Enzymatic hydrolysis of esterified diarrhetic shellfish poisoning toxins and pectenotoxins

Okadaic acid (OA) and dinophysistoxins-1 and -2 (DTX1, DTX2), the toxins responsible for incidents of diarrhetic shellfish poisoning (DSP), can occur as complex mixtures of ester derivatives in both plankton and shellfish. Alkaline hydrolysis is usually employed to release parent OA/DTX toxins, and analyses are conducted before and after hydrolysis to determine the concentrations of nonesterified and esterified toxins. Recent research has shown that other toxins, including pectenotoxins and spirolides, can also exist as esters in shellfish, but these toxins cannot survive alkaline hydrolysis. A promising alternative approach is enzymatic hydrolysis. In this study, two enzymatic methods were developed for the hydrolysis of 7-O-acyl esters, “DTX3,” and the carboxylate esters of OA, “diol-esters.” Porcine pancreatic lipase induced complete conversion of DTX3 to OA and DTXs within one hour for reference solutions. The presence of mussel tissue matrix reduced the rate of hydrolysis, but an optimized lipase concentration resulted in greater than 95% conversion within four hours. OA-diol-ester was hydrolyzed by porcine liver esterase and was completely converted to OA in less than 30 min, even in the presence of mussel tissue matrix. Esters and OA/DTX toxins were all monitored by LC–MS. Further experiments with pectenotoxin esters indicated that enzymatic hydrolysis could also be applied to esters of other toxins. Enzymatic hydrolysis has excellent potential as an alternative to the conventional alkaline hydrolysis procedure used in the preparation of shellfish samples for the analysis of toxins.     

Characterization of fatty acid esters of okadaic acid and related toxins in blue mussels (Mytilus edulis) from Norway

Marine algal toxins of the okadaic acid group can occur as fatty acid esters in blue mussels, and are commonly determined indirectly by transformation to their parent toxins by alkaline hydrolysis. Some data are available regarding the identity of the fatty acid esters, mainly of palmitic acid (16:0) derivatives of okadaic acid (OA), dinophysistoxin-1 (DTX1) and dinophysistoxin-2 (DTX2). Other fatty acid derivatives have been described, but with limited mass spectral data. In this paper, the mass spectral characterization of the [M-H](-) and [M+Na](+) ions of 16 fatty acid derivatives of each of OA, DTX1 and DTX2 is presented. The characteristic fragmentation of [M+Na](+) ions of OA analogues provided a useful tool for identifying these, and has not been described previously. In addition, a set of negative ion multiple reaction monitoring (MRM) methods was developed for direct determination of 16 fatty acid esters of OA, 16 fatty acid esters of DTX1 and 16 fatty acid esters of DTX2 in shellfish extracts. The MRM methods were employed to study the profiles of fatty acid esters of OA analogues in blue mussels and to compare these with fatty acid ester profiles reported for other groups of marine algal toxins.     

Identification of six species in the new genus Cronobacter (Enterobacter sakazakii) from culture using gas chromatographic analysis of fatty acid methyl esters

Capillary GC with flame ionization detection (FID) was used to determine the cellular fatty acid (CFA) profiles of six species in the new genus Cronobacter (Enterobacter sakazakii). The six different species are C. sakazakii, C. malonaticus, C. dublinensis, C. muytjensii, C. turicensis, and C. genomospecies. For GC-FID analysis, whole cell fatty acid methyl esters (FAMEs) from cells cultured on brain heart infusion (BHI) agar at 35 degrees C for 24 h were obtained by saponification, methylation, and extraction into hexane-methyl tert-butyl ether. A data set for 57 strains of Cronobacter species was prepared using fatty acid profiles from two or three replicates prepared on different days. Major fatty acids of the Cronobacter strains evaluated in this study were straight-chain C12:0, C14:0, C16:0, and unsaturated C18:1, omega7c, summed C16:1 omega7c/C16:1 omega6c, and summed C14:0 3-OH/iso-C16:1, and C17:0 omega cyclo 7-8. The CFA profiles for the Cronobacter species are similar, but there are several fatty acids-C12:0, C14:0, C16:0, C18:1 omega7c, and summed C16:1 omega7c/ C16:1 omega6c--that differ significantly among these six species. Analysis of FAMEs from Cronobacter strains grown on BHI agar by a rapid GC-FID method is a sensitive procedure for the identification of these organisms, and this analytical method provides a procedure for the differentiation of strains from closely related Cronobacter species.     

