Quantitative analysis of retinal glycerolipid molecular species acetylated by acetolysis

A method for the quantitative analysis of molecular species of 1,2-diacylglycerol acetates (1,2-DGAC) containing polyunsaturated fatty acids is described. Phosphatidylethanolamine (PE) isolated from frog retina was used to test the method. PE was converted to 1,2-DGAC by acetolysis. The molecular species of the 1,2-DGAC were resolved by reverse-phase high performance liquid chromatography (HPLC), detected by UV absorption spectroscopy at 210 nm, and identified by gas-liquid chromatography (GLC) of the fatty acid methyl esters (FAME). Molar response curves were generated for each DGAC molecular species that eluted as a single entity from HPLC by determining the moles of fatty acids in the molecular species collected and the response (peak area unit) of the UV detector. Each molecular species response curve was linear from about 10 pmoles to 4-8 nmoles, allowing the slope of each curve to be used as a molar absorptivity. This method provides a means for quantification of most of the molecular species of all glycerolipid classes.     

Characterization of the hydroxy fatty acid content ofBasidiomycotina

Fatty acids (FA) of nine fungal species belonging to the subphylumBasidiomycotina were identified by using capillary GC-MS, MS and HPLC. The identified fatty acids included 45 saturated (iso-, anteiso-, and 19 hydroxy acids) and 42 monoenoic acids (including 14 hydroxy acids); dienes and polyenes were represented by 13 fatty acids. The proportion of hydroxy acids in the total fatty acids in the fungal species ranged from 4.3 to 10.2%. Very long-chain fatty acids (C₂₄-C₃₀) were also determined. Four fatty acids 16:0 (8.8–14.3%), 18:1(11) (3.9–14.9%), 18:1(9) (7.7–19.0%) and 18:2(6) (7.6–19.4%), were found as major acids. Of the identified acids, 17 were detected inBasidiomycotina for the first time.     

沙蟋飞行肌中保幼激素滴度的昼夜节律

沙蟋Gryllus firmus是一种翅多型性的昆虫, 是研究种内迁飞和生殖调控的模式生物。本研究应用高效液相色谱仪（HPLC）、 气相色谱-质谱联用仪(GC-MS)对沙蟋长翅（有飞行能力）和短翅（无飞行能力）雌虫飞行肌内保幼激素（juvenile hormone, JH）和脂肪酸进行了定性定量分析。结果表明： 在温度28℃, 光周期16L∶8D条件下, 第5和第7日龄的沙蟋长翅雌虫飞行肌中JH的滴度具有明显的昼夜节律, 在飞行前（即关灯前）4 h, JH的滴度分别由386.52±68.40 ng/g和630.36±37.26 ng/g增加至1 327.53±277.98 ng/g和1 685.77±143.95 ng/g, 与短翅型SW相比分别增加了约3.4倍与2.7倍 (P＜0.05)。而相同日龄的短翅雌虫及第1日龄的两型雌虫均无明显的节律变化。进一步在第5和7日龄的长翅雌虫中发现了一个16C的脂肪酸--14-甲基十五烷酸, 也具有节律变化且与JH节律出现的时间相吻合, 而在无飞行能力的沙蟋中没有发现这种现象。实验还证明JH滴度的增加和节律不是由飞行肌的重量或者飞行肌重量比的变化所致。这些发现有助于探讨和了解保幼激素对飞行调控的内在机理。     

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.     

High Performance Liquid Chromatography of Hydroperoxides Formed by Autoxidation of Vegetable Oils

Trilinoleoylglycerol (TL) was autoxidized at 37°C in the dark. Monohydroperoxides (MHP) obtained from the oxidized products were analyzed by high performance liquid chromatography (HPLC). Several peaks which appeared in the chromatogram were identified by infrared (IR), gas chromatography mass spectrometry (GC-MS) and enzymatic hydrolysis. Some positional and geometrical isomers of their hydroperoxy fatty acid components were separated using both absorption and reversed phase systems. Furthermore, 1-hydroperoxylinoleoyl-2,3-dilinoleoyl-glycerol and 1,3-dilinoleoyl-2-hydroperoxylinoleoylglycerol were partly separated by HPLC using an absorption system. MHP obtained from autoxidized corn oil, safflower oil and soybean oil were separated into some peaks by HPLC, although resolution into the individual isomers was incomplete. When oxidized oils were subjected to HPLC analysis directly, a linear relationship was observed between the peak areas of MHP and peroxide value in the range of 10 ~ 50 meq/kg.     

