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Sex steroids are involved in many physiological and pathological processes. The determination of sex steroids is essential to understand the mechanisms of human health and diseases. Derivatization techniques could specifically enhance the sensitivities for sex steroids with a given functional group. However, no derivatization reagents are available for profiling multifunctional sex steroids, including phenolic estrogens, ketolic androgens, and ketolic progestogens, in a single analytical run. In the present study, a novel method involving ultraperformance liquid chromatography coupled with triple quadrupole mass spectrometry (UPLC-MS/MS) was developed for profiling both ketolic and phenolic sex steroids in human serum using an automated injection program combined with diverter valve switch and step analysis (AIDSA). The human serum, prepared through liquid-liquid extraction and subsequently derivatized using Girard P offline, was automatically injected twice under the automated injection program. For the first injection, Girard P-derivatized ketolic sex steroids were loaded onto the column, and subsequently, the second injection and online derivatization of the same sample using dansyl chloride were performed in the injector needle for 15 min. The dansyl-labeled phenolic sex steroids were then loaded onto the column. The diverter valve worked in coordination with the injection program to import the derivatized sex steroids and remove excess derivatization reagents. The two types of derivatives were individually analyzed in a step-by-step manner. In addition, online dansyl derivatization and Girard P derivative analyses were simultaneously implemented to shorten the total analysis time. This method was well validated and applied to determine the sex steroid levels in human serum.

analytical chemistry

Ultraperformance Liquid Chromatography–Tandem Mass Spectrometry Method for Profiling Ketolic and Phenolic Sex Steroids Using an Automated Injection Program Combined with Diverter Valve Switch and Step Analysis