Application of ESI and MALDI‐TOF MS for triacylglycerols analysis in edible oils

Triacylglycerols (TAG) are the most important group of compounds present in vegetable oils. These biomolecules, determining the physical, chemical and nutritional properties of the oils, are considered to be good fingerprints for quality and authenticity control. Therefore, TAGs characterization is a very important task in edible oil field, which has been undertaken by different analytical methods. The analysis of vegetable oils is still dominated by classic determinations, which are however laborious and time‐consuming and cannot be used routinely. More recently, advances in MS instrumentations coupled with online separation techniques and data processing have contributed to great expansion of MS in oil study, allowing the development of innovative analytical approaches that exhibit higher sensitivity, accuracy and rapidity in vegetable oils investigations. In the present contribution, a review of the most relevant applications of novel mass spectrometric techniques, such as ESI and MALDI, both alone and hyphenated with HPLC, used for analysis of the complex TAGs mixture of edible oils is illustrated.     

Antioxidant and aldose reductase inhibition activity of Ligustrum japonicum and Olea europaea L. leaf extracts

Ligustrum japonicum and Olea europaea leaf extracts prepared via domestic techniques were studied for antioxidant and aldose reductase inhibitory activity (ARI) in relation to individual phenol/flavonoid composition (HPLC‐various detection means) and content. On the same extract weight basis L. japonicum leaf extracts, poorer in total polar phenol (TPP) content and in most cases in flavonoids, were less efficient than olive leaf extracts in radical scavenging and in retarding lipid oxidation. On the same TPP content basis, the former were of comparable efficiency in radical scavenging but exhibited lower activity than the latter in bulk oils or liposomes. L. japonicum extracts were characterized by the presence of verbascoside and apigenin derivatives, whereas O. europaea extracts by that of oleuropein. Superiority of L. japonicum infusion versus O. europaea infusion was evidenced using the ARI activity assay either on the same extract weight or on TPP content basis. The former was also superior in comparison to oleuropein, tyrosol, and hydroxytyrosol activity. These findings suggest the presence of other active compounds besides the so far identified phenolics and enhance our view on the importance of Oleaceae plants as sources of bioactive compounds beyond O. europaea. Practical applications : Oleaceae plants are a unique source for the secoiridoid oleuropein or verbascoside that confer well documented biological properties. On continuation of our effort to valorize plants of this family as sources for functional constituents we examined, in parallel, leaf extracts of the less investigated species Ligustrum japonicum versus those of Olea europaea L. The data presented for the extracts obtained via domestic preparations techniques (infusions, decoctions, tinctures) are expected to attract the interest of those involved in herbal teas and dietary supplements sector.     

Characterization of volatile compounds and triacylglycerol profiles of nut oils using SPME‐GC‐MS and MALDI‐TOF‐MS

Several nut oil varieties mainly used as culinary and overall healthy food ingredients were subject of the present study. Headspace solid‐phase microextraction combined with gas chromatography‐mass spectrometry was employed in order to determine the qualitative composition of volatile compounds. Furthermore, matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry was used in order to assess the profiles and relative composition of the prevalent triacylglycerols (TAG) within the oils. The headspace of the majority of oil samples was dominated by high contents of acetic acid (up to 42%) and hexanal (up to 32%). As nut oils are typically gained by cold‐pressing from previously roasted nuts, characteristic pyrazine derivatives as well as degradation products of long‐chain fatty acids were detected. TAG analysis of these oils revealed a quite homogeneous composition dominated by components of the C₅₂ and C₅₄ group composed mainly of oleic (18:1), linoleic (18:2), stearic (18:0) and palmitic (16:0) acid residues representing together between 65 and 95% of the investigated nut oils. The TAG profiles showed characteristic patterns which can be used as ‘fingerprints’ of the genuine oils. Nut oils exhibiting quite similar fatty acid composition (e.g. hazelnut, pistachio and beech oil) could be clearly discriminated based on TAG showing significant differences between the oils.     

