Magnetic solid phase extraction followed by in-situ derivatization with core–shell structured magnetic graphene oxide nanocomposite for the accurate quantification of free testosterone and free androstenedione in human serum

Abstract

Circulating free androgens are important indicators for a variety of diseases, so accurate determination of these hormones in serum is of great clinical significance. However, there are still many challenges for the accurate quantification of free androgens using mass methods because of their very low levels and complex interferences in serum, as well as the high serum protein binding rate and high nonspecific binding (NSB) rate. Here, an HPLC–MS/MS method coupled with magnetic solid phase extraction (MSPE) and in-situ derivatization was developed to quantify two free androgens—free testosterone (FT) and free androstenedione (FA4)—in human serum simultaneously and accurately. Ultrafiltration was used to obtain free androgens in serum. To minimize the NSB rate and obtain accurate results, the ultrafiltration membrane was doubly modified with surfactant followed by a silane-coupling agent. Multiple pre-saturation with the tested samples was also used. With these strategies, the ultrafiltration recoveries were up to 95.4% for FT and 94.0% for FA4, so the NSB was negligible. After that, the extremely low levels of free androgens in ultrafiltrates were extracted and enriched using MSPE with core–shell structured ferroferric oxide coated with graphene oxide. Hydroxylamine hydrochloride was used to derivatize the analytes and the reaction took place on the surface of the adsorbent. All the extraction and derivatization conditions were optimized. Under such conditions, the assays were linear for FT within the range of 2–100 pg mL−1 and for FA4 within the range of 5–500 pg mL−1. The intra- and inter-run CV was less than 12.3% and 10.9% for FT and less than 7.2% and 8.3% for FA4, respectively. For the intra- and inter-run accuracies, the relative error of the mean was 9.9% and 8.4% for FT, and 11.5% and 7.3% for FA4, respectively. The total extraction recoveries with MSPE in-situ derivatization were 93.2% for FT, 93.8% for FA4 and 95.7% for the internal standard. The method was validated and was used to quantify the trace level of these two free androgens in serum samples from female patients suspected of having polycystic ovary syndrome (PCOS) accurately. It is expected to improve the diagnosis accuracy of PCOS when combined with other clinical indicators.