Comprehensive Single-Platform Lipidomic/Metabolomic Analysis Using Supercritical Fluid Chromatography-Mass Spectrometry.

Supercritical fluid chromatography (SFC) is a rapidly expanding technique in the analysis of nonpolar to moderately polar substances and, more recently, also in the analysis of compounds with higher polarity. Herein, we demonstrate a proof of concept for the application of a commercial SFC instrument with electrospray ionization-mass spectrometry (MS) detection as a platform for the comprehensive analysis of metabolites with the full range of polarities, from nonpolar lipids up to highly polar metabolites. The developed single-platform SFC-MS lipidomic/metabolomic method is based on two consecutive injections of lipid and polar metabolite extracts from biphase methyl tert-butyl ether extraction using a diol column and two different gradient programs of methanol-water-ammonium formate modifier. Detailed development of the method focused mainly on the pressure limits of the system, the long-term repeatability of results, and the chromatographic performance, including optimization of the flow rate program, modifier composition and gradient, and injection solvent selection. The developed method enabled fast and comprehensive analysis of lipids and polar metabolites from plasma within a 24 min cycle with two injections using a simple analytical platform based on a single instrument, column, and mobile phase. Finally, the results from SFC-MS analysis of polar metabolites were compared with widely established liquid chromatography MS analysis in metabolomics. The comparison showed different separation selectivity of metabolites using both methods and overall lower sensitivity of the SFC-MS due to the higher flow rate and worse chromatographic performance.

Direct Chiral Supercritical Fluid Chromatography-Mass Spectrometry Analysis of Monoacylglycerol and Diacylglycerol Isomers for the Study of Lipase-Catalyzed Hydrolysis of Triacylglycerols.

The fast and selective separation method of intact monoacylglycerol (MG) and diacylglycerol (DG) isomers using chiral supercritical fluid chromatography-mass spectrometry (SFC-MS) was developed and employed to study lipase selectivity in the hydrolysis of triacylglycerols (TGs). The synthesis of 28 enantiomerically pure MG and DG isomers was performed in the first stage using the most commonly occurring fatty acids in biological samples such as palmitic, stearic, oleic, linoleic, linolenic, arachidonic, and docosahexaenoic acids. To develop the SFC separation method, different chromatographic conditions such as column chemistry, mobile phase composition and gradient, flow rate, backpressure, and temperature were carefully assessed. Our SFC-MS method used a chiral column based on a tris(3,5-dimethylphenylcarbamate) derivative of amylose and neat methanol as a mobile phase modifier, which provides baseline separation of all the tested enantiomers in 5 min. This method was used to evaluate hydrolysis selectivity of lipases from porcine pancreas (PPL) and Pseudomonas fluorescens (PFL) using nine TGs differing in acyl chain length (14-22 carbon atoms) and number of double bonds (0-6) and three DG regioisomer/enantiomers as hydrolysis intermediate products. PFL exhibited preference of the fatty acyl hydrolysis from the sn-1 position of TG more pronounced for the substrates with long polyunsaturated acyls, while PPL did not show considerable stereoselectivity to TGs. Conversely, PPL preferred hydrolysis from the sn-1 position of prochiral sn-1,3-DG regioisomer, whereas PFL exhibited no preference. Both lipases showed selectivity for the hydrolysis of outer positions of DG enantiomers. The results show complex reaction kinetics of lipase-catalyzed hydrolysis given by different stereoselectivities for substrates.

LipidQuant 1.0: automated data processing in lipid class separation-mass spectrometry quantitative workflows.

SUMMARY: We present the LipidQuant 1.0 tool for automated data processing workflows in lipidomic quantitation based on lipid class separation coupled with high-resolution mass spectrometry. Lipid class separation workflows, such as hydrophilic interaction liquid chromatography or supercritical fluid chromatography, should be preferred in lipidomic quantitation due to the coionization of lipid class internal standards with analytes from the same class. The individual steps in the LipidQuant workflow are explained, including lipid identification, quantitation, isotopic correction and reporting results. We show the application of LipidQuant data processing to a small cohort of human serum samples. AVAILABILITY AND IMPLEMENTATION: The LipidQuant 1.0 is freely available at Zenodo https://doi.org/10.5281/zenodo.5151201 and https://holcapek.upce.cz/lipidquant.php. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

A systematic evaluation of the cucurbit[7]uril pharmacokinetics and toxicity after a single dose and short-term repeated administration in mice.

