Recent findings indicate that soy isoflavones and their metabolites may play a role in mitigating postmenopausal bone loss. equol using LC-MS/MS. The maximum plasma concentration (= 6 pairs) and dietary equol (= 6 pairs). Animals were matched on the basis of body weight and pairs were selected to ensure similar body weight average across treatment groups. Chemicals Daidzein was Mubritinib obtained from a commercial source (Indofine Chemical Co. Hillsborough NJ USA). Racemic equol (50% and 4 °C. The supernatants were used for analysis. Total unconjugated equol free equol equol monosulfate and equol disulfate concentrations were identified and quantified using a highly sensitive and specific electrospray ionization liquid chromatography-multiple reaction ion monitoring (MRM) mass spectrometry method as previously described37 with chromatography conditions detailed in earlier work35 and with the following modifications: equol monosulfate and disulfate were detected using their precursor to product ion transitions of 321/121 and 401/321 respectively. Identification and quantitation of equol and its sulfate conjugates were determined on the basis of comparison of MS/MS fragmentation pattern and retention time to reference material. LC-MS/MS method did not distinguish between 417/241 transition and concentrations were determined using the following Mubritinib equation which assumes that only equol sulfate and glucuronide conjugates are present: 1 The limit of detection for all analytes of interest was 5 nM. All parameters for metabolite identification including retention times and transitions are listed in Table 1. To validate calculation for equol glucuronide conjugates and ensure no other metabolites were present a control plasma sample was analyzed along with a sample from the dietary equol group. All pertinent transitions were monitored. Representative chromatograms of equol glucuronide and equol sulfates are shown in Figures ?Figures33 and ?and44. Figure 3 MS/MS chromatogram of control sample and product ion mass spectra of plasma samples from rats fed dietary equol (2 mg/mL). The presence of equol glucuronides was determined by monitoring the 417/241 transition using a triple-quadrupole mass spectrometer … Figure 4 Representative MS/MS chromatogram of a 5 μM isoflavone sulfate standard containing equol monosulfate and disulfate. Table 1 Equol Metabolites Detected in Plasma of Ovarectiomized Rats Fed a Single Oral Dose of Dietary Daidzein (= 4 Pairs 10 mg/mL) or Equol (= 5 Pairs 2 mg/mL) Pharmacokinetic Parameters The following pharmacokinetic parameters were determined using noncompartmental methods (WinNonlin Pro version 4.01 Pharsight Corp. Mountain View CA USA): = 4-6 Pairs)a Table 3 Pharmacokinetic CD80 Parameters of Equol Metabolites from a Single Oral Gavage of Dietary Equol Administered to Ovariectomized Rats (= 5-6 Pairs)a There were significantly higher maximum concentrations of all equol metabolites from dietary racemic equol compared to = 6 pairs) (10 mg/mL) or equol (= 6 pairs) (2 mg/mL) for plasma (A) total aglycone equol (B) unconjugated or free equol Mubritinib (C) equol monosulfate … The first appearance of equol metabolites (total equol and equol glucuronides) after administration of dietary daidzein occurred around 7 Mubritinib h (Figure ?(Figure5A D).5A D). This corresponds to findings from previous studies.15 17 38 In the dietary daidzein treatment group total equol unconjugated equol and equol glucuronide levels from dietary daidzein rose between 8 and 13 h and all metabolites peaked between 20 and 24 h consistent with earlier work assessing the bioavailability of daidzein and genistein conjugates in rats.15 None of the equol metabolites reached a plateau after 24 h (Figure ?(Figure5).5). There were few data to accurately assess HL CL/F and V/F of equol metabolite produced from dietary daidzein. Both findings suggest that a longer time frame is needed to adequately assess the pharmacokinetic profile of equol metabolites from daidzein consumption. Our results indicate there were higher levels of circulating equol metabolites with dietary racemic equol compared.