Policosanol characterization and accumulation during ripening of Tunisian Olea europaea L. fruits

Policosanol is a mixture of bioactive molecules shown to have beneficial effects in treating hypercholesterolemia. Food products enriched in policosanol are currently available in the US market. In the present study, eight policosanol components were identified by GC‐MS during the ripening of Meski olives. The quantitative characterization of these compounds was performed using GC‐FID. The results showed that the maximum level of total policosanol components (947.20 mg/100 g oil) was reached at the 26^th week after the flowering date of Meski olives. Hexacosanol and tetracosanol were the predominant policosanol components at Meski olive maturity. However pentacosanol, heptacosanol and tricosanol were less present in the olives and they accounted for 14% of the total policosanol at complete maturity of the fruit. The total policosanol content of Meski olives was higher than that of beeswax and whole sugar cane, which belong to the sources of dietary supplements containing policosanol. These findings indicate that olive is a potential source of these health‐enhancing compounds for functional foods and nutraceutical applications.     

木樨榄属植物中裂环烯醚萜类的提取方法及开发前景

裂环烯醚萜类化合物是木樨榄属植物标记性化合物,尤其是橄榄苦苷具有重要的药理活性。本文作者研究了作者裂环烯醚萜类化合物植物来源、化学结构特征和物化性质及药理作用,提出木樨榄属油橄榄叶提取物加工工艺和橄榄苦苷的检测方法,为我国油橄榄叶中橄榄苦苷的加工利用提供依据。     

Analysis of insect triacylglycerols using liquid chromatography‐atmospheric pressure chemical ionization‐mass spectrometry

An HPLC non‐aqueous reversed‐phase separation system was adapted for analyzing insect triacylglycerols (TAG). The method uses two conventional Nova‐Pak C18 columns connected in series, for a total length of 45 cm. The mobile phase gradient is mixed from acetonitrile and 2‐propanol, and the flow rate is programmed from 1.0 to 0.7 mL/min. TAG are detected by atmospheric pressure chemical ionization‐mass spectrometry. The method ensures efficient separation of isomers and analysis of high‐molecular‐weight TAG with equivalent chain lengths up to 72. The method performance is demonstrated on analysis of TAG isolated from the fat body of the bumblebee Bombus lucorum.     

Effects of thermal stressing on saturated vegetable oils and isolated triacylglycerols ‐ product analysis by MALDI‐TOF mass spectrometry,NMR and IR spectroscopy

It is well‐known that triacylglycerols in vegetable oils undergo slow oxidative modifications upon storage particularly at elevated temperatures. This has been shown primarily for oils with unsaturated fatty acid residues that are most sensitive towards oxidation. Saturated oils, however, were by far less investigated. In the present study saturated oils (coconut oil) as well as isolated triacylglycerols were exposed to defined thermal stressing and the resulting products were investigated in dependence on temperature and the heating period. Matrix‐assisted laser desorption and ionization spectrometry, ¹³C and ³¹P nuclear magnetic resonance spectroscopy and infrared spectroscopy were used for the characterization of the native as well as the thermally stressed oil samples. These methods were used since they provide both, fast and reliable information on oil composition and can be performed faster than other more established methods. We found that the degradation mechanism of saturated fatty acids is completely different from unsaturated fatty acids. Whereas unsaturated oils are primarily depleted under the cleavage of the double bonds, saturated oils undergo a conversion of one methylene group into a carbonyl group. This was independently demonstrated by all applied methods for the triacylglycerols as well as for the free fatty acids derived after saponification.     

