Separation of Triacylglycerols from Edible Oil Using a Liquid Chromatography–Mass Spectrometry System with a Porous Graphitic Carbon Column and a Toluene–Isopropanol Gradient Mobile Phase

This study presented a rapid and practical method of separating triacylglycerol (TAG) from edible oil using high‐performance liquid chromatography (LC) coupled with atmospheric‐pressure chemical ionization (APCI)/mass spectrometry (MS) system with a porous graphitic carbon column (150 mm × 2.1 mm, 5 μm) and a toluene–isopropanol–formic acid mobile phase. After investigating the experimental conditions, the gradient toluene–isopropanol mobile phase containing 0.1% formic acid was changed from 50:50 to 80:20 in 30 min; the column temperature was set to 35 °C, and APCI/MS was used in the positive‐ion acquisition mode. The TAG retention displayed a special order and was summarized to fit as follows: S‐ECN (special equivalent carbon number) = 2CN (carbon number) ? 3dB (double bond number) – 5uFA (unsaturated fatty‐acid number). Then, the LC–MS method was applied to separate TAG in 6 vegetable oils, resulting in the recognition of 27 TAG in corn oil, 21 TAGs in olive oil, 22 TAG in sunflower seed oil, 28 TAG in soybean oil, 25 TAG in sesame oil, and 31 TAG in peanut oil. The TAG separation through the LC–MS method was rapid, reproducible, and durable.     

The Updated Bottom Up Solution Applied to Atmospheric Pressure Photoionization and Electrospray Ionization Mass Spectrometry

The Updated Bottom Up Solution (UBUS) was recently applied to atmospheric pressure chemical ionization (APCI) mass spectrometry (MS) of triacylglycerols (TAG). This report demonstrates that the UBUS applies equally well to atmospheric pressure photoionization (APPI) MS and to electrospray ionization (ESI) MS. Critical Ratio 1 (CR1), the [MH]⁺/Σ[DAG]⁺ or [MNH₄]⁺/Σ[DAG]⁺ ratio, does not exhibit the same strongly sigmoidal shape as it does by APCI‐MS. CR1 varies more widely for APPI‐MS than by APCI‐MS, having a maximum value of 11.8, indicating a much greater effect of unsaturation on ion ratios in APPI‐MS than APCI‐MS. Critical Ratio 2, the [AA]⁺/[AB]⁺ ratio for Type II TAG or [AC]⁺/([AB]⁺+[BC]⁺) ratio for Type III TAG, allows quantification of regioisomers of TAG, and shows good agreement for APPI‐MS to regioisomer quantification determined by APCI‐MS. Critical Ratio 3, the [BC]⁺/[AB]⁺ ratio for Type III TAG, reveals new trends relating the degree of unsaturation by APPI‐MS, and shows that structural assignments made by ESI‐MS are in good agreement to APCI‐MS data. In addition to providing valuable structural information, the Critical Ratios also constitute a reduced data set that allows APPI‐MS or ESI‐MS mass spectra to be reconstructed when processed through the UBUS. Quantification by APPI‐MS of vitamin D in the gelcaps gave values of 42.90 ± 0.83 μg, or 1716 ± 33 international units, in good agreement with APCI‐MS.     

Comprehensive and quantitative profiling of lipid species in human milk,cow milk and a phospholipid‐enriched milk formula by GC and MS/MSALL

Here we present a workflow for in‐depth analysis of milk lipids that combines gas chromatography (GC) for fatty acid (FA) profiling and a shotgun lipidomics routine termed MS/MS^ALL for structural characterization of molecular lipid species. To evaluate the performance of the workflow we performed a comparative lipid analysis of human milk, cow milk, and Lacprodan^® PL‐20, a phospholipid‐enriched milk protein concentrate for infant formula. The GC analysis showed that human milk and Lacprodan have a similar FA profile with higher levels of unsaturated FAs as compared to cow milk. In‐depth lipidomic analysis by MS/MS^ALL revealed that each type of milk sample comprised distinct composition of molecular lipid species. Lipid class composition showed that the human and cow milk contain a higher proportion of triacylglycerols (TAGs) as compared to Lacprodan. Notably, the MS/MS^ALL analysis demonstrated that the similar FA profile of human milk and Lacprodan determined by GC analysis is attributed to the composition of individual TAG species in human milk and glycerophospholipid species in Lacprodan. Moreover, the analysis of TAG molecules in Lacprodan and cow milk showed a high proportion of short‐chain FAs that could not be monitored by GC analysis. The results presented here show that complementary GC and MS/MS^ALL analysis is a powerful approach for characterization of molecular lipid species in milk and milk products. Practical applications : Milk lipid analysis is routinely performed using gas chromatography. This method reports the total fatty acid composition of all milk lipids, but provides no structural or quantitative information about individual lipid molecules in milk or milk products. Here we present a workflow that integrates gas chromatography for fatty acid profiling and a shotgun lipidomics routine termed MS/MS^ALL for structural analysis and quantification of molecular lipid species. We demonstrate the efficacy of this complementary workflow by a comparative analysis of molecular lipid species in human milk, cow milk, and a milk‐based supplement used for infant formula. A workflow for milk lipid analysis based on combined gas chromatography and high‐resolution shotgun lipidomics. In‐depth structural characterization and quantification of molecular lipid species in milk and milk products.     