Cucurbit[n]urils are macrocyclic compounds capable of forming host-guest complexes with different molecules. In this study, we focused on cucurbit[7]uril (CB[7]) safety and pharmacokinetics. We investigated CB[7] cytotocixity in human renal cells ACHN using the xCELLigence system. We also determined maximum tolerated doses (MTD) and no observed adverse effect levels (NOAEL) after intramuscular (i.m.), intraperitoneal (i.p.), and intragastric (i.g.) administration in mice using clinical observation, blood biochemistry, and histopathology. At NOAELs, we studied its pharmacokinetics in plasma and kidneys. Finally, we performed a 7 day repeated-dose toxicity study at 50% of NOAEL after i.p. administration, assaying CB[7] concentration in plasma, brain, kidney, and liver; we also assessed the liver and kidney histopathology. In vitro, CB[7] did not show toxicity up to 0.94 mg/mL. MTDs in vivo were set at 300, 350, and 600 mg/kg, and NOAEL were established at 150, 100, and 300 mg/kg after i.m., i.p., and i.g. administration, respectively. Parenteral administration produced tissue damage mainly to the kidney, while i.g. administration caused only minor liver damage. Parenteral CB[7] administration led to fast elimination from blood, accompanied with kidney accumulation; absorption from the gastrointestinal tract was minimal. Short repeated i.p. administration was well tolerated. After initial CB[7] accumulation in blood and kidney, the concentrations stabilised and decreased during the experiment. Approximately 3.6% of animals showed signs of nephrotoxicity. Although CB[7] appears to be a promising molecule, nephrotoxicity may be the most critical drawback of its parenteral use, because the kidney represents the main organ of its elimination.

Effect of Oxime Encapsulation on Acetylcholinesterase Reactivation: Pharmacokinetic Study of the Asoxime-Cucurbit[7]uril Complex in Mice Using Hydrophilic Interaction Liquid Chromatography-Mass Spectrometry.

Oxime-based molecules are used for the treatment of patients to reactivate acetylcholinesterase (AChE) function after organophosphate intoxication. However, their efficacy is limited by low penetration through the blood-brain barrier and fast elimination. In this work, the cucurbit[7]uril (CB[7]) carrier was used for the encapsulation of the clinical agent asoxime to enhance brain bioavailability and the treatment window. We present a pharmacokinetic study of asoxime and the asoxime-CB[7] complex in an in vivo mouse model. Ultrahigh-performance liquid chromatography with electrospray ionization-mass spectrometry detection was developed to determine asoxime and CB[7] in biological fluids and tissues after thorough optimization of chromatographic conditions. The dihydroxypropane-silica stationary phase using hydrophilic interaction liquid chromatography conditions provided the best chromatographic performance. The final method was validated and applied for the pharmacokinetic study of mouse plasma, urine, bile, liver, kidney, and brain samples at different times after administration of asoxime and the asoxime-CB[7] complex. The results showed a greater than 3-fold increase in the area under the curve (AUC) in the brain for asoxime administered as a complex with CB[7] relative to that for the administration of asoxime alone. The effectiveness of the treatment strategy was evaluated using a reactivation study and a functional observatory battery. Protection of brain AChE activity is crucial for saving human lives or reducing the consequences of poisoning. The asoxime administered as a complex increased the brain activity by approximately 30% compared to that with atropine alone. CB[7] coadministration improved the AChE activity by 11%, which agrees with the higher asoxime AUC assessed in the pharmacokinetic study.

Nuclear phosphatidylinositol 4,5-bisphosphate islets contribute to efficient RNA polymerase II-dependent transcription.

