Preparation of standard LAOOH and ELAOOH isomers for MS/MS analysis

Standard LAOOH and ELAOOH isomers, which are essential in the development of analytical methods for LAOOH and ELAOOH, were prepared through 3 steps (Fig. 1B). In agreement with many previous studies, photo-oxidation (singlet oxygen-induced oxidation) was employed to prepare six LAOOH isomers, and peaks corresponding to LAOOH isomers were detected as the oxidation products on the UV (210 nm) chromatogram (Supplementary Information 1). Then by performing semipreparative HPLC-UV with silica (Condition 2, Table 1), the six LAOOH isomers (i.e., 9-10E, 12E-LAOOH (57.4 mg), 9-10E, 12Z-LAOOH (142.3 mg), 10-8E, 12Z-LAOOH (31.9 mg), 12-9Z, 13E-LAOOH (42.7 mg), 13-9E, 11E-LAOOH (100.3 mg), and 13-9Z, 11E-LAOOH (181.0 mg)) were individually isolated. Because 9-10E, 12Z-LAOOH and 9-10E, 12E-LAOOH were not entirely separated by silica columns, these isomers were completely separated by semipreparative HPLC-UV with an ODS column (Condition 3, Table 1). Each isolated LAOOH isomer was subjected to HPLC-UV (Condition 1, Table 1), and detected as a single peak individually (Fig. 2A). The geometric isomerism (E-Z configuration) and purity were verified by 1H NMR (Supplementary Information 2A–F). From these results, we obtained pure LAOOH isomers that were used as standard reference for quantitative analysis of LAOOH isomers in beverage and cosmetic products.

Figure 2 The UV chromatograms of LAOOH (A) and ELAOOH (B) isomers. The six isomers of LAOOH (50 µg/mL each) and ELAOOH (50 µg/mL each) were subjected to HPLC-UV with silica columns. Detailed analytical conditions are described in the Materials and Methods section. Full size image

Regarding the preparation of ELAOOH isomers, we initially attempted to isolate ELAOOH isomers from oxidized ELA using semipreparative HPLC-UV. However, unlike analysis of LAOOH isomers, it was very difficult to separate the six isomers of ELAOOH from each other by HPLC (data not shown). Therefore, we conceived the synthesis of ELAOOH by condensation reaction between carboxylic acid (i.e., LAOOH) and alcohol (i.e., ethanol). Since the hydroperoxyl group is unstable, we protected the hydroperoxyl group of LAOOH with MxP, yielding LAOO-MxP before the condensation reaction. Then, we successfully synthesized ELAOO-MxP by condensation reaction of LAOO-MxP and ethanol. Synthesized ELAOO-MxP was finally deprotected, and the resultant ELAOOH was chromatographically purified. Six ELAOOH isomers (i.e., 9-10E, 12E-ELAOOH (4.0 mg), 9-10E, 12Z-ELAOOH (9.3 mg), 10-8E, 12Z-ELAOOH (7.9 mg), 12-9Z, 13E-ELAOOH (8.6 mg), 13-9E, 11E-ELAOOH (5.5 mg), and 13-9Z, 11E-ELAOOH (7.8 mg)) were obtained and subjected to HPLC-UV (Condition 7, Table 1). Each isomer was detected as a single peak individually (Fig. 2B), inferring the high purity of the prepared ELAOOH isomers. The geometric isomerism (E-Z configuration) and purity were also verified by 1H NMR (Supplementary Information 3A–F). In the previous study, LAOO-MxP has been used for ester synthesis with glycerolipids (e.g., lysophosphatidylcholine and diacylglycerol)10,11,17, however it has been hardly applied to the condensation reaction with alcohol. Our study indicated that LOOH protected with MxP (e.g., LAOO-MxP) could also be applied for ester condensation with alcohols, and this technique is expected to be useful for preparing various LOOH standards.

MS/MS analysis of LAOOH and ELAOOH isomers

In the previous study, we revealed that in the presence of sodium ions during MS/MS analysis, LAOOH isomers yielded structure-diagnostic fragment ions that were highly useful in identifying the position of the hydroperoxyl group16,24. For example, mass spectrum of sodiated 13-9Z, 11E-LAOOH (i.e., m/z 335.2 [M + Na]+) revealed a neutral loss of 88 Da arising from fragmentation of the hydroperoxyl group. Moreover, similar results were not only observed in other fatty acid hydroperoxide (i.e., arachidonic acid hydroperoxide) but also in phospholipid hydroperoxide, triacylglycerol hydroperoxide, and squalene hydroperoxide17,26,27. From these studies, we concluded that utilization of sodium ion during MS/MS analysis is highly useful for analysis of several LOOH including fatty acid ester hydroperoxide. Hence, in this study, we postulated that this method would also be useful for analysis of ELAOOH isomers. Accordingly, direct infusion of the six LAOOH and ELAOOH standards into the MS/MS system was performed in the presence of the sodium ion to investigate their fragmentations. Unless specifically stated otherwise, spectral data were obtained under the optimized conditions. Each spectrum is representative of at least a triplicate analysis.