Lymphatic fatty acids from rats fed human milk and formula supplemented with fish oil

Absorption of long-chain polyunsaturated fatty acids from human milk and formula supplemented with fish oil was studied to determine if the distribution route into lymphatic triacylglycerol (TAG) and phospholipid (PL) varies with the dietary source. Rats were intraduodenally infused with human milk or formula containing graded amounts of fish oil (0, 0.5, or 1.0 g/100 mL), and the mesenteric lymph was collected. Arachidonic acid (20∶4n−6) levels in lymphatic TAG and PL were highest from animals fed human milk. In the animals infused with formula containing fish oil, as the amount of eicosapentaenoic acid (EPA, 20∶5n−3) infused increased, there was essentially an equal increase of EPA associated with both lymphatic TAG and PL. Animals intraduodenally infused with human milk or formula without fish oil had only minor levels (less than 1%) of EPA in the lymph. In the fish oil-treated animals, as the amount of docosahexaenoic acid (DHA, 22∶6n−3) infused increased, there was a 16-fold increase in DHA associated with lymphatic TAG, but only a 3-fold increase in DHA associated with lymphatic PL. The highest level of DHA in rats infused with human milk was observed in lymphatic PL. Hence, fish oil can be added to formula as a source of long-chain polyunsaturated fatty acids, but the distribution of fatty acids into lymphatic TAG and PL is not the same as that observed with human milk.     

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.     

Directed sequential synthesis of conjugated linoleic acid isomers from Δ7, 9 to Δ12, 14

Position and configuration isomers of conjugated linoleic acid (CLA), from 7, 9‐ through 12, 14‐C18:2, were synthesized by directed sequential isomerizations of a mixture of rumenic (cis‐9, trans‐11 C18:2) and trans‐10, cis‐12 C18:2 acids. Indeed, the synthesized conjugated fatty acids cover the range of unsaturated systems as found in milk fat CLA. The two‐step sequence consisted in initial sigmatropic rearrangement of cis/trans CLA isomers at 200 °C for 13 h under inert atmosphere (Helium, He), followed by selenium‐catalyzed geometrical isomerization of double bonds at 120 °C for 20 h under He. Product analysis was achieved by gas‐liquid chromatography using a 120 m polar capillary column coated with 70% cyanoalkylpolysiloxane equivalent polymer. Migration of conjugated systems was geometrically controlled as follows: the cis‐C_n, trans‐C_n+2 double bond system was rearranged through a pericyclic [1, 5] sigmatropic mechanism into a trans‐C_n‐1, cis‐C_n+1 unsaturated system, while the trans‐C_n, cis‐C_n+2 double bond system was rearranged through a similar pericyclic mechanism into a cis‐C_n+1, trans‐C_n+3 unsaturated system. Selenium‐catalyzed geometrical isomerization under mild conditions then allowed cis/trans double bond configuration transitions, resulting in the formation of all cis, all trans, cis‐trans and trans‐cis isomers. A sequential combination of the two reactions resulted in a facile controlled synthesis of CLA isomers, useful for the chromatographic identification of milk fat CLA, as well as for the preparation of CLA standard mixture.     