Development of a Capillary Electrophoresis-Based Enzyme Immunoassay with Electrochemical Detection for the Determination of Okadaic Acid and Dinophysistoxin2 in Shellfish Samples

A capillary electrophoresis-based enzyme immunoassay (CE-EIA) with electrochemical (EC) detection system was developed for the determination of two diarrheic shellfish poisoning (DSP) toxins okadaic acid (OA) and dinophysistoxin2 (DTX2). In this method, after the competitive immunoreaction in liquid phase, the horseradish peroxidase (HRP)-labeled antigen (Ag*) and the bound enzyme-labeled complex (Ag*-Ab) were separated and then the system of HRP catalyzing H₂O₂/o-aminophenol (OAP) reaction was adopted. The limit of detection (S/N = 3) was determined to be 0.05 and 0.07 ng/mL for OA and DTX2, respectively. The total analysis time was less than 40 min. The developed CE-EIA with EC detection system was capable of quantitatively detecting OA and DTX2 contents in the tested contaminated samples, and the results were compared with the same samples analyzed through enzyme-linked immunosorbent assay (ELISA). Consistent results between CE-EIA with EC detection and ELISA were found in most of the tested samples. The proposed system appeared to be more sensitive and faster than ELISA for determination of OA and DTX2 in shellfish meat extracts. Real shellfish samples were validated in recovery test, and the recoveries tested by the proposed method were 91.7–108.3% and 95.2–112.5% for OA and DTX2, respectively. The CE-EIA with EC detection provides a valid and sensitive analytical approach, not previously available, for the determination of OA and DTX2 in shellfish samples.     

Characterization of a lipid-rich fraction synthesized by Streptomyces avermitilis

Isolation of the macrocyclic lactone parasiticide avermectin and other closely related natural products produced by Streptomyces avermitilis also yields a lipid-rich fraction. The latter has been characterized by techniques based on gas-liquid chromatography (GLC) and mass spectrometry (MS). Initial examination of the lipid-rich fraction by direct probe electron-impact (EI) MS and packed-column GLC showed that it consists primarily of a mixture of triglycerides possessing C14-C17 acyl groups. Further examination of this fraction by capillary column GLC-MS demonstrated that it contains low levels of C15-C17 free fatty acids, squalene and diglycerides and, as the major components, at least ten mixed acyl triglycerides (total number of acyl carbon atoms ranging from 43 to 50). Prominent among the triglycerides were a C15-C15-C16 species, a C15-C16-C16 species and a C15-C16-C17 species. Capillary-column GLC and GLC-MS of the fatty acid methyl esters resulting from transesterification demonstrated that the major triglyceride acyl groups are anteiso-C15 (12-methyltetradecanoyl), iso-C16 (14-methylpentadecanoyl), n-C16 (hexa-decanoyl) and anteiso-C17 (14-methylhexadecanoyl). Lower levels of the methyl esters of the following fatty acids were observed: iso-C14 (12-methyltridecanoic), n-C14 (tetradecanoic), iso-C15 (13-methyltetradecanoic), n-C15 (pentadecanoic), iso-C17 (15-methylhexadecanoic) and n-C17 (heptadecanoic). Little evidence was seen for either unsaturated acyl groups or acyl groups of less than 13 or more than 18 carbon atoms. Desorption chemical ionization MS (ammonia reagent gas) analysis confirmed the nature of the lipid-rich fraction, and is an attractive one-step approach for determining the molecular weights and distribution of triglycerides in a mixture.     

