Dynamic Assessment of Functional Lipidomic Analysis in Human Urine

The development of enabling mass spectrometry platforms for the quantification of diverse lipid species in human urine is of paramount importance for understanding metabolic homeostasis in normal and pathophysiological conditions. Urine represents a non‐invasive biofluid that can capture distinct differences in an individual's physiological status. However, currently there is a lack of quantitative workflows to engage in high throughput lipidomic analysis. This study describes the development of a MS/MS^ALL shotgun lipidomic workflow and a micro liquid chromatography–high resolution tandem mass spectrometry (LC–MS/MS) workflow for urine structural and mediator lipid analysis, respectively. This workflow was deployed to understand biofluid sample handling and collection, extraction efficiency, and natural human variation over time. Utilization of 0.5 mL of urine for structural lipidomic analysis resulted in reproducible quantification of more than 600 lipid molecular species from over 20 lipid classes. Analysis of 1 mL of urine routinely quantified in excess of 55 mediator lipid metabolites comprised of octadecanoids, eicosanoids, and docosanoids generated by lipoxygenase, cyclooxygenase, and cytochrome P450 activities. In summary, the high‐throughput functional lipidomics workflow described in this study demonstrates an impressive robustness and reproducibility that can be utilized for population health and precision medicine applications.     

Adapted MS/MSALL Shotgun Lipidomics Approach for Analysis of Cardiolipin Molecular Species

Cardiolipin (Ptd₂Gro) is a complex, doubly charged phospholipid located in the inner mitochondrial membrane where it plays an essential role in regulating bioenergetics. Abnormalities in Ptd₂Gro content or composition have been associated with mitochondrial dysfunction in a variety of disease states. Here, we report the development of an adapted high‐resolution data‐independent acquisition (DIA) MS/MS^ALL shotgun lipidomic method to enhance the accuracy and reproducibility of Ptd₂Gro molecular species quantitation from biological samples. Utilizing the doubly charged molecular ions and the isotopic pattern with negative mode electrospray ionization mass spectrometry (ESI‐MS) using an adapted MS/MS^ALL approach, we profiled more than 150 individual Ptd₂Gro species, including monolysocardiolipin (MLPtd₂Gro). The method described in this study demonstrated high reproducibility, sensitivity, and throughput with a wide dynamic range. This high‐resolution MS/MS^ALL shotgun lipidomics approach could be extended to screening aberrations of Ptd₂Gro metabolism involved in mitochondrial dysfunction in various pathological conditions and diseases.     

An automated shotgun lipidomics platform for high throughput,comprehensive, and quantitative analysis of blood plasma intact lipids

Blood plasma has gained protagonism in lipidomics studies due to its availability, uncomplicated collection and preparation, and informative readout of physiological status. At the same time, it is also technically challenging to analyze due to its complex lipid composition affected by many factors, which can hamper the throughput and/or lipidomics coverage. To tackle these issues, we developed a comprehensive, high throughput, and quantitative mass spectrometry‐based shotgun lipidomics platform for blood plasma lipid analyses. The main hallmarks of this technology are (i) it is comprehensive, covering 22 quantifiable different lipid classes encompassing more than 200 lipid species; (ii) it is amenable to high‐throughput, with less than 5 min acquisition time allowing the complete analysis of 200 plasma samples per day; (iii) it achieves absolute quantification, by inclusion of internal standards for every lipid class measured; (iv) it is highly reproducible, achieving an average coefficient of variation of <10% (intra‐day), approx. 10% (inter‐day), and approx. 15% (inter‐site) for most lipid species; (v) it is easily transferable allowing the direct comparison of data acquired in different sites. Moreover, we thoroughly assessed the influence of blood stabilization with different anticoagulants and freeze‐thaw cycles to exclude artifacts generated by sample preparation. Practical applications: This shotgun lipidomics platform can be implemented in different laboratories without compromising reproducibility, allowing multi‐site studies and inter‐laboratory comparisons. This possibility combined with the high‐throughput, broad lipidomic coverage and absolute quantification are important aspects for clinical applications and biomarker research. This MS‐based automated shotgun lipidomics platform for comprehensive analysis of the blood plasma lipidome (covering 22 lipid classes) achieves high inter‐day and inter‐site reproducibility and accuracy and enables unprecedented large scale lipidomics studies.     

