直接进样电喷雾串联质谱法测定草鱼肌肉组织中磷脂

建立了直接进样电喷雾串联质谱测定草鱼肌肉组织中磷脂的方法.以Bligh Dyer法提取总脂质,采用流动注射泵直接进样的方式将样品导人电喷雾离子源,利用串联三重四级杆质谱的母离子扫描和中性丢失扫描功能,通过扫描磷脂的特征性子离子或中性质量丢失实现对磷脂酰胆碱、磷脂酰乙醇胺、磷脂酰肌醇、磷脂酰丝氨酸、磷脂酰甘油和磷脂酸六类磷脂的源内分离和鉴定.结果显示,在-定的浓度范围内,磷脂的浓度与磷脂直接进样电喷雾电离后形成准分子离子的响应值呈现良好的线性关系,回收率(67.1％～96.6％)和精密度可以满足生物样品分析的要求.采用本方法测定了草鱼肌肉组织中磷脂酰胆碱、磷脂酰乙醇胺、磷脂酰肌醇和磷脂酰丝氨酸4类磷脂的分子种及含量.本方法前处理简单,定性和定量分析快速准确,可以应用于其它生物样本脂质组学中磷脂的分析.     

Microfluidics-based electrospray ionization enhances the intrasource separation of lipid classes and extends identification of individual molecular species through multi-dimensional mass spectrometry: development of an automated high-throughput platform for shotgun lipidomics

Herein, we exploit the use of microfluidics and optimized Taylor cones for improved intrasource separation/selective ionization of lipid classes during electrospray ionization. Increased differential ionization of multiple phospholipid classes was achieved through microfluidics with chip-based ionization resulting in substantial enhancement of intrasource separation/selective ionization of phospholipid classes in comparison to the conventional ion source. For example, using myocardial lipid extracts, 3-fold improvements in intrasource separation/selective ionization of myocardial phospholipid classes were routinely realized in the negative-ion mode in the absence of LiOH or other basic modifiers in the infused sample solutions. Importantly, the relative ratios of ions corresponding to individual molecular species in each lipid class to a selected internal standard from myocardial extracts were nearly identical between the chip-based interface and the syringe-pump-driven capillary interface. Therefore, quantitation of individual lipid molecular species directly from biological extracts through comparisons with internal standards in each lipid class was readily accomplished with an accuracy and dynamic range nearly identical to those documented using the well-established direct syringe-pump-driven capillary interface. Collectively, the use of microfluidics and robotic sample handling substantially enhances intrasource separation of lipids in comparison to routine capillary interfaces and greatly facilitates the use of multi-dimensional mass spectrometry using shotgun lipidomics, thereby providing an automated and high-throughput platform for global analyses of cellular lipidomes.     

A shotgun tandem mass spectrometric analysis of phospholipids with normal-phase and/or reverse-phase liquid chromatography/electrospray ionization mass spectrometry

Electrospray ionization mass spectrometry is used in lipidomics studies. The present research established a top-down liquid chromatography/electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) shotgun analysis method for phospholipids (PLs) using a normal-phase column or a C30 reverse-phase column with the data-dependent MS/MS scanning mode. A normal-phase column can separate most of the major different classes of PLs. By using LC/ESI-MS/MS with a normal-phase column, approximately 50 molecular species were identified in a PL mixture from rat liver. When the reverse-phase column was used, the PLs could be separated depending on their hydrophobicity, essentially the length of their fatty acyl chains and the number of unsaturated bonds in them. The LC/ESI-MS/MS method using a C30 reverse-phase column was applied to phosphatidylcholine (PC) and phosphatidylethanolamine (PE) mixtures as test samples. Molecular species with the same molecular mass but with different pairs of fatty acyl chains were separately identified. As a result, about 60 PC and 50 PE species were identified. PLs from rat liver were subjected to LC/ESI-MS/MS using the C30 reverse-phase column and about 110 molecular species were identified. Off-line two-dimensional LC/ESI-MS/MS with the normal-phase and C30 reverse-phase columns allowed more accurate identification of molecular species by using one-dimensional C30 reverse-phase LC/ESI-MS/MS analysis of the collected fractions.     

