Gas chromatography of amino acids: Use of a fused-silica capillary column in the determination of plasma amino acids

A capillary chromatographic procedure using a fused silica column is described which can be used to quantitatively determine amino acids in plasma following the pre-chromatographic “clean-up” described in a recent paper [1]. In substituting this procedure for that involving a packed column, advantage has been taken of the greater resolving power to separate amino acids from background component peaks. In order to extend this advantage and provide a sound basis for quantitative analysis, the technique of cold on-column injection was employed. As a result, good precision of standard analysis was obtained with relative standard deviation (RSD) values for all amino acids of less than 4%. Application of the entire procedure to plasma samples yields RSD values of better than 10% for all amino acids with recoveries ranging from 72% to 104%. Simultaneous determination of plasma amino acid levels by gas chromatography (GC) using capillary columns and by classical ion exchange (CIE) showed reasonable agreement. Statistical evaluation showed no significant difference between twelve amino acids. Values for the remaining two, namely, phenylalanine and histidine are significantly different (p < 0.005). Comparison of the values obtained from GC capillary and packed columns reveals no significant difference between fourteen amino acids. Significant differences exist between results for phenylalanine and tyrosine (p < 0.001). It is concluded that there is good agreement between data obtained by GC capillary and CIE techniques and that differences between results for phenylalanine and histidine are method related.     

Liquid chromatographic determination of lysine, methionine, and threonine in pure amino acids (feed grade) and premixes: collaborative study

A total of 17 laboratories (including one author's laboratory) participated in a collaborative study for determination of lysine, methionine, and threonine in trade products or concentrated amino acid premixes. Thirteen samples, 4 pure amino acids and 6 premixes, including 3 Youden matched pairs, were analyzed. The applied liquid chromatographic (LC) method using cation-exchange resin and post-column derivatization with ninhydrin or o-phthaldialdehyde was shown to be accurate and specific for the analytes. Titration procedures, normally used for the assay of pure amino acids, are unspecific and the accuracy of the results can be affected by impurities. Repeatability relative standard deviations, RSDr, ranged from 0.84 to 1.17% for pure amino acids and from 0.50 to 1.68% for premixes; reproducibility relative standard deviations RSDR, ranged from 1.52 to 2.31% for pure amino acids and from 1.48 to 2.59% for premixes. Recoveries were between 97.5 and 102.8% of the expected amino acid assays. The method has been adopted Official First Action status by AOAC INTERNATIONAL.     

Determination of amino acids in urine by gas chromatography

This report describes a convenient means of reducing the complex matrix which is responsible for interference during gas chromatographic determination of amino acids in urine. The pre-chromatographic clean-up employs the principle of solid phase extraction using bonded silica incorporating cation exchange groups. This approach avoids the detrimental effects on amino acid recoveries associated with resin-based cation exchangers. In spite of significant reduction in the complexity of chromatograms, only the high efficiency and resolving power offered by the analytical capillary column (e. g. fused silica open tubular, FSOT) is sufficient for quantitative and analysis of amino acids in urine. Reproducibility data from the complete procedure are determined, coefficients of variation (CV) for most amino acids being better than 5% with a mean recovery of 96%.     

Solid-phase microextraction as a sample preparation strategy for the analysis of seleno amino acids by gas chromatography-inductively coupled plasma mass spectrometry

Solid-phase microextraction (SPME) is used as a sample preparation strategy for gas chromatographic (GC) analysis of the seleno amino acids, selenomethionine (SeMet), selenoethionine (SeEt) and selenocystine (SeCys). Acylation of the amino group and esterification of the carboxylic group in these compounds was performed with isobutylchloroformate to increase volatility. The amino acid derivatives were then extracted by silica fibers with polydimethylsiloxane (PDMS) coatings prepared by the sol-gel process. Investigations of extraction time, acid and salt addition, and polymer length (for the sol-gel process) were conducted with the goal of procedural optimization. Initial characterizations were conducted using gas chromatography with flame ionization detection (GC-FID). Inductively coupled plasma mass spectrometric detection was employed for final selenium detection. Sub-ppb detection limits were obtained for all analytes although relative standard deviations were higher than those typically obtained in solid-phase microextraction.     