As similar to our previous study24, each sodiated molecular ion of LAOOH isomer generated the significant hydroperoxyl group-derived fragment ions (Fig. 3A). Using a similar method, ELAOOH isomers were also subjected to MS/MS analysis in the presence of the sodium ion, which yielded the same fragment patterns with LAOOH. For example, 13-9Z, 11E-ELAOOH produced a characteristic fragment ion at m/z 275.1 ([M + Na−C 5 H 12 O]+) corresponding to a 88 Da loss from the sodiated molecular ion (m/z 363.2 [M + Na]+) (Fig. 3B). As we expected, the fragmentation patterns of ELAOOH observed in this study were consistent with our previously reported analysis of LAOOH16,24 as well as other LOOH species in the presence of sodium10,17,26. Therefore, the ability of sodium ions to promote ionization of hydroperoxyl group derived fragment ions can be considered effective regardless of the type of LOOH.

Figure 3 Q1 mass spectra and product ion mass spectra of LAOOH (A) and ELAOOH (B) isomers in the presence of the sodium ion (positive ion mode). LAOOH and ELAOOH isomers were dissolved in methanol containing 0.1 mM sodium acetate. The sample solutions (10 uM) were infused directly into a micrOTOF-Q II mass spectrometer at a flow rate of 150 µL/h. Detailed analytical conditions are described in the Materials and Methods section. Full size image

HPLC-MS/MS analysis of LAOOH and ELAOOH isomers

HPLC-MS/MS method for LAOOH was developed according to the previous method16 with slight modifications (Condition 9, Table 1) (Table 3). In the MRM chromatograms, each LAOOH isomer was completely separated (Fig. 4A) and detected at the following retention times: 14.0 min (10-8E, 12Z-LAOOH), 14.0 min (12-9Z, 13E-LAOOH), 14.0 min (13-9Z, 11E-LAOOH), 14.1 min (9-10E, 12Z-LAOOH), 14.2 min (13-9E, 11E-LAOOH), and 14.3 min (9-10E, 12E-LAOOH). By modifying the previously reported method where detection limits of LAOOH isomers were pmol levels16, detection limits were enhanced to 25 fmol. Then, we also successfully discriminated six ELAOOH isomers using HPLC-MS/MS with an ODS column (Condition 10, Table 1) (Fig. 4B) and MRM analysis based on fragments observed during product ion scanning of each isomer in the presence of the sodium ion (Table 3). Each ELAOOH isomer was detected at the following retention times: 10.5 min (12-9Z, 13E-ELAOOH), 10.6 min (9-10E, 12Z-ELAOOH), 10.6 min (10-8E, 12Z-ELAOOH), 10.7 min (13-9Z, 11E-ELAOOH), 11.0 min (9-10E, 12E-ELAOOH), and 11.3 min (13-9E, 11E-ELAOOH). To the best of our knowledge, this is the first complete discrimination of ELAOOH isomers using HPLC-MS/MS analysis. The detection limit of each ELAOOH isomer was 0.1–2.0 fmol. These methods may therefore be useful in the quantification of LAOOH and ELAOOH isomers on commodities samples and the evaluation of lipid oxidation mechanisms.

Figure 4 The MRM chromatograms of LAOOH (A) and ELAOOH (B) isomers. A mixture of six isomers of LAOOH (500 nM in methanol, 10 µL each) and a mixture of six isomers of ELAOOH (100 nM in methanol, 10 µL each) were analyzed by HPLC-MS/MS. Detailed analytical conditions are described in the Materials and Methods section. Full size image

