The resolution of MAcDG and lcTG subclass in complex lipid standard mixture by TLC-FID presented in the current study is consistent with that reported by Marshall at al. (2014) for the analysis of E. solidaginis larvae fat [20]. The concentration range observed to provide excellent resolution and quantitation using TLC-FID is 100–1000 μg/mL (0.1 to 1 mg/mL). Below 100 μg/mL the peak size and appearance was not consistent, and above 1000 μg/mL the peaks shape appeared large and broad that can interfere with the resolution of MAcDG from the different TG subclasses. Furthermore, the response is linear between 100 and 1000 μg/mL for the quantification of MAcDG as well as other TG subclasses. The amount of MAcDG in a sample can be quantified by TLC-FID from the average peak area of MAcDG (7 chromarods) present in each sample using the linear regression equation (y = mx + c) and calculated as follows [17]:

$$ MAcDG left(mu g/gkern0.5em sampleright)=frac{left(y-cright)x V}{m x W} $$

where y = average peak area of MAcDG, c and m = intercept and slope of the regression line, respectively, V = total volume of lipid solution (mL), and W = weight of sample used (g).

The output presented in this paper demonstrate the ability of the method for simple, rapid, and sensitive quantification of OH-MAcDG, MAcDG, mcTG and lcTG in biological samples. This demonstrated that hydroxylated MAcDG (OH-MAcDG) was resolved from other lipid classes using TLC-FID and presented opportunities for purification and further evaluation of the hydroxylated version of MAcDG, which is a more polar version of MAcDG, as well as determine possible functions and applications. It is well recognised that the bioactivities and physical properties of MAcDG is distinctly different from that of other TG subclasses. How hydroxylation further influences these properties is unknown. The application of TLC-FID to resolve and quantify polar and neutral lipid classes in complex biological samples ranging from animal (egg yolk, chicken fat, lamb fat, milk), marine (lobster, krill oil, red porgy wild, greater weever, piper gurnard) plant (sesame seeds, canola gum, macadamia nuts, olive oil), and edible fungus (mushrooms) origins have been widely reported [19, 33,34,35]. These applications demonstrate the versatility of this analytical technique for routine, simple, efficient, accurate, and sensitive analysis of neutral lipids. MAcDG can now be included as a subclass in routine neutral lipid analysis in these sources using TLC-FID. The proposed TLC-FID method provides a simple, comparatively inexpensive, sensitive, and rapid method to separate, identify and quantitate TG subclasses classes including MAcDG and OH-MAcDG in biological samples. In particular, the unique biochemical composition of MAcDG in biological samples including E. solidaginis can confer novel uses and application in the food science field (considering insects are emerging sources of dietary proteins and health-promoting functional lipids), healthcare (treating sepsis, asthma, arthritis, cancers and tumors), and biofuel industry (potential additive to improve performance of biofuels in cold climates [26, 36]. These uses suggest possible applications for MAcDG for which rapid, relatively inexpensive, accurate and sensitive analytical methods for quantification will be essential. TLC-FID has been demonstrated in enabling the separation and analysis of MAcDG and OH-MAcDG subclasses across a range of biological samples.

Under the C30-RPLC-HRAM-MS/MS conditions used in present study, the presence of OH group in OH-MAcDG appears to have increased the relative polarity of MAcDG-OH thereby reducing its retention on the C30-RP column relative to MAcDG and lcTG species in the standard mixture [37]. As such MAcDG-OH eluted before MAcDG and lcTG species, as shown in Fig. 1b. Furthermore, separation of mcTG and lcTG molecular species present in the standard mixture was based on their fatty acid chain lengths [26]. The hydrophobic interactions between the stationary phase and hydrophobicity of fatty acyl chains (based on chain length, number and position of double bonds of fatty acids) were known to resolve neutral lipid species present in the standard mixture [38]. McTG molecular species have shorter fatty acyl chains compared to lcTG species which makes the former less hydrophobic compared to lcTG. As such, mcTG molecular species were less retained under C30-RPLC conditions compared to the more hydrophobic lcTG species [39].