Quantitative determination of marine toxins associated with diarrhetic shellfish poisoning by liquid chromatography coupled with mass spectrometry

Quantitative determination by liquid chromatography (LC) coupled with mass spectrometry (MS) was achieved for the following 10 toxins found in association with diarrhetic shellfish poisoning: okadaic acid (OA), dinophysistoxin-1 (DTX1), 7-O-palmitoylokadaic acid (palOA), 7-O-palmitoyldinophysistoxin-1 (pa1DTX1), pectenotoxin-1 (PTX1), pectenotoxin-2 (PTX2), pectenotoxin-2 seco acid (PTX2SA), pectenotoxin-6 (PTX6), yessotoxin (YTX), and 45-hydroxyyessotoxin (YTXOH). Toxins in 2 g of the adductor muscle or the digestive glands of scallops, Patinopecten yessoensis, were extracted with 18 ml of methanol-water (9:1, v/v), freed of polar contaminants by partition between chloroform and water, and treated by solid-phase extraction on a silica cartridge column. Samples containing YTXOH were purified separately on a buffered reversed-phase column. Chromatographic separation was achieved by the following combinations of columns and mobile phases: a Symmetry C18 column with acetonitrile-0.05% acetic acid (7:3, v/v) for OA, DTX1, PTX6 and PTX2SA; a Develosil ODS column with the same mobile phase for PTX1 and PTX2; a Capcellpak column with methanol-2.5% acetic acid (98:2, v/v) for palOA and palDTX1; and an Inertsil ODS column with methanol-0.2 M ammonium acetate (8:2, v/v) for YTX and YTXOH. Carboxylic acid toxins were selectively monitored on [M-H]- ions, sulfated toxins on [M-Na]-ions, and neutral toxins on [M+NH4]+ ions. Average recoveries of the toxins spiked to tissue homogenates ranged from 70 to 134%. Detection limits in the muscle ranged from 5 to 40 ng/g and those in the digestive glands from 10 to 80 ng/g.     

Dynamic adsorption of diarrhetic shellfish poisoning (DSP) toxins in passive sampling relates to pore size distribution of aromatic adsorbent

Solid-phase adsorption toxin tracking (SPATT) technology was developed as an effective passive sampling method for dissolved diarrhetic shellfish poisoning (DSP) toxins in seawater. HP20 and SP700 resins have been reported as preferred adsorption substrates for lipophilic algal toxins and are recommended for use in SPATT testing. However, information on the mechanism of passive adsorption by these polymeric resins is still limited. Described herein is a study on the adsorption of OA and DTX1 toxins extracted from Prorocentrum lima algae by HP20 and SP700 resins. The pore size distribution of the adsorbents was characterized by a nitrogen adsorption method to determine the relationship between adsorption and resin porosity. The Freundlich equation constant showed that the difference in adsorption capacity for OA and DTX1 toxins was not determined by specific surface area, but by the pore size distribution in particular, with micropores playing an especially important role. Additionally, it was found that differences in affinity between OA and DTX1 for aromatic resins were as a result of polarity discrepancies due to DTX1 having an additional methyl moiety.     

Discovery of okadaic acid esters in the toxic dinoflagellate Dinophysis acuta from New Zealand using liquid chromatography/tandem mass spectrometry

The dinoflagellate Dinophysis acuta has been associated with various incidents of diarrhetic shellfish poisoning. A sample of Dinophysis acuta collected from New Zealand waters in 2002 was previously found to contain high levels of pectenotoxins, but only a very low level of the diarrhea-inducing okadaic acid (OA). After hydrolysis under basic conditions, however, the concentration of OA increased substantially, indicating the presence of conjugated forms of OA. Using various liquid chromatography/electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) techniques, a number of OA esters were detected in the original extract. The principal compound was identified as a C8 diol-ester of OA (OA-D8), which had been identified previously in another dinoflagellate, Prorocentrum lima. The retention time, as well as positive and negative ion MS, MS/MS and UV spectra of the D. acuta compound, matched exactly those of OA-D8 isolated from P. lima. In addition to OA-D8, several other novel OA esters were detected in the D. acuta but these have not yet been identified. This is the first report identifying the presence of OA esters in Dinophysis species.     

