Analysis of plasma free fatty acid cyanomethyl derivatives by GC-NPD for the diagnosis of mitochondrial fatty acid oxidation disorders

Summary Early diagnosis of fatty acid oxidation (FAO) disorders is important to reduce severe morbidity and mortality. Although analysis of plasma free fatty acids (FFAs) is frequently performed using stable isotope-dilution gas chromatography-mass-spectrometry (GC-MS), there are institutions where the required instrumentation is not available to support a rapid work-up of acutely ill patients. For this reason, we have developed a novel cyanomethyl derivatization method for FFAs which is followed by GC analysis of the resulting esters using nitrogen-phosphorus detector (NPD) for the rapid diagnosis of mitochondrial fatty acid oxidation disorders. FFAs were extracted from plasma and derivatized to the cyanomethyl ester by heating with bromoacetonitrile at 60°C for 30 min GC-NPD analysis was then performed. The mean recoveries of C6:0-C18:0FFAs were between 87% abd 96%. The method detection limits (S/N=3) were 0.1–0.5 ng for C6:0-C14:0 FFAs, and 0.001–0.01 ng for C16:0-C18:0 FFAs. We succesfully performed differential diagnosis of representative FAO disorders from the confimed patient's plasmas. This simple method offers cost-effective and time-saving alternative to GC-MS for the biochemical diagnosis of selected FAO disorders.     

Determination of polar impurities in biodiesels using solid-phase extraction and gas chromatography-mass spectrometry

This paper reports on a method for development and validation for simultaneous characterization and determination of oxygenated polar impurities--free fatty carboxylic acids (FFAs), partial glycerides (monoacylglycerides, MGs), residual glycerol and free sterols--in various biodiesels based on the combination of solid-phase extraction (SPE), silylation and GC/MS technologies. The effects of various SPE and silylation conditions on the method recoveries were evaluated. Using this integrated SPE-GC/MS method, 38 target polar compounds (13 FFAs, 17 glycerides and 8 sterols) in 9 biodiesels derived from 4 different feedstocks were successfully separated and quantified. It was found that the carbon chain length of FFAs was ranged from C(6) to C(24), with C(16) and C(18) being the most abundant in all biodiesels. The total FFAs concentration was consistent with the acid values (AVs) measured by standard method ASTM D974-04. MG congeners with carbon number of 18 (mono-C18) were most abundant in the biodiesel samples, followed by mono-C(16) and free glycerol. β-Sitosterol and campesterol were found to be the prevailing phytosterols in all pure vegetable oil-based biodiesels, while brassicasterol and stigmasterol was only significant in the biodiesel from canola oil and soybean oil, respectively, and abundant cholesterol was only detected in animal fat-based biodiesels.     

Serum fatty acid profiles and potential biomarkers of ankylosing spondylitis determined by gas chromatography–mass spectrometry and multivariate statistical analysis

Ankylosing spondylitis (AS) is a common chronic inflammatory rheumatic disease. Early and accurate detection is essential for effective disease treatment. Recently, research has focused on genomics and proteomics. However, the associated metabolic variations, especially fatty acid profiles, have been poorly discussed. In this study, the gas chromatography–mass spectrometry (GC‐MS) approach and multivariate statistical analysis were used to investigate the metabolic profiles of serum free fatty acids (FFAs) and esterified fatty acids (EFAs) in AS patients. The results showed that significant differences in most of the FFA (C12:0, C16:0, C16:1, C18:3, C20:4, C20:5, C22:5 and C22:6) and EFA (C12:0, C16:1, C18:0, C18:1, C18:2, C18:3, C20:4 and C22:6) concentrations were found between the AS patients and healthy controls (p < 0.05). Principal component analysis and partial least squares discriminant analysis were performed to classify the AS patients and controls. Additionally, FFAs C20:4, C12:0, C18:3 and EFAs C22:6, C12:0 were confirmed as potential biomarkers to identify AS patients and healthy controls. The present study highlights that differences in the serum FFA and EFA profiles of AS patients reflect the metabolic disorder. Moreover, FFA and EFA biomarkers appear to have clinical applications for the screening and diagnosis of AS. Copyright © 2014 John Wiley & Sons, Ltd.     

