A method for specific analysis of free fatty acids in biological samples by capillary gas chromatography.

The present report describes a simple method to selectively extract free fatty acids and analyze them by capillary gas-liquid chromatography. The procedure is based on the use of fumed silicon dioxide. In the presence of plasma, this material induces a rapid rise in the viscosity of the mixture and presents the ability to trap large particles such as emulsified lipids and lipoproteins. Albumin-bound fatty acids are thus left in the aqueous media. We present applications of our procedure for the analysis of free fatty acids in 0.2 ml of plasma from rat or human. By comparison with the method utilizing thin-layer chromatography for the separation of fatty acids and gas chromatography analysis, the present method has been found to be reliable and simple. The recovery of linoleic acid was 92.1 +/- 8.2%, a value which is about twice better than that obtained with the procedure using thin-layer chromatography. In particular, long-chain polyunsaturated fatty acids were better preserved. Our procedure does not require the use of organic solvents and its simplicity and reproducibility make it suitable for routine specific determination of the composition of free fatty acids in biological samples.     

气相色谱法测定啤酒中的游离脂肪酸

气相色谱法测定啤酒中辛酸到二十二碳酸共１１种游离脂肪酸,采用多级溶剂萃取及薄层色谱纯化技术进行样品制备,并采充氮措施抑制脂肪酸的氧化产生,此方法有较好的重复性和回收率。     

Determination of portal short-chain fatty acids in rats fed various dietary fibers by capillary gas chromatography

A simple, rapid and sensitive capillary gas chromatographic method was investigated to measure portal short-chain fatty acids (SCFAs). A 200-μl sample of portal plasma was denatured with sulfosalicyclic acid and then extracted with diethyl ether before the removal of protein precipitate. The resultant extract was concentrated by a transfer to 50 μl of 0.2 M NaOH, thus avoiding tedious further concentration steps. This reduced the sample volume to one-fourth. Since the ratio of acetic acid, a major SCFA, to other acids varies widely, ranging from 10-fold to 100-fold, acrylic and methacrylic acids were used as internal standards to simultaneously measure SCFAs having a carbon number of 2–6. As a result, good recovery (90.38–103.17%) and reproducibility (coefficient of variation 0.83–8.85%) were observed over a wide range. Furthermore, portal SCFAs in rats fed various dietary fibers were determined by the present method. We showed that the amounts not only of the major acids such as acetic acid and propionic acid, but also of the minor fermented products such as n-valeric acid and n-caproic acid, could be significantly changed by dietary manipulation. Thus, the present method is simple and reliable, and requires only a small amount of sample.     

Determination of amino acids in human serum by capillary gas chromatography

A simple and rapid method for the determination of serum amino acids by gas chromatography (GC) has been developed. Following deproteinization of serum with perchloric acid, free amino acids in the supernatant were converted into their N(O,S)-isobutoxycarbonyl methyl ester derivatives and measured by GC with flame ionization detection using a DB-17 capillary column. All the derivatives of the 22 protein amino acids were completely resolved as single peaks within 9 min by GC. The calibration curves were linear in the range 0.2–50 μg of each amino acid, and the correlation coefficients were above 0.998. By using this method, serum amino acids could be directly analysed without prior clean-up procedure such as ion-exchange column chromatography except for deproteinization of the samples, and without any interference from coexisting substances. Overall recoveries of amino acids added to serum samples were 88–108%. Analytical results for serum amino acids from normal subjects are presented.     

Determination of organic acids in seven wheat varieties by capillary gas chromatography

Fresh wheat tops were extracted with acidic 90% ethanol, and the ethanol was evaporated and a portion of the aqueous residue loaded onto DEAE-Sephadex. Organic acids were eluted with pyridinium formate and then lyophilized and the dried residue was derivatized with 1% trimethylchlorosilane in bis(trimethylsilyl)trifluoroacetamide. The acids were then quantitatively determined using capillary gas chromatography and identified using capillary gas chromatography-mass spectrometry. The acidic ethanol extraction of fresh plant tissue was quantitative for all acids except citric while losses in the remaining procedures were controlled by using an internal standard. The ion exchange chromatography made the greatest contribution to experimental error, imposing a minimum loading requirement of 0.1 mumol of each acid for adequate precision. Organic acid profiles were determined for seven wheat cultivars (Triticum aestivum cv Carazinho, Teal, Lance, Warigal, Isis, Maringa, and BH1146) grown on gravel in solution culture for 30 days. Profiles were simple, consisting of only malic, aconitic, and citric acids, with levels of each acid for all varieties falling within the range 2-5 mumol/g fresh tissue. Storage of samples led to a large increase in sampling error and increased the amount of extractable citric acid.     

Determination of methanol in whole blood by capillary gas chromatography with direct on-column injection

A gas chromatographic procedure was developed for the determination of methanol in small-volume whole blood samples. Samples (100–200 μl) were prepared by protein precipitation, with direct injection of the supernatant on a wide-bore capillary column. The recovery of methanol and acetonitrile (the internal standard) was approximately 90% and did not vary with sample volume. The assay was linear from 2 μg/ml (the limit of detection) through 1000 μg/ml and was highly reproducible (intra-day coefficient of variation <2.5%). Assay performance was assessed following exposure of rats to methanol. The results indicate that the present procedure is suitable for studies of methanol disposition in small rodent species.     

