Abstract
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.
References
Oduwole M, Peltoketo H, Huhtaniemi IT (2018) Front Endocrinol (Lausanne) 9:763
Grass J, Pabst M, Chang M, Altmann F (2011) Anal Bioanal Chem 400(8):2427–2438
Das N, Kumar TR (2018) J Mol Endocrinol 60(3):R131
Almeida BE, Oliveira JE, Ribela MT (2011) J Pharm Biomed Anal 54(4):681–686
Tan X, David A, Day J (2018) ACS Sensors 3(11):2327–2334
Hoofnagle A, Wener M (2009) J Immunol Methods 347:3–11
Jeelani R, Bluth MH (2017) Henry’s clinical diagnosis and management by laboratory methods, 23rd edn. Elsevier, New York
Guidelines, ICH Harmonised (2019) Bioanalytical Method Validation M10. 1–58
Li H, Ortiz R, Tran LT (2013) AAPS J 15:337–346
Zhang Q, Spellman DS, Song Y (2014) Anal Chem 86:8776–8784
Russo R, Rega C, Caporale A (2017) Rapid Commun Mass Spectrom 31(14):1184–1192
Hakola K, Van der Boogaart P, Mulders J (2005) J Microbiol Biotechnol 15(2):395–402
Thomas A, Geyer H, Kamber M, Schänzer W, Thevis M (2008) J Mass Spectrom 43:908–915
van de Merbel NC (2019) Bioanalysis 11(7):629–644
Zheng N, Zeng J, Arnold ME (2016) Anal Chim Acta 15(916):42–51
Gillette MA, Carr SA (2013) Nat Methods 10(1):28–34
Fung EN, Bryan P, Kozhich A (2016) Bioanalysis 8(8):847–856
Practice Committee of American Society for Reproductive Medicine, Birmingham, Alabama (2008) Fertil Steril 90(5 Suppl):S13–S20
Author information
Authors and Affiliations
Corresponding author
Ethics declarations
Conflict of interest
F.R, B.R, M.D and P.M is an employee of SCIEX, which operates in the field covered by the article.
Additional information
Publisher's Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Supplementary Information
Below is the link to the electronic supplementary material.
Rights and permissions
About this article
Cite this article
Rashid, F., Baghla, R., Kale, P. et al. Absolute Quantification of Follicle Stimulating Hormone (FSH) Using Its Signature Peptides and Enzymatic Digestion in Human Serum by UPLC/LC–MS/MS. Chromatographia 84, 793–802 (2021). https://doi.org/10.1007/s10337-021-04057-4
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s10337-021-04057-4