A comparative study of fatty acid profiles in ruminant and non‐ruminant milk

This is a comprehensive study of fatty acid (FA) profiles in milk from bovine, caprine, ovine, asine, and equine species. Milks from these species are universally common as constituents in a variety of different food and dairy products. We have obtained structural information on FAs, and discussed their correlation to health effects. The extracted fat from all species were derivatized to FA methyl esters for analysis by GC‐MS. Large differences in the lipid content and FA composition between ruminants and non‐ruminants were observed. Ovine milk showed the highest lipid content of all the animals tested, both ruminants and non‐ruminants. Among the ruminants, bovine milk was richer in saturated FAs (69.7%) than ovine‐ and caprine milk (57.5 and 59.9%, respectively). Ovine milk contained the highest amounts of monounsaturated FAs (39.1%) and also odd‐ and branched‐chain FAs (5.5%). Milk from the monogastric animals, mares and donkeys, were highest in polyunsaturated FAs with a content of 19.3 and 14.2%, respectively. The assumed health negative trans FAs were analyzed to be highest in the ruminant milk (0.7–1.0%). Milk from these species contained also the highest amount of the health beneficial CLA (0.4–0.7%). Practical applications: This is a comprehensive study of milk from five species analyzed under identical conditions. The different fatty acids and their derivatives are increasingly important components because of the contradictory reports on positive and negative effects on human health. New information on the composition of milk from different species is of great importance. The results may give valuable information to producers and nutritional advisors on the consumption of milk and milk products.     

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.     

Prediction of the goat milk fatty acids by near infrared reflectance spectroscopy

Near infrared reflectance spectroscopy (NIRS) was used to predict the goat milk fatty acid (FA) profile. The ability of cow milk broad‐based calibration equations to predict the goat milk FA profile was assessed. Three hundred twenty‐eight samples in the calibration set and 108 in the validation set were analyzed. We showed that the bias and unexplained error were significant for most of the FA despite an adequate standardized Mahalanobis distance (index to establish the boundaries of a population of samples) which allowed us to test the ability of bovine models to predict goat milk FA profiles. To better predict the goat milk FA composition, a specific goat model was investigated. The cross‐validation coefficient of determination (R²CV; proportion of variance explained by the model in cross‐validation) and residual predictive deviation (RPD; index which allows to standardize the standard error of prediction (SEP)) were >0.90 and 3, for saturated, monounsaturated, and unsaturated FA, total trans FA, isomer cis9trans11 of CLA, cis9‐, trans10‐, and trans11‐C18:1, respectively. Monitoring of the equation performance of milk FA included the calculation of the bias and unexplained error. Practical applications: In this work, we evaluated the ability of NIRS to predict FA composition of goat milk and tested the ability of using broad‐based cow milk calibration equations to monitor an independent set of goat milk samples. We tested the hypothesis that the calibration equations obtained for FA from cow milk could be applied on goat milk. As the spectra of goat milk are similar to those of cow milk when they are measured by the standardized Mahalanobis distance (index to establish the boundaries of a population of samples), we can accept the hypothesis. It will be possible to use the existing calibration equations for predicting FA profile on spectra of milk of a different specie. However, the rejection of the hypothesis would put in doubt the use of Mahalanobis distance as the unique index for testing the performance of calibration equations on different milk populations for predicting FA.     

<Superscript>1</Superscript>H NMR characterization of milk lipids: A comparison between cow and buffalo milk

Characterization of the lipid fraction of raw cow and buffalo milk samples, collected in different breeding areas in Apulia, a region of southern Italy, were performed by means of ¹H NMR. The aim of this work was to establish whether FA composition data obtained by ¹H NMR can be used in the differentiation of buffalo and cow milk samples according to species. A complete assignment of the signals present in the spectrum was attempted by COSY, heteronuclear coherence spectra. Quantification of FA was carried out by inserting the integrals of particular peaks in suitable calculations. Multivariate statistical analysis, conducted on the results of the quantification, permitted buffalo and cow milks to be distinguished.     

Simple,Rapid Lipidomic Analysis of Triacylglycerols in Bovine Milk by Infusion-Electrospray Mass Spectrometry

Bovine milk is a complex mixture of lipids, proteins, carbohydrates, and other factors of which lipids comprise 3–5% of the total mass. Rapid analysis and characterization of the triacylglycerols (TAG) that comprise about 95% of the total lipid is daunting given the numerous TAG species. In the attached methods paper, we demonstrate an improved method for identifying and quantifying TAG species by infusion-based “shotgun” lipidomics. Because of the broad range of TAG species in milk, a single internal standard was insufficient for the analysis and required sectioning the spectrum into three portions based upon mass range to provide accurate quantitation of TAG species. Isobaric phospholipid interferences were removed using a simple dispersive solid-phase extraction step. Using this method, > 100 TAG species were quantitated by acyl carbon number and desaturation level in a sample of commercially purchased bovine milk.     

