Separation of the molecular species of diacyl and alkenylacyl subclasses of the methyl ester of dinitrophenylethanolamine glycerophospholipid by high-performance liquid chromatography

A high-performance liquid chromatographic (HPLC) procedure for the separation of the molecular species of alkenylacyl and diacyl subclasses after the derivatization of ethanolamine glycero-phospholipids (EGP) to the methyl ester of the dinitrophenylated lipids is described. Methyl esters of dinitrophenylethanolamine glycerophospholipids were first separated into alkenylacyl, alkylacyl and diacyl subclasses by thin-layer chromatography, then each subclass was separated into individual molecular species by reversed-phase HPLC. By converting the EGP into UV-absorbing derivatives, it proved possible to utilize the specificity of spectrophotometric detection for the determination of the individual molecular species. Alkenylacyl and diacyl derivatives were resolved into fifteen to twenty different species in a single HPLC run. The method should facilitate studies on the metabolism of the polar head group of both molecular species of alkenylacyl and diacyl glycerophosphoethanolamine in a variety of tissues using radioactive precursors.     

Application of liquid chromatography-thermospray mass spectrometry in the analysis of glycerophospholipid molecular species

We report the application of high-performance liquid chromatographic (HPLC) separation with ultraviolet detection and direct, on-line, structural analyses by mass spectrometry of glycerobenzoate derivatives from complex mixtures of phospholipid molecular species. Individual phospholipids were resolved from total lipid extracts by thin-layer chromatography (TLC). Diradylglycerols were released from phospholipids by phospholipase-C treatment, converted to diradyl glycerobenzoates and subsequently separated by TLC into subclasses (alk-1-enylacyl, alkylacyl and diacyl types). The molecular species within each subclass were resolved by HPLC with an octadecyl reversed-phase column in acetonitrile-isopropanol (80:20, v/v). Individual peaks were quantitated at the picomole level by measuring absorbance at 230 nm. After post-column addition of methanol-0.2 M ammonium acetate (50:50, v/v), peaks were introduced through the thermospray interface into a VG Masslab 30-250 quadrupole mass spectrometer. Molecular species showed as base peaks the salt adducts of the molecular ion which permitted easy deduction of the overall fatty acyl composition. In addition, the diglyceride fragment of each species was found at [MH - 122]+ and two fragments formed by the loss of the fatty acyl groups (R) in the sn-1 or sn-2 position were found at [M - R1]+ and [M - R2]+, respectively. Since preferential release of either fatty acyl group was observed in positional isomers, the ratio of the intensity of these fragments gave information on the position of the fatty acyl groups in the individual HPLC peaks. We show that the use of on-line mass spectrometry, however, provides easy identification of all molecular species present in a complex phospholipid mixture, even when more than one molecular species is contained in an HPLC peak.     

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.     

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.     

Quantification of plasmalogen, alkylacyl and diacyl glycerophospholipids by micro-thin-layer chromatography

A method for the determination of plasmalogen, alkylacyl and diacyl glycerophospholipids based on mild alkaline deacylation and acid hydrolysis of plasmalogens on plates with subsequent micro-thin-layer chromatography on silica gel is presented. It is effective in quantifying alkyl and alkenyl analogues of phosphatidylethanolamine and phosphatidylcholine at concentrations up to 0.1% (of the total of the forms in individual classes of phospholipids.     

A simple method to identify ether lipids in spermatozoa samples by MALDI-TOF mass spectrometry

Plasmalogens (alkenylacyl glycerophospholipids) are important lipid constituents of many tissues and cells (e.g., selected spermatozoa). Since the molecular weights of plasmalogens overlap with that of diacyl- or alkyl acyl lipids, sophisticated mass spectrometry (MS; including MS/MS) analysis is normally used for the unequivocal identification of plasmalogens. We will show here that a simple matrix-assisted laser desorption/ionization time-of-flight mass spectrometer (without MS/MS capability) in combination with acidic hydrolysis and subsequent derivatization with 2,4-dinitrophenylhydrazine (DNPH) and/or digestion with phospholipase A₂ (PLA₂) is sufficient to determine the contributions of ether lipids in spermatozoa extracts. As neither diacyl nor alkylacyl lipids are sensitive to acids and do not react with DNPH, the comparison of the mass spectra before and after treatment with acids and/or DNPH addition readily provides unequivocal information about the plasmalogen content. Additionally, the released aldehydes are readily converted into the 2,4-dinitrophenylhydrazones and can be easily identified in the corresponding negative ion mass spectra. Finally, PLA₂ digestion is very useful in confirming the presence of plasmalogens. The suggested method was validated by analyzing roe deer, bovine, boar, and domestic cat spermatozoa extracts and comparing the results with isolated phospholipids.

