Determination of 13-O-demethyl tacrolimus in human liver microsomal incubates using liquid chromatography-mass spectrometric assay (LC-MS).
A simple, sensitive and specific liquid chromatography coupled electrospray ionization mass spectrometric (LC/ESI/MS) method for the determination of 13-O-demethylated metabolite (MI), one of the major metabolites of tacrolimus has been developed. The assay uses 32-demethoxyrapamycin (IS) as the internal standard; ethyl acetate as extraction solvent; a Hypersil-Keystone Beta Basic-18 reversed-phase column; and a gradient mobile phase of consisting 0.1% formic acid in water and methanol-acetonitrile (3:49, v/v). Mass detection is performed on a single quadrupole mass spectrometer equipped with an electrospray ionization (ESI) interface and operated in a positive ionization mode. MI in the microsomal incubates was quantitated by computing the peak area ratio (MI/IS) analyzed in single ion monitoring (SIM) mode (m/z: 804 and m/z: 901 for MI and IS, respectively). Precision of the assay was determined by calculating the intra-run and inter-run variation at three concentrations (15, 25, 80 ng/ml); the intra run relative standard deviation (R.S.D.) was less than 10% and ranged from 5.0 to 8.3%; and the inter-run R.S.D. was less than 10% and ranged from 4.6 to 9.6%. The limits of detection was 2 ng/ml. This assay has been used to evaluate the effect of three human immunodeficiency virus (HIV) protease inhibitors on the metabolism of tacrolimus in human liver microsomes.
Feng
Yan
Y
Department of Pharmaceutical Sciences, School of Pharmacy, School of Medicine, University of Pittsburgh, Pittsburgh, PA 15261, USA.
Zhang
Shimin
S
Poloyac
Samuel
S
Strom
Steve
S
Venkataramanan
Raman
R
D002853:Chromatography, Liquid / Q000379:methods*; D017320:HIV Protease Inhibitors / Q000494:pharmacology; D006801:Humans; D008862:Microsomes, Liver / Q000187:drug effects / Q000378:metabolism*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D016559:Tacrolimus / Q000031:analogs & derivatives* / Q000032:analysis / Q000378:metabolism
C503135:13-O-demethyl tacrolimus; D017320:HIV Protease Inhibitors; D016559:Tacrolimus