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

Charles River Laboratories Preclinical Services, Montreal, Canada.
Journal of pharmaceutical and biomedical analysis (Impact Factor: 2.98). 12/2010; 55(1):125-34. DOI: 10.1016/j.jpba.2010.12.031
Source: PubMed


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-d(4)). 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 0.025ng/mL to 10ng/mL (plasma) and 0.20ng/mL to 200ng/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 20mg dose of S-equol to three normal healthy volunteers.

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    • "The biological samples may sometimes have to be cleanedup and purified with the combination of the two or three above methods. Plomley et al. (2011) reported that the recovery of S-equol in the SPE method alone was only 30%. However, the recovery increased dramatically to 85% when the methods of PPT and SPE were combined. "
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