Long-chain fatty acids esterified by action of alkyl chloroformates and analysed by capillary gas chromatography

Long-chain fatty acids can be instantaneously converted into methyl, ethyl or chloroethyl esters by the action of corresponding alkyl chloroformates. The reaction is catalysed by pyridine and proceeds in acetonitrile in the presence of a small amount of the corresponding alcohol. The reproducibility is high and the yield exceeds 95%.     

毛细管气相色谱细胞脂肪酸测定法鉴定28种分枝杆菌的研究

用毛细管气相色谱对分枝杆菌属28种参考株的细胞挥发性脂肪酸进行测定,发现均含有较多量的脂肪酸C18:1、C18:0及本属特异性组分TBSA。利用色谱图中这三种脂肪酸峰高的比例关系,可将所测菌株分应8群。再借助具有种间特异性的脂肪酸C19:0、C21:0和C26:0将其中的1 7株(60％)鉴定到种,11株鉴定到含有2或3个种的群。本鉴定程序区分多数临床常见的致病菌(如人型、牛型结核杆菌及一些分枝杆菌复合体),经计算机对细胞脂肪酸模式的聚类分析,表明了这一程序的客观合理性。     

Determination of short-chain fatty acids in serum by hollow fiber supported liquid membrane extraction coupled with gas chromatography

A method based on hollow fiber supported liquid membrane extraction coupled with a gas chromatograph equipped with flame ionization detector (GC-FID) was developed for the determination of six short-chain fatty acids including acetic acid, propionic acid, i-butyric acid, n-butyric acid, i-valeric acid and n-valeric acid in serum. Hollow fiber supported liquid membrane extraction was employed for preconcentration and clean-up of the samples. The fatty acids were extracted from the acidic donor (diluted serum) into a liquid membrane formed in the wall of the hollow fiber with 10% tri-n-octylphoshphine oxide (TOPO) in di-n-hexyl ether, and then extracted back into a basic acceptor solution filled in the lumen of the hollow fiber. After being acidified with HCl, the acceptor was directly analyzed by GC-FID. The acceptor concentration, donor pH, membrane liquid and extracting time were optimized giving an enrichment factor up to 155 times. The good linearity (r(2)>0.980), reasonable recovery (87.2-121%), and satisfactory intra-assay (8.2-11.5%) and inter-assay (6.1-11.6%) precision illustrated the good performance of the present method. Limits of detection (LOD) ranged from 0.04 to 0.24 microM and limits of quantification (LOQ) varied from 0.13 to 0.80 microM.     

A non-organic and non-enzymatic extraction method gives higher yields of genomic DNA from whole-blood samples than do nine other methods tested.

We compared ten methods for extraction of DNA from whole blood. Nine methods require incubation with either enzymes or treatment of organic solvents or both. The 'Rapid Method' (RM) (Method 10) avoids the use of organic solvents (phenol/chloroform) and eliminates completely the use of proteinase K. Thus, the time and cost of DNA extraction are reduced significantly. This is accomplished by salting out and precipitation of the cellular proteins in saturated sodium chloride. This method takes less than an hour to completion, without compromising the yield or the quality of DNA. Using RM, we can make DNA from 0.1 ml of whole blood and as little as 0.5 ml of blood yields DNA sufficient to run a few Southern blots. The RM can also be applied to packed cells. The DNA is free of RNA, protein and degrading enzymes. The uncut DNA runs as a typical slow-migrating, high-molecular-weight and undegraded species in an agarose gel. The DNA is suitable for digestion by various restriction endonucleases. This procedure works equally well with fresh blood samples and with those that are stored at 4 degrees C and -70 degrees C. To our knowledge the RM reported here is the safest, fastest and most quantitative and economical method for preparation of DNA from whole blood and cells.     

Determination of chromium in urine samples by complexation-supercritical fluid extraction and liquid or gas chromatography

A method was developed for the determination of chromium as the Cr(acac)(3) complex in urine using SFE and chromatography. Quantitative extractions were achieved when the experiments were carried out under 3000 p.s.i. of pressure, at a temperature of 120 degrees C, with 2.0 ml of methanol, 30 min of static extraction and 5 min of dynamic extraction. The chromium was quantified by GC-FID detection. The calibration graph of Cr(acac)(3) solutions was linear between 0.50 and 43.0 microg ml(-1) of chromium (DL 0.18 microg ml(-1), R=0.9994). The same extracts were quantified by HPLC-340 nm detection. The calibration curve of the Cr(acac)(3) solutions was linear over a range of 0.013 to 60.0 microg ml(-1) (DL 0.02 microg ml(-1), R=0.9999). This method was applied to urine samples from 60 diabetic patients and 21 healthy volunteers. Chromium concentration ranges were 2.5-29.5 microg l(-1) for the diabetics and 5.9-12.3 microg l(-1) for the normal subjects.     