This paper describes a novel type of nuclear structure - nuclear lipid islets (NLIs). They are of 40-100 nm with a lipidic interior, and phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] molecules comprise a significant part of their surface. Most of NLIs have RNA at the periphery. Consistent with that, RNA is required for their integrity. The NLI periphery is associated with Pol II transcription machinery, including the largest Pol II subunit, transcription factors and NM1 (also known as NMI). The PtdIns(4,5)P2-NM1 interaction is important for Pol II transcription, since NM1 knockdown reduces the Pol II transcription level, and the overexpression of wild-type NM1 [but not NM1 mutated in the PtdIns(4,5)P2-binding site] rescues the transcription. Importantly, Pol II transcription is dependent on NLI integrity, because an enzymatic reduction of the PtdIns(4,5)P2 level results in a decrease of the Pol II transcription level. Furthermore, about half of nascent transcripts localise to NLIs, and transcriptionally active transgene loci preferentially colocalise with NLIs. We hypothesize that NLIs serve as a structural platform that facilitates the formation of Pol II transcription factories, thus participating in the formation of nuclear architecture competent for transcription.

HILIC/ESI-MS determination of gangliosides and other polar lipid classes in renal cell carcinoma and surrounding normal tissues.

Negative-ion hydrophilic liquid chromatography-electrospray ionization mass spectrometry (HILIC/ESI-MS) method has been optimized for the quantitative analysis of ganglioside (GM3) and other polar lipid classes, such as sulfohexosylceramides (SulfoHexCer), sulfodihexosylceramides (SulfoHex2Cer), phosphatidylglycerols (PG), phosphatidylinositols (PI), lysophosphatidylinositols (LPI), and phosphatidylserines (PS). The method is fully validated for the quantitation of the studied lipids in kidney normal and tumor tissues of renal cell carcinoma (RCC) patients based on the lipid class separation and the coelution of lipid class internal standard with the species from the same lipid class. The raw data are semi-automatically processed using our software LipidQuant and statistically evaluated using multivariate data analysis (MDA) methods, which allows the complete differentiation of both groups with 100% specificity and sensitivity. In total, 21 GM3, 28 SulfoHexCer, 26 SulfoHex2Cer, 10 PG, 19 PI, 4 LPI, and 7 PS are determined in the aqueous phase of lipidomic extracts from kidney tumor tissue samples and surrounding normal tissue samples of 20 RCC patients. S-plots allow the identification of most upregulated (PI 40:5, PI 40:4, GM3 34:1, and GM3 42:2) and most downregulated (PI 32:0, PI 34:0, PS 36:4, and LPI 16:0) lipids, which are primarily responsible for the differentiation of tumor and normal groups. Another confirmation of most dysregulated lipids is performed by the calculation of fold changes together with T and p values to highlight their statistical significance. The comparison of HILIC/ESI-MS data and matrix-assisted laser desorption/ionization mass spectrometric imaging (MALDI-MSI) data confirms that lipid dysregulation patterns are similar for both methods. Graphical abstract ᅟ.

Hydrophilic Interaction Liquid Chromatography-Mass Spectrometry Characterization of Gangliosides in Biological Samples.

The hydrophilic interaction liquid chromatography (HILIC) coupled to a negative-ion electrospray ionization tandem mass spectrometry (ESI-MS/MS) method has been developed for the identification of a wide range of gangliosides in biological samples. Gangliosides consist of a backbone of sphingoid base and a polar oligosaccharide chain containing at least one sialic acid. Gangliosides are extracted by chloroform-methanol-water mixture, where an upper aqueous layer containing gangliosides and other polar lipid subclasses is further purified by C18 solid-phase extraction. The optimization of chromatographic conditions includes the column selection, mobile-phase composition, pH value, buffer type, and concentration with the goal to achieve the best chromatographic resolution and MS sensitivity. The identification of gangliosides and other polar lipids is based on accurate m/z values of [M-H]- ions and fragment ions as well measured by high-resolution MS. The detailed interpretation of MS/MS spectra enables the generalization of fragmentation pathways, which is then used for the differentiation of a, b, and c series of gangliosides. The structural assignment is further confirmed by agreement with the predicted retention behavior in HILIC mode on the basis of the correlation among the ganglioside retention, the number of saccharide units, and their sequence. The final HILIC/ESI-MS/MS method is applied for the analysis of porcine brain, human kidney, lungs, plasma, and erythrocytes resulting in unambiguous identification of 145 ganglioside species from 19 subclasses, which represents the highest number of reported gangliosides. Moreover, 71 sulfatides and 59 polar phospholipids (phosphatidylserines, phosphatidylinositols, lysophosphatidylinositols, and phosphatidylglycerols) are detected within a 15 min run.

Correlation of lipidomic composition of cell lines and tissues of breast cancer patients using hydrophilic interaction liquid chromatography/electrospray ionization mass spectrometry and multivariate data analysis.

