Development of chiral liquid chromatography–tandem mass spectrometry isotope dilution methods for the determination of unconjugated and total S-equol in human plasma and urine

Liquid chromatography–tandem mass spectrometry (LC–MS/MS) methods for the determination of unconjugated and total (conjugated plus unconjugated) S-equol in human plasma and urine were developed and validated. The separation of R and S enantiomers was achieved with a Chiracel OJ-H column operated in a normal phase mode using ethanol/hexane mobile phase components. Ionization of S-equol by negative ion electrospray generated the [M−H]− ion whose response was augmented by post-column addition of ammonium hydroxide. A triple stage quadrupole mass spectrometer was used to measure the ion current generated from the dissociative transitions m/z 241 → m/z 121 (S-equol) and m/z 245 → m/z 123 (equol-d4). The determination of total S-equol included an additional deconjugation step involving incubation of the sample with sulfatase and glucuronidase. Average recovery for both unconjugated and total S-equol was 85% with no observable matrix effects. Linearity was established for unconjugated S-equol from 0.025 ng/mL to 10 ng/mL (plasma) and 0.20 ng/mL to 200 ng/mL (urine). The average coefficient of variation and accuracy per occasion was within ±15% of the theoretical concentration of S-equol. The method was used to measure the pharmacokinetics of S-equol in human plasma after an oral administration of a single 20 mg dose of S-equol to three normal healthy volunteers.