Quantification of free fatty acids in human stratum corneum using tandem mass spectrometry and surrogate analyte approach

The free fatty acids (FFAs) are one of the major components of the lipids in the stratum corneum (SC), the uppermost layer of the skin. Relative composition of FFAs has been proposed as a biomarker of the skin barrier status in patients with atopic dermatitis (AD). Here, we developed an LC‐ESI‐MS/MS method for simultaneous quantification of a range of FFAs with long and very long chain length in the SC collected by adhesive tape (D‐Squame). The method, based on derivatization with 2‐bromo‐1‐methylpyridinium iodide and 3‐carbinol‐1‐methylpyridinium iodide, allowed highly sensitive detection and quantification of FFAs using multiple reaction monitoring. For the quantification, we applied a surrogate analyte approach and internal standardization using isotope labeled derivatives of FFAs. Adhesive tapes showed the presence of several FFAs, which are also present in the SC, a problem encountered in previous studies. Therefore, the levels of FFAs in the SC were corrected using C12:0, which was present on the adhesive tape, but not detected in the SC. The method was applied to SC samples from patients with atopic dermatitis and healthy subjects. Quantification using multiple reaction monitoring allowed sufficient sensitivity to analyze FFAs of chain lengths C16–C28 in the SC collected on only one tape strip.     

碱催化法衍生化气相色谱/质谱法分析青海湖裸鲤鱼油中的脂肪酸

用碱催化法将青海湖裸鲤鱼油甲酯化,以气相色谱/质谱法分析鱼油中的脂肪酸。青海湖裸鲤可食用部分中鱼油含量为25.13%。从鱼油中共鉴定出47种脂肪酸,包括直链、单支链、多支链饱和脂肪酸,单不饱和、多不饱和脂肪酸,环丙烷基、呋喃基脂肪酸等。不饱和脂肪酸含量为73.6%,其中多不饱和脂肪酸含量为25.4%,以C18∶2(4.9%),C18∶3(3.1%),C20∶4(1.3%),C20∶5(二十碳五烯酸（EPA）, 9.4%)和C22∶6(二十二碳六烯酸（DHA）, 6.7%)为主。单不饱和脂肪酸含量为48.2%,主要由C16∶1(20.3%),C18∶1(25.9%)构成。饱和脂肪酸含量为25.7%,主要有C14∶0(3.4%),C16∶0 (19.4%)和C18∶0(1.1%)。青海湖裸鲤鱼油中还存在不常见的环丙烷基和呋喃基脂肪酸及多种奇数碳链和支链脂肪酸。因此,青海湖裸鲤是功能性脂肪酸的重要膳食来源。     

Simultaneous determination of six free fatty acids in alcohol extract of <Emphasis Type="Italic">P. japonicus</Emphasis> C. A. Mey. van <Emphasis Type="Italic">major</Emphasis> (Burk.) C. Y. Wu et K. M. Feng by gas chromatography-mass spectrometry

P. japonicus C. A. Mey. var. major (Burk.) C. Y. Wu et K. M. Feng is an important medicinal plant and has special beneficial effects on human health. Six types of free fatty acids (FFAs) were identified and quantified in the alcohol extract of P. japonicus C. A. Mey. var. major with a GC-MS method, including two fatty acids essential to the human body. All the six FFAs identified in alcohol extract were quantified using nonadecanoic acid as an internal standard. The results showed that the alcohol extract was abundant in four types of FFAs, unsaturated FFAs amounted to 47.04% of the total FFA content, and there were no trans FFAs. Palmitic acid (38.82%, 4.74 ± 0.09 mg/g) was the predominant fatty acid, followed by linoleic acid (27.92%, 2.31 ± 0.07 mg/g), stearic acid (14.14%, 1.54 ± 0.04 mg/g) and oleic acid (11.04%, 1.02 ± 0.03 mg/g).     

Lipids and lipophilic components of Viburnum opulus fruits during maturation

The variation of the composition of neutral and polar lipids and lipophilic components during maturation of guelder rose (V. opulus L., fam. Caprifoliaceae) fruits was investigated. During fruit ripening, all lipid groups are accumulated, the highest accumulation rate being observed in the first period of maturation. In nonpolar lipids, this is due to a substantial increase in the content of triacylglycerides, whereas the contents of free fatty acids, methyl esters of fatty acids, monoacylglycerides, and especially esters of triterpenoid compounds decrease. During early maturation, all acyl-containing classes of lipids contain large amounts of saturated fatty acids (mainly, 16:0), whose content decreases during ripening, while the contents of unsaturated 18:1 and 18:2 acids increase.     

Ammonia-treated N-(1-naphthyl) ethylenediamine dihydrochloride as a novel matrix for rapid quantitative and qualitative determination of serum free fatty acids by matrix-assisted laser desorption/ionization-Fourier transform ion cyclotron resonance mass spectrometry

