ng/ml and 1.50 ng/ml for tacrolimus and 13-ODMT, respectively, at the higher dose level.

Original languageEnglish (US)
Pages (from-to)32-37
Number of pages6
JournalJournal of Pharmaceutical and Biomedical Analysis
Publication statusPublished - Mar 20 2017



  • 13-O-desmethyl tacrolimus
  • IT-TOF
  • Mass spectroscopy
  • Nephrotoxicity
  • Organ rejection
  • Tacrolimus

ASJC Scopus subject areas

  • Analytical Chemistry
  • Pharmaceutical Science
  • Drug Discovery
  • Spectroscopy
  • Clinical Biochemistry

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