Quantification Of Phospholipids Research Articles

Quantifying Phospholipids in Organic Samples Using a Hydrophilic Interaction Liquid Chromatography-Inductively Coupled Plasma High-Resolution Mass Spectrometry (HILIC-ICP-HRMS) Method.

In this study, a novel method using hydrophilic interaction liquid chromatography (HILIC) coupled with inductively coupled plasma high-resolution mass spectrometry (ICP-HRMS) was introduced for the quantification of phospholipids in oil samples. The method employed a bridged ethyl hybrid (BEH) stationary phase HILIC column with a tetrahydrofuran (THF)/water mobile phase, enhancing the solubility and detection of phospholipids. During the study, a gradient/matrix effect on ICP-HRMS sensitivity was observed and successfully compensated for experimentally, ensuring reliable quantification results. This approach has proven effective for a wide range of different oil samples including vegetable oils, animal fats, and phospholipid supplements. Notably, this method allowed the direct quantification of phospholipids in oil samples, bypassing the need for prior sample preparation methods, such as solid phase extraction (SPE), thereby streamlining the analytical process. The precision, accuracy, and reduced need for extensive sample preparation offered by this method mark a significant advancement in lipids analysis. Its robustness and broad applicability have substantial implications for industries such as food and renewable energy production, where both efficient and accurate lipid identification and quantification are crucial.

Development and validation of a HPLC-UV method for the quantification of major phospholipids in milk

Phospholipids have gained increased interest in the past decades because of their numerous health benefits. Due to the amphiphilic nature of phospholipids, their quantitative analysis requires lengthy procedures. Hydrophilic interaction liquid chromatography (HILIC) with UV detector was used in the present study to effectively separate the major classes of phospholipids found in milk. Phospholipids were effectively separated on a diol-HILIC column using a gradient elution method using acetonitrile and 10 mM ammonium acetate (pH 5 adjusted using acetic acid). Good chromatographic separation of major phospholipid classes: phosphatidylethanolamine (PE), phosphatidylcholine (PC), and sphingomyelin (SM) was possible at 203 nm. The developed method was validated for both phospholipid standards and milk fat. The correlation coefficient for phospholipids using a linear regression equation for 1–50 μg/mL standard solutions and milk fat were >0.99 and >0.98, respectively. The relative standard deviation for intra-day and inter-day repeatability of phospholipids were less than 6 %, and recoveries fell within the 75-120% range. The developed method was applied for the quantification of phospholipids (PC, PE, and SM) in milk from indigenous and crossbred cattle. Gir, Sahiwal, and Karan Fries cows had no significant difference in PE and PC, while Gir cows exhibited significantly higher levels of SM.

Quantification of phospholipids and glycerides in human milk using ultra-performance liquid chromatography with quadrupole-time-of-flight mass spectrometry.

Human milk lipids, which are an important source of energy and affect growth and development of infants, require a comprehensive method for its qualitative and quantitative analysis. This work describes a method for the analysis of phospholipids, glycerides, free fatty acids and gangliosides in human milk by ultra-performance liquid chromatography using a C18 column with quadrupole-time-of-flight mass spectrometry (Q-TOF-MS). The lipids were extracted by liquid-liquid extraction and phospholipids were separated by solid phase extraction (SPE). The chromatographic columns with two different specifications (4.6 mm × 150mm, and 3mm × 50mm) were used to detect phospholipids and glycerides in human milk, respectively. The sphingolipids and glycerides were analyzed in positive ion mode, and the glycerophospholipids and free fatty acids were analyzed in negative ion mode. Both internal and external standards were used for absolute quantification in this experiment. 483 species of lipids, including phospholipids, glycerides, free fatty acids and gangliosides, in human milk were analyzed using UPLC-Q-TOF-MS with high sensitivity and good linearity, with coefficient of correlation above 0.99, the relative standard deviation of accuracy and precision less than 10%. The results in a large number of human milk samples showed that this method was suitable for qualitative and quantitative analysis of lipids in human milk, even for other mammalian milk and infant formulae.

Changes in Lysophospholipid Components in Ulcerative Colitis and Colitis-associated Cancer.

