HPLC with spectrophotometric or mass spectrometric detection for quantifying very-long chain fatty acids in human plasma and its association with cardiac risk factors

Author:

Shrestha Rojeet1ORCID,Chen Zhen1ORCID,Gao Zijun1,Chen Yifan1,Okada Emiko2,Ukawa Shigekazu3,Nakagawa Takafumi4,Nakamura Koshi5,Tamakoshi Akiko6,Chiba Hitoshi7,Hui Shu-Ping1

Affiliation:

1. Faculty of Health Sciences, Hokkaido University, Sapporo, Japan

2. Department of Nutritional Epidemiology and Shokuiku, National Institute of Biomedical Innovation, Health and Nutrition, Tokyo, Japan

3. Research Unit of Advanced Interdisciplinary Care Science, Graduate School of Human Life Science, Osaka City University, Osaka, Japan

4. The Hokkaido Centre for Family Medicine, Sapporo, Japan

5. Department of Public Health and Hygiene, Graduate School of Medicine, University of the Ryukyus, Nishihara, Okinawa, Japan

6. Department of Public Health, Faculty of Medicine, Hokkaido University, Hokkaido, Japan

7. Department of Nutrition, Sapporo University of Health Sciences, Sapporo, Japan

Abstract

Background We developed and compared two liquid chromatography methods, one with UV/Visible spectrophotometric detection (HPLC) and the other with mass spectrometric detection (LC-MS), for quantifying very-long chain fatty acids (VLCFA) in human plasma. Association of VLCFA with various cardiovascular risk factors were evaluated. Method Fasting blood samples were collected from 541 human volunteers (242 men and 299 women; mean age ±SD, 58.9 ± 12.4 years), including 429 and 112 individuals with and without hypertriglyceridemia, respectively. Esterified VLCFA were saponified and derivatized with 2-nitrophenylhydrazine. Separation of VLCFA species was achieved with C4 Mightysil column (HPLC) and Ascentis Express Phenyl-Hexyl column (LC-MS) followed by spectrophotometric and selected-reaction monitoring mode of mass spectrometric detection, respectively. Results The HPLC assay of VLCFA was precise with intra-assay imprecision of 2.5% to 6.9% and inter-assay imprecision of 3.2% to 9.5%. Moreover, there was an excellent correlation (r > 0.96) between HPLC and LC-MS methods. The 95 percentile reference intervals (RI; upper limit) of VLCFA were determined to be 41.3 µmol/L in healthy volunteers. Plasma VLCFA were significantly correlated with triglycerides (Spearman’s ρ = 0.306, P <  0.001) and total cholesterol (Spearman’s ρ = 0.251, P <  0.001). All species of VLCFA were significantly elevated in hypertriglyceridaemic individuals compared with control. Conclusion We established LC-based assays of VLCFA with either spectrophotometry or mass spectrometry as a detection system. Hypertriglyceridaemia is significantly associated with elevated concentration of each species of VLCFA.

Funder

Japan Society for the Promotion of Sciences (JSPS) KAKENHI Grants

by Ministry of Agriculture, Forestry and Fisheries, Japan, Research for Agriculture and Interdisciplinary Fields

Publisher

SAGE Publications

Subject

Clinical Biochemistry,General Medicine

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