Simple and Sensitive Method for the Quantitative Determination of Lipid Hydroperoxides by Liquid Chromatography/Mass Spectrometry

Chongsheng Liang; Siddabasave Gowda B Gowda; Divyavani Gowda; Toshihiro Sakurai; Iku Sazaki; Hitoshi Chiba; Shu-Ping Hui

doi:10.3390/antiox11020229

Simple and Sensitive Method for the Quantitative Determination of Lipid Hydroperoxides by Liquid Chromatography/Mass Spectrometry

Antioxidants (Basel). 2022 Jan 25;11(2):229. doi: 10.3390/antiox11020229.

Authors

Chongsheng Liang¹, Siddabasave Gowda B Gowda^{2

3}, Divyavani Gowda², Toshihiro Sakurai², Iku Sazaki¹, Hitoshi Chiba⁴, Shu-Ping Hui¹

Affiliations

¹ Graduate School of Health Sciences, Hokkaido University, Kita-12, Nishi-5, Kita-Ku, Sapporo 060-0812, Japan.
² Faculty of Health Sciences, Hokkaido University, Kita-12, Nishi-5, Kita-Ku, Sapporo 060-0812, Japan.
³ Graduate School of Global Food Resources, Hokkaido University, Kita-9, Nishi-9, Kita-Ku, Sapporo 060-0809, Japan.
⁴ Department of Nutrition, Sapporo University of Health Sciences, Nakanuma, Nishi-4-3-1-15, Higashi-Ku, Sapporo 007-0894, Japan.

Abstract

Lipid hydroperoxides (LOOH) are the initial products of the peroxidation of unsaturated lipids and play a crucial role in lipid oxidation due to their ability to decompose into free radicals and cause adverse effects on human health. Thus, LOOHs are commonly considered biomarkers of oxidative stress-associated pathological conditions. Despite their importance, the sensitive and selective analytical method for determination is limited, due to their low abundance, poor stability, and low ionizing efficiency. To overcome these limitations, in this study, we chemically synthesized eight fatty acid hydroperoxides (FAOOH), including FA 18:1-OOH, FA 18:2-OOH, FA 18:3-OOH, FA 20:4-OOH, FA 20:5-OOH, FA 22:1-OOH, FA 22:6-OOH as analytes, and FA 19:1-OOH as internal standard. Then, they were chemically labeled with 2-methoxypropene (2-MxP) to obtain FAOOMxP by one-step derivatization (for 10 min). A selected reaction monitoring assisted targeted analytical method was developed using liquid chromatography/tandem mass spectrometry (LC-MS/MS). The MxP-labelling improved the stability and enhanced the ionization efficiency in positive mode. Application of reverse-phase chromatography allowed coelution of analytes and internal standards with a short analysis time of 6 min. The limit of detection and quantification for FAOOH ranged from 0.1-1 pmol/µL and 1-2.5 pmol/µL, respectively. The method was applied to profile total FAOOHs in chemically oxidized human serum samples (n = 5) and their fractions of low and high-density lipoproteins (n = 4). The linoleic acid hydroperoxide (FA 18:2-OOH) and oleic acid hydroperoxide (FA 18:1-OOH) were the most abundant FAOOHs in human serum and lipoproteins. Overall, our validated LC-MS/MS methodology features enhanced detection and rapid separation that enables facile quantitation of multiple FAOOHs, therefore providing a valuable tool for determining the level of lipid peroxidation with potential diagnostic applications.

Keywords: 2-methoxypropene; chemical derivatization; human serum; lipid hydroperoxide; lipoprotein oxidation; liquid chromatography; mass spectrometry; unsaturated fatty acids.