High-resolution analysis by nano-electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry for the identification of molecular species of phospholipids and their oxidized metabolites

Nano-electrospray ionization (ESI) Fourier transform ion cyclotron resonance mass spectrometry (FTICRMS) was applied to identify the molecular species of phosphatidylethanolamine of Caenorhabditis elegans, which has a high concentration of phospholipids with a fatty acyl chain having an odd number of carbon atoms. The molecular species of diacyl phosphatidylethanolamine with one fatty acyl chain having an odd number of carbon atoms and one fatty acyl chain having an even number of carbon atoms was identified separately from alkyl-acyl phosphatidylethanolamine with an alkyl chain having an even number of carbon atoms and a fatty acyl chain having an even number of carbon atoms. Furthermore, nano-ESI-FTICRMS was applied to the direct identification of oxidized phosphatidylcholine from soybean. The mass peaks of individual molecular species of oxidative phosphatidylcholine, such as 34:3 diacyl phosphatidylcholine with peroxide (+2O) (m/z 788.544) or 34:2 diacyl phosphatidylcholine with peroxide (+2O) (m/z 790.560), can be separated from the peaks of the molecular species of the non-oxidative phospholipids. This suggests that the mass peaks with a difference of less than 0.1 mass units in their molecular weight can be separated and that their individual exact molecular compositions can be obtained by the FTICRMS analysis. The high resolution and high accuracy of FTICRMS are very effective in the analysis of molecular species of phospholipids and their derivatives.     

Quantitative determination of phospholipids in a pharmaceutical drug by scanning and video densitometry

This paper describes a convenient and practice method for quantitation of surfactant phospholipids (1,2-dipalmitoyl-3-sn-phosphatidyl choline [DPPC] and 1-palmitayl-2-oleyl-3-sn-phosphatidyl glycerol [POPG]) in a recombinant surfactant lyophile (Venticute) by high-performance, thin-layer chromatography (HPTLC) with video densitometry. DPPC and POPG were extracted from Venticute-lyophile using methanol. Separation from the other active ingredients and excipients was accomplished by HPTLC on silica gel F254 plates with a mixture of chloroform, methanol, glacial acetic acid, and water as development solvent. Postchromatographic derivatization by dipping in copper sulphate/phosphoric acid reagent and subsequent heating shows grey-brown bands on a light blue background. These were detected with the video densitometer in the VIS range, and with scanning densitometry at 365 nm. Linear calibration in a working range of 0.7-1.3 microg DPPC and 0.35-0.65 microg POPG was demonstrated by integrating the area under the peaks. Good results were obtained with recovery experiments. When compared to classical slit scanning densitometry, video densitometry represents a fast alternative to quantitate thin-layer chromatograms in surfactant phospholipid analysis.     

Direct enantiomeric resolution of disopyramide and its metabolite using chiral high-performance liquid chromatography. Application to stereoselective metabolism and pharmacokinetics of racemic disopyramide in man

A method for the simultaneous determination of disopyramide and mono-N-desisopropyldisopyramide enantiomers extracted from human plasma and urine is presented. Separation and quantitation were carried out using two columns coupled in series, and UV detection at 254 nm. First, the racemates of the two compounds were separated using a reversed-phase column, and then the enantiomers were separated using a stereoselective column packed with human alpha 1-acid glycoprotein. The mobile phase was 8 mM phosphate buffer, pH 6.20-2-propanol (92:8, v/v). The coefficients of variation (%) for the plasma daily determination were 6.7% for R(-)- and S(+)-disopyramide at drug levels of 1.5 micrograms/ml, and 8.5% and 7.7% for R(-)- and S(+)-mono-N-desisopropyldisopyramide, respectively, at drug levels of 0.375 micrograms/ml. The method has allowed the study of stereoselective metabolism and pharmacokinetics of disopyramide after oral administration as a racemate.     