RATIONALE: The goal of this work is the comparison of differences in the lipidomic compositions of human cell lines derived from normal and cancerous breast tissues, and tumor vs. normal tissues obtained after the surgery of breast cancer patients. METHODS: Hydrophilic interaction liquid chromatography/electrospray ionization mass spectrometry (HILIC/ESI-MS) using the single internal standard approach and response factors is used for the determination of relative abundances of individual lipid species from five lipid classes in total lipid extracts of cell lines and tissues. The supplementary information on the fatty acyl composition is obtained by gas chromatography/mass spectrometry (GC/MS) of fatty acid methyl esters. Multivariate data analysis (MDA) methods, such as nonsupervised principal component analysis (PCA), hierarchical clustering analysis (HCA) and supervised orthogonal partial least-squares discriminant analysis (OPLS-DA), are used for the visualization of differences between normal and tumor samples and the correlation of similarity between cell lines and tissues either for tumor or normal samples. RESULTS: MDA methods are used for differentiation of sample groups and also for identification of the most up- and downregulated lipids in tumor samples in comparison to normal samples. Observed changes are subsequently generalized and correlated with data from tumor and normal tissues of breast cancer patients. In total, 123 lipid species are identified based on their retention behavior in HILIC and observed ions in ESI mass spectra, and relative abundances are determined. CONCLUSIONS: MDA methods are applied for a clear differentiation between tumor and normal samples both for cell lines and tissues. The most upregulated lipids are phospholipids (PL) with a low degree of unsaturation (e.g., 32:1 and 34:1) and also some highly polyunsaturated PL (e.g., 40:6), while the most downregulated lipids are PL containing polyunsaturated fatty acyls (e.g., 20:4), plasmalogens and ether lipids. Copyright © 2016 John Wiley & Sons, Ltd.

Determination of triacylglycerol regioisomers using differential mobility spectrometry.

RATIONALE: Triacylglycerols (TG) contain three fatty acyls attached to the glycerol backbone in stereochemically numbered positions sn-1, 2 and 3. Isobaric TG with exchanged fatty acyl chains in positions sn-1/3 vs. sn-2 are referred to as regioisomers and the determination of their regioisomeric ratios is important for nutrition purposes. METHODS: Differential mobility spectrometry (DMS) coupled to electrospray ionization mass spectrometry (ESI-MS) is applied for the separation of simple unsaturated TG regioisomers extracted from porcine adipose tissue using their silver-ion molecular adducts. RESULTS: Four pairs of TG regioisomers containing combinations of unsaturated and saturated fatty acyl chains are successfully separated using DMS with 1-butanol or 1-propanol as the chemical modifier. Various experimental parameters are carefully optimized, such as the separation and compensation voltages applied to DMS electrodes, the type and flow rate of chemical modifier and the dwell time of analyte ions in the DMS cell. The optimized DMS approach is used for the characterization of TG regioisomers in less than one minute, compared to tens of minutes typical for silver-ion or reversed-phase high-performance liquid chromatography/mass spectrometry approaches. CONCLUSIONS: The application of this method for the characterization of TG regioisomers in porcine adipose tissue shows the method suitability for analyses of other animal fats.

High-Throughput and Comprehensive Lipidomic Analysis Using Ultrahigh-Performance Supercritical Fluid Chromatography-Mass Spectrometry.

New analytical approach for high-throughput and comprehensive lipidomic analysis of biological samples using ultrahigh-performance supercritical fluid chromatography (UHPSFC) with electrospray ionization-mass spectrometry (ESI-MS) is presented in this work as an alternative approach to established shotgun MS or high-performance liquid chromatography-MS. The lipid class separation is performed by UHPSFC method based on 1.7 µm particle-bridged ethylene hybrid silica column with a gradient of methanol-water-ammonium acetate mixture as a modifier. All parameters of UHPSFC conditions are carefully optimized and their influence on the chromatographic behavior of lipids is discussed. The final UHPSFC/ESI-MS method enables a fast separation of 30 nonpolar and polar lipid classes within 6 min analysis covering 6 main lipid categories including fatty acyls, glycerolipids, glycerophospholipids, sphingolipids, sterols, and prenols. Individual lipid species within lipid classes are identified based on positive and negative-ion full-scan and tandem mass spectra measured with high mass accuracy and high resolving power. Developed UHPSFC/ESI-MS method is applied for the analysis of porcine brain extract as a complex lipidomic sample, where 24 lipid classes containing 436 lipid species are identified. The method is validated for the quantitative analysis of lipid species in biological tissues using internal standards for each lipid class. This high-throughput, comprehensive and accurate UHPSFC/ESI-MS method is suitable for the lipidomic analysis of large sample sets in the clinical research.

