Method Development for Analysis of Phytoestrogens in Rat Serum by GC/MS-SIM

Jagroop Dahiya, Angelo DiCicco, Dayue Shang, Monica Dyck, Victor Verigin, Kenneth Breakell, Xiaoyan Jia and Helen Nicolidakis
Health Products & Food Branch
Health Canada Organic Residues Laboratory
3155 Willingdon Green, Burnaby V5G 4P2 British Columbia
and
G. Sarwar Gilani, Ivan Curran, and Gerard Cooke
Nutritional Research Division, Food Directorate
Health Products & Food Branch
2203C Banting Research Centre, Ross Avenue, Ottawa, Ontario K1A 0L2

Abstract

The amount of phytoestrogens in rat serum was measured by GC/MS-SIM. An isoflavone homologue, dihydroflavone (DHF) was used as the internal standard and was added to the rat serum samples before extraction and workup. After equilibration of the serum with 100 ng amounts of the internal standard, the sample was diluted with 10 volumes of 0.5 ml trimethyl- sulfate/L , and heated to 65 C before passage through a wetted solid phase C18 Bond Elute cartridge. The SPE cartridge was then washed with distilled water and the isoflavone and their conjugates were recovered by elution with methanol. The methanol extract was evaporated to dryness in SpeedVac, reconstituted with 0.5 ml acetate buffer (pH 4.5) and hydrolysed at 37 C overnight with 0.02 ml of Helix pomatia digestive juice, a mixed -glucouronidase-sulfatase preparation that had been passed through SPE cartridge to remove naturally occurring isoflavones in the enzyme preparation. After hydrolysis, isoflavones were isolated by SPE cartridge. Isoflavones TMS ethers were separated and quantified by GC/MS. Chromatographic separation was achieved on a DB-5 fused silica capillary column with helium as the carrier gas and with a temperature program from 260 to 300 C with increments of 10 C/min. Selected ion monitoring GC/MS of specific and charateristic ions in the electron ionization (70 eV) spectra of each isoflavone TMS ether derivatives permitted highly sensitive and specific quantification. The method was validated by repeated analysis of a pooled sample of serum obtained from healthy rats fed soy diets. Duplicate quality control samples were included with each analytic run, and one study sample selected at random was repeated within each analytic batch. The within day instrument reproducibility, expressed as %CV was 0.5% for daidzein, 1.0% for genistein, 0.5% for glycitein. The percent recovery in the samples spiked with standard mixture of crystalline isoflavones varied between 70 to 90 percent.