Use of ion pairing reagents for sensitive detection and separation of phospholipids in the positive ion mode LC-ESI-MS

Phospholipids make up one of the more important classes of biological molecules. Because of their amphipathic nature and their charge state (e.g., negatively charged or zwitterionic) detection of trace levels of these compounds can be problematic. Electrospray ionization mass spectrometry (ESI-MS) is used in this study to detect very small amounts of these analytes by using the positive ion mode and pairing them with fifteen different cationic ion pairing reagents. The phospholipids used in this analysis were phosphatidylethanolamine (PE), phosphatidylcholine (PC), phosphatidylglycerol (PG), phosphatidylserine (PS), phosphatidylinositol (PI), phosphatidic acid (PA), 1,2-diheptanoyl-sn-glycero-3-phosphocholine (DHPC), cardiolipin (CA) and sphingosyl phosphoethanolamine (SPE). The analysis of these molecules was carried out in the single ion monitoring (SIM) positive mode. In addition to their detection, a high performance liquid chromatography and mass spectrometry (HPLC-MS) method was developed in which the phospholipids were separated and detected simultaneously within a very short period of time. Separation of phospholipids was developed in the reverse phase mode and in the hydrophilic interaction liquid chromatography (HILIC) mode HPLC. Their differences and impact on the sensitivity of the analytes are compared and discussed further in the paper. With this technique, limits of detection (LODs) were very easily recorded at low ppt (ng L(-1)) levels with many of the cationic ion pairing reagents used in this study.     

Design,synthesis, and evaluation of water-soluble phospholipid analogues as inhibitors of phospholipase C from Bacillus cereus

The rate of hydrolysis of natural phospholipids by the phosphatidylcholine-preferring phospholipase C from Bacillus cereus (PLC(Bc)) follows the order phosphatidylcholine > phosphatidylethanolamine > phosphatidyl-l-serine. To probe the structural basis for this substrate specificity, a series of water-soluble, nonhydrolyzable substrate analogues were needed so their complexes with the enzyme could be studied via X-ray crystallography and isothermal titration calorimetry (ITC). Accordingly the water-soluble dithiophospholipids 2-10 having choline, ethanolamine, and l-serine headgroups were synthesized, and the inhibitory activity of each was determined in an assay using 1,2-dihexanoyl-sn-glycero-3-phosphocholine (C6PC) as the monomeric substrate. The 1,2-dibutanoyl dithiophosphocholine 2 was a weak inhibitor, whereas the related 1,2-dipentanoyl dithiophosphocholine 3 and the ethylene glycol dithiophosphocholines 4 and 5 were moderate inhibitors. The 1,2-omega-hydroxydiacyl dithiophosphocholines 6 and 7 were potent inhibitors, while the related compound 8, which had shorter acyl side chains, was a weak inhibitor. The dithiophosphoethanolamine 9 was a modest inhibitor, whereas the dithiophospho-l-serine 10 was a somewhat weaker inhibitor. Overall, the phospholipid analogues had increasing K(i) values according to the order 2 < 10 < 3 < 4 approximately 5 approximately 8 < 9 < 6 < 7 and increasing solubility according to the sequence 5 approximately 7 < 4 approximately 6 approximately 9 < 3 < 10 < 8 < 2.     

高效液相色谱法和蒸发光散射检测器分析大豆磷脂分子种

高效液相色谱法和蒸发光散射检测器分析大豆磷脂分子种     

Improved determination of individual molecular species of phosphatidylcholine in biological samples by high-performance liquid chromatography with internal standards.

Phosphatidylcholine isolated from samples of bile, liver and plasma was converted into 1,2-diradylglycerobenzoate molecular species by hydrolysis with phospholipase C and reaction with benzoic anhydride. Up to seventeen molecular species were separated and determined by reversed-phase high-performance liquid chromatography with detection at 230 nm. The major improvement introduced here was the use of distearoylphosphatidylcholine as the internal standard, which corrected the results for incomplete hydrolysis and benzoylation. Other improvements concerned the clean-up of benzoyl derivatives and the chromatographic separation. The analytical results obtained were validated by comparison with the results of either lipid phosphorus or gas chromatographic determinations.     