Effects of fatty acyl chain length, double-bond number and matrix on phosphatidylcholine responses in matrix-assisted laser desorption/ionization on an Orbitrap mass spectrometer.

RATIONALE: Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) is used for the fast qualitative and quantitative analysis of phosphatidylcholines (PC). Fatty acyl chain lengths and the number of double bonds (DB) affect relative responses of PC; hence the determination of correction factors of individual PC is important for the accurate quantitation. The signal intensity in MALDI-MS strongly depends on the matrix; therefore, the following matrices typically used in lipidomics are studied in the present work: 2,5-dihydroxybenzoic acid (DHB), 1,5-diaminonaphthalene (DAN) and 9-aminoacridine (9AA). METHODS: Series of PC with various fatty acyl chain lengths are synthesized for this study. PC concentrations over two orders of magnitude are studied with MALDI-MS. These experiments provide sets of calibration curves for each of the synthesized PC and the further analysis of parameters of calibration curves is performed. RESULTS: Correction factors for PC decrease with increasing fatty acyl chain length for all matrices. These dependences are steeper for unsaturated PC than for saturated ones. MALDI matrices also have a significant effect on this dependence. The weakest dependence on fatty acyl chain length is found for saturated PC in 9AA. In the case of the other matrices, the effect of fatty acyl chain length on the response is essential for both saturated and unsaturated PC. Calibration curves and parameters of calibration curves for both saturated and monounsaturated PC are fitted by a linear function with regression coefficients decreasing in the order 9AA > DAN > DHB. CONCLUSIONS: Differences in relative responses for PC in MALDI-MS measurements must be taken into account for accurate quantitation. Parameters of calibration curves can be used for the determination of PC concentrations using a single internal standard (IS). This method gives good results for the 9AA matrix, but the reproducibility of measurements for the DHB and DAN matrices is lower and the method can be used for a rough estimation only. These matrices are less convenient for the quantitation of PC.

Continuous comprehensive two-dimensional liquid chromatography-electrospray ionization mass spectrometry of complex lipidomic samples.

A new continuous comprehensive two-dimensional liquid chromatography-electrospray ionization mass spectrometry method has been developed for the lipidomic characterization of complex biological samples. The reversed-phase ultra-high-performance liquid chromatography with a C18 column (150 mm × 1 mm, 1.7 µm) used in the first dimension makes the separation of numerous lipid species differing in their hydrophobic part of the molecule, mainly fatty acyl chain lengths and the number and positions of double bonds, possible. Coeluted lipid species in the first dimension are resolved by the fast hydrophilic interaction liquid chromatography separation (50 mm × 3 mm, 2.7 µm, core-shell particles) of lipid classes according to their different polarities in the second dimension. Retention times in both dimensions, accurate m/z values, and tandem mass spectra provide high confidence in the identification of lipid species. The retention behavior of individual lipids in reversed-phase mode follows the equivalent carbon number pattern, which provides an additional tool for unambiguous identification. This analytical method is applied for the lipidomic characterization of total lipid extracts of human plasma and porcine brain samples, which resulted in the identification of 143 lipid species from four lipid categories and ten lipid classes.

Determination of lipidomic differences between human breast cancer and surrounding normal tissues using HILIC-HPLC/ESI-MS and multivariate data analysis.

