Age and Sex Effects of a Validated LC-MS/MS Method for the Simultaneous Quantification of Testosterone, Allopregnanolone, and Its Isomers in Human Serum

Overview

Journal Sci Rep

Specialty Science

Date 2024 Nov 13

PMID 39537691

Authors

Khalisa Amir Hamzah

Leisa-Maree Toms

Nathaniel Kucharski

Julia Orr

Peter Hobson

David S Nichols

Luke J Ney

Affiliations

Soon will be listed here.

Abstract

Despite the great relevance of the neurosteroid allopregnanolone and related isomers to various health conditions, quantification typically involves immunoassay, which suffers from serious issues with cross-reactivity of closely related molecules. This article describes the development and partial validation of a liquid chromatography coupled with tandem mass spectrometry assay for the simultaneous quantification of allopregnanolone, pregnanolone, isopregnanolone, epi-allopregnanolone, and testosterone in the human serum of healthy males and females aged 5-85 years. 1-amino-4-methylpiperazine (AMP) was used as a derivatisation reagent to enhance the ionisation signal. Linearity was calculated at 0.99 with a lower limit of quantification of 10.08 pg/mL for allopregnanolone, along with a linearity of 0.98 and a lower limit of quantification of 42.32 pg/mL for testosterone. Application of the method showed sex and age effects across the lifespan for both allopregnanolone and testosterone, whereas a comparative immunoassay for allopregnanolone was not able to detect differences in the same samples. Our partial validation of this method should provide a new tool for researchers to discover the role of allopregnanolone and its isomers in human health, and how it compares to testosterone and sex hormones.

References

Parker C . Radioimmunoassay. Annu Rev Pharmacol Toxicol. 1981; 21:113-32. DOI: 10.1146/annurev.pa.21.040181.000553. View

Mayne G, De Bloois E, Dabelea D, Christians U . Development and validation of an LC-MS/MS assay for the quantification of allopregnanolone and its progesterone-derived isomers, precursors, and cortisol/cortisone in pregnancy. Anal Bioanal Chem. 2021; 413(21):5427-5438. DOI: 10.1007/s00216-021-03523-0. View

Pineles S, Nillni Y, Pinna G, Webb A, Arditte Hall K, Fonda J . Associations between PTSD-Related extinction retention deficits in women and plasma steroids that modulate brain GABA and NMDA receptor activity. Neurobiol Stress. 2020; 13:100225. PMC: 7256058. DOI: 10.1016/j.ynstr.2020.100225. View

Ke Y, Gonthier R, Labrie F . A sensitive and accurate LC-MS/MS assay with the derivatization of 1-Amino-4-methylpiperazine applied to serum allopregnanolone, pregnenolone and androsterone in pre- and postmenopausal women. Steroids. 2016; 118:25-31. DOI: 10.1016/j.steroids.2016.11.007. View

Almeida F, Pinna G, Barros H . The Role of HPA Axis and Allopregnanolone on the Neurobiology of Major Depressive Disorders and PTSD. Int J Mol Sci. 2021; 22(11). PMC: 8197074. DOI: 10.3390/ijms22115495. View

Ney L, Gogos A, Hsu C, Felmingham K . An alternative theory for hormone effects on sex differences in PTSD: The role of heightened sex hormones during trauma. Psychoneuroendocrinology. 2019; 109:104416. DOI: 10.1016/j.psyneuen.2019.104416. View

van Mourik L, Toms L, He C, Banks A, Hobson P, Leonards P . Evaluating age and temporal trends of chlorinated paraffins in pooled serum collected from males in Australia between 2004 and 2015. Chemosphere. 2020; 244:125574. DOI: 10.1016/j.chemosphere.2019.125574. View

Lionetto L, de Andres F, Capi M, Curto M, Sabato D, Simmaco M . LC-MS/MS simultaneous analysis of allopregnanolone, epiallopregnanolone, pregnanolone, dehydroepiandrosterone and dehydroepiandrosterone 3-sulfate in human plasma. Bioanalysis. 2017; 9(6):527-539. DOI: 10.4155/bio-2016-0262. View

Pineles S, Nillni Y, Pinna G, Irvine J, Webb A, Arditte Hall K . PTSD in women is associated with a block in conversion of progesterone to the GABAergic neurosteroids allopregnanolone and pregnanolone measured in plasma. Psychoneuroendocrinology. 2018; 93:133-141. DOI: 10.1016/j.psyneuen.2018.04.024. View

10.