Determination of amino acids in the hepatic and brain tissue of the rat by gas chromatography

A procedure is described for the quantitative determination of amino acids in hepatic and brain tissue samples from the rat. Because the presence of certain matrix components in the tissue material led to interference with chromatographic analysis they were removed by a prechromatographic “clean-up” step. Quantitative analysis of amino acids, as their N-heptafluorobutyryl iso-butyl ester derivatives, was achieved by high resolution gas chromatography on an apolar fused silica open tubular column. Reproducibility data from the complete procedure are presented; coefficients of variation for arginine and histidine in hepatic tissue varied between 7.1 and 10.1% whereas those for most other amino acids were better than 5%, with a mean recovery of 90%.     

New procedure for isolation of amino acids based on selective hydrolysis of trimethylsilyl derivatives

A rapid procedure for the isolation of amino acids from physiological fluids by class separation suitable for gas chromatographic and gas chromatographic-mass spectrometric analysis is described. A physiological fluid such as plasma is adjusted to pH 2 and extracted with diethyl ether to remove organic acids and neutrals. After precipitation of proteins with trichloroacetic acid, the aqueous plasma is dried and derivatized by trimethylsilylation. Organic compounds like sugars and amino acids are rendered soluble in petroleum ether leaving inorganic salts when the soluble layer is transferred. Separation of sugars from amino acids is achieved by taking advantage of the different rates of aqueous hydrolysis of the trimethylsilyl (TMS) derivatives. Mixing the petroleum ether extract with a small volume of water results in two phases. The petroleum ether layer contains TMS-Sugar constituents of plasma and the aqueous layer contains free amino acids and amines. This procedure was used to isolate L-dopa, 3-O-methyldopa and tyrosine from human plasma in a quantitation assay using 18O-labelled amino acids and gas chromatography-mass spectrometry.     

Determination of (E)-2,3-dichloro-4-methoxyphenyl)-2-furanylmethanone-O-[2-(diethylamino) ethyl]oxime methanesulphonate (ANP-4364) in plasma using gas chromatography with electron-capture detection

A gas chromatographic method has been developed that permits the accurate and specific determination of the antianginal agent ANP-4364 in plasma. ANP-4364 is extracted with n-hexane containing ethyl chloroformate and, after a clean-up procedure, derivatized to the trichloroethyl carbamate, which is assayed on a gas chromatograph equipped with an electron-capture detector. Accurate determinations are possible over a concentration range from 1 to 50 ng/ml of ANP-4364 in plasma with a relative standard deviation of 7.5%. The minimum detectable concentration is 0.5 ng/ml. Plasma levels of ANP-4364 in dogs receiving oral (10 mg/kg) or intravenous (0.1 mg/kg) dosing have been determined.     

Gas chromatographic determination of carboxylic acid chlorides and residual carboxylic acid precursors used in the production of some penicillins.

An improved gas chromatographic method is described for the simultaneous determination of carboxylic acid chlorides and related carboxylic acids used in the production of some commercial semisynthetic penicillins. The acid chloride reacts with diethylamine to form the corresponding diethylamide. Carboxylic acid impurities are converted to trimethylsilyl esters. The two derivatives are separated and quantitated in the same chromatographic run. This method, an extension of the earlier procedure of Hishta and Bomstein (1), has been applied to the acid chlorides used to make oxacillin, cloxacillin, dicloxacillin, and methicillin (Figure 1); it shows promise of application to other acid chlorides. The determination is more selective than the usual titration methods, which do not differentiate among acids with similar pK's. Relative standard deviations of the acid chloride determination are 1.0-2.5%. Residual carboxylic acid can be repetitively determined within a range of 0.6% absolute.     

