Validation of a sequential extraction and liquid chromatography-tandem mass spectrometric method for determination of...

Androgens are key mediators of prostate development and function, a role that extends to the development of prostate diseases such as benign prostatic hyperplasia (BPH) and prostate cancer. In prostate, DHT is the major androgen and reduction and glucuronidation are the major metabolic pathways for DHT elimination. A streamlined method for quantitation of dihydrotestosterone (DHT), 5α-androstan-3α,17β-diol (3α-diol), and 3α-diol glucuronide (diol-gluc) was established and validated for use with archived prostate tissue specimens to facilitate examination of the roles of the underlying metabolism. This involved a sequential 70/30 hexane/ethyl acetate (hex/EtOAc) extraction of steroids, followed by an ethyl acetate extraction for diol-gluc. Derivatization of the hex/EtOAc fraction with2-fluoro-1-methylpyridinium p-toluene-4-sulfonate (FMP) was used to enhance sensitivity for hydroxyl steroids and liquid chromatography-tandem mass spectrometry (LC-MS/MS) was utilized for analysis of both fractions. The method was validated with calibration standards followed by recovery assessment from spiked samples of BPH and normal prostate. Lower limits of quantitation (LLOQ) were 50 pg/g, 20 pg/g and 100 pg/g for DHT, 3α-diol and diol-gluc, respectively for extracts from 50mg equivalents of tissue. Prepared samples were stable for up to three weeks at 4 °C and 37 °C. The method provides excellent sensitivity and selectivity for determination of tissue levels of DHT, 3α-diol, and diol-gluc. Furthermore, this protocol can easily be extended to other hydroxyl steroids, is relatively straightforward to perform and is an effective tool for assessing steroid levels in archived clinical prostate samples.