Determination of the Krebs cycle related keto acids of microorganisms by capillary gas chromatography on glass columns

A gas chromatographic technique for quantitative determination of the Krebs cycle related keto acids in microorganisms is described. The keto acids are converted to the corresponding methoximes and analyzed as the silyl esters by capillary gas chromatography on glass columns. The conditions have been chosen as to allow simultaneous determination of related naturally occurring oximino acids. The practical limit of sensitivity is about 2 μg of each keto acid.     

Determination of pyrrolidone carboxylate and gamma-glutamyl amino acids by gas chromatography.

Gas chromatographic methods for the quantitation of pyrrolidone carboxylate and γ-glutamyl amino acids are described. These intermediates of the γ-glutamyl cycle were separated by ion exchange chromatography and converted to their N-acyl-ester derivatives in a reaction with a mixture of 2,2,3,3,3-pentafluoro-1-propanol and pentafluoropropionic anhydride. The derivatives have excellent electron capture properties thus making possible their determination even in small amounts of material of biological origin. The method was applied for the determination of concentrations of pyrrolidone carboxylate in human urine and cerebrospinal fluid, and in the brain, liver, and kidney of the mouse. It was also used to demonstrate the formation in mouse tissues of several γ-glutamyl derivatives of amino acids after administration of the corresponding free amino acid.     

Determination of polychlorinated biphenyls in small-size serum samples by solid-phase extraction followed by gas chromatography with micro-electron-capture detection

An new method for the determination of polychlorinated biphenyls (PCBs) in serum samples of up to 1 ml has been developed. The procedure consisted in the solid-phase extraction (SPE) of the analytes on an Oasis cartridge and the subsequent on-line elimination of the fat by directly dropping of the eluate from the SPE cartridge on a multilayer column placed below the cartridge. This configuration allowed minimising of the sample manipulation as well as the time, solvent and sorbent consumption (i.e. complete sample preparation can be accomplished in about 1 h with only 3 ml of toluene and 300 mg of silica). The SPE plus clean-up method developed showed a satisfactory performance for the analysis of PCBs in rat serum samples providing similar recoveries (i.e. range 73-128% for most of the congeners selected) at the different spiking levels investigated (1.25, 0.50 and 0.25 ng/ml). Detection limits using a microelectron capture detector were in the range 0.01-0.30 ng/ml of serum and the relative standard deviations of the complete method better than 18% irrespective of the PCB concentration. The validated method has been applied to the evaluation for the first time of the PCB levels in serum samples of up to 1 ml from individuals of an Egyptian Vulture colony in Spain.     

Determination of plasma free fatty acids, free cholesterol, cholesteryl esters, and triacylglycerols directly from total lipid extract by capillary gas chromatography

An accurate capillary gas chromatographic method using different internal standards for determining free fatty acids, cholesterol, cholesteryl esters, and triacylglycerols in plasma and other biological sources is described. It is designed to give information about species composition and, consequently, more detailed information about changes in lipid metabolism of patients suffering from metabolic disorders. After plasma extraction the lipids, except phospholipids, are directly examined without any further derivatization. For free fatty acid determination the programmed temperature vaporizer (PTV) injector was heated from 40 degrees C (sample introduction) to 190 degrees C. In a second gas chromatographic run the PTV-injector system was heated from 60 degrees C (sample introduction) to 400 degrees C, enabling the determination of free cholesterol, cholesteryl esters, and triacylglycerol species, differing in the number of carbon atoms. Evaluation of the values obtained resulted in coefficients of variation (%) of 1.0-2.8, 2.0, 1.29-2.24, and 2.8, for free fatty acid standards, plasma free fatty acids, cholesterol and cholesteryl ester standards, and plasma total cholesterol, respectively. Free fatty acids, cholesterol, and cholesteryl esters were not influenced by storage of plasma at -24 degrees C up to 4 days prior to extraction. The results of the gas chromatographic method and the enzymatic methods correlated well. Determination by gas chromatography yielded higher total cholesterol and lower triacylglycerol values than those values obtained by enzymatic methods.     

Determination of the carbohydrate composition of mammalian glycoproteins by capillary gas chromatography/mass spectrometry

A technique to determine the carbohydrate composition of glycoproteins using capillary gas chromatography/mass spectrometry (electron impact) with selected ion monitoring is described. This method entails hydrolysis with methanolic-HCl followed by formation of trimethylsilyl methylglycoside derivatives, extraction of the carbohydrate derivatives into hexane, and GC/MS analysis. For those carbohydrates that are present in animal glycoproteins including fucose, mannose, galactose, glucosamine, galactosamine, and N-acetylneuraminic acid (sialic acid), the sensitivity of this assay was approximately 1-3 pmol and the assay was linear over a 100-fold range. The carbohydrate compositions determined on small quantities (1-10 pmol) of various glycoproteins including human transferrin and alpha-1 acid glycoprotein, fetuin, and ovalbumin were identical to their reported carbohydrate content and compositions. Major advantages of this technique include the time required to complete the sample preparation and analysis (less than 8 h), the sensitivity and specificity of the assay, and the fact that all carbohydrate moieties, including sialic acid, can be quantitated in a single hydrolysate of a glycoprotein.