In recent years, it has become clear that, in addition to normal cytokines, phospholipid mediators play an important role in the development, growth, infiltration, and metastasis of cancer and in the cancer microenvironment. A phospholipid analysis method using tandem mass spectrometry (LC-MS/MS) with high detection sensitivity has enabled quantification of phospholipids, even when using a very small sample. To date, we had applied this MS technology to colorectal cancer tissue. Therefore, in this study, this mass spectrometry technique was applied to ulcerative colitis (UC) and UC-related colorectal cancer, and an analysis was conducted with the aim of clarifying which lysophospholipids specifically change in each type of tissue. UC-associated colorectal cancer tissue and UC mucosa were collected from surgical specimens of colitic cancer (n=3). Cancerous and non-cancerous tissues were collected from surgical specimens from patients with sporadic colorectal cancer (n=11). After extraction from these tissues, the amounts of lysophospholipids were quantified by LC-MS/MS. In addition, lysophosphatidylserine (LPS) and lysophosphatidylinositol (LPI) were quantified for each molecular species of fatty acids. Compared to normal mucosa, LPI was increased 3.8-fold (p<0.001) and LPS 3.5-fold (p<0.001) in UC-related colorectal cancer. Molecular species of LPI which were increased in UC-related colorectal cancer were 18:0 (p=0.001), 16:0 (p=0.03) and 20:4 (p=0.004), and of LPS were 18:0 (p<0.001) and 22:6 (p=0.014). Lysophospholipids increased in colorectal cancer and in UC-associated colorectal cancer. In particular, LPI may have contributed significantly to colitis-associated carcinogenesis.

Correction to Relative Quantification of Phospholipids Based on Isotope-Labeled Methylation by Nanoflow Ultrahigh Performance Liquid Chromatography–Tandem Mass Spectrometry: Enhancement in Cardiolipin Profiling

Correction to Relative Quantification of Phospholipids Based on Isotope-Labeled Methylation by Nanoflow Ultrahigh Performance Liquid Chromatography–Tandem Mass Spectrometry: Enhancement in Cardiolipin Profiling

Phosphorus-31 nuclear magnetic resonance (31P NMR) for quantitative measurements of phospholipids derived from natural products: Effect of analysis conditions

In the present study the running conditions (sample pH, scan number, relaxation delay and NMR solvent) for the quantification of phospholipids by 31P NMR using commercial pure standards were optimised. A mixture of pure standards was run at different pH (7.3, 7.8, 9.5 and 12), scan numbers (64, 128, 192 and 512), relaxation delay times (1s, 2s, 3.5s and 10s) and NMR solvents (D2O/EDTA/sodium cholate or CDCl3/CD3OD/EDTA) to achieve this goal. Best 31P NMR conditions were found to be pH 7.3, a scan number of 192 and a relaxation delay of 3.5s, which were used for the quantification of natural samples (Krill oil, soybean lecithin and egg phospholipid) in D2O/EDTA/sodium cholate or CDCl3/CD3OD/EDTA. Phospholipids and lysophospholipids were highly resolved and detected in D2O/EDTA/sodium cholate compared to CDCl3/CD3OD/EDTA. A number of phospholipids (lysophoshatidylglycerol-2, LPG-2; lysophosphatidylethanolamine, LPE; N-acyl phosphatidylethanolamine, APE) were not detected in CDCl3/CD3OD/EDTA, but they were detected in D2O/EDTA/sodium cholate. Therefore, it is concluded that D2O/EDTA/sodium cholate is a better detection solvent for 31P NMR quantification of phospholipids extracted from natural sources.

Identification and quantification of phospholipids in strawberry seeds and pulp (Fragaria × ananassa cv San Andreas) by liquid chromatography with electrospray ionization and tandem mass spectrometry.

An extensive characterization and quantification of intact phospholipids (PLs) in strawberry (Fragaria × ananassa cv San Andreas) seed and pulp was carried out by hydrophilic interaction liquid chromatography (HILIC) and electrospray ionization (ESI) coupled to either Fourier-transform (FT) orbital-trap or linear ion-trap tandem mass spectrometry (LIT-MS/MS). More than 150 intact polar lipids including phosphatidylcholines (PCs), phosphatidylethanolamines (PEs), phosphatidylglycerols (PGs), phosphatidic acids (PAs), phosphatidylinositols (PIs), lysophosphatidylcholines (LPCs), and lysophosphatidylethanolamines (LPEs) were identified in negative ESI mode. PC 18:2/18:2 and 18:2/18:3 were found to be the major components of strawberry lipid extracts at concentrations of 230 ± 36 and 189 ± 32 μg/g, respectively, in seeds and at concentrations of 330 ± 50 and 140 ± 22 μg/g, respectively, in pulp. The lipidic extracts of both strawberry seeds and pulp exhibited the dominance of LPC 16:0/0:0 at a content of 132 ± 19 and 114 ± 16 μg/g, respectively, and LPC 0:0/18:2 at 236 ± 20 and 150 ± 20 μg/g, respectively. The other most abundant species of strawberry seeds and pulp were PE 18:2/18:2, 40 ± 9 and 190 ± 40 μg/g, followed by PI 16:0/18:2, 51 ± 15 and 24 ± 8 μg/g, respectively, while PG, PA, and LPE show comparable abundance below 10 μg/g. The most recurrent fatty acyl substituents of PLs were C18:3 (α-linolenic acid), C18:2 (linoleic acid), C18:1 (oleic acid), C18:0 (stearic acid), C16:0 (palmitic acid), and relatively high contents of a shorter chain such as C14:0 (myristic acid).

