Natural estrogens in dairy products: Determination of free and conjugated forms by ultra high performance liquid chromatography with tandem mass spectrometry

Abstract

Natural estrogens are synthesized by mammals in different amounts depending on the developmental stage and pregnancy/lactation period, and they may pass into milk, where they are mostly present as glucuronated and sulfated forms. In modern dairy practices, about 75% of milk is produced from pregnant cows; therefore, the amount of hormones that may pass into milk could be of concern. While estrogen determination in milk has been investigated in depth, the individual determination of estrogens and their conjugated forms in dairy products has not been fully addressed. The aim of this work was to develop and assess a sensitive method, using the peculiar retention properties of graphitized carbon black, to extract natural free estrogens and their major conjugated metabolites, without any enzymatic cleavage, from yogurt, cheese, and butter. The free and conjugated estrogens were eluted in two distinct fractions from the solid-phase extraction cartridge and analyzed separately by ultra high performance liquid chromatography coupled to tandem mass spectrometry. Recoveries were higher than 80% for all the three sample typologies. The highest matrix effects were observed for butter, which was richest in lipid content, but was below 30%. A survey on some commercial dairy products suggests that production processes decreased estrogen content.

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]. In this sense, Guoliang et al applied a simple solvent extraction procedure followed by HPLC-FD with a previous derivatization with ethyl-acridinesulfonyl chloride for the determination of E 1, E 2 and E 3 among other type of compounds.…”

“…However, no data related with the sensitivity of the methodology were indicated for this specific matrix. A similar approach was carried out by Chavaliere et al [18] who applied an ultrasound assisted solvent extraction using 1 g of cheese sample and a mixture of 5 mL of MeOH/water (80:20, v/v) containing 1% (v/v) trifluoroacetic acid as extraction solvent, followed by a SPE clean-up step using graphitized carbon black, for the extraction of…”

“…However, and probably because of the complexity of dairy products, the analysis of these compounds in such products has not been so widely tackled as for other simpler matrices like, for example, water samples [15,16]. In fact, only some phyto-, natural and synthetic estrogens have been analyzed in a few occasions in cheese samples (cow cheese) [6,7,[17][18][19], while the analysis of the presence of these groups of compounds in kefir samples has not been reported in the bibliography. In these studies, different extraction techniques were applied including liquid-liquid extraction (LLE) [7,19], LLE combined with solid-phase extraction (SPE) [16,18] and the application of miniaturized techniques as hollowfiber liquid-phase microextraction (HF-LPME) [17].…”

Mycoestrogen determination in cow milk: Magnetic solid‐phase extraction followed by liquid chromatography and tandem mass spectrometry analysis

“…The estimation of LOD and LOQ was conducted as already reported . Briefly, a first LOD and LOQ estimation was performed considering the SD of the intercept (σ) and the slope of the calibration graphs ( S ), according to the formulas LOD = 3 σ/S and LOQ = 10 σ/ S .…”

Dispersive liquid–liquid microextraction as an effective preanalytical step for the determination of estradiol in human urine

“…The most common and reliable analytical techniques for estrogen analysis are separation methods, in particular HPLC [13][14][15][16]. To a lesser extent, MEKC [17,18] and GC [19,20] are also reported.…”