Multi-targeted Screening of Phytoestrogens in Food, Raw Material, and Dietary Supplements by Liquid Chromatography with Tandem Mass Spectrometry Ana Mornar 1 & Tajana Buhač 1 & Daniela Amidžić Klarić 1 & Ilija Klarić 2 & Miranda Sertić 1 & Biljana Nigović 1 Received: 19 July 2019 /Accepted: 22 September 2019 # Springer Science+Business Media, LLC, part of Springer Nature 2019 Abstract This work was carried out to develop a high-performance liquid chromatography tandem mass spectrometry method for simul- taneous determination of 11 marker compounds in phytoestrogen-rich food and dietary supplements (raw materials and formu- lated products) prepared from various sources: soy, red clover, chastetree, hop, and flax. The method was fully validated and showed detection limits varying from 0.2 to 1.5 μg/mL, average recoveries ranging from 95 to 105%, and relative standard deviation values lower than 4.98%. Evidently lower amounts of phytoestrogens were found in food samples than in the dietary supplements. Calculations for the actual daily dose of total phytoestrogens ranged from 0.01 to 6.61 mg among the different products. All derived total phytoestrogen amounts turned out to be below the total content stated by manufacturers (less than 63% of the claimed specifications). Moreover, two samples did not contained phytoestrogens at all. The most eye-catching batch-to- batch deviations were represented by chasteberry-based products (relative standard deviation higher than 9.8%). Keywords Phytoestrogens . Multi-target analysis . Liquid chromatography tandem mass spectrometry . Dietary supplements Introduction Phytoestrogens are non-steroidal polyphenolic chemical enti- ties, which can be divided into several sub-groups according to their chemical structure. The most studied phytoestrogens are isoflavones, prenylflavonoids, other flavonoids, and lignans. They have been extensively studied for many years in the scope of revealing protective effect against a number of complex diseases, including for instance reduced post- menopausal symptoms. Soybeans, red clover, chastetree, hop, and flax have all been found to contain a wide range of phytoestrogenic compounds, and a large number of dietary supplements contain their extracts as principal ingredients. However, clinical studies evaluating the efficacy of these die- tary supplements have produced very different results (Messina 2014; Glisic et al. 2018). It is possible that findings from several studies may have been misinterpreted, because of the use of unstandardized dietary supplements. Taking into consideration these assumptions, it is of great importance to establish selective, sensitive, and accurate analytical method for assay of phytoestrogens in dietary supplements to ensure the reliability of pharmacological and clinical research, to un- derstand their beneficial effects to human health, as well as to enhance product quality control and assessment. A wide range of analytical methods for determination of phytoestrogens in dietary supplements have been reported. The techniques used can be classified as chromatographic and non-chromatographic. High-performance liquid chroma- tography (HPLC), as the main tool in the former group, com- bined with different detection systems, has been the technique * Ana Mornar amornar@pharma.hr Tajana Buhač tajana.buhac@zg.t-com.hr Daniela Amidžić Klarić daniela.amidzic@gmail.com Ilija Klarić klaric67@gmail.com Miranda Sertić msertic@pharma.hr Biljana Nigović bnigovic@pharma.hr 1 Faculty of Pharmacy and Biochemistry, University of Zagreb, Ante Kovačića 1, 10 000 Zagreb, Croatia 2 Public Health Brčko DC, R. Dž. Čauševića 1, Brčko DC, Bosnia and Herzegovina https://doi.org/10.1007/s12161-019-01653-x Food Analytical Methods (2020) 13:482–495 /Published online: 21 2019 November