The comprehensive approach for the lipidomic characterization of human breast cancer and surrounding normal tissues is based on hydrophilic interaction liquid chromatography (HILIC)-electrospray ionization mass spectrometry (ESI-MS) quantitation of polar lipid classes of total lipid extracts followed by multivariate data analysis using unsupervised principal component analysis (PCA) and supervised orthogonal partial least square (OPLS). This analytical methodology is applied for the detailed lipidomic characterization of ten patients with the goal to find the statistically relevant differences between tumor and normal tissues. This strategy is selected for better visualization of differences, because the breast cancer tissue is compared with the surrounding healthy tissue of the same patient, therefore changes in the lipidome are caused predominantly by the tumor growth. A large increase of total concentrations for several lipid classes is observed, including phosphatidylinositols, phosphatidylethanolamines, phosphatidylcholines, and lysophosphatidylcholines. Concentrations of individual lipid species inside the abovementioned classes are also changed, and in some cases, these differences are statistically significant. PCA and OPLS analyses enable a clear differentiation of tumor and normal tissues based on changes of their lipidome. A notable decrease of relative abundances of ether and vinylether (plasmalogen) lipid species is detected for phosphatidylethanolamines, but no difference is apparent for phosphatidylcholines.

Metabolomic analysis of the human placenta reveals perturbations in amino acids, purine metabolites, and small organic acids in spontaneous preterm birth.

Spontaneous preterm delivery presents one of the most complex challenges in obstetrics and is a leading cause of perinatal morbidity and mortality. Although it is a common endpoint for multiple pathological processes, the mechanisms governing the etiological complexity of spontaneous preterm birth and the placental responses are poorly understood. This study examined placental tissues collected between May 2019 and May 2022 from a well-defined cohort of women who experienced spontaneous preterm birth (n = 72) and healthy full-term deliveries (n = 30). Placental metabolomic profiling of polar metabolites was performed using Ultra-High Performance Liquid Chromatography/Mass Spectrometry (UHPLC/MS) analysis. The resulting data were analyzed using multi- and univariate statistical methods followed by unsupervised clustering. A comprehensive metabolomic evaluation of the placenta revealed that spontaneous preterm birth was associated with significant changes in the levels of 34 polar metabolites involved in intracellular energy metabolism and biochemical activity, including amino acids, purine metabolites, and small organic acids. We found that neither the preterm delivery phenotype nor the inflammatory response explain the reported differential placental metabolome. However, unsupervised clustering revealed two molecular subtypes of placentas from spontaneous preterm pregnancies exhibiting differential enrichment of clinical parameters. We also identified differences between early and late preterm samples, suggesting distinct placental functions in early spontaneous preterm delivery. Altogether, we present evidence that spontaneous preterm birth is associated with significant changes in the level of placental polar metabolites. Dysregulation of the placental metabolome may underpin important (patho)physiological mechanisms involved in preterm birth etiology and long-term neonatal outcomes.

Characterization of triacylglycerol enantiomers using chiral HPLC/APCI-MS and synthesis of enantiomeric triacylglycerols.

In this work, the first systematic characterization of triacylglycerol (TG) enantiomers in real samples using chiral high-performance liquid chromatography (HPLC) with atmospheric pressure chemical ionization mass spectrometry (APCI-MS) is performed. Our chiral HPLC/APCI-MS method is based on the use of two cellulose-tris-(3,5-dimethylphenylcarbamate) columns connected in series using a gradient of hexane-2-propanol mobile phase. All TG enantiomers containing 1-8 DBs and different fatty acyl chain lengths are separated using our chiral HPLC method except for TGs having a combination of saturated and di- or triunsaturated fatty acyls in sn-1 and sn-3 positions. In our work, the randomization reaction of monoacyl TG standards is used for the preparation of all TG enantiomers and regioisomers in a mixture, while the stereospecific esterification of 1,2- or 2,3-isopropylidene-sn-glycerols by selected fatty acids is used for the synthesis of TG enantiomers. The composition of TG enantiomers and regioisomers in hazelnut oil and human plasma samples is determined. Unsaturated fatty acids are preferentially esterified in sn-2 position in hazelnut oil, while no significant preference of saturated or unsaturated fatty acyls is observed in case of human plasma sample. Fatty acids with the higher number of DBs are preferred in sn-1 position of TG enantiomers in hazelnut oil unlike to moderate sn-3 preference in human plasma. The characterization of cholesteryl esters from TG fraction of human plasma sample using our chiral HPLC/APCI-MS method is presented as well.

Retention behavior of isomeric triacylglycerols in silver-ion HPLC: effects of mobile phase composition and temperature.