The fatty acids of the sponge Hymeniacidon sanguinea from the Black Sea

The fatty acid composition of the sponge Hymeniacidon sanguinea from the Black Sea has been determined by methods involving silver ion HPLC and GC-MS. More than a hundred different fatty acids were identified, of which many were similar to those in sponges from tropical seas. By contrast, some of the fatty acids identified, including trans-6-hexadecenoic acid and 5,15-tetracosadienoic acid, may not have been found previously in sponges and other marine sources, and perhaps are new to science.     

Gas chromatography-mass spectrometry of cis-9,10-epoxyoctadecanoic acid (cis-EODA). I. Direct evidence for cis-EODA formation from oleic acid oxidation by liver microsomes and isolated hepatocytes

Oleic acid, cis-9-octadecenoic acid, is the major fatty acid in mammals. Its oxide, cis-9,10-epoxyoctadecanoic acid (cis-EODA), has been identified in blood and urine of humans, its origin is, however, still unknown. Lipid peroxidation and enzyme-catalyzed epoxidation of oleic acid are two possible sources. In the present article, we investigated by HPLC and GC-MS whether cis-EODA is formed enzymatically from oleic acid by the cytochrome P450 (CYP) system. Oleic acid, cis-EODA and its hydratation product threo-9,10-dihydroxyoctadecanoic acid (threo-DiHODA) were quantitated by HPLC as their p-bromophenacyl esters. For structure elucidation by GC-MS, the pentafluorobenzyl (PFB) esters of these compounds were isolated by HPLC and converted to their trimethylsilyl ether derivatives. Liver microsomes of rats, rabbits and humans oxidized oleic acid into cis-EODA. This is the first direct evidence for the enzymatic formation of cis-EODA from oleic acid. The epoxidation of oleic acid was found to depend on CYP, NADPH+H(+), and O(2). cis-EODA was measurable in incubates of liver microsomes for up to 30 min of incubation. Maximum cis-EODA concentrations were reached after 5-7 min of incubation and found to depend upon oleic acid concentration. Isolated rat hepatocytes hydratated cis-EODA into threo-DiHODA which was further converted to unknown metabolites. However, from incubation of oleic acid with these cells we could not detect threo-DiHODA or cis-EODA. Our study suggests that circulating and excretory cis-EODA may originate, at least in part, from CYP-catalyzed epoxidation of oleic acid. GC-MS of intact cis-EODA as its PFB ester in the negative-ion chemical ionization mode should be useful in investigating the physiological role of cis-EODA in man.     

High performance liquid chromatography preparation of the molecular species of GM1 and GD1a gangliosides with homogeneous long chain base composition

A semi-preparative, analytical high performance liquid chromatographic (HPLC) procedure is described for the isolation of molecular species of GM1 and GD1a gangliosides containing a single long chain base, C18 or C20 sphingosine, C18 or C20 sphinganine, each in its natural erythro or unnatural threo form. The threo forms were obtained from 2,3-dichloro-5,6-dicyanobenzoquinone/NaBH4 -treated gangliosides. The ganglioside molecular species separated by HPLC were analyzed for carbohydrate, fatty acid, and long chain base composition. In particular, long chain bases were submitted to gas-liquid chromatographic-mass spectrometric analyses as their trimethylsilyl (TMS) or N-acetyl-TMS derivatives, and chain length, presence or absence of C4-C5 double bond, and C-3 steric configuration were ascertained. The final preparations of individual molecular species of GM1 and GD1a gangliosides were more than 99% homogeneous in their saccharide moiety, contained a single long chain base (homogeneity higher than 99%), and had a fatty acid composition primarily of stearic acid (92 to 97%). All the individual molecular species of GM1 and GD1a gangliosides were also prepared in radioactive form by selective tritiation at C-3 of the long chain base. Their specific radioactivity ranged from 1.3 to 1.45 Ci/mmol. The availability of these molecular species of gangliosides is expected to facilitate studies aimed at ascertaining the role played by the hydrophobic portion in the functional behavior of gangliosides.     

The synthesis of astaxanthin esters,independent of the formation of cysts,highly correlates with the synthesis of fatty acids in Haematococcus pluvialis

The compositions and contents of astaxanthin esters and fatty acids in four types of Haematococcus pluvialis cells were studied by HPLC and GC-MS. Results showed that the synthesis and accumulation of astaxanthin was independent of the formation of cysts, but was highly correlated with the synthesis and accumulation of fatty acids, though it is an well known phenomenon that the accumulation of astaxanthin is usually accompanied by the formation of cyst. The red cysts contain more than 30% of fatty acids, with 81% of the unsaturated fatty acids. Taken together, besides a resource of astaxanthin, H. pluvialis would be a good resource of valuable fatty acids.     