Multiresidue method for determination of algal toxins in shellfish: single-laboratory validation and interlaboratory study

A method that uses liquid chromatography with tandem mass spectrometry (LC/MS/MS) has been developed for the highly sensitive and specific determination of amnesic shellfish poisoning toxins, diarrhetic shellfish poisoning toxins, and other lipophilic algal toxins and metabolites in shellfish. The method was subjected to a full single-laboratory validation and a limited interlaboratory study. Tissue homogenates are blended with methanol-water (9 + 1), and the centrifuged extract is cleaned up with a hexane wash. LC/MS/MS (triple quadrupole) is used for quantitative analysis with reversed-phase gradient elution (acidic buffer), electrospray ionization (positive and negative ion switching), and multiple-reaction monitoring. Ester forms of dinophysis toxins are detected as the parent toxins after hydrolysis of the methanolic extract. The method is quantitative for 6 key toxins when reference standards are available: azaspiracid-1 (AZA1), domoic acid (DA), gymnodimine (GYM), okadaic acid (OA), pectenotoxin-2 (PTX2), and yessotoxin (YTX). Relative response factors are used to estimate the concentrations of other toxins: azaspiracid-2 and -3 (AZA2 and AZA3), dinophysis toxin-1 and -2 (DTX1 and DTX2), other pectenotoxins (PTX1, PTX6, and PTX11), pectenotoxin secoacid metabolites (PTX2-SA and PTX11-SA) and their 7-epimers, spirolides, and homoYTX and YTX metabolites (45-OHYTX and carboxyYTX). Validation data have been gathered for Greenshell mussel, Pacific oyster, cockle, and scallop roe via fortification and natural contamination. For the 6 key toxins at fortification levels of 0.05-0.20 mg/kg, recoveries were 71-99% and single laboratory reproducibilities, relative standard deviations (RSDs), were 10-24%. Limits of detection were <0.02 mg/kg. Extractability data were also obtained for several toxins by using successive extractions of naturally contaminated mussel samples. A preliminary interlaboratory study was conducted with a set of toxin standards and 4 mussel extracts. The data sets from 8 laboratories for the 6 key toxins plus DTX1 and DTX2 gave within-laboratories repeatability (RSD(R)) of 8-12%, except for PTX-2. Between-laboratories reproducibility (RSDR) values were compared with the Horwitz criterion and ranged from good to adequate for 7 key toxins (HorRat values of 0.8-2.0).     

杏仁油的物化性能及其脂肪酸组成的分析

用３种不同溶剂萃取杏仁得到杏仁油,并对其物化常数进行测定。杏仁油用饱和氢氧化钾 甲醇皂化,再用甲醇 硫酸（体积比为４∶１）甲酯化后,将乙醚萃取液作气相色谱分析。太原杏仁油中脂肪酸的主要成分为油酸（Ｃ１８∶１,质量分数约６８％）和亚油酸（Ｃ１８∶２,质量分数约２５％）,少量棕榈酸（Ｃ１６∶０）、棕榈烯酸（Ｃ１６∶１）和硬脂酸（Ｃ１８∶０）,微量花生酸（Ｃ２０∶０）。     

Sulfoquinovosyldiacylglycerins from <Emphasis Type="Italic">Scaphechinus mirabilis</Emphasis>

A sulfolipid, the structure of which was established by NMR spectroscopy, electrospray-ionization mass spectrometry (ESI-MS), and GC–MS, was isolated for the first time from the sea urchin Scaphechinus mirabilis. The sulfolipid was identified as a sulfoquinovosyldiacylglycerin (SQDG) and was the sum of related compounds. The fatty acids (FAs) of the SQDGs included saturated 14:0–24:0 FAs (95.8% of total FAs) and mono-unsaturated 20:1-24:1 FAs (4.2%). The principal FAs were saturated 14:0 (33.1%) and 16:0 (54.2%). ESI-MS/MS detected 15 molecular species of SQDGs, among which the contents of 14:0/14:0 and 16:0/16:0 were relatively high although 16:0/14:0 dominated.     