Factors influencing the electrospray intrasource separation and selective ionization of glycerophospholipids

The external electric field induces a separation of cations from negative electrolyte ions in the infusate while differential ionization of molecular species that possess differential electrical propensities can be induced in either the positive- or negative-ion mode during the electrospray ionization process. These physical and electrical processes that occur in the electrospray ion source have been used to selectively ionize lipid classes possessing different electrical propensities that are now known as "intrasource separation and selective ionization". However, the chemical principles underlying charge-dependent alterations in ionization efficiencies responsible for the selective ionization of lipid classes are not known with certainty. Herein, we examined the multiple factors that contribute to intrasource separation and selective ionization of lipid classes under optimal instrumental conditions. We demonstrated that many different lipid classes could be selectively ionized in the ion source and that intrasource resolution of distinct molecular constituents was independent of lipid concentration, flow rate, and residual ions under most experimental conditions. Moreover, the presence of alkaline conditions facilitates the selective ionization of many lipid classes through a mechanism independent of the design of the ESI ion source. Collectively, this study provides an empirical foundation for understanding the chemical mechanisms underlying intrasource separation and selective ionization of lipid classes that can potentially be used for global analysis of cellular lipidomes without the need for chromatographic separation.     

Total structure characterization of unsaturated acidic phospholipids provided by vicinal di-hydroxylation of fatty acid double bonds and negative electrospray ionization mass spectrometry

In the present work, the unsaturated fatty acid substituents of some phosphatidic acid, phosphatidylserine, phosphatidylinositol, and phosphatidylglycerol species were converted to their 1,2-di-hydroxy derivatives by OsO₄. The subsequent electrospray ionization tandem low-energy mass spectrometry analysis of the deprotonated species allowed positional determination of the double bonds by the production of specific product-ions. The product-ions are formed by charge-remote and charge-proximate homolytic cleavages as well as charge-directed heterolytic cleavages and rearrangements. The commercial availability of pure species of the phospholipids in question was limited, and a number of species were therefore synthesized. The developed method was used to fully characterize the two isobaric phosphatidylglycerol species 16:0/16:1Δ⁹ and 16:0/16:1Δ¹⁰ extracted from the bacteria Methylococcus capsulatus. The presence of these fatty acids was supported by a gas-chromatography mass spectrometry investigation of the dimethyloxazoline derivatives of the species of the lipid extract. The present work demonstrates that a total structure characterization of acidic unsaturated phospholipids in isolate or in mixtures is accomplished by vicinal di-hydroxylation of olefinic sites and subsequent electrospray ionization mass spectrometry of the derivatized phospholipids.     

Structural Characterization of Lipopeptides from Brevibacillus brevis HOB1

Brevibacillus brevis HOB1 was isolated from the formation water of an oil field and found to produce lipopeptides. The separation of lipopeptides was successfully achieved by reversed-phase high-performance liquid chromatography (HPLC) leading to nine separated peaks. The chemical structures of these lipopeptides were studied by means of electrospray ionization mass spectrometry (ESI-MS), gas chromatography-mass spectrometry (GC/MS), HPLC and electrospray ionization tandem mass spectrometry (ESI-MS/MS). As the results, all the lipopeptides had peptide parts with the same amino acid composition of Asp, Glu, Val, and Leu in the molar ratio 1:1:1:4, while the lipid part was composed of C₁₃–C₁₅ β-hydroxy fatty acids. As the sequence of fraction 1 was determined to be N-Glu-Leu-Leu-Val-Asp-Leu-Leu-C, the same as surfactin, they were proposed to be surfactin isoforms. Fraction 4 (C₁₅ surfactin) exhibited a good surface activity of 26.8 mN/m with CMC of 9?×?10^?6 M. Surfactin is a powerful biosurfactant possessing biological activities. As far as we know, Br. brevis is a new surfactin-producing species.     

Structural characterization and identification of dibenzocyclooctadiene lignans in Fructus Schisandrae using electrospray ionization ion trap multiple-stage tandem mass spectrometry and electrospray ionization Fourier transform ion cyclotron resonance multiple-stage tandem mass spectrometry

The electrospray ionization ion trap multiple-stage tandem mass spectrometry (ESI-MSⁿ) and electrospray ionization Fourier transform ion cyclotron resonance multiple-stage tandem mass spectrometry (ESI-FT-ICR-MSⁿ) have been applied successfully to the direct investigation of a number of dibenzocyclooctadiene lignan constituents from the methanol extracts of the Fructus Schisandrae in the positive ion mode. The detailed structural characterization of the same skeleton and different peripheral substituents had been studied and the precise elemental compositions of ions at high mass resolution had been obtained. So the fragmentation mechanisms could be clarified. And the lignan components in Schisandra chinensis (Turcz.) Baill. fruits (SCF) and Schisandra sphenanthera Rehd. et Wils. fruits (SSF) were identified by comparing the structural information and fragmentation mechanisms. Then a pair of isobaric compounds was differentiated. Meanwhile these two similar fruits were distinguished. The research results demonstrated that ESI-MSⁿ technique is a sensitive, selective and effective tool for the direct analysis and rapid determination of constituents in complex mixtures from nature products. And these should be useful for the identification of similar compounds and differentiation of similar species from Chinese herbs.     

