Determination of triacylglycerol oxidation mechanisms in canola oil using liquid chromatography-tandem mass spectrometry

Abstract

Triacylglycerol (TG), the main component of edible oil, is oxidized by thermal- or photo- oxidation to form TG hydroperoxide (TGOOH) as the primary oxidation product. Since TGOOH and its subsequent oxidation products cause not only the deterioration of oil quality but also various toxicities, preventing the oxidation of edible oils is essential. Therefore understanding oxidation mechanisms that cause the formation of TGOOH is necessary. Since isomeric information of lipid hydroperoxide provides insights about oil oxidation mechanisms, we focused on dioleoyl-(hydroperoxy octadecadienoyl)-TG (OO-HpODE-TG) isomers, which are the primary oxidation products of the most abundant TG molecular species (dioleoyl-linoleoyl-TG) in canola oil. To secure highly selective and sensitive analysis, authentic OO-HpODE-TG isomer references (i.e., hydroperoxide positional/geometrical isomers) were synthesized and analyzed with HPLC-MS/MS. With the use of the method, photo- or thermal- oxidized edible oils were analyzed. While dioleoyl-(10-hydroperoxy-8E,12Z-octadecadienoyl)-TG (OO-(10-HpODE)-TG) and dioleoyl-(12-hydroperoxy-9Z,13E-octadecadienoyl)-TG (OO-(12-HpODE)-TG) were characteristically detected in photo-oxidized oils, dioleoyl-(9-hydroperoxy-10E,12E-octadecadienoyl)-TG and dioleoyl-(13-hydroperoxy-9E,11E-octadecadienoyl)-TG were found to increase depending on temperature in thermal-oxidized oils. These results prove that our methods not only evaluate oil oxidation in levels that are unquantifiable with peroxide value, but also allows for the determination of oil oxidation mechanisms. From the analysis of marketed canola oils, photo-oxidized products (i.e., OO-(10-HpODE)-TG and OO-(12-HpODE)-TG) were characteristically accumulated compared to the oil analyzed immediately after production. The method described in this paper is valuable in the understanding of oil and food oxidation mechanisms, and may be applied to the development of preventive methods against food deterioration.

Keywords: Industry; Technology.

Fig. 1

Triacylglycerol (TG) oxidation mechanisms and the structures of triacylglycerol hydroperoxide isomers (a). Dioleoyl-linoleoyl glycerol (I) is oxidized to dioleoyl-(hydroperoxy octadecadienoyl) glycerol (OO-HpODE-TG, II–VII) by radical and/or singlet-oxygen oxidation. Hydroperoxide positions and geometrical structures depend on the TG peroxidation mechanisms. Schemes for the reference preparation of OO-HpODE-TG isomers (b). The references were synthesized from dioleoyl glycerol and HpODE isomers

Fig. 2

The product ion mass spectra of reference OO-HpODE-TG isomers (A–L). Reference OO-HpODE-TG isomers were dissolved in methanol containing 0.1 mM sodium acetate (1 µM) and infused directly into the MS/MS system at a flow rate of 10 μL/min. Insets show the speculated fragmentation patterns of OO-HpODE-TG isomers

Fig. 3

MRM chromatograms of reference OO-HpODE-TG isomers (a). OO-HpODE-TG isomer references (5 pmol each) were analyzed with MRM (m/z 938.0 > 768.8 for isomers bearing 9-HpODE, m/z 938.0 > 809.8 for isomers bearing 10-HpODE, m/z 938.0 > 808.8 for isomers bearing 12-HpODE and m/z 938.0 > 849.8 for isomers bearing 13-HpODE). Peaks 1–12 and their retention time were as follows: (1) 1,3-OO-2-(13-ZE-HpODE)-TG (12.6 min); (2) 1,3-OO-2-(12-HpODE)-TG (13.4 min); (3) 1-(13-ZE-HpODE)-2,3-OO-TG (14.5 min); (4) 1-(12-HpODE)-2,3-OO-TG (15.0 min); (5) 1,3-OO-2-(10-HpODE)-TG (15.9 min); (6) 1,3-OO-2-(13-EE-HpODE)-TG (16.1 min); (7) 1,3-OO-2-(9-EZ-HpODE)-TG (17.0 min); (8) 1,3-OO-2-(9-EE-HpODE)-TG (18.0 min); (9) 1-(13-EE-HpODE)-2,3-OO-TG (18.4 min); (10) 1-(10-HpODE)-2,3-OO-TG (18.8 min); (11) 1-(9-EZ-HpODE)-2,3-OO-TG (20.5 min); (12) 1-(9-EE-HpODE)-2,3-OO-TG (21.6 min). Calibration curves of reference OO-HpODE-TG isomers (1,3-OO-2-(HpODE)-TG (b) and 1-(HpODE)-2,3-OO-TG (c)). Different amounts of OO-HpODE-TG isomers (0.25–50 pmol) were analyzed by optimized LC–MS/MS MRM. MRM chromatograms of thermal- (d–g) and photo- (h–k) oxidized oil. Oxidized oils were 100-fold diluted with hexane, and a portion (50 µL) was analyzed using LC–MS/MS MRM. Peak numbers refer to the OO-HpODE-TG isomers described above

Fig. 4

MS/MS/MS analysis of photo-oxidized oil. The ion m/z 938.0 was selected as a first precursor ion, and m/z 768.8 (OO-(9-HpODE)-TG (a); m/z 809.8 (OO-(10-HpODE)-TG (b); m/z 808.8 (OO-(12-HpODE)-TG (c); and m/z 849.7 (OO-(13-HpODE)-TG (d); were selected as a second precursor ion. Each chromatogram is a total ion current chromatogram of the product ion generated from the second precursor ion. MS/MS/MS spectra (I–IV) demonstrating product ions generated from the second precursor ion. Photo-oxidized oil were 100-fold diluted with hexane, and a portion (50 µL) was analyzed under optimized LC–MS/MS/MS conditions

Fig. 5

Concentrations of 1,3-OO-2-(HpODE)-TG and 1-(HpODE)-2,3-OO-TG isomers in oxidized oil. Fresh oil was oxidized under the following conditions: 25 °C, shade (a, b); 100 °C, shade (c, d); 140 °C, shade (e, f); 180 °C, shade (g, h); 25 °C, 1000 lux (i, j); 25 °C, 10,000 lux (k, l); 25 °C, 100,000 lux (m, n). Oxidized oils were 100-fold diluted with hexane, and a portion (50 µL) was analyzed under optimized LC–MS/MS MRM conditions

Fig. 6

(A and B) Concentrations of OO-HpODE-TG isomers in marketed canola oil. Marketed oils were analyzed immediately after opening. Oils were 100-fold diluted with hexane and a portion (50 µL) was analyzed under optimized LC–MS/MS MRM conditions