性激素,包括雄激素,雌激素,和孕激素,是各种疾病的重要生物标志物。性激素的定量通常通过LC-MS/MS进行。目前,大多数方法需要液-液萃取或固相萃取进行样品制备。然而,这些预处理容易损害LC-MS/MS通量。为了改进当前的标准做法,我们研究了冷诱导的性激素提取相分离。用乙腈沉淀蛋白质并用水调节溶液组成后,将样品在-30°C下储存10分钟以产生两个不同的相:在富含水的层之上的富含乙腈的层。在这个过程中,疏水性激素自发地分离到上层。这种简单可靠的基于冷诱导相分离的LC-MS/MS方法用于同时检测雌酮,雌二醇,雌三醇,睾丸激素,雄烯二酮,脱氢表雄酮,黄体酮,和血清中的17-羟孕酮。该方法的验证表明性能令人满意,包括可接受的线性度,准确度,精度,和可操作性。与主流液液萃取法相比,这种新方法在吞吐量方面表现出显著的进步,这将样品制备的时间成本从90分钟缩短到40分钟。我们建议这种方法可以成为常规临床实验室中性激素分析的绝佳替代方法。
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.