Quantification of the Molecular Species of TAG and DAG in Lesquerella (Physaria fendleri) Oil by HPLC and MS

Ten diacylglycerols (DAG) and 74 triacylglycerols (TAG) in the seed oil of Physaria fendleri were recently identified by high‐performance liquid chromatography (HPLC) and mass spectrometry (MS). These acylglycerols (AG) were quantified by HPLC with evaporative light scattering detector and electrospray ionization mass spectrometry of the lithium adducts of the AG in the HPLC fractions of lesquerella oil. The MS¹ ion signal intensities of molecular ions [M + Li]⁺ in HPLC fractions of an HPLC peak were used to estimate the ratios of AG in the HPLC peak. The ratios of TAG with the same mass in HPLC fractions were estimated by the ratios of the sums of MS² ion signal intensities from the neutral loss of the three fatty acids [M + Li ? FA]⁺. The ratio of DAG with the same mass were estimated by the ratio of the sums of two MS² ion signal intensities [M + Li ? FA]⁺ and [FA + Li]⁺ from the two different FA of a DAG. We have estimated the contents of ten molecular species of DAG and 74 molecular species of TAG in P. fendleri oil using this new method. The content of ten DAG combined was about 1 % and 74 TAG was about 98 %. The contents of DAG in decreasing order were: LsLs (0.25 %), LsLn (0.25 %), LsO (0.24 %), and LsL (0.11 %); and the contents of TAG in decreasing order were: LsLsO (31.3 %), LsLsLn (24.9 %), LsLsL (15.8 %), LsL‐OH20:2 (4.3 %), LsO‐OH20:2 (2.8 %), and LsLn‐OH20:2 (2.5 %).     

Membrane phospholipids and sterols in microfiltered milk fat globules

Native milk fat globules of various mean diameters, ranging from d₄₃ = 2.3 µm to 8.0 µm, were obtained using microfiltration of raw whole milk. After milk fat globule washing, the milk fat globule membrane (MFGM) was separated by manual churning. After total lipid extraction and separation of polar lipids, their phospholipid (PL) and sterol composition was measured using thin‐layer chromatography, methyl ester analyses by gas chromatography, and gas chromatography coupled to mass spectrometry. The main PL species were phosphatidylethanolamine, phosphatidylcholine and sphingomyelin. The respective fatty acid composition of each PL species was measured. Many different minor bioactive sterols were detected in the MFGM, e.g. lanosterol, lathosterol, desmosterol, stigmasterol and β‐sitosterol. No significant differences in the PL and sterol profile were found between MFGM extracted from small and large milk fat globule fractions.     

Composition and Structure of Single Cell Oil Produced by Schizochytrium limacinum SR31

Schizochytrium limacinum sp. was commercially utilized to produce single cell oil (SCO), but its detailed lipid profile remains unknown. In order to analyze the composition and structure of SCO produced by Schizochytrium limacinum SR31, the SCO extracted at stationary growth phase was separated into neutral lipids (NL), polar lipids and glycolipids by using silica gel column chromatography. The result of lipid classes in NL revealed that triacylglycerol (TAG) was the primary glyceride (93.81 %). Fatty acid (FA) analysis by gas chromatography–mass spectrometer (GC–MS) proved that palmitic acid (29.78 %) and docosahexaenoic acid (25.64 %) were the major FA. Quadrupole time of flight mass spectrometry in electrospray ionization mode coupled with ultra‐performance liquid chromatography was carried out to investigate TAG structures. More than 80 species were identified. Positional distribution of FA in TAG might contribute to further study of the FA shifts during the lipid turnover stage and nutritional evaluation of the SCO.     