Complementary approach for analysis of phospholipids by liquid chromatography hyphenated to elemental and molecular mass spectrometry.

Phospholipids are one of the most important lipid categories with multiple functions in biological systems. Their analysis can contribute to a better understanding of metabolomic and kinetic processes in living cells. Comprehensive methods based on liquid chromatography coupled to mass spectrometry are available for phospholipid identification and quantification. However, quantification of phospholipids using electrospray ionization-mass spectrometry with internal standards is still challenging due to several reasons. In particular, the detector response of phospholipid species differs with variation of the head group as well as the fatty acid chain length and double bond number. Inductively coupled plasma-tandem mass spectrometry (ICP-MS/MS) provides an alternative approach for their absolute quantification with universal detector response for phosphorus independent of its chemical form and proportional to its quantity. Therefore, a quantification method based on compound-independent calibration using hydrophilic interaction liquid chromatography (HILIC) coupled to ICP-MS/MS was developed. An inverse gradient system was implemented for constant mobile phase composition after HILIC separation, which provides steady plasma ionization conditions. Isobaric phosphorus interferences were decreased by using the oxygen reaction mode of the triple quadrupole based ICP-MS/MS instrument. Complementary molecular information was obtained by ESI-high-resolution MS and MS/MS. The applicability of this approach was demonstrated in a proof of concept by complementary analysis of a total lipid extract of baker's yeast.

31P NMR Quantification of Phospholipids and Lysophospholipids in Food Emulsions

For food emulsions containing enzymatically modified egg yolk, the conventional Folch extraction does not fully recover the polar lysophospholipids. This can be overcome by repeated methanol extractions. After solvent evaporation, the extracted (lyso)phospholipids are solubilized into mixed micelles with cholate as a detergent. The solubilized (lyso)phospholipids can be accurately quantified by 31P NMR with recoveries ranging between 96% and 108%. Detection at a high (16.4 T) relative to a mainstream (9.4 T) magnetic field strength did not offer a significant advantage since the slow molecular tumbling of the mixed micelles increased line widths. This was due to field-strength-dependent chemical shift anisotropy relaxation. Method precision is similar at 9.4 and 16.4 T, with within-laboratory reproducibilities of 7-22% and 12-25%, respectively. The method can be implemented as a routine analytical procedure at 9.4 T (400 MHz NMR spectrometer), and the limits of detection and quantification are adequate for the verification of the standard of identity of a mayonnaise prepared with enzymatically modified egg yolk.

Enrichment of phospholipids using magnetic Fe3O4/TiO2 nanoparticles for quantitative detection at single cell levels by electrospray ionization mass spectrometry

Quantitative detection of phospholipids at the single cell level remains in challenge. Herein, the TiO2-coated Fe3O4 nanoparticles were synthesized to selectively enrich trace phospholipids from single cell, which were then eluted using 1.5% ammonia/methanol (w/w) for sensitive detection by electrospray ionization mass spectrometry. Under the optimal experimental conditions, eighteen phospholipids in single cell samples were detected and identified by MS/MS experiments. The limit-of-detections (LODs) were 0.012 μg/L for phosphatidylcholine (PC, 34:1) and 0.014 μg/L for phosphatidylcholine (PC, 36:2) in PBS matrix, with the linear range of 0.05–50 μg/L (R2 ≥ 0.999). The recovery rates of 94.90–104.00% were obtained, with the relative standard deviations (RSDs ≤ 6.90%). Quantitative determination of PC in real unicellular samples was also achieved, with the concentration of 1.82–2.11 μg/L for PC(34:1) and 1.25–1.65 μg/L for PC(36:2) in six types of single cell, opening up possibilities for quantitative analysis of trace compounds in complex bio-samples. A set of 6 types of tumor cells were analyzed and further differentiated by the partial least squares-discriminant analysis (PLS-DA). Conclusively, a facile method for the direct quantification of phospholipids in single cell samples has been developed, showing potential applications for advanced investigation of phosphorylated substance at the single cell level.