Determination of 1,2-benzisoxazole-3-acetamidoxime hydrochloride (PE-257) in plasma using electron-capture gas chromatography

A gas chromatographic method has been developed that permits the accurate and specific determination of a new psychotropic agent, PF-257, in plasma. PF-257 is extracted with ethyl acetate from alkaline plasma and, after a clean-up procedure, derivatized with heptafluorobutyric anhydride to form 3-[(5-n-heptafluoropropyl-1,2, 4-oxadiazol-3-yl)methyl]-1,2-benzisoxazole (HOMB). The HOMB is assayed on a gas chromatograph equipped with an electron-capture detector. Accurate determinations of PF-257 are possible in the concentration range from 1-40 ng/ml with a relative standard deviation of 6.8%. The minimum detectable concentration in plasma is 0.1 ng/ml. Plasma levels of PF-257 in rats receiving intravenous or oral dosing (10 mg/kg) were determined.     

二维阀切换离子色谱法测定海带中游离氨基酸

建立一种测定海带中游离氨基酸的阀切换高效阴离子交换色谱耦合脉冲安培检测器法。采用一根阳离子交换柱对氨基酸进行富集,而后经阀切换至氨基酸分析柱Amino Pac~PA-10(250 mm×2 mm)上分离并进入安培检测器检测。在最佳分离条件下,20种氨基酸的质量浓度在0.1~20.0 mg/L范围内与其色谱峰面积线性关系良好,线性相关系数r~20.99,20种氨基酸的检出限为0.01 mg/L,加标回收率为83.12%~117.34%,测定结果的相对标准偏为1.02%~13.05%(n=8)。该方法样品前处理简单,无基底杂质干扰,适用于海带样品中游离氨基酸的测定。     

Isolation and desalting with cation‐exchange chromatography for compound‐specific nitrogen isotope analysis of amino acids: application to biogeochemical samples

We have established a procedure for removing interfering materials from extracts of geological and biological samples, in order to determine precise compound‐specific nitrogen isotopic compositions of amino acids. We employed cation‐exchange chromatography of protein and non‐protein amino acids prior to derivatization for gas chromatographic separation. The average recovery of a standard amino acid solution was better than 94%, without nitrogen isotope fractionation during the cation‐exchange chromatography. We applied the procedure to various environmental samples including ‘hard’ (calcareous, siliceous, rock and sediment samples) and ‘soft’ materials (aggregated microbial samples and biological soft tissue samples). We conclude that cation‐exchange chromatography is a pre‐treatment procedure which should be widely useful for the determination of compound‐specific nitrogen isotopic compositions of amino acids. Copyright © 2010 John Wiley & Sons, Ltd.     

Rapid method for the determination of 16 organochlorine pesticides in sesame seeds by microwave-assisted extraction and analysis of extracts by gas chromatography-mass spectrometry

A method for the multiresidue analysis of 16 organochlorine insecticides in sesame seeds has been developed. The method is based on the microwave-assisted extraction (MAE) of the sesame seeds by the use of a water-acetonitrile mixture followed by Florisil clean-up of the extracts and subsequent analysis by gas chromatography-mass spectrometry (GC/MS) in the selected ion monitoring (SIM) mode. MAE operational parameters (extraction solvent, temperature and time, extractant volume) were optimized with respect to extraction efficiency of the target compounds from sesame seeds with 46% oil content. Recoveries >80% with relative standard deviations (RSD) <12% were obtained for all compounds under the selected parameters. The Florisil clean-up step proved sufficient for the removal of co-extracted substances so that no adverse effect on the chromatographic system was observed. Limit of quantification (LOQ) values were in the range of 5-10 microg/kg. The proposed method was applied in the analysis of sesame seed samples imported to Greece.     

Isothermal gas chromatographic determination of nanogram amounts of chlorimipramine, chlorpromazine and their N-desmethyl metabolites in plasma using nitrogen-selective detection

A gas chromatographic method using nitrogen-selective detection for the quantitative determination of nanogram amounts of chlorimipramine, chlorpromazine and their nor1 and nor2 derivatives in plasma is described. Derivatization with trifluoroacetic anhydride of nor1 and nor2 metabolites allowed the chromatographic separation of these compounds. A three-step solvent extraction procedure was performed using n-heptane containing 1% isoamyl alcohol and n-hexane and compared with a plasma clean-up procedure using C18 Sep-Pak cartridges. The two procedures were characterized by similar degrees of precision. The use of C18 Sep-Pak cartridges, however, produced a significant time and material saving over the conventional extraction method.     