The MS/MS spectrum of MAcDG 16:0/18:1/2:0 [M + NH4]+ at m/z 654.57 is shown in Fig. 2a. The diagnostic sn-3 acetyl moiety at m/z 577.52 corresponds to the neutral loss of [CH3COONH4+] or 77 Da from m/z 654.57 [MAcDG 16:0/18:1/2:0 + NH4]+ molecular ion [40]. The composition and positions of fatty acids (FA) in TG molecular species including MAcDG and OH-MAcDG present in the standard mixture were identified based on the neutral loss of the fatty acid fragments as ammonium adducts, and presence of the fatty acid ketene ions [FA + H-H2O]+ in the MS/MS spectra [26, 41]. As such for MAcDG 16:0/18:1/2:0 (Fig. 2a), the fragment at m/z 355.28 correspond to the neutral loss of 18:1 + NH3 (sn-2 FA) or 299 Da, while the fragment at m/z 381.30 correspond to the neutral loss of 16:0 + NH3 (sn-1 FA) or 273 Da from m/z 654.57 [MAcDG 16:0/18:1/2:0 + NH4]+ ion [42]. Distinction between sn-2 and sn-1 fatty acids is based on the relative abundance of these two FA fragments, with relative abundance of sn-2 FA fragment [18:1 + NH3] lower than that of sn-1 FA fragment [16:0 + NH3], which is a trend typical of sn-1 and sn-2 FAs in TG [43]. Furthermore, fragment ions at m/z 239.24 and 265.25 in Fig. 2a were assigned to fatty acid ketene ions [16:0 + H-H2O]+ and [18:1 + H-H2O]+ corresponding to sn-1 FA and sn-2 FA, respectively. The distinction between sn-2 and sn-1 positions on TG molecular species including MAcDG and OH-MAcDG could also be identified by the relative abundance of these fatty acid ketene ions [26]. The relative abundance of [18:1 + H-H2O]+ ketene ion at m/z 265.26 corresponding to sn-2 FA was higher than that of [16:0 + H-H2O]+ ion at m/z 239.24 for sn-1 FA of MAcDG 16:0/18:1/2:0, which is a trend typical of sn-1 and sn-2 FA ketene ions in TG species (Fig. 2a). Thus, the trend based on relative abundance of sn-1 and sn-2 [FA + H-H2O]+ ketene ions is opposite to that observed for fragments formed by neutral loss of FAs from [MAcDG 16:0/18:1/2:0 + NH4]+ ion, and is consistent with the literature on determining TG molecular species [41, 42].