Robust and Reliable Quantification of Phospholipids in Edible Oils Using 31P NMR Spectroscopy

AbstractUpon storage, crude plant oils will form a solid sediment called gum, which consists mainly of phospholipids (PL). PL are removed during the production of edible oils by a process called degumming. A higher yield is recognized as a major advantage of enzymatic degumming over traditional processes. Robust and accurate PL quantification methods are needed to develop and monitor enzymatic degumming processes. Several techniques, such as atomic emission spectroscopy, liquid chromatography, and thin‐layer chromatography, have been applied for the quantification of PL in edible oils. In the past decade, 31P NMR spectroscopy has been shown to have advantages over these techniques because of the possibility of the simultaneous, fast, and accurate quantification of different PL directly in the oil. This article demonstrates the application of 31P NMR spectroscopy as a method for the quantification of all relevant PL and phosphorous‐containing degradation products in crude and refined oils. In addition, the validation results show that this method is robust because the limit of detection is as low as 5 μmol/100 g oil. Variations of less than 5% were obtained for all P‐containing compounds present in the oils at concentrations above 100 μmol/100 g oil.

Selective Enrichment of Phosphopeptides and Phospholipids from Biological Matrixes on TiO2 Nanowire Arrays for Direct Molecular Characterization by Internal Extractive Electrospray Ionization Mass Spectrometry.

Rapid analysis of phosphopeptides and phospholipids in biological matrixes is of significant interest in multiple disciplines of life sciences. Herein, trace phospholipids in human plasma, whole blood, and undiluted human urine as well as phosphopeptides in protein digest were selectively captured on a homemade array of TiO2 nanowires for sensitive characterization by internal extractive electrospray ionization mass spectrometry (TiO2-iEESI-MS). Sequential release of captured chemicals from TiO2 array was achieved by tuning pH of the extraction solvent. A single sample analysis, including sample loading, chemical extraction and MS detection, was accomplished within 3 min. As far as the quantification of phospholipids, acceptable linearity ( R2 ≥ 0.9985) and relative standard deviations (RSDs ≤ 8.9%) were obtained within the range of 0.1-500 μg L-1 for LysoPC(14:0) and LysoPC(16:0) in raw urine samples. Limit of detection (LOD) ≤ 0.025 μg L-1 and recovery rates of 94.8-101.6% were obtained for these phospholipids. As far as the quantification of phosphopeptides, R2 ≥ 0.9994 and RSDs ≤ 9.2% within the range of 0.3-200 μg L-1 were obtained for two phosphopeptides in nonphosphopeptides mixtures. LODs ≤ 0.09 μg L-1 and recovery rates of 83.4-107.0% were obtained for these phosphopeptides. On the basis of the orthogonal partial least-squares discriminant analysis, TiO2-iEESI-MS patterns from the blood of 46 patients with ovarian cancer were confidently discriminated from the MS patterns of 46 healthy volunteers. Our results indicate the strong potential of TiO2-iEESI-MS approach for the selective detection of trace phosphopeptides and phospholipids in various biological matrixes with high sensitivity, high specificity, low sample consumption, and high throughput.

Rapid profiling and quantification of phospholipid molecular species in human plasma based on chemical derivatization coupled with electrospray ionization tandem mass spectrometry

In this study, we developed a novel strategy using solid-phase extraction (SPE) coupled with shotgun mass spectrometry (MS) based on trimethylsilyldiazomethane (TMSCHN2) stable-isotope derivatization for rapid profiling and accurate quantification of phospholipids (PLs) in human plasma. HybridSPE-Phospholipid (HybridSPE-PL, zirconia coated silica stationary phase) was used for sample pretreatment via the Lewis acid-base interaction between zirconia and phosphate moiety of PLs. This step allows rapid enrichment and recovery of PLs from human plasma. Afterward, PLs were derivatized with TMSCHN2, which leads to methylation of hydroxyl and amino groups in PLs and allows highly sensitive PL analysis by shotgun MS in positive ionization mode (limit of detection decreased up to 116.67 fold compared to underived PLs). We developed an accuracy quantification method for determination of PL molecular species in biological samples. Two or more PL standards were selected for each PL class and derivatized with TMSCHN2 without stable-isotope coding. They were then used as the internal standards. PLs in biological samples were isotopic derivatized via acid-catalyzed H/D exchange and methanolysis of TMSCHN2. For accurate quantification, a calibration curve for each class of PLs was typically constructed by using the internal standards to normalize the non-uniformity response caused by the differential fragmentation kinetics resulting from the distinct chemical constitution of individual PL species in the biological samples. This newly developed method was used to comprehensively analyze PL molecular species in human plasma samples. It is a promising methodology for rapid profiling and accurate quantification of complex lipid molecules in biological samples.