Determination of the adulteration of butter

The adulteration of butter is a serious problem due to economic advantages taken by replacing expensive milk fat with cheaper oil without informing the customers. The authentication of milk fat methods include analysis of bulk components, especially triacylglycerols, fatty acids, sterols and tocopherols. Fatty acid and sterol composition was analysed by using GC‐MS. TAG and tocopherol profiles were examined by HPLC with diode array (DAD) and fluorescence detectors (FLDs). In addition, identification of selected TAG of butter fat was conducted by LC‐atmospheric pressure chemical ionisation (APCI)/MS technique. The lipid composition of 16 different butters available on Polish market were investigated. The cholesterol content in butter fat ranged from 176.8 to 264.8 mg/100 g of fat and in two samples of milk fat β‐sitosterol was found. The total saturated fatty acid (SFA) content in milk fat was 67.1–73.5%, monounsaturated fatty acid 24.5–30.5% and polyunsaturated fatty acid was 1.2–2.0%. Abnormalities in fatty acid profiles, e.g. high concentration of linoleic fatty acid, were found in two butters. These abnormalities were also determined in TAG profiles. The examination of tocopherols in butter fat confirmed that two products were adulterated by the addition of plant oils because they contained δ‐tocopherol which is typical for plant origin foodstuffs. The methods described are useful for investigating milk fat adulterations, and the most efficient are analysis of sterols and tocopherols composition. Practical applications: The described methods are useful for investigating adulteration of milk fat. Traditional strategies rely on examination of fatty acids methyl esters and TAG; these methods have some disadvantages. Due to the variability of fatty acid composition of milk fat and because TAG analysis is complex and time consuming, FA analysis is not an efficient approach for butter authentication. The most efficient method for butter authentication is qualitative and quantitative analysis of sterols and tocopherols. This analysis will determine if components of plant origin were used for butter production.     

Separation of asymmetric triacylglycerols into their enantiomers by recycle high‐performance liquid chromatography

A recycle HPLC system equipped with a polysaccharide‐based chiral column was used for the enantiomeric separation of asymmetric triacylglycerols (TAGs). When the chiral columns were screened by the resolution of 1,2‐dipalmitoyl‐3‐oleoyl‐rac‐glycerol separation was achieved only with the cellulose tris‐ (3,5‐dimethylphenylcarbamate) chiral selector. The resolution of other TAG enantiomers was also examined, and 1,2‐dioleoyl‐3‐palmitoyl‐rac‐glycerol, 1,2‐dipalmitoyl‐3‐linoleoyl‐rac‐glycerol, 1,2‐dipalmitoyl‐3‐eicosapentaenoyl‐rac‐glycerol, 1,2‐dipalmitoyl‐3‐docosahexaenoyl‐rac‐glycerol, and 1,2‐docosahexaenoyl‐3‐palmitoyl‐rac‐glycerol were resolved into their respective enantiomers. However, neither 1,2‐dioleoyl‐3‐linoleoyl‐rac‐glycerol, consisting of only unsaturated fatty acids, nor 1,2‐dipalmitoyl‐3‐stearoyl‐rac‐glycerol, consisting of only saturated fatty acids, was resolved. In addition, 1,2‐eicosapentaenoyl‐3‐palmitoyl‐rac‐glycerol was not resolved clearly, even in the recycle runs. These results suggest that asymmetric TAGs having both a palmitic acid moiety and an unsaturated fatty acid moiety at the sn‐1 or sn‐3 positions might be resolved on a cellulose tris‐ (3,5‐dimethylphenylcarbamate) chiral column.     

Enhanced Structuring of Fat with Reduced Saturates Using Mixed Molecular Compounds

Molecular compound formation between multicomponent triacylglyceride (TAG) mixtures was characterized using palm oil mid fraction enriched in sn‐1,3‐dipalmitoyl‐2‐oleoylglycerol (cPOP) and a commercial fraction enriched in sn‐1,3‐dioleyl‐2‐palmitoylglycerol containing a wide range of TAGs (cOPO). Compound formation occurred at a 1:1 (w/w) ratio of cPOP to cOPO corresponding to an equal parts ratio of two different families of TAGs. One family of TAGs consisted of a grouping of all TAGs composed of a saturated–oleic–saturated (StUSt) positioning of fatty acids. The second family consisted of a grouping of all TAGs composed of all oleic–saturated–oleic and saturated–saturated–oleic fatty acids (UStU, StStU). An increase in solid fat content, peak melting temperature, and crystalline domain size and a change in crystalline microstructure were observed at this ratio corresponding to an overall equality in the amount of cPOP to cOPO. This indicates that it is possible to achieve that same level of solid fat content and hardness as a mixture containing more saturates, simply by slightly decreasing the saturates and substituting an oleic monounsaturated fatty acid containing TAG mixture such that a molecular compound is formed. The increase in full‐width at half‐maximum indicated a decrease in crystalline order and/or decrease in crystal domain size. The characteristic spherulitic crystalline microstructure of OPO changed to small granular crystals upon addition of cPOP. This discovery may allow for the incorporation of a larger proportion of healthy monounsaturated fats into foods while decreasing the use of saturated and trans fats.     

