Multiclass analytical method for the determination of natural/synthetic steroid hormones, phytoestrogens, and mycoestrogens in milk and yogurt

Abstract

Within this study, a new method enabling monitoring of various estrogenic substances potentially occurring in milk and dairy products was proposed. Groups of compounds fairly differing in physico-chemical properties and biological activity were analyzed: four natural estrogens, four synthetic estrogens, five mycoestrogens, and nine phytoestrogens. Since they may pass into milk mainly in glucuronated and sulfated forms, an enzymatic hydrolysis was involved prior to the extraction based on the QuEChERS methodology. For the purification of the organic extract, a dispersive solid-phase extraction (d-SPE) with sorbent C18 was applied. The final analysis was performed by ultra-high-performance liquid chromatography (UHPLC) coupled with triple quadrupole tandem mass spectrometry (MS/MS). Method recovery ranged from 70 to 120% with a relative standard deviation (RSD) value lower than 20% and limits of quantification (LOQs) in the range of 0.02-0.60 μg/L (0.2-6.0 μg/kg dry weight) and 0.02-0.90 μg/kg (0.2-6.0 μg/kg dry weight) for milk and yogurt, respectively. The new procedure was applied for the investigation of estrogenic compounds in 11 milk samples and 13 yogurt samples from a Czech retail market. Mainly phytoestrogens were found in the studied samples. The most abundant compounds were equol and enterolactone representing 40-90% of all estrogens. The total content of phytoestrogens (free and bound) was in the range of 149-3870 μg/kg dry weight. This amount is approximately 20 times higher compared to non-bound estrogens.

Cocktails of Mycotoxins, Phytoestrogens, and Other Secondary Metabolites in Diets of Dairy Cows in Austria: Inferences from Diet Composition and Geo-Climatic Factors

“…Phytoestrogens can act as endocrine disruptors, impairing reproductive functions, generating temporal infertility, and potentially reducing the productive efficiency of dairy herds [ 9 , 28 , 29 , 30 , 31 ]. Interestingly, mycoestrogens (such as ZEN, alternariol (AOH), and their modified forms) and PEs (such as isoflavones) have synergistic effects [ 32 , 33 , 34 ], which must be considered in the context of a complete risk assessment on livestock reproductive performance [ 35 , 36 , 37 , 38 ]. Fungal and plant growth as well as concentrations of secondary metabolites in the dietary components and finally in the complete rations are influenced by multiple factors such as plant species/varieties, infecting/colonizing fungal species/varieties, climatic conditions, geography, parasitic/symbiotic interactions, use of pesticides, and other agricultural practices utilized [ 39 , 40 , 41 , 42 , 43 , 44 , 45 , 46 ].…”

Multiresidue determination of estrogens in different dairy products by ultra-high-performance liquid chromatography triple quadrupole mass spectrometry

“…As it was indicated in the introduction section, only a few studies have been published regarding the evaluation of estrogenic compounds in milk or milk derivatives [6,7,[17][18][19][26][27][28][29] except for kefir samples, which has not been previously analyzed. Concerning cheese samples, it should be indicated that it has only been previously analyzed to determine phyto-, natural and synthetic estrogens, but not mycoestrogens [6,7,17,18].…”

“…The mobile phase used for the analysis of phyto-and mycoestrogens consisted of MeOH (A) and water (B), while for natural and synthetic estrogens MeOH/ACN (50/50, v/v) (A) and 2 mM ammonium hydroxide (B) were used, based on a previous study [29]. The gradient applied was the same for all groups of analytes: 10% A with a flow rate of 0.3 mL/min (initial), 10-40% A (0.5 min), 99.9% A with an increase of the flow until 0.4 mL/min (7.5 min), 99.9% A (2 min), 10% A with a decrease of the flow to 0.3 mL/min (2 min), 10% A (2 min).…”

“…also from matrices different than fruits or vegetables like mussels [24,25], sewage sludge [22] or fish [25], among others. However, up to now, and to the best of our knowledge, the QuEChERS method has only been used for the extraction of some estrogenic compounds from milk or dairy products such as yogurt in very few occasions [26][27][28][29]. However, none of them have determined such compounds in cheese or kefir samples using the QuEChERS method.…”

Mycotoxins, Phytoestrogens and Other Secondary Metabolites in Austrian Pastures: Occurrences, Contamination Levels and Implications of Geo-Climatic Factors

“…Negative effects of PEs on the reproduction of ruminants have been associated with pasture legumes such as clovers ( Trifolium spp) and lucerne/alfalfa ( Medicago sativa ) [ 27 ]. In the context of the reproductive performance of livestock, it seems important that co-occurrences of fungal metabolites and PEs are taken into consideration [ 29 , 30 ].…”