EPT1 (selenoprotein I) is critical for the neural development and maintenance of plasmalogen in humans

Ethanolamine phosphotransferase (EPT)1, also known as selenoprotein 1 (SELENOI), is an enzyme that transfers phosphoethanolamine from cytidine diphosphate-ethanolamine to lipid acceptors to produce ethanolamine glycerophospholipids, such as diacyl-linked phosphatidylethanolamine (PE) and ether-linked plasmalogen [1-alkenyl-2-acyl-glycerophosphoethanolamine (plasmenyl-PE)]. However, to date there has been no analysis of the metabolomic consequences of the mutation of EPT1 on the concentration of ethanolamine glycerophospholipids in mammalian cells. We studied a patient with severe complicated hereditary spastic paraplegia, sensorineural-deafness, blindness, and seizures. Neuroimaging revealed hypomyelination, followed by brain atrophy mainly in the cerebellum and brainstem. Using whole exome sequencing, we identified a novel EPT1 mutation (exon skipping). In vitro EPT activity, as well as the rate of biosynthesis of ethanolamine glycerophospholipids, was markedly reduced in cultures of the patient's skin fibroblasts. Quantification of phospholipids by LC-MS/MS demonstrated reduced levels of several PE species with polyunsaturated fatty acids, such as 38:6, 38:4, 40:6, 40:5, and 40:4. Notably, most plasmenyl-PE species were significantly decreased in the patient's cells, whereas most plasmanylcholine [1-alkyl-2-acyl-glycerophosphocholine (plasmanyl-PC)] species were increased. Similar findings regarding decreased plasmenyl-PE and increased plasmanyl-PC were obtained using EPT1-KO HeLa cells. Our data demonstrate for the first time the indispensable role of EPT1 in the myelination process and neurodevelopment, and in the maintenance of normal homeostasis of ether-linked phospholipids in humans.

Relative Quantification of Phospholipids Based on Isotope-Labeled Methylation by Nanoflow Ultrahigh Performance Liquid Chromatography-Tandem Mass Spectrometry: Enhancement in Cardiolipin Profiling.

In this study, lipid analysis based on isotope-labeled methlylation (ILM) was performed by nanoflow ultrahigh performance liquid chromatography-eletrospray ionization-tandem mass spectrometry (nUPLC-ESI-MS/MS) for enhanced detection and quantification of targeted phospholipids. ILM depends on methylation of phosphate groups by (trimethylsilyl)diazomethane, and the ILM based quantitation with reversed phase nUPLC-ESI-MS/MS provides advantages in PL profiling such as enhanced detectability of methylated PLs owing to increased hydrophobicity and substantial increase in resolution due to the increase of retention. Efficacy of ILM in nUPLC-ESI-MS/MS analysis was evaluated in the selected reaction monitoring (SRM) method by varying the mixing ratio of H-/D-methylated PL standards, which resulted in the successful quantification of 24 species, including phosphatidic acid (PA), phosphatidylserine (PS), phosphatidylglycerol (PG), ceramide-1-phosphate (Cer1P), phosphoinositides, and cardiolipin (CL), with ∼6.6% variation in the calculated ratio of H-/D-methylated PLs. The method was applied to the lipid extracts from a DU145 cell line after D-allose treatment, resulting in the quantification of 83 PLs of which results were not statistically different from those obtained by conventional quantification methods. Morever, detection and quantification of CLs and PAs were evidenced to be highly effective when used with the ILM method as 43 CLs and 20 PAs from cellular lipid extracts were analyzed while only 18 CLs and 12 PAs were identified when conventional methods were carried out. This proves the ILM combined with LC-MS to be a promising method for analysis of the aforementioned classes of lipids. Overall, the study highlighted the applicability of targeted quantification by the ILM method in lipidomic analysis and demonstrated an improvement in the detection of less abundant anionic PLs.