Postparturition changes in the triacylglycerols of cow colostrum

The changes in the triacylglycerol (TAG) composition of colostrum fat of three cows were studied. In addition to the determination of fatty acid composition by gas chromatography, the distribution of TAG according to the acyl carbon number (ACN) and molecular weight was analyzed utilizing both supercritical fluid chromatography (SFC) and ammonia negative-ion chemical ionization mass spectrometry (MS). Colostrum TAG contained substantially less stearic and oleic acids and more myristic and palmitic acids than the normal Finnish milk fat. The major trends in the changes of fatty acids and TAG were similar for each cow, although clear differences between individuals were found. During the first week of parturition, the proportions of short-chain fatty acids (C₄–C₁₀) typically increased as well as those of stearic and oleic acids, whereas the relative amounts of C₁₂–C₁₆ acids decreased, especially those of myristic and palmitic acids. Distinct changes occurred also in TAG distributions: the proportions of molecules with ACN 38–40 increased and those with ACN 44–48 decreased. Although there were distinct differences between individuals shortly after delivery, both the fatty acid compositions and TAG distributions of the milk samples of the cows started to resemble each other after one week. The theoretical profiles of colostrum TAG calculated based on the fatty acid compositions differed clearly from the ACN distributions analyzed by SFC and MS. Thus, the analysis of TAG is essential, because the changes in molecular species composition of colostrum TAG cannot be estimated according to the fatty acid analysis alone.     

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.     

Lipase‐mediated hydrolysis of flax seed oil for selective enrichment of α‐linolenic acid

Polyunsaturated fatty acids (PUFA) are important ingredients of human diet because of their prominent role in the function of human brain, eye and kidney. α‐Linolenic acid (ALA), a C18, n‐3 PUFA is a precursor of long chain PUFA in humans. Commercial lipases of Candida rugosa, Pseudomonas cepacea, Pseudomonas fluorescens, and Rhizomucor miehei were used for hydrolysis of flax seed oil. Reversed phase high performance liquid chromatography followed by gas chromatography showed that the purified oil contained 12 triacylglycerols (TAGs) with differences in fatty acid compositions. Flax seed oil TAGs contained α‐linolenic acid (50%) as a major fatty acid while palmitic, oleic, linoleic made up rest of the portion. Among the four commercial lipases C. rugosa has preference for ALA, and that ALA was enriched in free fatty acids. C. rugosa lipase mediated hydrolysis of the TAGs resulted in a fatty acid mixture that was enriched in α‐linolenic to about 72% yield that could be further enriched to 80% yield by selective removal of saturated fatty acids by urea complexation. Such purified ALA can be used for preparation of ALA‐enriched glycerides. Practical applications : This methodology allows purifying ALA from fatty acid mixture obtained from flax seed oil by urea complexation.     

Mammary Uptake of Fatty Acids Supplied by Intravenous Triacylglycerol Infusion to Lactating Dairy Cows

Supplementing dairy cows with n-3 fatty acid-rich feeds does not easily increase quantities in milk fat. Previous results demonstrated very long-chain n-3 fatty acids are primarily transported in the PL fraction of blood, making them largely unavailable to the mammary gland for enrichment of milk fat. Our objective was to compare mammary uptake of fatty acids of increasing chain length and unsaturation delivered intravenously as TAG emulsions. Late lactation dairy cows were assigned to a completely randomized block design. Treatments were intravenous TAG emulsions enriched with oleic acid (OLA), linoleic acid (LNA), alpha-linolenic acid (ALA), or docosahexaenoic acid (DHA) and were delivered continuously at 16 mL/h for 72 h. Each treatment supplied 30 g/day of the target fatty acid. Treatment did not affect feed intake, milk yield, or milk composition, but all treatments reduced intake and yield. The proportion of DHA increased in plasma FFA, TAG, and PL with infusion. Increases of n-3 fatty acids, ALA, EPA, and DHA, were evident in the plasma PL fraction, suggesting re-esterification in the liver. Transfer efficiencies were 37.8 ± 4.1, 27.6 ± 5.4, and 10.9 ± 4.1 %, and day 3 total milk fatty acyl yields were 37.0 ± 3.4, 10.8 ± 0.4, and 3.3 ± 0.3 g for LNA, ALA, and DHA. Variation in oleic acyl yield prevented calculation of OLA transfer efficiency. Mammary uptake of fatty acids was reduced with increased chain length and unsaturation. Both liver and mammary mechanisms may regulate transfer of long-chain polyunsaturates.     