Toms L, Harden F, Paepke O, Hobson P, Ryan J, Mueller J . Higher accumulation of polybrominated diphenyl ethers in infants than in adults. Environ Sci Technol. 2008; 42(19):7510-5. DOI: 10.1021/es800719v. View

11.

Matuszewski B, Constanzer M, Chavez-Eng C . Strategies for the assessment of matrix effect in quantitative bioanalytical methods based on HPLC-MS/MS. Anal Chem. 2003; 75(13):3019-30. DOI: 10.1021/ac020361s. View

12.

Higashi T, Aiba N, Tanaka T, Yoshizawa K, Ogawa S . Methods for differential and quantitative analyses of brain neurosteroid levels by LC/MS/MS with ESI-enhancing and isotope-coded derivatization. J Pharm Biomed Anal. 2015; 117:155-62. DOI: 10.1016/j.jpba.2015.08.040. View

13.

Im E, Lew B, Lee M, Lee J, Paeng K, Chung B . Simultaneous determination of androgens and prostaglandins in human urine using ultra-high-performance liquid chromatography-tandem mass spectrometry. J Chromatogr B Analyt Technol Biomed Life Sci. 2019; 1109:45-53. DOI: 10.1016/j.jchromb.2019.01.022. View

14.

Rasmusson A, Pinna G, Paliwal P, Weisman D, Gottschalk C, Charney D . Decreased cerebrospinal fluid allopregnanolone levels in women with posttraumatic stress disorder. Biol Psychiatry. 2006; 60(7):704-13. DOI: 10.1016/j.biopsych.2006.03.026. View

15.

De Nicola A, Meyer M, Garay L, Kruse M, Schumacher M, Guennoun R . Progesterone and Allopregnanolone Neuroprotective Effects in the Wobbler Mouse Model of Amyotrophic Lateral Sclerosis. Cell Mol Neurobiol. 2021; 42(1):23-40. PMC: 11441202. DOI: 10.1007/s10571-021-01118-y. View

16.

Ney L, Felmingham K, Bruno R, Matthews A, Nichols D . Simultaneous quantification of endocannabinoids, oleoylethanolamide and steroid hormones in human plasma and saliva. J Chromatogr B Analyt Technol Biomed Life Sci. 2020; 1152:122252. DOI: 10.1016/j.jchromb.2020.122252. View

17.

Evans J, Sun Y, McGregor A, Connor B . Allopregnanolone regulates neurogenesis and depressive/anxiety-like behaviour in a social isolation rodent model of chronic stress. Neuropharmacology. 2012; 63(8):1315-26. DOI: 10.1016/j.neuropharm.2012.08.012. View

18.

Karrman A, Mueller J, van Bavel B, Harden F, Toms L, Lindstrom G . Levels of 12 perfluorinated chemicals in pooled australian serum, collected 2002-2003, in relation to age, gender, and region. Environ Sci Technol. 2006; 40(12):3742-8. DOI: 10.1021/es060301u. View

19.

Pibiri F, Nelson M, Guidotti A, Costa E, Pinna G . Decreased corticolimbic allopregnanolone expression during social isolation enhances contextual fear: A model relevant for posttraumatic stress disorder. Proc Natl Acad Sci U S A. 2008; 105(14):5567-72. PMC: 2291140. DOI: 10.1073/pnas.0801853105. View

20.

van Broekhoven F, Verkes R . Neurosteroids in depression: a review. Psychopharmacology (Berl). 2002; 165(2):97-110. DOI: 10.1007/s00213-002-1257-1. View