The blood free fatty acids (FFAs), which provide energy to the cell and act as substrates in the synthesis of fats, lipoproteins, liposaccharides, and eicosanoids, involve in a number of important physiological processes. In the present study, matrix-assisted laser desorption/ionization-Fourier transform ion cyclotron resonance mass spectrometry (MALDI-FTICR MS) with ammonia-treated N-(1-naphthyl) ethylenediamine dihydrochloride (ATNEDC) as a novel MALDI matrix in a negative ion mode was employed to directly quantify serum FFAs. Multiple point internal standard calibration curves between the concentration ratios of individual fatty acids to internal standard (IS, C_17:0) versus their corresponding intensity ratios were constructed for C_14:0, C_16:1, C_16:0, C_18:0, C_18:1, C_18:2, C_18:3, C_20:4, and C_22:6, respectively, in their mixture, with correlation coefficients between 0.991 and 0.999 and limits of detection (LODs) between 0.2 and 5.4 μM, along with the linear dynamic range of more than two orders of magnitude. The results indicate that the multiple point internal standard calibration could reduce the impact of ion suppression and improve quantification accuracy in the MALDI mode. The quantitative results of nine FFAs from 339 serum samples, including 161 healthy controls, 118 patients with hyperglycemia and 60 patients without hyperglycemia show that FFAs levels in hyperglycemic patient sera are significantly higher than those in healthy controls and patients without hyperglycemia, and elevated FFA levels are also associated with increased levels of fasting blood glucose (FBG) in hyperglycemic patient sera. Serum FFAs were identified on the basis of the observed accurate molecular masses and reliable isotope distributions obtained by MALDI-FTICR MS.     

血浆游离脂肪酸代谢轮廓柱前衍生定量方法在糖尿病患者中医虚证分型中的应用

以α-溴代苯乙酮为衍生化试剂,十七酸为内标物,建立了糖尿病患者血浆中游离脂肪酸(FFA)代谢谱分析法,实现了6种主要FFA及6种微量FFA的定量分析。用此方法分析了75位临床糖尿病患者的血浆FFA代谢谱,并通过线性判别分析(LDA)对气虚和气阴两虚两种中医虚证进行了关联分析,正判率为94.3%。逐步判别分析结果表明,花生四烯酸(C20:4)和油酸(C18:1)承载了这两种虚证的重要信息,可作为潜在的标志物。利用代谢组学技术研究血浆FFA代谢谱与中医虚证的相关关系对规范证候临床诊断,提高中医药诊疗体系的可信度与可重复性具有重要意义。     

Lipids from <Emphasis Type="Italic">Halostachys caspica</Emphasis> and <Emphasis Type="Italic">Halocharis hispida</Emphasis>

The composition of lipids from the aerial parts of two species of halophytes from the family Chenopodiaceae, Halostachys caspica C. A. Mey. and Halocharis hispida Bge. was determined. Neutral lipids (NL, 62.1 and 54.2%, respectively) dominated the total lipids (TL) of these plants. More than a third of the NL were esters of aliphatic alcohols and phytosterols (FAE). Fatty acids 16:0, 18:1, and 18:2 dominated the acids of FAE; 16:0, 18:1, and 18:3, the phospholipids. The principal fatty acids of glycolipids were unsaturated acids (68.3 and 75.1%) with linolenic acid dominating (44.9 and 43.5%). Presented at the 7th International Symposium on the Chemistry of Natural Compounds, Tashkent, October 16–18, 2007. Translated from Khimiya Prirodnykh Soedinenii, No. 3, pp. 276–278, May–June, 2009.     

Characterization of internal lipids from wool

The internal lipids of wool were isolated after solubilizing the wool keratin with a mixture of papain and dithioerythritol 1.4. Thin layer chromatography of the internal lipids was applied to classify the extracted components. The internal wool lipids gave 11 spots of which some were identified as cholesterol and free fatty acids, C-16 and C-18 being predominant. A very small amount of triglycerides and cholesterol esters were also found. A characteristic difference between internal wool lipids and wool wax appears to be the limited number of well defined components of which free cholesterol and fatty acids constitute the main bulk. Furthermore another feature of keratin membrane lipids is the extremely reduced phospholipid: cholesterol ratio of 0.3. The internal lipids originate mainly from the cell membrane complex of the fiber. The existence of a chemically modified bilayer membrane structure without the essential phospholipids must be taken into consideration.     

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.     

Polyunsaturated fatty acids of <Emphasis Type="Italic">Cortusa turkestanica</Emphasis> seed oil

The composition of free and bound lipids from seeds of Cortusa turkestanica A. Lozinsk (Primulaceae) was studied. The fatty acid composition of principal acyl-containing classes was established using GC/MS. It was found that triacylglycerines isolated from free and bound lipids contained the usual acids and rarely encountered polyunsaturated 18:3 ω-6 (γ-linolenic, 8.8 and 3.2%) and 18:4 ω-3 (stearidonic, 0.7 and 0.4%) acids and were enriched in linoleic acid (18:2, ω-6, 58.2 and 60.8%, respectively). It was found that polyunsaturated 18:3 ω-6 and 18:4 ω-3 acids were missing in the free form and were found among polar substances of bound lipids.     