Intestinal digestion of fish oils and ω‐3 concentrates under in vitro conditions

A comparative study of the in vitro bioaccesibility of ω‐3‐oils (salmon oil, SO; tuna oil, TO; enriched‐ω‐3 oil as triacylglycerols (TAGs), ω‐3‐TAG; and enriched‐ω‐3 oil as ethyl esters (EEs), ω‐3‐EE) was performed after treatment with pancreatin (pancreatic lipase as major lipolytic enzyme) at pH 7.5. Aliquots were taken at different times of digestion for analyzing the evolution of lipid products. The micellar phase (MP) formed at 120 min of digestion was isolated, its total lipid content was extracted and its composition in lipid products was analyzed. The rate of hydrolysis of ω‐3‐TAG concentrates was continuous throughout the time of reaction (51% hydrolysis of TAGs at 120 min), whereas the digestion of SO and TO was initially faster but stopped after 10 min of reaction (35 and 38% hydrolysis of TAGs at 120 min of SO and TO, respectively). A poor hydrolysis of EEs took place for the ω‐3‐EE oil (around 7% hydrolysis of EEs at 120 min). The MP of ω‐3‐TAG oil, SO, and TO mainly consisted of free fatty acids (FFAs) and MAGs. The MP from digested ω‐3‐EE oil consisted of FFAs and undigested EEs. Therefore, the highest degree of hydrolysis and inclusion of lipid products in the micellar structure was found for the ω‐3‐TAG oil, but compared to fish oils long times of digestion were required. This experience also shows for the first time the MP composition from ω‐3‐concentrates in the form of EEs. Practical applications: Commercial ω‐3 sources can be found as purified fish oil or concentrates in the form of TAGs, FFAs, and EEs. Despite differences exist regarding their intestinal metabolism, there is lack of information about the specific composition in lipolytic products of the absorbable fraction (MP) from ω‐3‐TAG or ω‐3‐EE concentrates. This comparative study showed that (i) the in vitro bioaccesibility of ω‐3‐polyunsaturated fatty acid (PUFA) seems to be better as ω‐3‐TAG concentrates than purified fish oils, but after long times of digestion; and (ii) the in vitro hydrolysis of ω‐3‐PUFA as EEs seems to be poor, at least after the activity of the major lipolytic enzyme of pancreatin, namely pancreatic lipase. Furthermore, the inclusion of EEs within micellar structures seems to be limited. These results contribute to the knowledge of the intestinal lipolysis of ω‐3 sources by showing the composition of the MP on lipid products for the first time.     

Characterization of triacylglycerols in madeira laurel oil by HPLC-atmospheric pressure chemical ionization-MS

Madeira laurel oil was fractionated by liquid extraction combined with TLC, and TAGs were analyzed by HPLC coupled with atmospheric pressure chemical ionization-MS (APCI-MS). Eluted molecular species compositions of the eluted TAG in the complex natural mixture were determined by GC identification of FAME and byLC-atmospheric pressure chemical ionization (APCI)-MS analysis of the lipid. The APCI-MS spectra of most TAG exhibited [M+H]⁺ and [M−RCOO]⁺ ions, which defined the M.W. and the molecular association of fatty acyl residues, respectively. Despite the relatively high degree of saturation, with a saturated/unsaturated ratio of 0.70, no totally saturated TAG nor mixed asymmetric TAG with two saturated FA (SSM or SSU, where S is saturated, M is monounsaturated, and U is unsaturated) were found. This type of molecular structure provides a possible explanation for the relatively low m.p. (12–15°C) and also the high oxidative resistance observed.     