A systematic study of the retention behavior of isomeric triacylglycerols (TGs) in silver-ion HPLC on a ChromSpher Lipids column has been performed between 10 to 40°C using the most widespread hexane- and dichloromethane-based mobile phases. The randomization of mono-acyl TG standards and the random esterification of glycerol with fatty acids are employed to produce mixtures of TG isomers. The mobile phase composition has no influence on the general retention pattern, but significant differences in the retention order of double bond (DB) positional isomers in hexane and dichloromethane mobile phases are described and compared with the previous literature data. Saturated TGs with fatty acyl chain length from C7:0 to C22:0 are partially separated using the hexane mobile phase but not at all with the dichloromethane mobile phase. The hexane mobile phase enables at least partial resolution of TG regioisomers with up to seven DBs, while the resolution of only ALA/AAL and ALnA/AALn isomers is achieved with the dichloromethane mobile phase. The effect of temperature differs significantly depending on the mobile phase composition. Retention times of TGs increase with increasing temperature in the hexane mobile phase, while an opposite effect is observed for the dichloromethane mobile phase.

Nontargeted quantitation of lipid classes using hydrophilic interaction liquid chromatography-electrospray ionization mass spectrometry with single internal standard and response factor approach.

The identification and quantitation of a wide range of lipids in complex biological samples is an essential requirement for the lipidomic studies. High-performance liquid chromatography-mass spectrometry (HPLC/MS) has the highest potential to obtain detailed information on the whole lipidome, but the reliable quantitation of multiple lipid classes is still a challenging task. In this work, we describe a new method for the nontargeted quantitation of polar lipid classes separated by hydrophilic interaction liquid chromatography (HILIC) followed by positive-ion electrospray ionization mass spectrometry (ESI-MS) using a single internal lipid standard to which all class specific response factors (RFs) are related to. The developed method enables the nontargeted quantitation of lipid classes and molecules inside these classes in contrast to the conventional targeted quantitation, which is based on predefined selected reaction monitoring (SRM) transitions for selected lipids only. In the nontargeted quantitation method described here, concentrations of lipid classes are obtained by the peak integration in HILIC chromatograms multiplied by their RFs related to the single internal standard (i.e., sphingosyl PE, d17:1/12:0) used as common reference for all polar lipid classes. The accuracy, reproducibility and robustness of the method have been checked by various means: (1) the comparison with conventional lipidomic quantitation using SRM scans on a triple quadrupole (QqQ) mass analyzer, (2) (31)P nuclear magnetic resonance (NMR) quantitation of the total lipid extract, (3) method robustness test using subsequent measurements by three different persons, (4) method transfer to different HPLC/MS systems using different chromatographic conditions, and (5) comparison with previously published results for identical samples, especially human reference plasma from the National Institute of Standards and Technology (NIST human plasma). Results on human plasma, egg yolk and porcine liver extracts are presented and discussed.

Ultrahigh-performance supercritical fluid chromatography for intraclass separation of lipids: Investigation of general principles.

Powerful chromatographic techniques are required for lipidomic analyses due to the extreme complexity of natural lipidomes. In the past few years, ultrahigh-performance supercritical fluid chromatography (UHPSFC) has proven to be a good alternative to conventional LC methods for comprehensive lipidomic analysis. The goal of this work was to study UHPSFC intraclass separation of lipids according to the fatty acyl composition. The effects of column chemistry, mobile phase composition and gradient, flow rate, back pressure, temperature, and column coupling on intraclass separation of lipids were widely investigated and discussed. In general, UHPSFC exhibited interclass selectivity together with intraclass separation of lipids according to their total number of double bonds and acyl chain lengths. Moreover, separations of diacylglycerol and lysophosphatidylcholines regioisomers were achieved in some cases. The nature of the stationary phase showed the most prominent effect on UHPSFC intraclass selectivity, while other chromatographic conditions were used for partial improvement in resolution of lipid species. An octadecyl-based stationary phase showed excellent separation of nonpolar lipid species, including triacylglycerol isobars; however, it provided poor peak shapes and limited retention time reproducibility for polar lipids. Diol- and 1-aminoanthracene-based columns provided the best inter- and intraclass resolution of most lipids. The main benefit for UHPSFC separation of complex lipid samples is the combination of the acyl chain/double bond intraclass separation of lipids with excellent lipid class selectivity, which can facilitate mass spectrometry detection and quantitation of trace species without ion suppression effects.