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

Abstract 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 (LLE) or solid phase extraction (SPE) 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 (CIPS) for sex hormone extraction. After protein-precipitation with acetonitrile (ACN) and adjusting the solution constitution with water, samples were stored at -30° C for 10 minutes to generate two distinct phases: an ACN-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 CIPS-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 to the mainstream LLE-based method, this new method exhibits significant progress in throughput, which shortens the time cost of sample preparation from 90 to 40 minutes. We propose that this method can be an excellent alternative for sex hormone analysis in routine clinical laboratories.