Characterization of phosphatidylethanolamine as a lithiated adduct by triple quadrupole tandem mass spectrometry with electrospray ionization

Structural characterization of the glycerophosphoethanolamine (GPE) molecule as a lithiated adduct ion by collisionally activated dissociation (CAD) tandem mass spectrometry with electrospray ionization is described. Abundant fragment ions reflecting polar head group and fatty acid constituents were observed in the product ion spectrum of GPE, which permits an unambiguous structural determination, including the regiospecificity of fatty acyl substituents. The pathways leading to the formation of fragment ions are proposed. The suggested mechanisms are supported by the tandem mass spectra of various deuterated analogs and source CAD of GPE followed by CAD tandem mass spectrometry. Identification of GPE molecular species and specific GPE subclasses in a biological mixture by tandem mass spectrometry with various constant neutral loss scannings is also described.     

Characterization of complex,heterogeneous lipid A samples using HPLC‐MS/MS technique III. Positive‐ion mode tandem mass spectrometry to reveal phosphorylation and acylation patterns of lipid A

In this study, we report the detailed analysis of the fragmentation patterns of positively charged lipid A species based on their tandem mass spectra obtained under low‐energy collision‐induced dissociation conditions of an electrospray quadrupole time‐of‐flight mass spectrometer. The tandem mass spectrometry experiments were performed after the separation of the compounds with a reversed‐phase high performance liquid chromatography method. We found that both, phosphorylated and nonphosphorylated lipid A molecules can be readily ionized in the positive‐ion mode by adduct formation with triethylamine added to the eluent. The tandem mass spectra of the lipid A triethylammonium adduct ions showed several product ions corresponding to inter‐ring glycosidic cleavages of the sugar residues, as well as consecutive and competitive eliminations of fatty acids, phosphoric acid, and water following the neutral loss of triethylamine. Characteristic product ions provided direct information on the phosphorylation site(s), also when phosphorylation isomers (ie, containing either a C1 or a C4′ phosphate group) were simultaneously present in the sample. Continuous series of high‐abundance B‐type and low‐abundance Y‐type inter‐ring fragment ions were indicative of the fatty acyl distribution between the nonreducing and reducing ends of the lipid A backbone. The previously reported lipid A structures of Proteus morganii O34 and Escherichia coli O111 bacteria were used as standards. Although, the fragmentation pathways of the differently phosphorylated lipid A species significantly differed in the negative‐ion mode, they were very similar in the positive‐ion mode. The complementary use of positive‐ion and negative‐ion mode tandem mass spectrometry was found to be essential for the full structural characterization of the C1‐monophosphorylated lipid A species.     

Mass spectrometry characterization of endophytic bacterium Curtobacterium sp. strain ER1/6 isolated from Citrus sinensis

The bacteria of the genus Curtobacterium are usually seen as plant pathogen, but some species have been identified as endophytes of different crops and could as such present a potential for disease control and plant growth promotion. We have therefore applied the desorption electrospray ionization mass spectrometry imaging (DESI‐MSI) in the direct analysis of living Curtobacterium sp. strain ER1/6 colonies to map the surface metabolites, and electrospray ionization tandem mass spectrometry (ESI‐MS/MS) for characterization of these compounds. Several colony‐associated metabolites were detected. The ESI‐MS/MS showed characteristic fragmentations for phospholipids including the classes of glycerophosphocholine, glycerophosphoglycerol, and glycerophosphoinositol as well as several fatty acids. Although a secure identification was not obtained, many other metabolites were also detected for this bacteria species. Principal component analysis showed that fatty acids were discriminatory for Curtobacterium sp. ER1/6 during inoculation on periwinkle wilt (PW) medium, whereas phospholipids characterize the bacterium when grown on the tryptic soy agar (TSA) medium.     

