Cold-induced phase separation for the simple and reliable extraction of sex hormones for subsequent LC-MS/MS analysis

Sex hormones, including androgens, estrogens, and progestogens, are important biomarkers for various diseases. Quantification of sex hormones is typically conducted by LC-MS/MS. At present, most methods require liquid-liquid extraction or solid phase extraction for sample preparation. However, these pretreatments are prone to compromise LC-MS/MS throughput. To improve on the current standard practices, we investigated cold-induced phase separation for sex hormone extraction. After protein precipitation with acetonitrile and adjusting the solution constitution with water, samples were stored at −30°C for 10 min to generate two distinct phases: an acetonitrile-rich layer on top of a water-rich layer. During this process, the hydrophobic sex hormones spontaneously separate into the upper layer. This simple and reliable cold-induced phase separation-based LC-MS/MS methodology was used here to simultaneously detect estrone, estradiol, estriol, testosterone, androstenedione, dehydroepiandrosterone, progesterone, and 17-hydroxyprogesterone in serum. Validation of this method indicated satisfactory performance, including acceptable linearity, accuracy, precision, and tractability. Compared with the mainstream liquid-liquid extraction-based method, this new method exhibits significant progress in throughput, which shortens the time cost of sample preparation from 90 to 40 min. We propose that this method can be an excellent alternative for sex hormone analysis in routine clinical laboratories.Supplementary key words: cold-induced phase separation, LC-MS/MS, sex hormone, steroid, solid phase extraction, acetonitrile, liquid-liquid extraction, dansyl chloride, derivatization, estradiol

Sex hormones include androgens, estrogens, and progestogens in vivo. In the past decade, these compounds have been utilized for early diagnosis of various diseases, such as infertility, polycystic ovary syndrome, breast cancer, and adrenal tumors (1, 2). Recently, their potential effect for severe acute respiratory syndrome coronavirus 2 treatment has attracted much attention (3). Thereupon, accurate quantification of sex hormones possesses essential value for diverse fields of clinical science.In modern clinical laboratories, LC-MS/MS has become the preferred platform for analyzing endogenous sex hormones (4). Numerous methodologies have been developed recently (5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16). As excellently summarized in the latest reviews, to achieve profiling of sex hormones, most, if not all, of the published methods introduced liquid-liquid extraction (LLE) or solid phase extraction (SPE) for sample preparation (17, 18). Taking our MS center as a typical example, the methods in use to detect sex hormones in serum samples are both accompanied with LLE (19, 20). The inherent nitrogen-drying process significantly compromises the method throughput. For improvement, we are constantly looking for other advanced extraction strategies.In 1994, Gu et al. (21) found that the homogenous solution of acetonitrile (ACN)/water was able to spontaneously separate into two phases after storing below −1.32°C, which finally resulted an ACN-rich layer on the top (organic phase) and a water-rich layer down below (aqueous phase). During such process, hydrophobic components would distribute into the organic phase, whereas hydrophilic targets would preserve in aqueous phase. In other words, an ACN/water-based “in situ LLE” can be well realized through a simple cooling down process. In the past few years, this mechanism (naming as “cold-induced phase separation (CIPS),” “low-temperature-induced phase separation,” “homogeneous liquid-liquid microextraction,” “low-temperature partition,” and others) has been mainly utilized for drug/pesticide-residue analysis in food/environmental science (22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32). However, its applicability for pretreatment of biotargets remains to be clarified.In this study, by importing CIPS for the extraction of sex hormones, we put forward a novel and efficient LC-MS/MS method to simultaneously quantify estrogens (estrone [E1], estradiol [E2], and estriol [E3]), androgens (testosterone [T], androstenedione [AD], dehydroepiandrosterone [DHEA]), and progestogens (progesterone [P] and 17-hydroxyprogesterone [17-OHP]) in serum. For extraction, samples are allowed to stand under subzero temperature after protein precipitation by ACN. Along with phase separation, targets are enriched into the upper layer owing to their hydrophobicity. As the components of this layer are ACN and water, it can be transferred for LC-MS/MS analysis directly. To the best of our knowledge, it is the very first time to realize sex hormone profiling without traditional LLE/SPE processes. Under optimized condition, counting derivatization and postcleanup procedure, the whole sample preparation can be completed within 40 min for this CIPS approach, which is much faster than previous methods (up to 90 min, as shown in “Conventional LLE-based method with slight modifications for sex hormone analysis” in the supplemental data section). We expect that the new method can be a promising alternative for daily sex hormone analysis in routine clinical laboratories.