Analysis of EPA and DHA and distinction between fish oils and concentrates

Fish oil is the major dietary source of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). Adequate dietary intake of these n‐3 PUFA is beneficial to reduce the risks of cardiovascular mortality, prostate cancer, and neurological disorders in children and adults. There is a surge in demand for fish oil in the functional food market. Microencapsulation of fish oil is the trend to improve its stability and sensory quality. The EPA and DHA content of the fish oil products may vary markedly from the label due to their susceptibility to oxidation. Quick and reliable methods other than the AOAC BF₃ method have been exploited to quantify EPA and DHA in the encapsulated fish oil and the microencapsulated powdered products such as infant formula. This article describes a method to differentiate the ethyl‐ester form from the TAG form of EPA and DHA in encapsulated fish oil which may be a mixture of natural triacylglycerol enriched with ethyl esterified EPA and DHA. A method is recommended due to the difference in apparent potency of these two esterified forms, which may be a concern in infant formula and elsewhere.     

Lipids and human milk

To support the growth and development of the breast‐fed infant, human milk provides the dietary essential fatty acids, linoleic acid (LA; 18:2n‐6), α‐linolenic acid (ALA, 18:3n‐3), as well as longer‐chain polyunsaturated fatty acids including arachidonic acid (20:4n‐6) and docosahexanoic (DHA 22:6n‐3). The linoleic acid, alpha‐linolenic acid, DHA and arachidonic acid concentration of pasteurized and unpasteurized human milk remains stable during the first month of storage at –20°C and –80°C. However after the first month, a slow decrease in concentration progresses until the end of 6 months of storage at both temperatures. The levels of n‐6 and n‐3 fatty acids, particularly linoleic acid, alpha‐linolenic acid and DHA, in human milk vary widely within and among different populations, and are readily changed by maternal dietary intake of the respective fatty acid. The present paper reviews recent understanding from key researchers of maternal diet and human milk fat composition and form our work the effect of milk fat composition on storage conditions. It is important to understand that maternal diet can affect human milk fat composition and subsequently infant development and growth.     

Regiospecific analysis of triacylglycerols

Isobaric triacylglycerols which differ in the sn‐1(3) and sn‐2 positions of acyl chains are regioisomers. The objective of this review is to describe the resolution of regioisomers by silver‐ion chromatography, RP–HPLC, and by GLC. Furthermore, the aim of this review is to discuss the determination of regioisomers solely by mass spectrometry and by chromatography‐mass spectrometry (GLC and electron ionization MS, silver‐ion chromatography and APCI–MS or ESI–MS, RP–HPLC and APCI–MS, and NP–HPLC and ESI–MS).     

n‐3 PUFA Induce Microvascular Protective Changes During Ischemia/Reperfusion

Ischemia/reperfusion (I/R) injury can occur in consequence of myocardial infarction, stroke and multiple organ failure, the most prevalent cause of death in critically ill patients. I/R injury encompass impairment of endothelial dependent relaxation, increase in macromolecular permeability and leukocyte‐endothelium interactions. Polyunsaturated fatty acids (n‐3 PUFA), such as eicosapentaenoic acid (EPA, 20:5n‐3) and docosahexaenoic acid (DHA, 22:6n‐3) found in fish oil have several anti‐inflammatory properties and their potential benefits against I/R injury were investigated using the hamster cheek pouch preparation before and after ischemia. Before the experiments, hamsters were treated orally with saline, olive oil, fish oil and triacylglycerol (TAG) and ethyl ester (EE) forms of EPA and DHA at different daily doses for 14 days. Fish oil restored the arteriolar diameter to pre ischemic values during reperfusion. At onset and during reperfusion, Fish oil and DHA TAG significantly reduced the number of rolling leukocytes compared to saline and olive oil treatments. Fish oil, EPA TAG and DHA TAG significantly prevented the rise on leukocyte adhesion compared to saline. Fish oil (44.83 ± 3.02 leaks/cm²), EPA TAG (31.67 ± 2.65 leaks/cm²), DHA TAG (41.14 ± 3.63 leaks/cm²), and EPA EE (30.63 ± 2.25 leaks/cm²), but not DHA EE (73.17 ± 2.82 leaks/cm²) prevented the increase in macromolecular permeability compared to saline and olive oil (134.80 ± 1.49 and 121.00 ± 4.93 leaks/cm², respectively). On the basis of our findings, we may conclude that consumption of n‐3 polyunsaturated fatty acids, especially in the triacylglycerol form, could be a promising therapy to prevent microvascular damage induced by ischemia/reperfusion and its consequent clinical sequelae.