Analysis of long-chain fatty acyl coenzyme a thioesters by negative ion fast-atom bombardment mass spectrometry and tandem mass spectrometry

Long-chain acyl Coenzyme A (CoA) is essentially composed of three major chemical groups, fatty acyl-, phosphopantetheino-, and 3′, 5′,-adenosine diphospho-moieties. The negative ion fast-atom bombardment mass spectrometry spectra of long-chain acyl CoA thioesters were characterized by the formation of abundant [M ? H]^? and two distinct classes of fragment ions, one class which retained the acyl group and another class which is related to CoA that contains the phosphopantethene and adenine. The ions which retained the acyl group in the spectrum of palmitoyl CoA appeared at m/z 675, 657, 595, and 577 and were found to decompose by loss of alkylketene observed at m/z 357 and 339. Those ions which retained the adenine group were observed at m/z 426 and 408. In contrast to these ions observed following fast-atom bombardment ionization, tandem mass spectrometry of the [M ? H]^?, from palmitoyl CoA (m/z 1004), yielded the adenine-containing ions as major products and the acyl-containing ions were of low abundance or not detected. These results suggested that the formation of many characteristic ions observed in direct FAB analysis occurred during the desorption process. The unique relationship between ions which involved the transition from acyl-containing ions to only CoA-containing ions by the loss of alkylketene allowed the development of tandem mass spectrometry protocols for the analysis of acyl CoA mixtures. Precursor scans of either m/z 357 or 339 yielded the identification of each species in a complex mixture. Identification of specific species was obtained with a neutral loss scan of the mass for a specific alkylketene.     

Development of a multi-residue analytical method,based on liquid chromatography–tandem mass spectrometry,for the simultaneous determination of 46 micro-contaminants in aqueous samples

A multi-residue analytical method based on high-performance liquid chromatographic separation, electrospray ionization with tandem mass spectrometric detection (HPLC/MS–MS) was developed for the simultaneous analysis of 46 basic, neutral and acidic compounds covering a wide range of polarity (log K_OW < 0–5.9). The compound list included selected iodinated contrast media, analgesics, anti-inflammatories, stimulants, beta-blockers, antibiotics, lipid regulators, anti-histamines, psychiatric drugs, herbicides, corrosion inhibitors and the gastric acid regulator pantoprazole. The main feature of the presented method was a simultaneous solid phase extraction (SPE) of all analytes followed by simultaneous separation and detection by HPLC/MS–MS with electrospray ionization in both positive and negative polarization within the same chromatogram. Optimization of electrospray drying gas temperature resulted in using a temperature gradient on the ion source. Six different polymeric sorbents for SPE were compared with respect to recoveries, taking into account the specific surface of each sorbent. Method quantitation limits (MQL) in surface and seawater ranged from 1.2 to 28 ng/L, in wastewater from 5.0 to 160 ng/L, respectively. In order to demonstrate the applicability of the method, river water, treated wastewater and seawater were analyzed.     

Comparison of flow injection analysis electrospray mass spectrometry and tandem mass spectrometry and electrospray high-field asymmetric waveform ion mobility mass spectrometry and tandem mass spectrometry for the determination of underivatized amino acids

Twenty proteinogenic amino acids (AAs) were determined without derivatization using flow injection analysis followed by electrospray ionization mass spectrometry and tandem mass spectrometry (ESI-MS and ESI-MS/MS) and electrospray ionization high-field asymmetric waveform ion mobility mass spectrometry and tandem mass spectrometry (ESI-FAIMS-MS and ESI-FAIMS-MS/MS), in positive and negative ionization modes. Three separate sets of ESI-FAIMS conditions were used for the separation and detection of the 20 AAs. Typically ESI-FAIMS-MS showed somewhat improved sensitivity and significantly better signal-to-noise ratios than ESI-MS mainly due to the elimination of background noise. However, the difference between ESI-FAIMS-MS and ESI-MS/MS was significantly less. ESI-FAIMS was able to partially or completely resolve all the isobaric amino acid overlaps such as leucine, isoleucine and hydroxyproline or lysine and glutamine. Detection limits for the amino acids in ESI-FAIMS-MS mode ranged from 2 ng/mL for proline to 200 ng/mL for aspartic acid. Overall, ESI-FAIMS-MS is the preferred method for the quantitative analysis of AAs in a hydrolyzed yeast matrix.     

