Detection of Conjugated Soy Metabolites in Urinary and Tissue Samples after Methanol Extraction

^{HEBRON C. CHANG1,2*, JIAYUAN HAN1 AND RONALD L. PRIOR2}

1. Department of Biotechnology, Asia University, Taichung, Taiwan (R.O.C.)
2. Arkansas Children’s Nutrition Center, Little Rock, Arkansas, U. S. A.

(Received: August 11, 2008; Accepted: October 13, 2008)

ABSTRACT

   Glucuronide conjugates of soy isoflavone metabolites genistein, daidzein, glycitein and equol in urinary samples from women, female pigs and female rats were pretreated with methanol instead of solid phase extraction or liquid-liquid extraction after enzymatic digestion. The samples were analyzed using an LC-ESI-MS/MS ion trap system. Analysis of rat liver sample using this procedure was also attempted. By comparison the elution sequences with results from previous studies, glucuronide conjugates including 7-gluc-Dai, 7-gluc-Gly, 4’-gluc-Dai, 7-gluc-Gen, then 4’-gluc-Gen were identified sequentially in women urine. However, 5-gluc-Gen was also observed in female porcine urine. In rat urine, sul-Dai was observed after 7-gluc-Dai, while 7-gluc-Equ and 4’-gluc-Equ were observed after 7-gluc-Gen. The metabolite profiles in rat liver tissue samples were found with 7-gluc-Dai and 7-gluc-Gen. However, glucuronide conjugates of equol and sulfate conjugate of daidzein were only found in rat urine. Interestingly, quantification studies performed by LC-APCI/MS and LC-ECD showed that the aglycone and conjugated glycitein were both found in porcine and rat urine, but only conjugated glycitein was found in woman urine.

Key words: methanol pretreatment, urine, tissue, glucuronide conjugate