Structure of a major glycolipid from Thermus oshimai NTU-063

The structure of a major glycolipid isolated from the thermophilic bacteria Thermus oshimai NTU-063 was elucidated. The sugar and fatty acid compositions were determined by GC-MS and HPLC analysis on their methanolysis and methylation derivatives, respectively. After removal of both O- and N-acyl groups by alkaline treatment, the glycolipid was converted to a fully acetylated tetraglycosyl glycerol derivative, the structure of which was then determined by NMR spectroscopy (TOCSY, HSQC, HMBC). Thus, the complete structure of the major glycolipid from T. oshimai NTU-063 was established as beta-Glcp-(1-->6)-beta-Glcp-(1-->6)-beta-GlcpNAcyl-(1-->2)-alpha-Glcp-(1-->1)-glycerol diester. The N-acyl groups on the 2-amino-2-deoxy-glucopyranose residue are C15:0 and C17:0 fatty acids, whereas the fatty acids of glycerol diester are more heterogeneous including both straight and branched fatty acids from C15:0 to C18:0.     

Lipids of Daucus carota cell suspension cultures

In carrot cell suspension cultures greater amounts of phospholipid were detected than in carrot root material, but the major phospholipid classes were the same in both materials, and their fatty acid composition was very similar. In contrast to the cultured cells, no significant amounts of free fatty acids and monoglycerides, as well as diglycerides, could be detected in the carrot root. The fatty acid composition of the major lipid classes from cell cultures is reported for the first time in this report. The degree of unsaturation was higher in triglycerides and phospholipids than in free fatty acids. In a study of phospholipid biosynthesis, [³H]-glycerol was shown to be incorporated into 4-phospholipids (PE, PC, PG, PS⁷) to different extents. The highest specific activity was observed in PC and PG. Five molecular species were isolated from each of the 4 phospholipids and analyzed by GC-MS and LSC.     

Analysis of creatine, creatinine, creatine-d3 and creatinine-d3 in urine, plasma, and red blood cells by HPLC and GC-MS to follow the fate of ingested creatine-d3

Creatine, which is increasingly being used as an oral supplement, is naturally present in the body. Studies on the fate of a particular dose of creatine require that the creatine be labeled, and for studies in humans the use of a stable isotopic label is desirable. The concentrations of total creatine and total creatinine were determined using HPLC. Creatine and creatinine were then separated using cation exchange chromatography and each fraction was derivatized with trifluoroacetic anhydride and the ratio of the deuterated:undeuterated species determined using GC-MS. Ratios of creatine:creatine-d(3), and creatinine:creatinine-d(3), and the concentrations of each of these species, were able to be determined in urine, plasma and red blood cells. Thus, the uptake of labeled creatine into plasma and red blood cells and its excretion in urine could be followed for a subject who ingested creatine-d(3). Creatine-d(3) was found in the plasma and red blood cells 10 min after ingestion, while creatine-d(3) and creatinine-d(3) were found in the urine collected after the first hour.     

The synthesis of astaxanthin esters, independent of the formation of cysts, highly correlates with the synthesis of fatty acids in Haematococcus pluvialis

The compositions and contents of astaxanthin esters and fatty acids in four types of Haematococcus pluvialis cells were studied by HPLC and GC-MS. Results showed that the synthesis and accumulation of astaxanthin was independent of the formation of cysts, but was highly correlated with the synthesis and accumulation of fatty acids, though it is an well known phenomenon that the accumulation of astaxanthin is usually accompanied by the formation of cyst. The red cysts contain more than 30% of fatty acids, with 81% of the unsaturated fatty acids. Taken together, besides a resource of astaxanthin, H. pluvialis would be a good resource of valuable fatty acids. Supported by the National Natural Science Foundation of China (Grant No. CNSF30570183), Chinese Academy of Science (KSCX2-YW-G-027) and Yunnan Provincial Sciences and Technology Department, China (2007AD009)     

Potato tubers exhibit both homolytic and heterolytic hydroperoxide fatty acid-cleaving activities

The action of a crude potato-tuber extract on 9- and 13-hydroperoxides of linoleic and linolenic acids was investigated. HPLC analysis revealed that 50% of the 9-hydroperoxide isomers and almost all the 13-hydroperoxide isomers were rapidly enzymically metabolized. No degradation of fatty acid hydroperoxides was observed with a thermally denatured enzymic extract. GC-MS identification of the volatiles formed by the reaction revealed that no volatiles were detected from the 9-hydroperoxide isomers, whereas 13-hydroperoxide of linolenic acid was cleaved into (Z)-3-hexenal, pentenols or dimers of pentene.     