Modified RP-LC of Phenylthiocarbamyl Amino Acid Adducts in Plasma Acetonitrile Extracts Using Multiple Internal Standards and Photo-Diode UV Detection

Among the techniques available for quantitative analysis of physiological amino acids, systems using optical detection are of low specificity because of possible interference at the analytical wavelength. Another disadvantage is problems of sample extraction from complex biological matrices, for example plasma. This paper describes reversed-phase LC of phenylthiocarbamyl (PTC) amino acids in plasma deproteinated by addition of acetonitrile. Specificity was monitored by photo-diode UV detection and accuracy was assessed by a plasma spiking procedure with more than one internal standard. Dual-wavelength spectrophotometry (254 and 283 nm) was also used for separate measurement of co-eluting adducts of tryptophan and ornithine. This method enables the quantification, with high reproducibility, of a total of twenty-three plasma amino acids from fasting healthy subjects. LOQ values are satisfactory for all the amino acids (average 6 μmol L^?1). However, the method does not enable analysis of aspartate and overall homocystine, present at very low concentrations, in all plasma samples. This PTC–amino acid chromatographic method is inexpensive, reliable, and suitable for clinical research and therapeutic drug monitoring, but adaptation to dual on-line detection is required to improve its sensitivity.     

Determination of (15R)- and (15S)-15-methylprostaglandin E2 in human plasma with picogram per milliliter sensitivity by column-switching high-performance liquid chromatography

(15R)-15-Methylprostaglandin E2 (PGE2) is a pro-drug under evaluation for the treatment of acute upper gastrointestinal hemorrhage and gastrointestinal cytoprotection. It is converted in acid (e.g., gastric fluid) to its active 15S epimer. Both epimers are found in human plasma at low pg/ml levels following oral dosing. A high-performance liquid chromatographic (HPLC) method was developed for the simultaneous analysis of (15R)- and (15S)-15-methyl-PGE2 in human plasma. The method combined off-line solid-phase extraction and reversed-phase HPLC clean-up with panacyl bromide derivatization and subsequent analysis using a heteromodal column-switching technique. Assay linearity was demonstrated over a range of 10-200 pg/ml for both 15-methyl-PGE2 epimers (r greater than or equal to 0.995). There were no significant inter-day differences in assay results for either epimer at 50 and 25 pg/ml (p greater than 0.05), with pooled estimates of precision at these levels producing relative standard deviations of less than or equal to 8% and less than or equal to 12%, respectively. The method quantitation limit (signal-to-noise ratio 5:1) for both epimers was 10 pg/ml when processing 3 ml of plasma. The analysis procedure was shown to be useful for quantifying at or below 10% of the (15R)-15-methyl-PGE2 maximum plasma concentration following a 50-micrograms oral dose in three human volunteers. For the same three subjects, however, the plasma concentration of (15S)-15-methyl-PGE2 did not exceed the quantitation limit of 10 pg/ml.     

Multiresidue method for determination of 90 pesticides in fresh fruits and vegetables using solid-phase extraction and gas chromatography-mass spectrometry

A multiresidue method for analysis of 90 pesticides with different physico-chemical properties in fruits and vegetables was developed. The method involves a rapid and small-scale extraction procedure with acetone using vortex mixing. Solid-phase extraction (SPE) on a highly cross-linked polystyrene divinylbenzene column (LiChrolut EN) was used for clean-up and pre-concentration of the pesticides from the water-diluted acetone extracts. For most fruit and vegetable samples this partial clean-up was sufficient, but some of them with more co-extracting substances need further clean-up (cereals, spinach, carrots, etc.). Diethylaminopropyl (DEA) modified silica was used for efficient removal of interferences caused by various organic acids, sugars, etc. The pesticide residues were determined by gas chromatography with a mass selective detector (GC-MS). The majority of pesticide recoveries for various fruits and vegetables were >80% in the concentration range from 0.01 to 0.50 mg/kg, except for the most polar pesticides (methamidophos, acephate, omethoate) which cannot be determined by this method. The limit of quantitation for most of the pesticides was 0.01 mg/kg with majority of relative standard deviations (R.S.D.s) below 10%.     

