Ultra-high-performance liquid chromatography-tandem mass spectrometry for the analysis of free and conjugated natural estrogens in cow milk without deconjugation

Abstract

A sensitive liquid chromatography/electrospray ionization-tandem mass spectrometry method for the determination of free and conjugated estrogens in cow milk is described. Milk samples were diluted with water and then extracted and cleaned up by solid-phase extraction using graphitized carbon black as adsorbent material, without any enzymatic cleavage, derivatization, and/or protein precipitation step. Two fractions were collected (free and conjugated estrogens) and analyzed separately. Mass spectrometry analysis was performed in negative ion mode using selected reaction monitoring acquisition mode. For all compounds, the coefficients of determination (R(2)) ranged between 0.9892 and 0.9997. Analytical recoveries were in the range of 86-109% for free estrogens and 85-118% for conjugates, with relative standard deviations below 10%, and the method detection limit ranged between 3 and 80 ng L(-1). Finally, the developed method was used to determine the presence of free and conjugated estrogens in six retail milk samples (five cow milk samples and one goat milk sample) and one goat milk sample provided by a local shepherd. Estrone was found to be the major free estrogen present in commercial milk samples, and estrone 3-sulfate showed the highest concentration among the target conjugated estrogens. Estriol was also observed in some analyzed milk samples, but the concentrations were always below the limit of quantification.

Analysis of Naturally Occurring Steroid Hormones in Infant Formulas by HPLC-MS/MS and Contribution to Dietary Intake

“…This approach was preferred in order to simplify the analysis and also because of the relatively high proportion of conjugated species of steroid hormones existing in milk [25,26,27]. The conjugated forms are not biologically active, but they can be transformed into their free active form in the human gut.…”

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

“…Taking into account that our group has already successfully employed GCB as adsorbent in SPE of free and conjugated estrogens and estrogens and mycoestrogens from milk , we decided to test GCB MNPs for the extraction of mycoestrogens from milk.…”

Recent advancements in analytical methods for the determination of steroidal estrogen residues in environmental and food matrices

“…5 It remains among the most widely used techniques and many publications can be found in the recent literature which utilize mass spectrometric determination of estrogens. 40,43 Methods using liquid chromatography with UV and fluorescence detections are also reported in recent literature. 40,43 Methods using liquid chromatography with UV and fluorescence detections are also reported in recent literature.…”

“…16,22,[24][25] Fluorescence is a highly sensitive detection method, and yet it suffers from matrix interferences when it comes to complicated environmental samples. [40][41][42][43] Even though MS is a well-established detection method, it still provides room for innovation. 12,17 Hyphenated tandem mass spectrometric determination is considered as the most popular MS method for the determination of estrogens in environmental and food samples.…”