The proposed C30-RPLC-MS/MS method also allowed for resolution and identification of OH-MAcDG molecular species in the complex standard mixture. Structurally, while OH-MAcDG and MAcDG are distinguished from non-acetylated TG species including scTG, mcTG and lcTG by the presence of sn-3 acetyl groups, OH-MAcDG differs from MAcDG by having one hydroxyl group on the sn-2 fatty acid chain. These structural differences were seen in the mass spectra of MAcDG and OH-MAcDG molecular species present in the standard mixture under C30-RPLC-MS/MS conditions (Fig. 2a-b). For example, the MS/MS spectra of OH-MAcDG 16:0/18:0(OH)/2:0 m/z 672.58 is shown in Fig. 2b. The neutral loss of [CH3COONH4+] or 77 Da from [TG + NH4]+ ions is representative of MAcDG molecular species [40]. In contrast OH-MAcDG molecular species are distinguished from MAcDG by the neutral loss of [CH3COONH4+ + H2O] or 95 Da from [TG + NH4]+ ions [44]. Accordingly, the fragment at m/z 577.52 corresponding to neutral loss of [CH3COONH4+ + H2O] or 95 Da from m/z 672.58 [OH-MAcDG 16:0/18:0(OH)/2:0 + NH4]+ ion is diagnostic of the sn-3 acetate moiety in OH-MAcDG 16:0/18:0(OH)/2:0 (Fig. 2b). Furthermore, relative abundances of fatty acid fragments arising from neutral loss from [TG + NH4]+ ions and fatty acid ketene ions were used to assign sn-1 and sn-2 fatty acid composition of OH-MAcDG species. Consistent with conventions in the literature for assigning the sn1 and sn2 fatty acids based on relative abundance [43], fragments at m/z 355.28 (18:0 sn-2) and m/z 381.30 (16:0 sn-1) correspond to neutral loss of [18:0 + NH3] and [16:0 + NH3] from [OH-MAcDG 16:0/18:0(OH)/2:0 + NH4]+ ion, respectively (Fig. 2b). As previously alluded to, the relative abundance of fragment corresponding to sn-2 FA is lower compared to sn-1 FA fragment [43]. In a similar fashion, the fragments at m/z 239.24 and m/z 265.25 were diagnostic of fatty acid ketene ions [16:0 + H-H2O]+ and [18:0 + H-H2O]+ respectively, with the relative abundance of the sn-2 FA ketene ion higher than the sn-1 FA ketene ion (Fig. 2b). In summary, neutral loss of 77 Da and 95 Da from [TG + NH4]+ ions was used to distinguish between MAcDG and OH-MAcDG molecular species present in the standard mixture.

In contrast, non-acetylated TG including scTG, mcTG and lcTG present in the standard mixture did not form fragments corresponding to neutral loss of 77 Da and 95 Da from [TG + NH4]+ ions under C30-RPLC-HRAM-MS/MS conditions, which provided a basis for their identification relative to acylated TG (MAcDG and OH-MAcDG). For example, the MS/MS spectra of mcTG 8:0/8:0/8:0 m/z 488.39 is shown in Fig. 2c. The neutral loss of 161 Da is representative of C8:0 FA loss from m/z 488.39 [mcTG 8:0/18:0/8:0 + NH4]+ ion, which corresponds to the fragment at m/z 327.25 (Fig. 2c), while the fragment at m/z 127.11 corresponds to the fatty acid ketene ion [8:0 + H-H2O]+ at sn-1, sn-2 and sn-3 positions of mcTG 8:0/8:0/8:0 [26]. The same approach was applicable for structural elucidation of lcTG molecular species present in the standard mixture. Accordingly, the m/z 551.50 fragment in Fig. 2d corresponds to the neutral loss of FA 16:0 (273 Da) from m/z 824.68 [lcTG 16:0/16:0/16:0 + NH4]+ ion, while fatty acid ketene ion [16:0 + H-H2O]+ was at m/z 127.11 [26, 28]. Application of C30-RPLC-MS/MS to identify and quantify neutral lipid molecular species in biological samples have been reported in the literature. Narvaez et al. (2016) applied a RPLC-MS/MS method in positive ion mode to resolve and analyze neutral lipids including TG in rat liver and rat plasma lipid extracts [22]. A similar application has been used for analysis of TG composition of Calu-3 cells, rat blood and human skin tissues [45]. However, none of these techniques have included the analysis of MAcDG and OH-MAcDG. The method reported herein utilizing a C30-RPLC-HRAM-MS/MS technique to analyze neutral lipids including TG was successful in detecting MAcDG in wild cervid meats (moose and caribou) [46]. Taken together, the proposed C30-RPLC-HRAM-MS/MS method allows for accurate detection and quantification of TG molecular species including OH-MAcDG and MAcDG with good resolution, sensitivity, and throughput during routine lipidomics.