Planar solid phase extraction--a new clean-up concept in multi-residue analysis of pesticides by liquid chromatography-mass spectrometry

Efficient clean-up is indispensable for preventing matrix effects in multi-residue analysis of pesticides in food by liquid and gas chromatography coupled to mass spectrometry. As a completely new approach, highly automated planar chromatographic tools were applied for powerful clean-up, called high-throughput planar solid phase extraction (HTpSPE). Thin-layer chromatography (TLC) was used to completely separate pesticides from matrix compounds and to focus them into a sharp zone, followed by extraction of the target zone by the TLC-MS interface. HTpSPE resulted in extracts nearly free of interference and free of matrix effects, as shown for seven chemically representative pesticides in four different matrices (apples, cucumbers, red grapes, tomatoes). Regarding the clean-up step, quantification by LC-MS provided mean recovery (against solvent standards) of 90-104% with relative standard deviations of 0.3-4.1% (n=5) for two spiking levels of 0.1 and 0.5 mg/kg. Clean-up of one sample was completed in a manner of minutes, while running numerous samples in parallel at reduced costs, with very low sample and solvent volumes.     

Determination of erythrocyte amino acids by gas chromatography

Erythrocyte amino acid levels were determined, by gas chromatography, in a group of 34 normal human adults. No significant sex or age correlations were noted. A method for the quantitative gas chromatographic analysis of free amino acids in erythrocytes is described. Following hemolysis and deproteinization the amino acids were isolated on a cation-exchange resin. Glutathione was removed from the amino acid mixture by adsorption on an anion-exchange resin. Following conversion to their N-acetyl-n-propyl esters, 19 amino acids were separated and quantitated by gas chromatography on a single column in 18 min. Typical reproducibility data indicate that a coefficient of variation of 2-5% is attainable.     

Rapid determination of minority organic acids in honey by high-performance liquid chromatography

A rapid high-performance liquid chromatographic method for the determination of organic acids in honey is reported. Malic, maleic, citric, succinic and fumaric acids were identified and quantified in 15 min. First time repeatibility, reproducibility and recoveries were determined out for these acids in honey samples. Maleic acid was also quantified for first time by a chromatographic method. The organic acids were removed from honey by using a solid-phase extraction procedure with anion-exchange cartridges. Previously, the solution of honey was adjusted to pH 10.50 with 0.1 M NaOH and stirred for 15 min at room temperature. Then, this solution was adjusted to pH 5.00 with 0.1 M H2SO4. This procedure was carried out to avoid interferences in the baseline. The chromatographic separation was achieved with only one Spherisorb ODS-2 S5 column thermostated at 25 degrees C. Metaphosphoric acid (pH 2.20) was used as mobile phase at a flow-rate of 0.7 ml/min. Organic acids were detected with a UV-vis detector (215 nm). The precision results showed that the relative standard deviations of the repeatability and reproducibility were < or =3.20% and < or =4.86%, respectively. The recoveries of the organic acids ranged from 62.9 to 99.4%. Under optimum conditions the detection limits ranged from 0.0064 to 7.57 mg/kg and the quantification limits ranged from 0.025 to 10.93 mg/kg.     

固相萃取-气相色谱法测定茶叶中残留的92种农药

建立了茶叶中92种农药多残留的气相色谱分析方法。茶叶样品用乙腈一次性提取后,有机磷类农药经Envi-Carb固相小柱净化,用10 mL乙腈-甲苯(体积比为3∶1)洗脱剂淋洗,气相色谱-火焰光度检测器(GC-FPD)检测;有机氯类和拟除虫菊酯类农药经串联Envi-Carb和NH2固相小柱净化,用5 mL乙腈-甲苯(体积比为3∶1)洗脱剂淋洗,GC-电子捕获检测器(ECD)检测。采用外标法定量。添加回收试验的结果表明:92种农药的平均回收率为80.3%～117.1%,相对标准偏差为1.5%～9.8%。方法的检出限为0.0025～0.10 mg/kg。该方法的灵敏度、准确度和精密度均符合农药残留测定的技术要求。