Single-Laboratory Validation of UHPLC-MS/MS Assays for Red Clover Isoflavones in Human Serum and Dietary Supplements

Abstract

Background: Extracts of red clover (Trifolium pratense L.) containing estrogenic and pro-estrogenic isoflavones are used in dietary supplements primarily for the management of menopausal symptoms in women.

Objective: A UHPLC-MS/MS assay was developed and validated for the quantitative analysis of the six major red clover isoflavones in dietary supplements and in human serum in support of clinical trials.

Methods: Enzymatic deconjugation of isoflavone glucuronides and sulfate conjugates in human serum specimens was carried out followed by protein precipitation. Isoflavones in red clover dietary supplements were acid hydrolyzed to release aglycons from glycosides. UHPLC separations (< 4 min) were combined with MS/MS using collision-induced dissociation, selective reaction monitoring and deuterated internal standards to measure biochanin A, formononetin, daidzein, genistein, irilone, and prunetin.

Results: The method was validated with respect to selectivity, specificity, accuracy, linearity, precision, LOD, and LOQ. The calibration curves for all analytes were linear (R2 > 0.998). The mean recovery for low-, medium- and high-quality control standards ranged between 80% and 108%. The precision of the method was assessed using coefficients of variation, which were <15%.

Conclusions: The UHPLC-MS/MS method is fast, precise, sensitive, selective, accurate, and applicable to the quantitative analysis of red clover isoflavones in different matrices.

Highlights: This validated UHPLC-MS/MS assay is applicable to the rapid quantitative analysis of red clover isoflavones in human serum and in dietary supplements.

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Figures

Figure 1.

Chemical structures of red clover isoflavones and deuterated internal standards.

Figure 2.

UHPLC-MS/MS chromatograms of red clover isoflavones extracted from (A) hydrolyzed human serum obtained from a woman volunteer 5 h after consuming an ethanolic extract of the aerial parts of red clover that had been standardized to 120 mg isoflavones; (B) a commercial dietary supplement (RCC003); and (C) isoflavone standards spiked into blank human serum at the medium QC level.