Analysis of triacylglycerols in refined edible oils by isocratic HPLC‐ESI‐MS

A simple, fast and reproducible reversed‐phase high performance liquid chromatography (HPLC) method coupled to electrospray ionization mass spectrometry (ESI‐MS) for the analysis of triacylglycerols (TAGs) species in the commercial edible oils has been developed. The TAGs species were separated using isocratic 18% isopropanol in methanol and a Phenomenex C18 column. The ESI‐MS conditions were optimized using flow injection analysis of standard TAG. Fifteen, fourteen, and sixteen TAGs were separated and identified in corn oil, rapeseed oil, and sunflower oil, respectively. The presence of intense protonated molecular (M + H⁺), ammonium (M + ${\rm NH}_{4}^{ + } $ ), and sodium (M + Na⁺) adducts ions and their respective diacylglycerols ions in the ESI‐MS spectra showed correct identification of TAGs. Some minor potassium adducts (M + K⁺) were also found. In addition, the identity of the fatty acid, position of each fatty acid, and the location of the double bond in the fatty acid moiety were explained. It was found that this isocratic method is useful for fast screening and identification of triacylglycerols in lipids.     

Characterization of the effects of β‐carotene on the thermal oxidation of triacylglycerols using HPLC‐ESI‐MS

RP HPLC method coupled to ESI‐MS was used for the analysis and characterization of the oxidation of model triacylglycerols (TAGs) in presence of β‐carotene. β‐Carotene was added to the TAGs and oxidized in the Rancimat at 110°C. The samples were separated isocratically using a mixture of isopropanol with methanol and a Phenomenex C18 column. β‐Carotene degradation was measured using high performance TLC. We found that β‐carotene plays an important role during the thermal degradation of high oleic acid model TAGs. Half of the β‐carotene was degraded before 3 h of thermal treatment. β‐Carotene significantly increases the peroxide value of the TAGs after the third hour, suggesting a pro‐oxidant action. However, different TAGs show different activity toward thermal treatment and β‐carotene. The LLL was found to be less stable, OLL and OLO were stable till 10 and 12 h respectively, while POO, OOO, and OSO were the stable TAGs till 14 h. In TAGs, replacing linoleic acid by oleic acid, the stability of the corresponding TAG was found to increase by 2 h. A new class of oxidized TAGs was reported for the first time, together with previously reported species. The proposed mechanism of formation and identification of the newly identified species have been explained. Among the oxidized species of TAGs, mono‐hydroperoxides, bis‐hydroperoxides, epoxy‐epidioxides, and epoxides were the major compounds identified.     

Solid/liquid extraction and isolation by molecular distillation of hydroxytyrosol from Olea europaea L. leaves

Molecular distillation, or short‐path distillation (SPD), is particularly appropriate for processing of low‐volatility compounds, which are easily altered at high temperature. Olea europaea L. leaves constitute an olive tree by‐product very interesting for their natural antioxidants content. In this research, molecular distillation technology has been applied to obtain high‐value‐added compounds by the SPD fractionation of an olive tree leaf extract. The process consists of two stages: (a) ethanolic extraction of the olive leaves, followed by incorporation of the extract into glycerine and (b) molecular distillation of the glycerine enriched in olive leaf extract compounds (terpenic and phenolic compounds). Four molecular distillation tests under different conditions were carried out. Results showed that 80.9% 3,4‐dihydroxy‐phenylethanol (hydroxytyrosol) was recovered from the glycerine admixture under a pressure of 1.50–2.00 mbar, a temperature of 190 °C and a feed rate of 15 mL/min.     

