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
ABSTRACT 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. "
ABSTRACT: Glucuronidation and sulfation represent two major pathways in phase II drug metabolism in humans and other mammalian species. The great majority of drugs, for example, polyphenols, flavonoids and anthraquinones, could be transformed into sulfated and glucuronidated conjugates simultaneously and extensively in vivo. The pharmacological activities of drug conjugations are normally decreased compared with those of their free forms. However, some drug conjugates may either bear biological activities themselves or serve as excellent sources of biologically active compounds. As the bioactivities of drugs are thought to be relevant to the kinetics of their conjugates, it is essential to study the pharmacokinetic behaviors of the conjugates in more detail. Unfortunately, the free forms of drugs cannot be detected directly in most cases if their glucuronides and sulfates are the predominant forms in biological samples. Nevertheless, an initial enzymatic hydrolysis step using β-glucuronidase and/or sulfatase is usually performed to convert the glucuronidated and/or sulfated conjugates to their free forms prior to the extraction, purification and other subsequent analysis steps in the literature. This review provides fundamental information on drug metabolism pathways, the bio-analytical strategies for the quantification of various drug conjugates, and the applications of the analytical methods to pharmacokinetic studies. Copyright © 2013 John Wiley & Sons, Ltd.Biomedical Chromatography 10/2013; DOI:10.1002/bmc.2912 · 1.66 Impact Factor
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ABSTRACT: S-equol is a natural product that is produced by the microbial biotransformation of daidzein, an isoflavone found in soy. Evidence suggests that the health benefits of soy may be related to one's ability to produce S-equol, thus S-equol is being developed for the treatment of vasomotor symptoms in postmenopausal women. The toxicokinetics of S-equol were evaluated in Sprague-Dawley rats and cynomolgus monkeys; S-equol was rapidly absorbed with C(max) occurring between 0.5 h and 1.0 h in the rat and 3h in the monkey. AUC was linear over the doses tested with no differences between male and female animals. Conjugated S-equol was the major metabolite in plasma with less than 1% present as the unconjugated form. S-equol showed a weak induction of liver cytochrome P450s in vivo, and did not significantly inhibit the major human cytochrome P450s in vitro. S-equol was highly protein bound (>95%) in rat, monkey and man in a concentration-independent manner. Orally administered S-equol did not significantly change uterine weight or morphology in either the rat or monkey even at the highest doses tested. These studies show that S-equol has pharmacokinetic parameters suitable for drug development with a low potential for uterotropic effects.Food and chemical toxicology: an international journal published for the British Industrial Biological Research Association 03/2012; 50(5):1741-8. DOI:10.1016/j.fct.2012.02.039 · 2.61 Impact Factor
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ABSTRACT: This study performed quantitative identifications of phytoestrogenic compounds, such as free and conjugated isoflavones, lignans, coumestrol and various flavonoids, on six different vegetable samples by a triple quadrupole liquid chromatography-tandem mass spectroscopy (LC-MS/MS) technique following different pretreatments, such as conventional extraction (CE), acid hydrolysis (AH), enzymatic hydrolysis (EH) and enzymatic and acid hydrolysis (EAH). A comparison of sample preparation methods used as pretreatment revealed that the EH method was more effective for the identification of total isoflavone content, and the EAH method was more effective for identification of the total lignans, other flavonoids and total phytoestrogenic compound content in vegetables. It was found that secoisolariciresinol (235.5-913.8 mu g/100 g vegetable, wet weight) was the major phytoestrogenic compound in all vegetable samples. Considering the sample preparation methods that determined the highest concentration values, the total amount of phytoestrogenic compounds in green bean, carrot, cauliflower, white cabbage, iceberg lettuce and artichoke were found to be 921.7, 239.6, 228.3, 349.5, 252.7 and 259.2 mu g/100 g (wet weight), respectively.Journal of Food Composition and Analysis 05/2012; 26(1-2). DOI:10.1016/j.jfca.2012.01.002 · 2.26 Impact Factor