Quantitation of phenacyl esters of retinal fatty acids by high-performance liquid chromatography.

A high-performance liquid chromatographic (HPLC) method for the separation and quantitation of retinal fatty acids containing long-chain polyunsaturated fatty acids is described. Fatty acids from frog retinal lipids were converted to the corresponding phenacyl derivatives which were separated on a C18 reversed-phase column and detected at 242 nm. Molar absorptivities (peak area units/nmol) of up to seventeen fatty acid phenacyl derivatives were determined and used for quantitation of fatty acids separated by HPLC. Compared with gas chromatography, the HPLC method gave a similar molar percent distribution of the fatty acids and was twenty to fifty times more sensitive. This HPLC method provides a useful means for the study of chemistry and metabolism of long-chain polyunsaturated fatty acids in retina and other tissues where amounts of material may be limited or recovery of individual components desirable.     

Isocratic mixed mode liquid chromatographic separation of phospholipids with octadecylsilane-silica stationary phases

Molecular species of phosphatidylcholine, phosphatidylethanolamine and phosphatadic acid were resolved by isocratic reversed phase high performance liquid chromatography (HPLC) using mobile phases of methanol-isopropanol containing para-toluenesulfonic acid (p-tsa). Separation by both non-polar fatty acid chain length and by polar head group functionality was achieved concurrently upon a commercially available octadecylsilane (C18) column endcapped with trimethylsilane (C1) groups. Using a mobile phase of 97.5:2.5 methanol:isopropanol with 7OmMpara-toluenesulfonic acid (p-tsa) at a pH of approximately 1, twelve phospholipid species comprised of four tail group classes (dilauroyl-,dimyristoyl-, dipamitoyl- and distearoyl-) and three head group speciations (phosphatidylcholine, phosphatidylethanolamine and phosphatadic acid) were separated. The column was then exposed to the acidic mobile phase for 48 hours continuously during which the bound phase underwent severe acid-induced hydrolysis, after which the separation of the twelve analytes resulted in the separation of the phospholipid species by non-polar tail group alone. The experimental results are discussed in terms of potential separation mechanisms including dependency of the separation on adsorption of the counter ion into the stationary phase, residual acidic silanol group interactions, and potential interactions of the surface active phospholipids with C1 groups.     

Non-intuitive separation of vanilla compounds using rapid resolution high-performance liquid chromatography

A method is presented for modeling the retention peak migration in rapid resolution high-performance liquid chromatography (HPLC) depending on experimental parameter values. It allows time reduction on the determination of the experimental conditions for optimal resolution (especially for untrained chromatographers). Separation for 18 species present in a conventional vanilla formulation was not possible in a single chromatogram, due to a systematic error in defining single peak migration with the usual assumptions. This was achieved by the means of two runs under different experimental conditions. Prediction of the peak inversion for quantitation purposes in a given mixture is now possible and can help to avoid misidentification on set-ups with UV-ELSD or other non-specific detectors.     

Separation and quantification by high-performance liquid chromatography with light scattering detection of the main wheat flour phospholipids during dough mixing in the presence of phospholipase

Phospholipids (PL) are minor components of wheat flour involved in baking quality and exogenous phospholipids are used as emulsifiers giving better loaf volume and crumb grain. Few biochemical data are available on the phospholipid evolution during mixing, probably because of the time-consuming methods proposed for their extraction, separation and quantification. In the present study, the extraction, separation and quantification of the main wheat flour phospholipids were carried out. Total lipids (2% dry mass of wheat flour) were extracted from flour or dough by a mixture of chloroform-methanol-water (1:1:1 (v/v)). The phospholipids were separated from the lipid extract on silica cartridge by solid-phase extraction (SPE) procedure under a 1.5-4 mmHg vacuum, at a 0.8 mL min(-1) flow rate (1 mmHg = 133.322 Pa). The recovery of the lipid extract was 100%, whereas the SPE yield for the PLs was 50%. The resulting fraction was then submitted to HPLC with evaporative light scattering detection on a Diol stationary phase allowing the separation and quantification of each class of phospholipids, in less than 16 min. The developed method allowed to quantify the phospholipid amounts from eight wheat flours as well as their evolution during mixing in the presence of phospholipase.     

