University of Oulu

Heidi Hautajärvi, Janne Hukkanen, Miia Turpeinen, Sampo Mattila, Ari Tolonen, Quantitative analysis of 4β- and 4α‑hydroxycholesterol in human plasma and serum by UHPLC/ESI-HR-MS, Journal of Chromatography B, Volumes 1100–1101, 2018, Pages 179-186, ISSN 1570-0232,

Quantitative analysis of 4β- and 4α‑hydroxycholesterol in human plasma and serum by UHPLC/ESI-HR-MS

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Author: Hautajärvi, Heidi1,2; Hukkanen, Janne3; Turpeinen, Miia3;
Organizations: 1Admescope Ltd., Typpitie 1, 90620 Oulu, Finland
2Faculty of Technology University of Oulu, POB 4300, 90014 Oulu, Finland
3Medical Research Center Oulu, Oulu University Hospital and University of Oulu, Oulu, Finland
Format: article
Version: accepted version
Access: open
Online Access: PDF Full Text (PDF, 1.3 MB)
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Language: English
Published: Elsevier, 2018
Publish Date: 2020-10-02


Cholesterol oxidation product 4β‑hydroxycholesterol (4β‑OHC) may possibly be used as an endogenous biomarker of CYP3A enzyme activity and as CYP3A4 is involved in the metabolism of approximately 50% of the drugs in clinical use, the monitoring of CYP3A activity by 4β‑OHC plasma or serum levels, may be of clinical significance. The plasma and serum concentrations of 4α‑hydroxycholesterol (4α‑OHC), an isomer of 4β‑OHC, increase during uncontrolled storage conditions and therefore serve as an indicator of proper handling of samples.

A sensitive and simple high-throughput method for the simultaneous quantification of both 4α‑OHC and 4β‑OHC in human plasma and serum was developed utilizing ultrahigh performance liquid chromatography coupled with high resolution mass spectrometry (UHPLC/ESI-HR-MS). The chromatographic analysis was carried out on a Waters HSS T3 C18 reversed phase column with a mobile phase composed of 0,1% formic acid with 200 mg/l sodium acetate, and methanol. 4β‑OHC and 4α‑OHC and also internal standard d7‑4β‑OHC were monitored using HR-MS as sodium adducts, which could not be used as a precursor ions in conventional tandem mass spectrometry methods due to their extensive stability in collision for MS/MS. The use of HR-MS detection enabled avoiding laborious sample derivatization, which is required with triple quadrupole mass spectrometer-based methods to achieve adequate analytical sensitivity for 4β‑OHC, as the underivatized molecule is otherwise poorly ionized to other molecular ions than sodium adduct. Chromatographic separation of 4α‑OHC and 4β‑OHC was obtained and confirmed with standard samples prepared in blank surrogate matrix. The lower limits of quantitation in the assay were 0.5 ng/ml for 4β‑OHC, and 2 ng/ml for 4α‑OHC. Endogenous levels of 4β‑OHC can vary between 10 and 100 ng/ml depending on the possible induction or inhibition of CYP3A4, whereas the levels of 4α‑OHC can vary between 5 and 100 ng/ml, depending on the storage conditions of the samples. Thus, the sensitivity of the assay developed allows for the simultaneous measurement of endogenous levels of 4α‑OHC and 4β‑OHC cost-effectively and with high throughput.

The method was successfully used for the determination of 4β‑OHC and 4α‑OHC concentrations in clinical plasma and serum samples collected before and after treatment with a known CYP3A4 inducer rifampicin. The endogenous levels in clinical human samples before treatment varied between 13.4 and 31.9 ng/ml for 4β‑OHC, and between 3.53 and 5.65 ng/ml for 4α‑OHC, and a three-fold increase in 4β‑OHC plasma levels was observed after the rifampicin treatment, while 4α‑OHC levels remained unaffected.

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Series: Journal of chromatography. B, Analytical technologies in the biomedical and life sciences
ISSN: 1570-0232
ISSN-E: 1873-376X
ISSN-L: 1570-0232
Volume: 1100-1101
Pages: 179 - 186
DOI: 10.1016/j.jchromb.2018.09.028
Type of Publication: A1 Journal article – refereed
Field of Science: 1182 Biochemistry, cell and molecular biology
Funding: This work was supported in part by the grant from the Emil Aaltonen Foundation and the Finnish Foundation for Cardiovascular Research, the Diabetes Research Foundation. HH and AT work for Admescope Ltd.
Copyright information: © 2018 Elsevier B.V. All rights reserved. This manuscript version is made available under the CC-BY-NC-ND 4.0 license