TG subclass and molecular species composition in E. solidaginis is of interest due to its role as a model system in cold stress tolerance. In this model system, MAcDG have been demonstrated to provide cryoprotection during exposure to low temperature stress [20]. This work has also led to subsequent studies recently on the potential of producing specialized oil seed crops with superior levels of MAcDG for applications in the production of biofuels specific for cold temperatures or northern climates [15, 47]. For example, Liu et al. (2015) reported that EaDAcT genetically modified camelina and soybean accumulated MAcDG at up to 70 mol% of the total seed oil produced by the resultant crops. A similar strategy of genetic modification increased MAcDG content to 85 mol% in field-grown transgenic camelina [15]. Furthermore, recent interests in insects as a potential source of proteins and functional ingredients for food and animal feed to improve population health and wellbeing has gained recognition, for which the larvae of E. solidaginis could be a promising food source [48, 49]. TG in E. solidaginis larvae lipidome have been reported to contain about 36 mol% MAcDG compared to long chain TG [20]. However, no report was done on the molecular species composition of MAcDG in E. solidaginis. MAcDG is associated with several health benefits in human and these include treating sepsis [10], tumor growth and cancers [11], rheumatoid arthritis [12] and asthma [13].

In the E. solidaginis chromatogram, a clear separation of these TD subclasses was observed based on hydrophobicity, chain lengths and molecular weight as follows: MAcDG< scTG< mcTG< lcTG (Fig. 3b). No OH-MAcDG specie was observed in E. solidaginis lipidome, which is in line with the literature [20]. The C30-RPLC separated the more polar MAcDG species from non-acetylated TG species (scTG, mcTG and lcTG), which allowed for facile mass spectrometric identification and quantification (Fig. 3b). The MS/MS spectra of MAcDG 18:1/16:1/2:0 [M + H]+ at m/z 652.56 is shown in Fig. 4a. The fragment at m/z 575.51 corresponds to the neutral loss of 77 Da [CH3COONH4+] from m/z 652.56 [MAcDG 18:1/16:1/2:0 + NH4]+ ion. This neutral loss 77 Da is representative of the sn-3 acetyl moiety [CH3COONH4+] characteristic of all MAcDG species. The two fatty acid fragments were at m/z 381.31 (16:1 sn-2) and m/z 353.27 (18:1 sn-1) corresponding to neutral loss of [16:1 + NH3] and [18:1 + NH3] from [MAcDG 18:1/16:1/2:0 + NH4]+ ion, respectively (Fig. 4a). Furthermore, corroboration of the fatty acid composition of MAcDG 18:1/16:1/2:0 was also based on the diagnostic fatty acid ketene ions at m/z 237.22 (sn-2 FA) and m/z 265.26 (sn-1 FA) which correspond to [16:1 + H-H2O]+ and [18:1 + H-H2O]+ ions, respectively (Fig. 4a). Assignment of sn-1 FA and sn-2 FA of MAcDG 18:1/16:1/2:0 was made based on the relative abundance of the neutral loss FA fragments and fatty acid ketene ions as elaborated previously in the discussion. A similar approach was applied to elucidate the structures of other MAcDG species detected in E. solidaginis larvae lipidome (Fig. 4b & Fig. S-1 – S-3). Significantly, the proposed C30-RPLC-HRAM-MS/MS method provided facile resolution of MAcDG and TG molecular species present as isomers in E. solidaginis larvae lipidome. For example, the MS/MS spectra of MAcDG species present in E. solidaginis larvae were distinguished from scTG, mcTG and lcTG species by the neutral loss of [CH3COONH4+] or 77 Da from [TG + NH4]+ ion, which is diagnostic of the sn-3 acetyl moiety of all MAcDG species (Fig. 4a-e). Using the approach based on the relative abundances of neutral loss of the fatty acid fragments and fatty acid ketene ions explained above, as well as in the literature [28], the two isomers at m/z 874.79 [TG 52:3 + NH4]+ reported in Fig. 4d-e were assigned as lcTG 18:1/18:1/16:1 and lcTG 18:2/18:1/16:0 molecular species respectively.

