Comprehensive Profiling of Free and Conjugated Estrogens by Capillary Electrophoresis–Time of Flight/Mass Spectrometry

Abstract

The biological activity of estrogens is tightly regulated by regioselective phase I/II metabolic transformations that are critical to human health. Current methods for analysis of urinary estrogens are limited by complicated sample pretreatment and/or inadequate specificity for free estrogens and their glucuronide/sulfate conjugates that vary widely in their intrinsic polarity. In this work, direct speciation of intact estrogen conjugates and their regioisomers is demonstrated using capillary electrophoresis-time-of-flight/mass spectrometry (CE-TOF/MS) when using an alkaline buffer system with negative ion mode detection. This method allows for resolution of weakly acidic native estrogens, anionic estrogen conjugates and their positional isomers without significant matrix-induced ion suppression effects in human urine. Identification of unknown estrogen metabolites using CE-TOF/MS is supported by accurate mass together with their characteristic relative migration times, which can be predicted based on two intrinsic physicochemical properties of an ion. CE-TOF/MS offers a promising strategy for comprehensive profiling of estrogens and other classes of steroid conjugates that is needed for deeper insight into the etiology and treatment of chronic disorders associated with impaired estrogen metabolism.

Mass spectrometric based approaches in urine metabolomics and biomarker discovery

“…Therefore, in comparison to GC-MS and LC-MS, the number of investigations employing CE-MS in urine metabolome analysis is limited, with more applications in fingerprinting rather than the quantitative targeted platform (Ramautar et al, 2009;Ramautar et al, 2011;Ramautar, Somsen & de Jong, 2009, 2013. For example, a CE-ToF method was developed for the profiling and the targeted analysis of free estrogens and their glucuronide and sulfate conjugates (Kuehnbaum & Britz-McKibbin, 2011). In spite of adequately resolving free and conjugated estrogens in alkaline buffer, the method could not detect endogenous estrogens present in sub-micromolar concentrations in human urine due to inadequate sensitivity.…”

“…In spite of adequately resolving free and conjugated estrogens in alkaline buffer, the method could not detect endogenous estrogens present in sub-micromolar concentrations in human urine due to inadequate sensitivity. Moreover, the incorporation of an additional SPE step resulted in the removal of the conjugated forms from enriched premenopausal female urine extracts (Kuehnbaum & Britz-McKibbin, 2011).…”

Capillary electrochromatography and nano‐liquid chromatography coupled to nano‐electrospray ionization interface for the separation and identification of estrogenic compounds

“…In this respect, estrogens analysis has been described using conventional chromatographic techniques (GC and HPLC). On the contrary, only few studies have described the use of miniaturized techniques . In this sense, Kuehnbaum and Britz‐McKibbin developed a CE‐TOF/MS method to analyze free and conjugated estradiol (E 2 ) and estriol (E 3 ) in human urine.…”

“…On the contrary, only few studies have described the use of miniaturized techniques . In this sense, Kuehnbaum and Britz‐McKibbin developed a CE‐TOF/MS method to analyze free and conjugated estradiol (E 2 ) and estriol (E 3 ) in human urine. Sirén et al.…”

“…On the contrary, only few studies have described the use of miniaturized techniques [2,[15][16][17][18][19][20]. In this sense, Kuehnbaum and Britz-McKibbin [15] developed a CE-TOF/MS method to analyze free and conjugated estradiol (E 2 ) and estriol (E 3 ) in human urine. Sirén et al [16] used the partial filling technique in micellar electrokinetic chromatography (PF-MEKC) MS to determine metabolic profiles by analyzing endogenous low-hydrophilic steroids, including 17␤-E 2 .…”