New N-acylamino acids and derivatives from renewable fatty acids: gelation of hydrocarbons and thermal properties

This work reports the synthesis of new fatty N-acylamino acids and N-acylamino esters from the C16:0, C18:0, C18:1, and C18:1(OH) fatty acid families and demonstrates the activity of these compounds as organogel agents. Compounds were heated and dissolved in various solvents (n-hexane, toluene, and gasoline). Only saturated C16:0 and C18:0 derived from alanine were able to form gels in toluene, and saturated C16:0 derived from phenylalanine showed gelation in n-hexane. This is the first evidence that fatty N-acylamino esters and N-acylamino acid derivatives of l-serine and fatty acids C16:0, C18:0, and C18:1 are able to form gels with hexane. This observation confirms the importance of the hydroxyl group in the segment derivative of l-serine in forming good gels.     

气相色谱法鉴别缓慢生长分枝杆菌

采用毛细管气相色谱法对９种缓慢生长分枝杆菌的化学成分进行了分析。结果表明,不同菌种的色谱图存在明显的差异。对７５株临床菌株进行色谱法鉴别,并与传统生物学鉴定法作了对比。     

Survey of the distribution of red tide toxins (okadaic acid and dinophytoxin-1) in the Dalian Bay sea area of China by micellar electrokinetic capillary chromatography.

Two kinds of diarrhoetic shellfish toxins, okadaic acid (OA) and dinophytoxin-1 (DTX-1) were determined by micellar electrokinetic capillary chromatography (MEKC) with ultraviolet detection. A detection limit of 3.25 microg/mL for both of them was achieved. The UV absorbance of these toxins measured at 200 nm showed good linearity in the range of 6.25-200 microg/mL with R = 0.992 for OA and 0.997 for DTX-1. Three kinds of shellfish (Chlamys farreri, Mytilus edulis and Ruditaps philippinarum) collected from eight locations (sampling in the intertidal zone) along the Dalian Bay sea area of China were surveyed in February and May of 2000. Results indicated that three kinds of shellfish were contaminated by OA and DTX-1. Based on per gram of hepatopancreas in February, the contamination contents ranged from 0 to 1.26 microg for OA and from 0 to 1.82 microg for DTX-1, and in May, the contents ranged from 0 to 1.45 microg for OA and 0 to 2.56 microg for DTX-1. Among the eight locations, Hei Shi Jiao and Long Wang Tang were the most contaminated areas. Of the three kinds of shellfish, Mytilus edulis was the most significant species in accumulating OA and DTX-1.     

气相色谱测定牦牛骨中脂肪酸的甲酯化方法比较

利用气相色谱（GC）技术,采用酸水解提取脂质,比较了6种甲酯化法（乙酰氯-甲醇法、H₂SO₄-甲醇法、HCl-甲醇法、KOH-甲醇法、KOH-甲醇+H₂SO₄-甲醇法和KOH-甲醇+HCl-甲醇法）对脂肪酸测定的影响,优选牦牛骨中脂肪酸测定的最佳方法。37种脂肪酸标准样品在0.28~250.00 mg/L范围内线性关系良好,相关系数均大于0.99（除C4：0外）。碱酯化法和酸碱结合法几乎无法测出牦牛骨中的脂肪酸,其测得的总脂肪酸含量小于0.20 g/100 g。乙酰氯-甲醇法测得的总脂肪酸含量（13.61 g/100 g）显著高于H₂SO₄-甲醇法（总脂肪酸含量为11.68 g/100 g）和HCl-甲醇法（总脂肪酸含量为3.18 g/100 g）测得的结果。乙酰氯-甲醇法和H₂SO₄-甲醇法的日内和日间精密度分别为0.27%~8.60%和0.34%~2.64%,两种方法中脂肪酸的回收率为83.06%~105.54%。结果表明,酸水解-乙酰氯-甲醇法是牦牛骨中脂肪酸测定的最佳方法。C18：1n9c、C16：0、C18：0和共轭亚油酸（CLA）是牦牛骨的主要脂肪酸,其总和达脂肪酸总量的85%以上,饱和脂肪酸与不饱和脂肪酸含量比值约为1：2。牦牛骨中脂肪酸的研究为骨资源脂质的有效利用提供了重要依据。     