Detection and quantification of the sulfated disaccharides in chondroitin sulfate by electrospray tandem mass spectrometry

A new method of identifying and quantifying the disaccharide building blocks of glycosaminoglycans is introduced. The polysaccharides are subjected to an enzymatic digestion that releases the sulfated disaccharides. The disaccharides are then identified using a combination of electrospray ionization mass spectrometry and tandem mass spectrometry. Quantification of the isomeric disaccharides is also achieved by tandem mass spectrometry, using a recently developed methodology which quantifies mixtures of isomers without the use of chromatography or prior separation. Using mass spectrometry to characterize the components of glycosaminoglycans significantly reduces both sample consumption and analysis time of traditional methods.     

Phosphorylcholine-containing lipid molecular species profiling in biological tissue using a fast HPLC/QqQ-MS method

A fast reversed-phase liquid chromatography-electrospray ionization triple quadrupole mass spectrometry method was developed for the molecular species profiling of glycerophosphocholine (GPC) and sphingomyelin (SM) in total lipid extracts. A two-stage mass spectrometry strategy was adopted to analyze in detail the composition of lipid molecular species. Precursor ion analysis was first conducted to obtain the preliminary composition profile of the phosphorylcholine-containing lipid. The product ion spectra were sequentially acquired for each recorded signal to determine the molecular structure of the lipid. A total of 150 GPCs and 12 SMs were identified in the fetal mouse lung with relative amounts ranging from 13.7?% to less than 0.002?% (normalizing by the total signal response). A column packed with core–shell particles was used to obtain excellent chromatographic separation with a shorter time demand in a conventional high-performance liquid chromatography system. Considering the compromise between the chromatographic efficiency and the electrospray signal response, the optimization of the mobile phase improves the chromatographic plate number to approximately 40,000 and the detection limits to less than 0.001?mg/L. The applicability of the method was validated through a study of chemically induced early lung maturation. The metabolic alteration in the fetal mouse lung was clearly reflected in the GPC and SM composition with several characteristics of the molecular structure that related to the character of the phospholipid layer upon the epithelial lining of alveoli and the relevant cell function. The results indicated that this analytical strategy is reliable for comprehensive molecular species profiling of GPC and SM and might be extended to the analysis of other phospholipids.

Diels–Alder derivatization for sensitive detection and characterization of conjugated linoleic acids using LC/ESI-MS/MS

The utility of Diels–Alder derivatization with 4-phenyl-1,2,4-triazoline-3,5-dione (PTAD) for liquid chromatography/electrospray ionization tandem mass spectrometry of conjugated linoleic acids (CLAs) was examined. PTAD rapidly reacted with the CLAs, and the resulting derivatives were highly responsive in electrospray ionization mass spectrometry operating in the positive-ion mode. The derivatives produced characteristic product ions during tandem mass spectrometry, which enabled the sensitive detection [limit of detection 18 fmol (signal-to-noise ratio of 5)] and the identification of the conjugated diene position. The PTAD derivatization also significantly increased the reversed-phase liquid chromatography separation selectivity for the most biologically active CLA isomers: cis-9,trans-11-CLA and trans-10,cis-12-CLA. The PTAD derivatization was applied to analyses of food and biological samples; the major CLAs in milk and beef fat samples were successfully identified, and trace amounts of CLAs in human saliva were detected with a simple pretreatment and short analysis time.     

Structural elucidation of molecular species of pacific oyster ether amino phospholipids by normal-phase liquid chromatography/negative-ion electrospray ionization and quadrupole/multiple-stage linear ion-trap mass spectrometry

Although marine oysters contain abundant amounts of ether-linked aminophospholipids, the structural identification of the various molecular species has not been reported. We developed a normal-phase silica liquid chromatography/negative-ion electrospray ionization/quadrupole multiple-stage linear ion-trap mass spectrometric (NPLC-NI-ESI/Q-TRAP-MS³) method for the structural elucidation of ether molecular species of serine and ethanolamine phospholipids from marine oysters. The major advantages of the approach are (i) to avoid incorrect selection of isobaric precursor ions derived from different phospholipid classes in a lipid mixture, and to generate informative and clear MSⁿ product ion mass spectra of the species for the identification of the sn-1 plasmanyl or plasmenyl linkages, and (ii) to increase precursor ion intensities by “concentrating” lipid molecules of each phospholipid class for further structural determination of minor molecular species. Employing a combination of NPLC-NI-ESI/MS³ and NPLC-NI-ESI/MS², we elucidated, for the first time, the chemical structures of docosahexaenoyl and eicosapentaenoyl plasmenyl phosphatidylserine (PS) species and differentiated up to six isobaric species of diacyl/alkylacyl/alkenylacyl phosphatidylethanolamine (PE) in the US pacific oysters. The presence of a high content of both omega-3 plasmenyl PS/plasmenyl PE species and multiple isobaric molecular species isomers is the noteworthy characteristic of the marine oyster. The simple and robust NPLC-NI-ESI/MSⁿ-based methodology should be particularly valuable in the detailed characterization of marine lipid dietary supplements with respect to omega-3 aminophospholipids.     

