Abstract: The mechanisms of lipids separation are as following: different lipid classes are separated by adsorption mechanisms and eluted out of the column in normal-phase liquid chromatography (NPLC), and individual molecular species are separated based on hydrophobicity in reversed-phase liquid chromatography (RPLC). In RPLC, the elution sequence of lipid molecules is determined by both the chain length and the degree of unsaturation in the fatty-acyl chains. To avoid co-elution of molecular species, an online, normal-phase and reversed-phase two-dimensional (2D) liquid chromatography (LC) quadrupole time-of-flight mass spectrometry (Q TOF-MS) system was developed for the lipid profiling of Madin-Darby canine kidney (MDCK) cells and the investigation of the lipid changes in MDCK cells treated with aristolochic acid (Ⅰ). Different lipid classes in MDCK cells were separated in the first dimension of the two-dimensional liquid chromatograph system and lipid molecular species were further separated in the second dimension followed by mass spectrometry detection, so that the ion suppression effects were reduced while the detection sensitivity was improved.
All lipids in MDCK cells were identified with high accuracy mass values measured by Agilent 6530 accurate mass Q TOF-MS. The abundant molecular species were confirmed by targeted MS/MS，meanwhile the retention time and low abundance lipid molecules were identified with m/z value and the retention time based on the correlation between the equivalent carbon number (ECN) and the retention time. The measured accurate-masses were applied for preliminary identification using the online database with a mass tolerance of less than ±0.005 on the basis of the predicted elemental composition. 1416 endogenous lipid species from 13 lipid classes were identified by accurate masses, tandem mass spectra and the retention time. 11 exogenous lipid standards from different classes, including FA 17∶0, Hemi BMP(17∶0), LPG(17∶1 ), PG(14∶0/14∶0), Sphingosyl PE(d17∶1/12∶0), PE(14∶0/14∶0), LPE(17∶1), PS(14∶0/14∶0), LPC(17∶0), PC(14∶1/14∶1), SM(d18∶1/17∶0), were selected to be separated in five fractions for the evaluation of this method. The linear regression coefficients (R²=0.9917-0.998 3), the limit of detection (2.5 μg/L) and the relative standard deviation of peak area (0.9%-7.7%) and retention time (0.01%-0.11%) were all satisfactory.
To investigate the lipid changes in MDCK cells dosed with aristolochic acid (Ⅰ), 16 MDCK cell samples (each containing 5×10⁵ cells) were randomly separated into a dosed group (n=8) and a control group (n=8). All 16 samples were detected by the 2D LC/MS method. The dosed group and control group were alternately injected to reduce systemic error. During the sequence, one blank sample was injected after every three injections, and no significant carryover of lipids was observed. MS data of all 16 samples were extracted by Mass-Hunter Qualitative Analysis software and analyzed by Mass Profiler Professional software. Through setting threshold parameters, the software presented a list of potential biomarkers whose absolute fold-change of peak area was larger than 2 and p value less than 0.05. By the above-mentioned approach, 15 changed lipid species were confirmed, as their concentrations in the dosed group were 2.4 times of those in the control group. The results would contribute to the study on therapeutic and toxicological mechanisms of aristolochic acids and revealed that this two-dimensional liquid chromatography quadrupole time-of-flight mass spectrometry method was a promising tool for lipidomics research.