Directly resistively heated-column gas chromatography for the evaluation of cow milk fat purity

Cow milk fat purity is currently evaluated by triacylglycerol (TAG) analysis. The method is based on the gas chromatographic analysis of TAG according to their total number of carbon atoms, followed by the application of formulae deriving from multiple linear regressions. The original procedure was set up by using a packed column, but different successive works showed its suitability also with the adoption of more modern adjustments. Recently, the use of high-speed gas chromatography has been spreading. In this work, the suitability of the directly resistively heated-column gas chromatography [Ultrafast module (UFM)-GC] for the evaluation of milk fat purity by the Official EU method was tested. Pure milk fat, together with mixtures of milk fat containing two levels of four foreign fats (coconut fat, sunflower oil, lard and beef tallow), were analyzed by both the conventional capillary GC method and UFM-GC. Sheep, goat and buffalo milk fats were also analyzed. The reference material CRM-519 was used for calibration. Repeatability and reproducibility values were always below the limits reported in the Official method, demonstrating the suitability of UFM-GC for the evaluation of milk fat purity. The investigation on sheep, goat and buffalo milk fat confirmed that the S ranges, calculated for cow milk fat, are not applicable to the evaluation of genuineness of non-bovine milk fats.     

Identification and Characterization of Phospholipids with Very Long Chain Fatty Acids in Brewer’s Yeast

Yeast lipids and fatty acids (FA) were analyzed in Saccharomyces pastorianus from seven breweries and in the dietary yeast supplement Pangamin. GC–MS identified more than 30 FA, half of which were very‐long chain fatty acids (VLCFA) with hydrocarbon chain lengths of ≥22 C atoms. Positional isomers ω‐9 and ω‐7 were identified in FA with C18–C28 even‐numbered alkyl chains. The most abundant ω‐7 isomer was cis‐vaccenic acid. The structure of monounsaturated FA was proved by dimethyl disulfide adducts (position of double bonds and cis geometric configuration) and by GC–MS of pyridyl carbinol esters. Ultra‐high performance liquid chromatography‐tandem mass spectrometry with negative electrospray ionization identified the phospholipids phosphatidylethanolamine, phosphatidylinositol and phosphatidylcholine, with more than 150 molecular species. Wild‐type unmutated brewer's yeast strains conventionally used for the manufacture of food supplements were found to contain VLCFA.     

Detection of cow milk admixture to buffalo milk

A method has been devised that gives the distribution of various fatty acids of pure and adulterated buffalo milk with cow milk. Gas chromatography (GC) was used for the qualitative and quantitative determination of fatty acids of authentic buffalo milk, cow milk and buffalo milk adulterated with cow milk. The milk fat was separated by fractional crystallization at -20 C into 2 fractions, i.e., semisolid and mother liquor. The concentration of fatty acids in the mother liquor changed significantly for 14:0, 16:0 and 18:1 as adulteration levels were increased. The fatty acids of the semisolid fractions change in the proportion of 16:0, 18:0 and 18:1 when cow milk is mixed with buffalo milk. By applying simple regression equations for these acids, adulteration of buffalo milk with 5% cow milk could be distinguished.     

Triglyceride composition of ewe,cow, and goat milk fat

The separation and identification of the components in milk fat, which are mainly triglycerides, is a challenge due to its complex composition. A reverse-phase high-performance liquid chromatography (HPLC) method with gradient elution and light-scattering detection is described in this paper for the triglyceride analysis in ewes’ milk fat. Triglyceride identification was carried out by combining HPLC, gas-liquid chromatography (GLC), and the calculated equivalent carbon numbers of several triglyceride standards. Quantitation of partially resolved peaks in the HPLC chromatogram was accomplished by applying a peak deconvolution program. Forty-four fatty acids were identified by GLC analysis, but only 19 were used for the following prediction of triglyceride molecular species; 181 triglycerides were identified, some of which were grouped at the same peak and needed application of the deconvolution program. Consequently, coefficients of variation were close to or lower than 5%. Moreover, the triglyceride composition of ewe, cow, and goat milk fat were compared by using these methods. These results show that ewe milk fat is richer in short- and medium-chain triglycerides, and cow milk fat is richer in long-chain and unsaturated triglycerides.     

Rapid detection of vegetable oils in milk fat by on‐line LC‐GC analysis of β‐sitosterol as marker

On‐line liquid chromatography‐gas chromatography (LC‐GC) has been applied to the detection of vegetable oils in milk fat using β‐sitosterol as marker. The method involves transesterification of the fat, pre‐separation of the sterol fraction from other lipid constituents and on‐line transfer to the capillary GC system. The on‐line approach avoids time‐consuming sample preparation steps prior to GC analysis. The suitability of this analytical approach was tested with model mixtures of milk fat with cotton and rapeseed oil. The method allows the detection of adulterations at low levels. Simultaneous quantification of cholesterol in milk fat is also possible. Considering the rapid sample preparation, the described method can be applied for screening of large sample numbers.     