The utility of the proposed of C30-RPLC-MS/MS for resolving isomers of MAcDG from that of other TG subclasses present in E. solidaginis larvae was also demonstrated. For example, MAcDG 18:0/18:1/2:0 [M + H]+ m/z 682.59 eluted at 20.64 min under C30-RPLC conditions (Fig. 5b) is an isomer of scTG 16:0/18:0/4:0 [M + H]+ m/z 682.59 which eluted at 21.44 min (Fig. 5a). These isomers were easily resolved based on their MS/MS fragmentation patterns (Fig. 5a-b). For scTG 16:0/18:1/4:0, the three fatty acid composition were assigned as follows: the fragments at m/z 577.53 is diagnostic of 4:0 fatty acid at sn-3, m/z 409.34 for 16:0 at sn-1 and m/z 383.32 for 18:1 at the sn-2 positions of the glycerol moiety. This correspond to the neutral loss of [4:0 + NH3], [16:0 + NH3] and [18:1 + NH3] from m/z 682.59 [scTG 16:0/18:2/2:0 + NH4]+ ion, respectively (Fig. 5a). Assignment of the sn-1, sn-2 and sn-3 FAs of scTG 16:0/18:0/4:0 was based on the relative abundance of these fragments with sn-1 FA > sn-2 FA > sn-3 FA, a trend typical of TG species [26, 28]. Furthermore, diagnostic fatty acid ketene ions at m/z 239.24 and m/z 265.26 correspond to [16:0 + H-H2O]+ and [18:1 + H-H2O]+, which is representative of sn-1 FA and sn-2 FA respectively (Fig. 5a). In contrast, the structure of MAcDG 18:0/18:1/2:0 [M + H]+ m/z 682.59 in Fig. 5b was distinguished from scTG 16:0/18:1/4:0 by the fragment at m/z 605.56 which correspond to neutral loss of 77 Da from m/z 682.59 [MAcDG 18:0/18:1/2:0 + NH4]+ ion. Assignment of the sn-1 and sn-2 FAs in MAcDG 18:0/18:1/2:0 was based on the relative abundance of the fragments associated with the neutral loss of each fatty acid and the fatty acid ketene ions (Fig. 5b). A similar approach was used to assign all TG and MAcDG molecular species including isomers present in E. solidaginis (Fig. 45 & Fig. S1-S2). This work shows the advantage of C30-RPLC-HESI-HRAM-MS/MS as a superior platform for resolving isomers of MAcDG from other TG subclasses in biological samples.

Application of C30-RPLC-HESI-HRAM-MS/MS for separation and identification of MAcDG molecular species in larvae of E. solidaginis and comparisons with the literature

TG subclass and molecular species composition in E. solidaginis is of interest due to its role as a model system in cold stress tolerance. In this model system, MAcDG have been demonstrated to provide cryoprotection during exposure to low temperature stress [20]. This work has also led to subsequent studies recently on the potential of producing specialized oil seed crops with superior levels of MAcDG for applications in the production of biofuels specific for cold temperatures or northern climates [15, 47]. For example, Liu et al. (2015) reported that EaDAcT genetically modified camelina and soybean accumulated MAcDG at up to 70 mol% of the total seed oil produced by the resultant crops. A similar strategy of genetic modification increased MAcDG content to 85 mol% in field-grown transgenic camelina [15]. Furthermore, recent interests in insects as a potential source of proteins and functional ingredients for food and animal feed to improve population health and wellbeing has gained recognition, for which the larvae of E. solidaginis could be a promising food source [48, 49]. TG in E. solidaginis larvae lipidome have been reported to contain about 36 mol% MAcDG compared to long chain TG [20]. However, no report was done on the molecular species composition of MAcDG in E. solidaginis. MAcDG is associated with several health benefits in human and these include treating sepsis [10], tumor growth and cancers [11], rheumatoid arthritis [12] and asthma [13]. The distribution of MAcDG and TG subclasses in E. solidaginis presented in the current work were consistent with those presented by Marshall et al. (2014) who demonstrated that the level of lcTG and MAcDG in E. solidaginis was 36 and 29% respectively, and varied with seasons under cold temperature stress following analysis using TLC-FID [20]. The output from this method shows for the first time the molecular species composition of MAcDG compared with other TG subclasses in E. solidaginis. This is important considering E. solidaginis can survive at cold temperatures as low as − 80 °C and is a well recognised model system for studying cold temperature stress in different biological systems. MAcDG has been previously reported to confer cryoprotective properties to E. solidaginis during temperature stress survival. Information on the molecular species composition of MAcDG will allow further work by researchers in the scientific community to improve the understanding of the mechanisms associated with MAcDG metabolism during cold temperature stress acclimation, therapies associated with sepsis, asthma, arthritis in patients (Fig. 7). Furthermore, insect larvae are an emerging source of high-quality dietary lipids. This information could be useful in the evaluation of E. solidaginis as a source of dietary MAcDG or functional lipids (Fig. 7). In summary, the proposed C30-RPLC-HRAM-MS/MS method appears to be a suitable approach for the analysis of MAcDG and other TG subclasses, molecular species, isomers, and fatty acid composition in E. solidaginis lipidome, that could also be applied for routine lipidomics analysis of other biological samples.

