A Heating-Assisted Integrated Purification-Enrichment-Derivatization Paper Chip for Rapid MS/MS Quantification of Serum Steroid Hormones.

Rapid and reliable simultaneous quantification of multiple steroid hormones is important for the diagnosis and monitoring of endocrine disorders such as polyendocrine metabolic ovarian syndrome (PMOS), formerly known as polycystic ovary syndrome (PCOS). Existing methods are susceptible to ion suppression in complex serum matrices and therefore usually require laborious sample preparation and time-consuming chromatographic separation, limiting their application in high-throughput screening and rapid testing. In this study, we developed an integrated purification-enrichment-derivatization paper chip, termed iPED chip, which integrates diatomaceous earth-assisted online cleanup at the tail end, thermal enrichment at the paper tip, and in situ hydroxylamine derivatization. A programmable induction heating device was also developed to provide a localized, controllable, and noncontact thermal field for the paper reaction zone, thereby promoting analyte enrichment and reaction. Coupled with tandem mass spectrometry, the iPED chip-MS/MS method enabled rapid simultaneous quantification of testosterone (T), androstenedione (4-AD), dehydroepiandrosterone (DHEA), and 17α-hydroxyprogesterone (17-OHP) in serum within 20 min. Method characterization demonstrated effective enrichment of the target hormones, broad linearity over 0.078-40 μg/L in spiked calf serum, low limits of detection of 0.042-0.093 μg/L, recoveries of 95-106%, and RSDs below 10%. In spiked hormone-depleted human serum samples at clinically relevant levels, the results agreed well with those obtained by conventional LC-MS/MS, supporting the reliability of iPED chip-MS/MS and its potential for rapid clinical testing and large-scale biofluid screening.