On-line sample preparation using restricted-access media in the analysis of the soy isoflavones, genistein and daidzein, in rat serum using liquid chromatography electrospray mass spectrometry

Soy isoflavones are the subject of many investigations in experimental anim als and humans regarding possible modulation of endocrine activity and chem oprevention of carcinogenesis. Genistein and daidzein, the principal biolog ically active isoflavones in soy, were measured using on-line solid-phase e xtraction (SPE) and liquid chromatography electrospray mass spectrometry (L C/ES-MS) detection in serum of rats consuming a common open-formula (NIH 31 ) chow that contained approximately 30 mu g each of genistein and daidzein per gram of feed and a specially designed 'soy-free' chow that contained ap proximately 60-fold lower isoflavones. The use of a restricted-access/rever se phase trap cartridge and automated column switching permitted rapid and robust analytical performance with many injections of plasma onto a reverse phase LC column, Enzymatic deconjugation and a single centrifugation step were the only sample preparation steps required, The limit of detection for the isoflavones, based on the MS responses observed in serum from male and female rats consuming the soy-free chow, was 0.020 mu M The method, which uses deuterated isoflavones as internal standards, was determined to be acc urate using spiked control serum (102-110% of added amounts) and precise us ing spiked control serum and incurred serum (<6% relative standard deviatio n), The average genistein and daidzein levels were determined in female (0. 62 and 0.25 mu M, respectively) and male rats (0.35 and 0.20 mu M, respecti vely) consuming the standard diet. The sex difference observed for serum ge nistein concentrations was statistically significant (p < 0.0001). These re sults underscore the potential impact of standard open-formula diets on the results from rodent bioassays of biological activity, Copyright (C) 2000 J ohn Wiley & Sons, Ltd.