Quantitation of anacetrapib, stable-isotope labeled-anacetrapib (microdose), and four metabolites in human plasma using liquid chromatography tandem mass spectrometry

• A sensitive and selective method for the simultaneous quantitation of anacetrapib (I) and microdosed [13C515N]-anacetrapib (II) in human plasma was developed and validated.
• Two qualified assays for four hydroxy metabolites of anacetrapib (M1–M4) in human plasma were evaluated and established.
• The validated and exploratory assays were used to support a clinical study to evaluate the absolute bioavailability, food effect, and multiple dose pharmacokinetics of anacetrapib.

An ultra-high performance liquid chromatography/tandem mass spectrometry (UPLC–MS/MS) method for the simultaneous determination of (4S,5R)-5-[3,5-bis (trifluoromethyl)phenyl]-3-{[4′-fluoro-5′-isopropyl-2′-methoxy-4-(trifluoromethyl)biphenyl-2-yl] methyl}-4-methyl-1,3-oxazolidin-2-one (anacetrapib, I) and [13C515N]-anacetrapib, II in human plasma has been developed to support a clinical study to determine the absolute bioavailability of I. The analytes and the stable-isotope labeled internal standard ([13C715N2H7]-anacetrapib, III) were extracted from 100 μL of human plasma by liquid–liquid extraction using 20/80 isopropyl alcohol/hexane (v/v). The chromatographic separation of the analytes was achieved using Waters BEH Shield RP 18 (50 × 2.1 mm × 1.7 μm) column and mobile phase gradient of 0.1% formic acid in water (Solvent A) and 0.1% formic acid in acetonitrile (Solvent B) at 0.6 mL/min flow rate. The MS/MS detection was performed on AB Sciex 5000 or AB 5500 in positive electrospray ionization mode, operated in selected reaction monitoring mode. The assay was validated in the concentration range 1–2000 ng/mL for I; and a lower curve range, 0.025–50 ng/mL for II.In addition to the absolute bioavailability determination, it was desired to better elucidate the pharmacokinetic behavior of several hydroxylated metabolites of I. Toward this end, two exploratory assays for the hydroxy metabolites of I were qualified in the concentration range 0.5–500 ng/mL. All metabolites were separated on a Supelco Ascentis Express Phenyl-Hexyl (50 × 2.1 mm, 2.7 μm) column. Metabolite M4 was analyzed in the negative mode with a mobile phase consisting of a gradient mixture of water (A) and acetonitrile (B). The other three metabolites, M1–M3 were analyzed in the positive mode using a mobile phase gradient of water with 0.1% formic acid (A) and acetonitrile with 0.1% formic acid (B).The assays were utilized to support a clinical study in which a microdosing approach was used to determine the pharmacokinetics of anacetrapib and its metabolites.