EXPRESS: HPLC with Spectrophotometric or Mass Spectrometric detection for quantifying Very-Long Chain Fatty Acids in Human Plasma and its Association with Cardiac Risk Factors.

Shrestha, Rojeet; Chen, Zhen; Gao, Zijun; Chen, Yifan; Okada, Emiko; Ukawa, Shigekazu; Nakagawa, Takafumi; Nakamura, Koshi; Tamakoshi, Akiko; Chiba, Hitoshi; Hui, Shu-Ping

BACKGROUND We developed and compared two liquid chromatography (LC) 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 was determined to be 41.3 μmol/L in healthy volunteers. Plasma VLCFA were significantly correlated with triglycerides (Spearmanâs ï²=0.306, p<0.001) and total cholesterol (Spearmanâs ï²=0.251, p<0.001). All species of VLCFA were significantly elevated in hypertriglyceridemic individuals compared to control. CONCLUSION We established LC-based assays of VLCFA with either spectrophotometry or mass spectrometry as a detection system. Hypertriglyceridemia is significantly associated with elevated concentration of each species of VLCFA.