Absolute Quantification of Follicle Stimulating Hormone (FSH) Using Its Signature Peptides and Enzymatic Digestion in Human Serum by UPLC/LC–MS/MS

In vitro quantification of follicle stimulating hormone (a Macroheterogeneity protein) in human serum using mass spectrometry is challenging and as yet unreported due to its presence in low abundance and high matrix interferences. In the present work, we identified three signature peptides for quantification of FSH using LC–MS/MS and more importantly optimized the whole serum digestion procedure enabled by Solid phase extraction (SPE) for peptide extraction. Signature peptides “AYPTPLR”, “VTVMGGFK” and “ELVYETVR”, were identified as substantial and abundant sequences for maximum sensitivity and selectivity. Tryptic digestion procedure was optimized for Human serum samples spiked with FSH in various concentrations coupled with SPE protocol to enrich signature peptide for maximal recovery. The lower limit of quantification (LLOQ) of FSH using the MRM method was 25 mIU mL −1 , with a linear dynamic range of 25–5000 mIU mL −1 in human serum with a correlation coefficient of > 0.99. Precision and accuracy of the three batches were found to be 12.5% and 98.8% at LLOQ, 12.4% and 99.6% at low quality control (LQC), 12.5% and 97.7% at middle quality control (MQC), and 12.5% and 95.5% at high quality control (HQC), respectively. Signature peptide-based highly selective and sensitive LC–MS/MS method developed in human serum may open future applications for absolute quantification of FSH in clinical samples.