The Role of Endogenous Lipids in the Emulsifying Properties of Cocoa.

This paper describes a study in which the emulsifying properties of cocoa material with and without its lipid fraction were explored. This study was motivated by the commercial interest in naturally-occurring particulate emulsifiers as opposed to the chemically modified emulsifying particles presently available for commercial use. The hypothesis was that endogenous lipids from cocoa were responsible for driving the formation of stable oil-in-water (o/w) emulsions. The data presented includes relative quantification of phospholipids from different commercially available cocoa material using 31P NMR spectroscopy and analyses of the emulsifying power of delipidified cocoa material. The commercially available cocoa material comprised several phospholipids, with phosphatidylcholine being the most abundant in all samples. Dispersions of delipidified cocoa material were found to drive the formation of o/w emulsions despite the absence of lipids. We therefore concluded that the emulsifying behavior of cocoa material is not entirely reliant upon the endogenous lipids. This suggests that cocoa material may have a new and potentially widespread use in industrial food preparation and may inform manufacturing strategies for novel food grade emulsifiers.

Lipidomic profiling of dried seahorses by hydrophilic interaction chromatography coupled to mass spectrometry

Dried seahorse is a precious raw food material for cooking soups. In this study, a lipidomics strategy using the techniques of solid-phase extraction (SPE) and hydrophilic interaction chromatography–tandem mass spectrometry (HILIC–QTOF/MS) was developed for extraction, visualization, and quantification of phospholipids in dried seahorses. The parameters of SPE were optimized, and 1mL of sample and chloroform/methanol (1:2, v/v) were found to be the best loading volume and eluting solvent, respectively. Afterwards, each phospholipid class was successfully separated on a HILIC column and analyzed by mass spectrometry. A total of 50 phospholipid molecular species were identified and determined, including 15 phosphatidylcholines (PCs), 14 phosphatidylethanolamines (PEs), 12 phosphatidylinositols (PIs) and 9 phosphatidylserines (PSs). In comparison to previously methods, this strategy was robust and efficient in extraction, characterization, and determination of phospholipids. The dried seahorse was found to contain large amounts of polyunsaturated fatty acyl phospholipids which are beneficial to human health.

Characterization and relative quantification of phospholipids based on methylation and stable isotopic labeling

Phospholipids (PLs), one of the lipid categories, are not only the primary building blocks of cellular membranes, but also can be split to produce products that function as second messengers in signal transduction and play a pivotal role in numerous cellular processes, including cell growth, survival, and motility. Here, we present an integrated novel method that combines a fast and robust TMS-diazomethane-based phosphate derivatization and isotopic labeling strategy, which enables simultaneous profiling and relative quantification of PLs from biological samples. Our results showed that phosphate methylation allows fast and sensitive identification of the six major PL classes, including their lysophospholipid counterparts, under positive ionization mode. The isotopic labeling of endogenous PLs was achieved by deuterated diazomethane, which was generated through acid-catalyzed hydrogen/deuterium (H/D) exchange and methanolysis of TMS-diazomethane during the process of phosphate derivatization. The measured H/D ratios of unlabeled and labeled PLs, which were mixed in known proportions, indicated that the isotopic labeling strategy is capable of providing relative quantitation with adequate accuracy, reproducibility, and a coefficient of variation of 9.1%, on average. This novel method offers unique advantages over existing approaches and presents a powerful tool for research of PL metabolism and signaling.

High Throughput Identification and Quantification of Phospholipids in Complex Mixtures.

We present a fully automatic method, autoP, for identification and quantification of lipids in complex lipid mixtures from 1D (31)P and 2D (1)H-(31)P NMR spectra. The (31)P chemical shifts in lipids are highly sensitive to experimental conditions such as pH and temperature, so the present method uses the much more unambiguous (1)H chemical shifts for assignment and (31)P intensities for quantification. By using 2D (1)H-(31)P total correlation spectroscopy (TOCSY) correlation experiments, we demonstrate that approximately 20 different lipids can be automatically and unambiguously assigned and quantified by this automatic method.

Simple and rapid quantification of phospholipids for supramolecular membrane transport assays.

We introduce a simple (1)H NMR method for quantification of the phospholipid content of liposomes. The method is validated by comparison with the established Stewart assay, which revealed significant uncertainties in phospholipid quantification of established liposome preparations used in supramolecular membrane transport assays.