Fatty Acids from the Sponge <Emphasis Type="Italic">Tedania dirhaphis</Emphasis>

The fatty acid (FA) composition of total lipids from the marine sponge Tedania dirhaphis from the Sea of Okhotsk was studied. GC and GC-MS identified 50 acids, particular attention being paid to components with 14–22 C atoms. Acids 16-Me-19:0, 10,14-Me₂-15:1(Δ6), 18:1(Δ6), 18:1(Δ8), and 22:1(Δ16) were observed and identified for the first time in sponges. The main FA in lipids from T. dirhaphis was 28:3(Δ5,9,21), the relative content of which reached 63.3%.__________Translated from Khimiya Prirodnykh Soedinenii, No. 3, pp. 233–236, May–June, 2005.     

Lipids and flavolignans from <Emphasis Type="Italic">Silybum marianum</Emphasis>

Lipids from the air-dried aerial part (AP) and seeds of Silybum marianum (L.) Gaerth. (Asteraceae) were studied. The class and fatty acid compositions of neutral lipids (AP, seeds) and glyco- and phospholipids (AP) were determined. Neutral lipids (NL) with a complicated set of lipophilic components, mainly triterpenols, sterols, and their esters predominated in the AP. The fatty acids of the AP were dominated by 16:0, 18:2 (glycolipids), and 18:3 (neutral lipids, phospholipids); of seed NL, by 18:2 and 18:1. The content and composition of flavolignans isolated from defatted seeds and the content of total protein in the meal were found.     

Simultaneous Determination of Six Free Fatty Acids in the Alcohol Extract of Tie Bang Chui with Gas Chromatography-Mass Spectrometry

A GC-MS method was developed for the determination of free fatty acids (FFAs) in the alcohol extract of Tie Bang Chui. Six major types of FFAs were identified. All the six FFAs identified in alcohol extract were quantified with nonadecanoic acid as an internal standard. The results showed that the alcohol extract was abundant in three types of FFAs, linoleic acid (38.14%, 8.49 ± 0.22 mg/g) was the predominant fatty acid, followed by palmitic acid and oleic acid, unsaturated FFAs amounted to 71.35% of the total FFA content, and there was no trans FFA present.     

Lipids from extracts ofChlorella vulgaris

The composition of ethanolic (I) and gasoline (II) extracts of the cultivated microalgaChlorella vulgaris has been studied. With the aid of CC, TLC, qualitative reactions, GLC, and UVS the following classes of compounds have been detected in them. From the neutral lipids: hydrocarbons, carotenoids, traces of sterols and their esters, fatty acid esters, tri- and diacylglycerols, free fatty acids, and chlorophylls; and from the polar lipids: di- and monogalactosylglycerols, phosphatidylethanolamine, lecithin, phosphatidylinositol, phosphatidylserine, and three sphingosine bases. The polar lipids I and II made up 52.4 and 50.2% of the total, respectively. As compared with extract I, extract II was somewhat enriched with neutral lipids, including provitamins of the A group and vitamins of the F group. In the fatty acids of chlorella, 19 components were detected, the main ones being the 16:0 acid and 18:2 and 18:3 acids.Institute of the Chemistry of Plant Substances, Academy of Sciences of the Uzbek SSR, Tashkent. Translated from Khimiya Prirodnykh Soedinenii, No. 3, pp. 284–288, May–June, 1986.     

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）。     

Detection and identification of <Emphasis Type="Italic">Bacteriovorax stolpii</Emphasis> UKi2 sphingophosphonolipid molecular species

Bacteriovorax stolpii is a predator of larger gram-negative bacteria and lives as a parasite in the intraperiplasmic space of the host cell. This bacterium is unusual among prokaryotes in that sphingolipids comprise a large proportion of its lipids. We here report the presence of 18 molecular species of B. stolpii UKi2 sphingophosphonolipids (SPNLs). (31)P NMR spectroscopy and analysis of P(i) released by a differential hydrolysis protocol confirmed the phosphonyl nature of these lipids. The SPNLs were dominated by those with 1-hydroxy-2-aminoethane phosphonate (hydroxy-aminoethylphosphonate) polar head groups; aminoethylphosphonate was also detected in minor SPNL components. The long-chain bases (LCBs) were dominated by C(17) iso-branched phytosphingosine; C(17) iso-branched dihydrosphingosine was also present in some SPNLs. The N-linked fatty acids were predominantly iso-branched and most contained an alpha-hydroxy group (C(15) alpha-hydroxy fatty acid was the major fatty acid). Minor molecular species containing nonhydroxy fatty acids were also detected. The definitive iso-structures of the predominant fatty acids and LCBs present in the B. stolpii SPNLs were established using (13)C and (3)H nuclear magnetic resonance spectroscopy; less than 20% were unbranched. Detection and analyses of intact compounds by MS-MS were performed by a hybrid quadrupole time-of-flight (Q-TOF-II) MS equipped with an electrospray ionization source. Analyses of peracetylated derivatives verified the structural assignments of these lipids.