Lipidomic Analysis: From Archaea to Mammals

Lipids are among the most important organic compounds found in all living cells, from primitive archaebacteria to flowering plants or mammalian cells. They form part of cell walls and constitute cell storage material. Their biosynthesis and metabolism play key roles in faraway topics such as biofuel production (third‐generation biofuels produced by microorganisms, e.g. algae) and human diseases such as adrenoleukodystrophy, Zellweger syndrome, or Refsum disease. Current lipidomic analysis requires fast and accurate processing of samples and especially their characterization. Because the number of possible lipids and, more specifically, molecular species of lipids is of the order of hundreds to thousands, it is necessary to process huge amounts of data in a short time. There are two basic approaches to lipidomic analysis: shotgun and liquid chromatography–mass spectometry. Both methods have their pros and cons. This review deals with lipidomics not according to the type of ionization or the lipid classes analyzed but according to the types of samples (organisms) under study. Thus, it is divided into lipidomic analysis of archaebacteria, bacteria, yeast, fungi, algae, plants, and animals.     

Use of BPX‐70 60‐m GC columns for screening the fatty acid composition of industrial cookies

The fatty acid (FA) composition of food is requested for labelling purposes and food composition tables. Suitable analytical methods for labelling purposes must be able to efficiently identify and accurately quantify FA as swiftly and cheaply as possible. This study evaluated a middle‐length highly polar column, the BPX‐70 60‐m column, to balance analysis efficiency and duration. The use of a 60‐m column led to the loss of data on minor FA but a gain in analysis time. The column was evaluated by analysing the FA composition of ten cookies made with different kinds of fats, including milk fat, and pure and/or partially hydrogenated vegetable oils. The FA elution order in this GC phase has been poorly documented in the literature compared to equivalent highly polar CPSil‐88 and SP‐2460 GC phases. Co‐elutions and overlaps on the BPX‐70 column were studied and commented upon. Overall, the BPX‐70 60‐m column could be used for rapid screening of the FA composition of simple foods. Analysis of the FA composition of a complex matrix, such as a dairy product, and specific analysis of trans‐FA required a longer highly polar column, possibly after fractionation by silver‐ion liquid chromatography. Compared to other GC phases, the BPX‐70 enabled effective isolation of 18:3 isomers although these isomers co‐eluted with 20:1 isomers on other highly polar GC phases. However, some CLA isomers co‐eluted with other FA on this column, and a specific analysis of these special FA would require another phase and/or different chromatographic conditions.     

An Efficient Glycoblotting‐Based Analysis of Oxidized Lipids in Liposomes and a Lipoprotein

Although widely occurring lipid oxidation, which is triggered by reactive oxygen species (ROS), produces a variety of oxidized lipids, practical methods to efficiently analyze oxidized lipids remain elusive. Herein, it is shown that the glycoblotting platform can be used to analyze oxidized lipids. Analysis is based on the principle that lipid aldehydes, one of the oxidized lipid species, can be captured selectively, enriched, and detected. Moreover, 3‐methyl‐1‐p‐tolyltriazene (MTT) methylates phosphoric and carboxylic acids, and this MTT‐mediated methylation is, in combination with conventional tandem mass spectrometry (MS/MS) analysis, an effective method for the structural analysis of oxidized lipids. By using three classes of standards, liposomes, and a lipoprotein, it is demonstrated that glycoblotting represents a powerful approach for focused lipidomics, even in complex macromolecules.     

Development of an Automated Multi-Injection Shotgun Lipidomics Approach Using a Triple Quadrupole Mass Spectrometer

Shotgun lipidomics is a well-suited approach to monitor lipid alterations due to its ability to scan for varying lipid types on a global, class and individual species level. However, the ability to perform high-throughput shotgun lipidomics has remained challenging due to time-consuming data processing and hardware limitations. To increase the throughput nature of shotgun lipidomics, an automated shotgun lipidomics approach is described utilizing conventional low flow gradient liquid chromatography (LC) analysis (post-injection) coupled with multiple sample injections per sample (on a lipid scan per injection basis). The proposed automated multi-injection approach resulted in a reproducible lipid scanning period of 2.5 min (in a 4.5 min total data acquisition period), thereby providing a sufficient scanning period for performing either mass spectrometric or tandem mass spectrometric analyses. In addition to being simple, robust and reproducible, this approach was also constructed to be cost-effective by using common LC instrumentation and customizable as the data acquisition period can be tailored to perform different scan types, period lengths and scan numbers. Combined with a strategy to create multiple lipid-specific aliquots per sample, the overall approach provides a simple and efficient platform to perform high-throughput lipid profiling.