Fig. 7
figure 7

Flow diagram showing potential applications of the proposed multimodal approach for analysis of monoacetyldiacylglycerides in food ingredients, patients, health, and disease assessments during routine lipidomics. TAGs = triglycerides

Strength and limitations of the multimodal approach using high resolution C30-RPLC-HESI-HRAM-MS/MS and TLC-FID for routine MAcDG analysis

Strengths

The multimodal approach proposed in this paper is advantageous compared to other approaches in the literature in that TLC-FID facilitate quick, cost effective, simple resolution of MAcDG from other neutral lipids including subclasses of TG, (mcTG, scTG, lcTG), as well as polar lipids using silica rods and a non-polar solvent system. The flame ionization detection (FID) facilitates rapid, cost effective quantitation of total MAcDG in the sample. This approach demonstrated for the first time the resolution of hydroxylated MAcDG from other TG subclasses. Applying C30-RPLC-HESI-HRAM-MS/MS allow determination of the molecular species composition including the fatty acids composition of MAcDG in biological samples. Most lipidomics analysis currently use C18 RPLC columns to resolve TG and other neutral lipids during routine lipidomics (Fig. 7). We used C30 RPLC instead, due to the superior resolving power of C30 stationary phase in separating TG and other lipid isomers. When combined with accurate mass tandem mass spectrometry, this is a powerful platform to resolve MAcDG molecular species, fatty acids and isomers including positional isomers as demonstrated in this paper. This is very important because MAcDG was not typically analyzed as a component of routine or global lipidomics. Generally, a targeted approach was employed that often involved pre-concentration steps.

Limitations

Currently, there is no available commercial MAcDG standard for use in the scientific community. This is a major drawback in the method particularly during extraction and quantification. Having a unique molecular version of MAcDG would allow analysts to spike their test samples to better assess recovery, during extraction, and perform more accurate and precise quantification. The hydroxylated version of MAcDG presented in this paper is currently being explored as a suitable internal standard to fill this purpose. Furthermore, MAcDG is required for use of this method during lipidomics, particularly when using the TLC FID method. This means that analytical labs will need to purify or fractionate extracts of samples from E. solidaginis, transgenic camelina or soybeans known to have up to 70 mol% MAcDG [15] for use as pure standards in the analysis. Small quantities will be required for the LC-MS based analysis, but larger quantities (microgram quantities) will be required for the TLC-FID based analysis reported in this study. This is another limitation of the proposed method that could be an impediment to routine applications and adoption in lipidomics laboratories. We hope by highlighting these limitations and benefits that this will provide an impetus for other scientists in the scientific community to devise improved strategies using the information presented in this paper as a foundation to improve or enhance this method. Currently, work in our research group is showing MAcDG to be more common in biological samples than is currently appreciated in the scientific literature. Many times, they are incorrectly analyzed as different molecular species of short chain TG.

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