Analysis of Biologically Active Constituents in Centella asiatica by Microwave-Assisted Extraction Combined with LC–MS

Centella asiatica (L.) Urban is a traditional herbal medicine used in Asiatic countries, and is commonly used to treat various wounds, leprosy, tuberculosis and lupus diseases. In this work, a new method based on microwave assisted extraction followed by liquid chromatography–diode array detection–electrospray ionization multistage tandem mass spectrometry analysis has been developed for the identification and quantification of biologically active constituents in C. asiatica, including asiatic acid, asiaticoside and madecassoside. The separation was performed on an Agilent Eclipse C₁₈ column (150 mm × 4.6 mm i.d., 5 μm) with gradient elution of acetonitrile and 0.1% aqueous acetic acid within 50 min. Detection was performed at 205 nm. The calibration curves showed good linearity (r ² > 0.9992). The limits of detection ranged from 1.2 to 1.6 μg mL^?1. The intra- and inter-day precision was less than 3% and the recovery of the assay was in the range of 95.4–106.8%. The method was successfully applied to the quantification of the three constituents in different samples of C. asiatica. The results indicated that the developed method could be considered to be a simple, rapid and reliable method for the quality evaluation of C. asiatica. The samples were also analyzed on a liquid chromatography–electrospray ionization–time-of-flight mass spectrometry system to confirm the identification results.     

Precise and global identification of phospholipid molecular species by an Orbitrap mass spectrometer and automated search engine Lipid Search

In the present research, we have established a new lipidomics approach for the comprehensive and precise identification of molecular species in a crude lipid mixture using a LTQ Orbitrap mass spectrometer (MS) and reverse-phase liquid chromatography (RPLC) combination with our newly developed lipid search engine “Lipid Search”. LTQ Orbitrap provides high mass accuracy MS spectra by Fourier-transform (FT) mass spectrometer mode and can perform rapid MSⁿ by ion trap (IT) mass spectrometer mode. In this study, the negative ion mode was selected to detect fragment ions from phospholipids, such as fatty acid anions, by MS2 or MS3. We selected the specific detection approach by neutral loss survey-dependent MS3, for the identification of molecular species of phosphatidylcholine, sphingomyelin and phosphatidylserine. Identification of molecular species was performed by using both the high mass accuracy of the mass spectrometric data obtained from FT mode and structural data obtained from fragments in IT mode. Some alkylacyl and alkenylacyl species have the same m/z value as molecular-related ions and fragment ions, thus, direct acid hydrolysis analysis was performed to identify alkylacyl and alkenylacyl species, and then the RPLC–LTQ Orbitrap method was applied. As a result, 290 species from mouse liver and 248 species from mouse brain were identified within six different classes of phospholipid, only those in manually detected and confirmed. Most of all manually detected mass peaks were also automatically detected by “Lipid Search”. Adding to differences in molecular species in different classes of phospholipids, many characteristic differences in molecular species were detected in mouse liver and brain. More variable number of saturated and monounsaturated fatty acid-containing molecular species were detected in mouse brain than liver.     

A simple and universal method for the separation and identification of phospholipid molecular species

One of the major challenges in lipidomics is to obtain as much information about the lipidome as possible. Here, we present a simple yet universal high-performance liquid chromatography/tandem mass spectrometry (HPLC/MS/MS) method to separate molecular species of all phospholipid classes in one single run. The method is sensitive, robust and allows lipid profiling using full scan mass spectrometry, as well as lipid class specific scanning in positive and negative ionisation mode. This allows high-throughput processing of samples for lipidomics, even if different types of MS analysis are required. Excellent separation of isobaric and even isomeric species is achieved, and original levels of lyso-lipids can be determined without interference from lyso-lipids formed from diacyl species by source fragmentation. As examples of application of this method, more than 400 phospholipid species were identified and quantified in crude phospholipid extracts from rat liver and the parasitic helminth Schistosoma mansoni.