液相色谱-串联质谱法同时测定贝类中大田软海绵酸、鳍藻毒素、蛤毒素和虾夷扇贝毒素

建立了同时测定贝类中大田软海绵酸(okadaic acid, OA)及其衍生物鳍藻毒素(dinophysistoxin-1, DTX-1)、蛤毒素(pectenotoxin-2, PTX-2)和虾夷扇贝毒素(yessotoxin, YTX)的液相色谱-串联质谱分析方法。样品经甲醇提取,固相萃取柱净化,C18色谱柱分离,经含甲酸和甲酸铵的乙腈-水溶液为流动相梯度洗脱,选择反应监测(SRM)模式检测,正、负离子切换扫描,基质标准校正,外标法定量。结果表明,OA、DTX-1和YTX的线性范围为2.0～200.0 μg/L,定量限(以信噪比(S/N)≥10计)为1.0 μg/kg; PTX-2的线性范围为1.0～100.0 μg/L,定量限为0.5 μg/kg;几种化合物的添加平均回收率为83.1%～105.7%,相对标准偏差(RSD)为3.16%～9.29%。成功应用本法对黄海灵山湾海域采集的贝类样品进行了分析,发现部分样品中含有大田软海绵酸、鳍藻毒素、蛤毒素和虾夷扇贝毒素。     

Gas-liquid chromatographic determination of fatty acid composition of cholesteryl esters in human serum using silica Sep-Pak cartridges

A simple and fast analytical procedure for separation and purification of cholesteryl esters of human serum is described. A single lipid extract, together with spiked cholesteryl pentadecanoate, as an internal standard, was passed through a Silica Sep-Pak cartridge. 1.5% diethyl ester in light petroleum was used to elute cholesteryl esters from the column. The separation was verified with thin-layer chromatography on silica gel using light petroleum-diethyl ether-glacial acetic acid (80:20:1) as a solvent. A very clean thin-layer chromatogram of cholesteryl esters without any additional spots of other lipids was obtained. The cholesteryl esters were quantitated by analyzing their fatty acid composition as methyl esters by gas-liquid chromatography. The coefficients of variation were 0.8--4.9% for the major fatty acids (C16:0, C16:1, C18:1, C18:2, C20:4) and 6.7--30.8% for the minor fatty acids (C18:0 and C20:0). The recoveries for cholesteryl palmitate, cholesteryl oleate and cholesteryl linoleate were 90.7, 92.3 and 91.0%, respectively.     

Comparative investigation of the fatty acid compositions of the phospholipids of marine invertebrates

The fatty acid (FA) compositions of the phosphatidylcholines (PCs) and phosphatidylethanolamines (PEs) of the muscle tissues of 13 species from seven types of marine invertebrates have been studied. In the PCs and PEs of all the animals the main FAs were the 20:53, 20:46, 16:0, 18:1, and 18:0 acids, although the representatives of each type had their own specific features in their FA composition. The main FAs in the PCs and PEs in the majority of animals investigated coincided, although there were exceptions. The most promising sources for the directed isolation of PCs or PEs enriched with certain FAs have been determined. The highest level of polyenic FAs and the highest unsaturation index were found in representatives of theCoelenterata andEchinodermata (stars) types. It was shown that the amount of polyenic acids as a fraction of the sum of the acyl and alkenyl radicals in the majority of animals investigated was greater in the PCs than in PEs.Institute of Marine Biology, Far Eastern Scientific Center, Academy of Sciences of the USSR, Vladivostok. Far Eastern State University, Vladivostok. Translated from Khimiya Prirodnykh Soedinenii, No. 1, pp. 24–29, January–February, 1984.     

