Quantification of Steroids in Human Plasma

Liquid chromatography-mass spectrometry (LC-MS) methods for quantification of steroids in plasma typically use triple-quadrupole MS. How does LC-MS analysis using high resolution (HR) technology compare?

Overview: Steroid hormones are endogenous metabolites that are synthesized by various enzymes from cholesterol. The steroid backbone of cholesterol does not contain amino groups which are routinely derivatized to enhance compound detectability during analysis. Various ionization sources have been used to determine steroid hormones, with triple-quadrupole MS the most common. There is evidence that LC-MS analysis using high resolution (HR) technology can produce analyses of drugs and peptides that are as sensitive and accurate as triple-quadrupole MS.

Method: Eight steroids were quantitatively analyzed for clinical research purposes using a Thermo Scientific™ Q Exactive™ Focus MS. Plasma samples were prepared by centrifugation of donor whole blood withdrawn in heparin tubes. Two quality control samples were prepared, one female plasma and one male plasma. Plasma samples (100 μL) were diluted with 100 μL of 5% phosphoric acid containing the internal standards in water. LC-HRMS analyses were performed in HR full scan acquisition, allowing an overview of all ions such as steroid metabolites.

Name Formula m/z theor
[M+H]+
RT
[min]
Lower Cs
[nM]
Upper Cs
[nM]
Cortisol C21H30O5 363.21660 8.5 2.19 1400
Corticosterone C21H30O4 347.22169 9.8 0.47 60
11-Deoxycortisol C21H30O4 347.22169 10.1 0.16 20
Deoxycortisol C21H30O3 331.22677 11.8 0.16 20
Androstenedione C19H26O2 287.20056 10.7 0.31 40
Testosterone C19H28O2 289.21621 11.3 0.13 80
17-hydroxyprogesterone C21H30O3 331.22677 11.2 0.16 20
Progesterone C21H30O2 315.23186 13.3 0.50 160

Results: The Q Exactive Focus MS demonstrated precision and sensitivity comparable to triple quadrupole MS instruments. Various steroid isomers can be detected in full scan but also in MS/MS acquisition, showing that there is a need for an efficient chromatographic system. With sensitive and selective full scan data, the Q Exactive Focus HRMS can be used for more in-depth investigations when requested by researchers or biochemists.

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