Triacylglycerols from viper bugloss (Echium vulgare L.) seed bio‐oil

Triacylglycerols (TAG) in viper bugloss oil were isolated from raw pressed oil by silicic acid column chromatography. The obtained blend of TAG was separated by silver ion thin‐layer chromatography (TLC Ag⁺) into nine fractions, varying in terms of unsaturation level and molecular polarity. The composition of TAG in viper bugloss oil was determined by HPLC coupled with a diode‐array detector and an evaporative light‐scattering detector. The results showed that the first three fractions were combinations of TAG containing palmitic, oleic and linoleic acids. Fractions 4 and 6 contained TAG of a similar acid composition as above, but with the addition of γ‐linolenic acid. The remaining fractions (7–9) were the most varied in acid composition. They were found to contain 26–39% palmitic acid, 12–15% oleic acid, 13–41% linoleic acid 8–24% γ‐linolenic acid, 1.5–5.5% α‐linolenic acid and 1–5% stearidonic acid. The analysis of fatty acid allocation in TAG of viper bugloss lipids revealed that linoleic acid (ranging from 2 to 100%) was the only acid found in all isolated fractions. In the investigated oil, the predominant TAG included: LnLnG (11.38%), LnLnSt (11.17%), LnGSt (7.71%), LnStSt (6.19%) and LnLnLn (5.44%). Almost 86% of the TAG contained α‐linolenic acid, while γ‐linolenic and stearidonic acids amounted to 49 and 38%, respectively.     

油橄榄果渣中山楂酸和齐墩果酸的提取工艺

以油橄榄果渣为原料,对其主要有效成分山楂酸(MA)和齐墩果酸(OA)的提取工艺进行研究。以MA、OA的提取率和浸膏得率为评价指标,采用正交实验优选油橄榄果渣中MA、OA的最佳提取工艺,得到的最佳条件为:以φ(CH3CH2OH)=90%的乙醇为提取剂,料液比〔即油橄榄果渣质量与溶剂体积的比(g/mL),下同〕1∶10,回流提取3 h,提取1次,在该条件下MA、OA提取率分别为91.27%、94.73%,浸膏得率为23.38%。     

Comparative analysis of phytosterol components from rapeseed (Brassica napus L.) and olive (Olea europaea L.) varieties

Phytosterols occur in relatively high concentration in the seeds of rapeseed (Brassica napus L.) and in lower concentration in olive (Olea europaea L.) oil. The aim of this research was to investigate some new rapeseed varieties and olive genotypes that are grown in Northwest Turkey and to compare the phytosterol contents of both crops. For rapeseed, the data were collected in the growing seasons 2004–2005 from a field experiment with 19 new rapeseed varieties and three replications. For olives, 21 different varieties were used in the 2004–2005 and 2005–2006 growing seasons. The separation and identification of free phytosterols and the analysis of their contents were successfully achieved using the capillary column‐gas chromatographic method. According to the obtained results, for rapeseed, sitosterol (1.54–2.36 g/kg) was the major component of total phytosterols, followed by campesterol (0.02–1.58 g/kg) and brassicasterol (0.26–0.58 g/kg). Regarding the olive varieties, the sitosterol content changed between 1.03 and 2.01 g/kg, followed by avenasterol ranging from 0.07 to 0.44 g/kg. The brassicasterol, campesterol and stigmasterol contents did not affect the total amount of sterols. The total phytosterol content ranged between 4.25 and 11.37 g/kg for rapeseed and 1.29 and 2.38 g/kg for olives.     

HPLC‐APCI analysis of triacylglycerols in milk fat from different sources

Triacylglycerols (TAG) from milk‐fat from different sources (cow, goat and human milks) were characterised using non‐aqueous reversed phase high‐performance liquid chromatography–atmospheric‐pressure chemical ionisation, coupled to MS/MS (RP HPLC‐APCI MS/MS). The fatty‐acid content of all samples was also established by methyl transesterification and GC‐MS analysis. Optimisation of the HPLC gradient, combined with APCI tandem MS, enables TAGs complex mixtures to be analysed without prior separation. More than 160 different glycerides were identified; between 50 and 70 compounds were identified in the chromatograms of each sample. This method also enabled the principal TAG regioisomers to be recognized. The study focused on the investigation of the structure of TAGs containing very‐long‐chain PUFA, namely all cis‐ 4,7,10,13,16,19‐ DHA (DHA, C_{22:6, n‐3}) and all cis‐5,8,11,14,17‐eicosapentaenoic acid (EPA, C_{20:5, n‐3}), both in human and in n‐3‐enriched cow's milks. Ten TAGs containing DHA were identified in human milk and only one in milk from cows fed an n‐3 enriched diet.