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A method has been developed for the analysis of phytoestrogens and their conjugates in human urine using liquid chromatography electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS). Stable isotopically labeled [13C3]daidzein and [13C3]genistein were synthesized and used as internal standards for isotope dilution mass spectrometry. Free aglycons and intact glucuronide, sulfate, diglucuronide, disulfate, and mixed sulfoglucuronide conjugates of isoflavones and lignans were observed in naturally incurred urine samples. Sample pretreatment was not necessary, other than addition of internal standards and pH adjustment. Urine was injected directly onto the analytical column. The limits of detection were generally <50 ng/ml, precision was generally <10% CV for conjugates. Total hydrolyzed daidzein and genistein were measured against quality assurance urine sample and were accurate to within 12%. The accuracy of conjugate measurement can not be ascertained, as no reference samples are available. The mean sum of daidzein and its conjugates was within 20% of the hydrolyzed value. Concentrations of the free aglycons of up to 22% of genistein and 18% of daidzein were observed. The average pattern was ca. 54% 7-glucuronide, 25% 4′-glucuronide, 13% monosulfates, 7% free daidzein, 0.9% sulfoglucuronides, 0.4% diglucuronide, and <0.1% disulfate. Selective enzymatic deconjugation with glucuronidase and mixed glucuronidase/sulfatase were used to validate the accuracy of the quantitation of the intact daidzein conjugates. There were no apparent sex differences, or conditioning effects on the conjugation profile of isoflavones after chronic dosing. [Copyright &y& Elsevier]

Intestinal nitrosation produces ATNCs (Apparent Total N-nitroso Compounds) and these have been linked with an increased risk of colon cancer from eating red meat. Modern LC-MS instrumentation makes direct detection of ATNC components in faecal water a possibility. The difficulty is in determining which of the many compounds present are N-nitrosamines before embarking on efforts to characterise them. We have assumed that any in vivo nitrosation of alimentary tract contents will be non-specific and depend on the amount and basicity of amine present, with concentration of nitrosating agent being the limiting factor. By further nitrosating faecal waters (and ileostomy fluids) we can increase the amount of ATNC and readily access these compounds. The amount and the number of nitrosamines generated depend on the concentration of individual amines present. By derivatisation separately using both 14N and 15N labelled nitrite, we demonstrated that inspecting chromatograms in parallel for a unit mass difference provides a novel and practicable means for identifying unknown ATNC in faecal and ileostomy samples. MS procedures were linked with the traditional approaches of thermal energy analyser (TEA), preparative HPLC, visualisation of nitrosamines with Griess reagent and degradation of N-nitroso compounds by UV irradiation. We have demonstrated that this approach is repeatable and have used it to identify 30 putative N-nitroso compounds (as protonated parent masses [M + H]+ at: 242, 258, 312, 313, 333, 348, 365, 377, 382, 386, 392, 412, 414, 421, 434, 442, 466, 467, 483, 493, 572, 582, 625, 636, 637, 656, 662, 752, 808, and 870.