Structural Determination of Fatty Acid Components in the Seed Oils of Five Species of Euphorbiaceae

The fatty acid components of the seed oils of five species of Euphorbiaceae collected in Xishuanbanna district, Yunnan province—Aleurites montana (Lour.) Wis., Mallotus paniculatus (Lam.) Muell-Arg., Ostodes paniculata Bl., Mcaranga denticulata (Bl.) Muell-Arg., and Trewia nudiflora L.- have been analyzed. Long-chain fatty acids were first converted to corresponding 2-substituted 4,4-dimethyloxazoline derivatives (DMOX) by condensing with 2-amino- 2-methylpropanol (AMP) and subsequently analyzed by GC-MS. The position of unsaturation (double and triple bonds) and substituents (e.g., hydroxyl) in the aliphatic chain can easily be located by interpreting the mass spectra recorded in this way. The qualitative as well as quantitative results can be provided for all constituents in a single GC-MS run. This me thod proves simple and efficient, with good reliability, and is well suitable for the structure determination without reference specimens.     

Fatty acid amides from freshwater green alga Rhizoclonium hieroglyphicum

Freshwater green algae Rhizoclonium hieroglyphicum growing in the Ural Mountains were examined for their fatty acid amides using capillary gas chromatography-mass spectrometry (GC-MS). Eight fatty acid amides were identified by GC-MS. (Z)-9-octadecenamide was found to be the major component (2.26%).     

Use of derivatization to improve the chromatographic properties and detection selectivity of physiologically important carboxylic acids

In this review, tagging techniques with reagents used for ultraviolet-visible (UV-Vis), fluorescent (FL), chemiluminescent (CL) and electrochemical detection (ED) for higher carboxylic acids in HPLC are evaluated in terms of the tagging reactions, handling, flexibility, stability of the reagents and the corresponding derivatives, sensitivity and selectivity. Emphasis is given to the applications of these tagging techniques to biologically important carboxylic acids of relatively high molecular mass including free fatty acids, prostaglandins, leukotrienes and thromboxanes etc. Some typical examples are described. Although RIA and GC-MS are powerful techniques for the highly sensitive determination of carboxylic acids, tagging for these techniques is not included in this review because recent progress in tagging methods has been mainly concerned with HPLC detection.     

The synthesis of astaxanthin esters,independent of the formation of cysts,highly correlates with the synthesis of fatty acids in <Emphasis Type="Italic">Haematococcus pluvialis</Emphasis>

The compositions and contents of astaxanthin esters and fatty acids in four types of Haematococcus pluvialis cells were studied by HPLC and GC-MS. Results showed that the synthesis and accumulation of astaxanthin was independent of the formation of cysts, but was highly correlated with the synthesis and accumulation of fatty acids, though it is an well known phenomenon that the accumulation of astaxanthin is usually accompanied by the formation of cyst. The red cysts contain more than 30% of fatty acids, with 81% of the unsaturated fatty acids. Taken together, besides a resource of astaxanthin, H. pluvialis would be a good resource of valuable fatty acids.     

Fatty acids of lichen species from Tian Shan Mountains

Eight lichen species growing in the Tian Shan Moutains were investigated for their fatty acid composition using capillary GC-MS. Seventy-three fatty acids were identified: 24 saturated, 28 monoenoic, 5 dienoic, 11 trienoic and eight tetra-, penta- and hexaenoic. Very long-chain fatty acids,viz. 26:0, 26:1(11), 26:3(3), 27:1, 28:1 and 30:1 were also identified. For the first time 36 fatty acids were identified in these lichens.     

Improved detection of polyunsaturated fatty acids as phenacyl esters using liquid chromatography-ion trap mass spectrometry

The fatty acid composition of Shewanella pealeana was determined by the analysis of fatty acid methyl esters via gas chromatography-mass spectrometry (GC-MS) and fatty acid 2-oxo-phenylethyl esters via high-performance liquid chromatography-mass spectrometry (LC-MS) combined with ultra violet (UV) detection. There was good agreement between the percentage composition of components determined by GC-MS and LC-UV analyses. However, LC-MS analysis using Atmospheric Pressure Chemical Ionization (APCI) demonstrated dramatically enhanced detection of unsaturated fatty acid 2-oxo-phenylethyl esters. The degree of enhancement was proportional to the degree of unsaturation. Tests with a pure polyunsaturated fatty acid (PUFA) standard gave an absolute detection limit in full scan mode of 200 pg. In samples, the selectivity of MS over UV gave a significantly lower detection limit due to lack of chemical interferences. In 'Selected Reaction Monitoring' (SRM) mode, the detection limit was 5 pg. This was essentially independent of whether the sample is a standard or complex mixture of fatty acids. Tandem mass spectrometry was used to support structural information and to enhance the ability to target specific fatty acids. Several PUFAs which were not evident from GC-MS analysis were detected and identified by APCI LC-MS, including some rare or novel PUFAs from S. pealeana and a menhaden oil standard. Detailed analysis of bacterial fatty acid composition by either GC-MS or APCI LC-MS is highly preferable to analysis systems based solely on retention time identification.