High-performance liquid chromatography/electrospray ionization ion-trap tandem mass spectrometric analysis and quantification of phosphatidylcholine molecular species in the serum of cystic fibrosis subjects supplemented with docosahexaenoic acid

Since phosphatidylcholine (PC) is the most abundant phospholipid (PL) class in human serum, its concentration represents an important marker for the evaluation of lipid absorption and metabolism. High-performance liquid chromatography coupled on-line with electrospray ionization ion-trap tandem mass spectrometry (HPLC/ESI-MS/MS) was successfully applied to the quantitative analysis of PC molecular species from serum of cystic fibrosis (CF) subjects before and after supplementation with docosahexaenoic acid (DHA). Seven molecular species of PC (containing C16:0/C20:4, C16:0/C22:6, C18:0/C20:4, C18:0/C22:6, C16:0/C18:1, C16:0/C18:2 and C18:0/C18:2, respectively) were quantified using MS in the negative scan mode with 1,2-diundecanoyl-sn-glycero-phosphocholine as the internal standard. The molecular species containing DHA, C16:0/C22:6 and C18:0/C22:6, increased from 41.3 +/- 31.7 and 33.1 +/- 18.2 to 85.4 +/- 20.4 and 52.1 +/- 20.7 microg/mL serum, respectively, after a 3-month supplementation. Interestingly, the species containing arachidonic acid (C18:0/C20:4 and C16:0/C20:4) decreased from 115 +/- 55 and 139 +/- 57 to 58.1 +/- 22.5 and 70.5 +/- 28.1, respectively. HPLC/ESI-MS/MS allowed the direct analysis of the lipid extract without previous purification of PLs, thus it is a useful analytical support in CF research in order to understand the extent of lipid dysfunctions typical of CF or other diseases. The present method might also be used for quantitative analysis of each serum phospholipid class molecular species. However, the instrument response was found to be very dependent on the phospholipid class considered, and thus the use of appropriate standards for each class of PLs is recommended.     

Occurrence of oleic and 18:1 methyl-branched acyl chains in lipids of Rhodobacter sphaeroides 2.4.1

The fatty acids (FAs) composition of lipids extracted from Rhodobacter sphaeroides 2.4.1 was investigated by gas chromatography–mass spectrometry (GC–MS) analysis of the corresponding FA methyl esters (FAMEs), obtained through trans-esterification of the original lipid species. A GC stationary phase based on a highly polar ionic liquid (IL) was selected, aimed to enhance the separation of isomeric FAMEs with particular emphasis on positional and geometrical isomers of monounsaturated 16:1 and 18:1 fatty acyl chains. The occurrence of 18:1 cis-Δ⁹ (oleic) acid, a positional isomer of the well-known and most predominant 18:1 cis-Δ¹¹ (cis-vaccenic) acid, has been demonstrated here for the first time. Furthermore a methyl branched 18:1 FA was also identified and its structure tentatively assigned as 11-methyl-Δ¹²-octadecenoic acid (most likely as trans isomer). The unprecedented observation about 18:1 cis-Δ⁹ FA occurrence in R. sphaeroides 2.4.1 is, even indirectly, supported by a biosynthetic pathway postulated with the aid of the Kyoto Encyclopedia of Genes and Genomes (KEGG) database. The concurrent presence of 16:1 cis-Δ⁷ and 18:1 cis-Δ⁹ FAs suggested the existence of parallel and/or complementary processes to those invoked for the formation of most common 16:1 cis-Δ⁹ and 18:1 cis-Δ¹¹ FAs. A further route was hypothesized for the trans FAs biosynthesis in wild-type cells of R. sphaeroides.