Evaluation of lipid oxidation mechanisms of food and cosmetic samples

Subsequently, we implemented analytical methods for LAOOH and ELAOOH isomers to obtain a comprehensive analysis of commodities. Some alcoholic beverages such as beer and whisky are known to contain fatty acids and fatty acid ethyl esters19,28,29. Lipid peroxidation of these fatty acids and fatty acid ethyl esters have been reported to affect the flavor of foods. Therefore, we focused on liquor as a food (beverage) sample in this study. We analyzed five different marketed liquor samples, including four whisky and one brandy. The typical chromatograms of liquor are shown in Fig. 5. LAOOH and ELAOOH isomers were detected in one whisky sample and brandy despite being analyzed immediately after opening (Figs 5 and 6). LAOOH isomers that were detected in liquors were the isomers characteristic of photo-oxidation (i.e., 9-10E, 12Z-LAOOH, 10-8E, 12Z-LAOOH, 12-9Z, 13E-LAOOH, and 13-9Z, 11E-LAOOH). On the other hand, the ELAOOH isomers formed by both photo-oxidation (i.e., 9-10E, 12Z-ELAOOH, 10-8E, 12Z-ELAOOH, 12-9Z, 13E-ELAOOH, and 13-9Z, 11E-ELAOOH) and auto-oxidation (i.e., 9-10E, 12Z-LAOOH, 9-10E, 12E-LAOOH, 13-9Z, 11E-LAOOH, and 13-9E, 11E-LAOOH) were detected in liquor samples (Fig. 6). The detection of auto-oxidation products of ELA but not LA in whisky 3 and brandy might have been due to the higher detection sensitivity of ELAOOH. The detection limits of ELAOOH isomers are about 25 times higher than LAOOH isomers; thus, ELAOOH isomers characteristic of auto-oxidation (i.e., 9-10E, 12E-ELAOOH and 13-9E, 11E-ELAOOH) were detected. Such results suggested that both photo- and auto-oxidation had already occurred in marketed liquor before opening (e.g., during the manufacturing process and/or sales). Also, the concentration of fatty acid and fatty acid ethyl ester in whisky is mainly affected by the manufacturing process30, and thus may explain the presence/absence of LAOOH and ELAOOH in whisky samples in this study. Among the measured liquor samples, brandy contained a relatively high concentration of ELAOOH isomers, and therefore the brandy analyzed in this study might have contained certain compounds that acted as a prooxidant. Considering that LAOOH and ELAOOH isomers characteristic of photo-oxidation were detected, photosensitizers that induce photo-oxidation might have been contained. Formation of LOOH contributes to the emergence of offensive odor and taste17,31, and so, preventing the generation of LOOH in liquor (i.e., suppression of photo-oxidation) could be essential for maintaining the quality of liquor.

Figure 5 The MRM chromatograms of LAOOH (A) and ELAOOH (B) isomers in brandy sample. Extract from brandy sample (10 µL) was analyzed by HPLC-MS/MS. Detailed analytical conditions are described in the Materials and Methods section. Full size image

Figure 6 The concentrations of LAOOH (A) and ELAOOH (B) isomers of marketed liquor samples (Mean ± SD (n = 3)). Extract from liquor samples (four whiskies and one brandy) were analyzed by HPLC-MS/MS with MRM mode. Detailed analytical conditions are described in the Materials and Methods section. Full size image

In addition to liquor, cosmetics (e.g., skin cream) are also known to contain fatty acids and fatty acid ethyl esters2,4,21. Furthermore, the oxidation of lipids contained in cosmetics could lead to the deterioration of quality. Thomsen et al. reported that odor-causing substances (e.g., hexanal) are formed in cosmetics depending on storage temperature, light-exposure, and time4. However, the oxidation mechanisms of lipids in cosmetics have not been evaluated, furthermore, the influence of storage conditions on oxidation mechanisms have not been assessed. In this study, in order to evaluate the influence of the storage temperature and period on oxidation mechanisms, skin cream samples stored under dark at different temperatures (−5 °C, 25 °C, and 40 °C) for different period of times (2, 4, 6, and 15 months) were analyzed. LAOOH and ELAOOH isomers were then extracted and analyzed by the HPLC-MS/MS method. In contrast to liquor samples, auto-oxidized LAOOH isomers (i.e., 9-10E, 12E-LAOOH, 9-10E, 12Z-LAOOH, 13-9E, 11E-LAOOH, and 13-9Z, 11E-LAOOH) and ELAOOH isomers (i.e., 9-10E, 12E-ELAOOH, 9-10E, 12Z-ELAOOH, 13-9E, 11E-ELAOOH, and 13-9Z, 11E-ELAOOH) were mainly detected in skin cream (Figs 7 and 8). This result suggested that auto-oxidation occurred in skin cream under dark and confirmed the influence of storage temperature and term on lipid oxidation. When the skin cream was stored at −5 °C, each concentration of LAOOH isomers and ELAOOH isomers were not altered despite the difference in storage period (Fig. 8). On the other hand, the LAOOH isomers and ELAOOH isomers in skin cream stored at 25 °C were increased depending on the stored periods. As auto-oxidation of lipids tend to proceed at high temperature, it is most likely that low-temperature storage (i.e., −5 °C), inhibits the oxidation process. In contrast, the concentrations of LAOOH and ELAOOH isomers in skin cream stored at 40 °C tended to either decrease throughout the storage time or increase for a short time period and then later decrease during storage. This result seems to be due to the lipid oxidation being accelerated by high temperature and then LOOH being decomposed. In fact, the sample stored at 40 °C for 15 months exhibited significant change in color and smell. From these results, we found that it was possible to evaluate lipid oxidation mechanisms (i.e., auto-oxidation) in cosmetics with the use of the developed method, and to determine the possibly effective defense method (i.e., storage at low temperature) to suppress the progress of oxidation.

Figure 7 The MRM chromatograms of LAOOH (A) and ELAOOH (B) isomers in cosmetic samples. Extract from cosmetic sample (10 µL) was analyzed by HPLC-MS/MS. Detailed analytical conditions are described in the Materials and Methods section. Full size image