An LC-MS/MS workflow to characterize 16 regio- and stereoisomeric trihydroxyoctadecenoic acids

Abstract

Trihydroxyoctadecenoic acids (TriHOMEs) are linoleic acid-derived oxylipins with potential physiological relevance in inflammatory processes as well as in maintaining an intact skin barrier. Due to the high number of possible TriHOME isomers with only subtle differences in their physicochemical properties, the stereochemical analysis is challenging and usually involves a series of laborious analytical procedures. We herein report a straightforward analytical workflow that includes reversed-phase ultra-HPLC-MS/MS for rapid quantification of 9,10,13- and 9,12,13-TriHOME diastereomers and a chiral LC-MS method capable of resolving all sixteen 9,10,13-TriHOME and 9,12,13-TriHOME regio- and stereoisomers. We characterized the workflow (accuracy, 98-120%; precision, coefficient of variation ≤6.1%; limit of detection, 90-98 fg on column; linearity, R2 = 0.998) and used it for stereochemical profiling of TriHOMEs in bronchoalveolar lavage fluid (BALF) of individuals with chronic obstructive pulmonary disease (COPD). All TriHOME isomers were increased in the BALF of COPD patients relative to that of smokers (P ≤ 0.06). In both COPD patients and smokers with normal lung function, TriHOMEs with the 13(S) configuration were enantiomerically enriched relative to the corresponding 13(R) isomers, suggesting at least partial enzymatic control of TriHOME synthesis. This method will be useful for understanding the synthetic sources of these compounds and for elucidating disease mechanisms.

Keywords: chiral chromatography; fatty acid biosynthesis; fatty acid/biosynthesis; fatty acid/oxidation; inflammation; lipid mediators; liquid chromatography-tandem mass spectrometry; methods/high-performance liquid chromatography; trihydroxy linoleates.

Copyright © 2018 Fuchs et al.

Figures

Fig. 1.

Overview of the analyzed TriHOME isomers. The numbering of the TriHOME isomers (5a/b–12a/b) corresponds to the system proposed when their structures were originally reported (8). InChIKeys are provided in supplementary Table S2. R = (CH2)7COOCH3.

Fig. 2.

Reversed-phase UHPLC-MS/MS chromatogram of a solution containing TriHOME standard mix A and standard mix B resolving the four 9,10,13-TriHOME enantiomer pairs and the four 9,12,13-TriHOME enantiomer pairs. Peaks were assigned to the corresponding isomers based upon individual injections of standard mixes AI, AII, BI, and BII. A: Chromatogram of 9,10,13-TriHOME, MS/MS transition: m/z 329.1 → 139.0. B: Chromatogram of 9,12,13-TriHOME, MS/MS transition: m/z 329.1 → 211.0. Compound nomenclature is provided in Fig. 1.

Fig. 3.

Chiral LC-MS resolution of a mix of TriHOME standard mix A and standard mix B containing all eight 9,10,13-TriHOME and eight 9,12,13-TriHOME isomers. Peaks were assigned to the corresponding isomer based upon individual injections of standard mixes AI, AII, BI, and BII (each containing one enantiomer pair of 9,10,13-TriHOME and 9,12,13-TriHOME) followed by injections of pure enantiomers 5a–12a. A: Chromatogram of 9,10,13-TriHOME isomers, MS/MS transition: m/z 329.1 → 139.0. B: Chromatogram of 9,12,13-TriHOME isomers, MS/MS transition: m/z 329.1 → 211.0.

Fig. 4.

The concentration in BALF of the 9,10,13-TriHOME and 9,12,13-TriHOME diastereomers of female smokers with normal lung function versus smoking female COPD patients. Values are shown as the median with interquartile range. Significance was tested by a nonparametric Mann-Whitney test, with significance levels: ns, not significant (P = 0.06); *P < 0.05; **P < 0.01; ***P < 0.001.

Fig. 5.

Average ee of TriHOMEs with (S) configuration at carbon 13 in BALF of female smokers with normal lung function and smoking female COPD patients. Error bars indicate 95% confidence intervals (healthy smokers, n = 19; COPD smokers, n = 11).