Quantitation and characterization of phospholipids in pharmaceutical formulations by liquid chromatography-mass spectrometry

A simple and fast method for phospholipid analysis was developed using high-performance liquid chromatography-mass spectrometry with an atmospheric pressure ionization interface. Separation of the phospholipid molecular species was achieved using a linear gradient of a mixture of chloroform-10 mM ammonium acetate-methanol (30:5:65) on a silica column. Optimization of the mass spectrometer conditions has allowed the method to separate and detect the phospholipids mainly as protonated molecular species. In comparison to existing LC-MS methods, improvement in the total analysis time and sensitivity were achieved. Separation of all major phospholipid molecular classes was achieved in less than 6 min. Marked improvement was observed in the linearity of the response of the phospholipids studied providing a linear response over three orders of magnitude. Data supporting the validation of this method for the characterization of major phospholipids molecular species are also presented.     

Analysis of aminophospholipid molecular species by methyl-beta-cyclodextrin modified micellar electrokinetic capillary chromatography with laser-induced fluorescence detection

Micellar electrokinetic capillary chromatography (MEKC) with laser-induced fluorescence detection is used for the analysis of three classes of aminophospholipids: phosphatidylethanolamine (PE), phosphatidylserine (PS), and lysophosphatidylethanolamine (LPE) molecular species. 3-(2-Furoyl) quinoline-2-carboxaldehyde (FQ), a fluorogenic dye, was employed for labeling of these phospholipids. The FQ-labeled lipid species were then separated by sodium deoxycholate MEKC modified with methyl-beta-cyclodextrin. Baseline resolution of each class of phospholipids was achieved within 7 min. The migration time in each class increased with the carbon number of their side aliphatic chain. Separation efficiencies of approximately 3x10(5) plates were observed for most of these species. Concentration detection limits (3 sigma) were from 10(-9) to 10(-10) M for PE and LPE species and from 10(-8) to 10(-9) M for PS species. The relative standard deviations for migration time and peak area were less than 0.9% and 4.5%, respectively, for seven PE species. This method was applied to the separation of PE isolated from HT29 human colon cancer cells and roughly 30 PE species were resolved.     

Analysis of 1,2(2,3)- and 1,3-positional isomers of diacylglycerols from vegetable oils by reversed-phase high-performance liquid chromatography

Separation of 1,2(2,3)- and 1,3-positional isomers of diacylglycerols (DAG) from vegetable oils by reversed-phase high-performance liquid chromatography (RP-HPLC) is investigated. The method is based on isocratic elution using 100% acetonitrile and UV detection at 205 nm. The following elution order of DAG molecular species is identified: 1,3-dilinolein < 1,2-dilinolein < 1,3-dimyristin < 1-oleoyl-3-linoleoyl-glycerol < 1,2-dimyristoyl-rac-glycerol < 1(2)-oleoyl-2(3)-linoleoyl-glycerol < 1-linolenoyl-3-stearoyl-glycerol < 1(2)-linolenoyl-2(3)-stearoyl-glycerol < 1,3-diolein < 1-palmitoyl-3-oleoyl-glycerol < 1,2-dioleoyl-sn-glycerol < 1(2)-palmitoyl-2(3)-oleoyl-glycerol < 1-linoleoyl-3-stearoyl-glycerol < 1,3-dipalmitin < 1(2)-linoleoyl-2(3)-stearoyl-glycerol < 1-oleoyl-3-stearoyl-glycerol < 1,2-dipalmitoyl-rac-glycerol < 1-palmitoyl-3-stearoyl-sn-glycerol < 1,3-distearin < 1,2-distearoyl-rac-glycerol. Linearity is observed over three orders of magnitude. Limits of detection and quantitation range 0.2-0.7 microg/mL for 1,3-dilinolein to 0.6-1.9 microg/mL for 1,2-dioleoyl-sn-glycerol, respectively. Precision and accuracy of the method are also demonstrated. The method is developed to separate mixtures of DAG molecular species produced from edible oils.