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A selective and sensitive method for the determination of protein and non-protein amino acids in biological fluids by capillary gas chromatography (GC) has been developed. The amino acids in the samples were directly converted into their N(O,S)-isobutoxycarbonyl methyl ester derivatives and measured by GC with nitrogen-phosphorus selective detection (NPD) using a DB-17ht capillary column. Using this method, the derivatives of the 21 protein amino acids and the 25 non-protein amino acids provided excellent NPD responses and were quantitatively and reproducibly resolved within 28 min. The lower detection limits of these amino acids, at a signal-to-noise ratio of 3, were ca. 6-150 pg injected. The calibration curves for each amino acid in the range of 0.02-2 micrograms were linear and sufficiently reproducible for quantitative analysis. This method was successfully applied to small urine and serum samples without prior clean-up; there was no evidence of interference from coexisting substances. Overall recoveries of amino acids added to urine and serum samples were 83-112%. The intra-assay and inter-assay R.S.D. of amino acids in these samples were 0.3-8.9% (n = 3) and 1.9-15.8% (n = 3), respectively.

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 micrograms 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.

A selective and sensitive method was developed for the determination of volatile N-nitrosamines by gas chromatography (GC). After denitrosation of N-nitrosamines with hydrobromic acid, the resulting secondary amines were converted into their N-diethylthiophosphoryl derivatives and then measured by GC using a DB-1701 capillary column with flame photometric detection. The calibration graphs for N-nitrosamines in the range 0.05-1 nmol were linear and sufficiently reproducible for quantitative determination. This method was successfully applied to cigarette smoke samples without prior clean-up. N-Nitrosamines and secondary amines were completely separated by extraction with diethyl ether containing 25% 2-propanol. Overall recoveries of N-nitrosamines added to cigarette smoke samples were 83-110%. By using this method, N-nitrosamines in these samples could be determined without any interference from coexisting substances. Analytical results for the contents of N-nitrosamines and secondary amines in mainstream and sidestream smokes of several cigarettes are presented.

A rapid, selective and sensitive method for the determination of pamidronate in human plasma and urine samples by gas chromatography (GC) has been developed. After deproteinization of the sample with trichloroacetic acid, pamidronate was converted into its N-isobutoxycarbonyl methyl ester derivative and measured by GC with flame photometric detection (FPD), using a HP-1 capillary column. The derivative preparation and GC analysis were accomplished within 30 min. The derivative was sufficiently volatile and stable, giving a single and symmetrical peak, and provided an excellent FPD response. The detection limit of pamidronate, at a signal-to-noise ratio of 3, was ca. 100 pg injected, and the calibration curve for this compound in the range 20-1000 ng was linear and sufficiently reproducible for quantitative determination. This method could be successfully applied to plasma and urine samples without a preliminary clean-up except for deproteinization with trichloroacetic acid, and pamidronate could be measured without any influence from coexisting substances. Overall recoveries of pamidronate added to plasma and urine samples were 93-97%. The coefficients of variation for intra-assay and inter-assay of pamidronate in these samples were 1.0-7.9% (n = 3) and 4.1-8.3% (n = 6), respectively.

A simple and rapid method for the determination of the 22 protein amino acids by capillary gas chromatography is described. The amino acids were converted into their N(O,S)-isobutoxycarbonyl (isoBOC) methyl ester derivatives and measured by GC with flame ionization detection using a DB-17 capillary column. Arginine was converted into ornithine by arginase treatment prior to the isoBOC reaction. The N(O,S)-isobutoxycarbonylation of amino acids with isobutyl chloroformate was completed within 15 s by sonication in aqueous alkaline media. All of the derivatives were quantitatively resolved as single symmetrical peaks within 9 min by GC on a single column. The detection limits of amino acids were 0.2-4.0 ng per injection, and the calibration curves were linear in the range 0.2-50 micrograms for each amino acid. This method was successfully applied to protein hydrolysate samples. The analytical results of amino acid compositions of several proteins are presented.

A selective and sensitive gas chromatographic method for the determination of saturated and unsaturated aliphatic aldehydes in foods has been developed. After extraction of the sample with acetonitrile, aldehydes were converted into their thiazolidine derivatives by the reaction with cysteamine, and then measured by gas chromatography with flame photometric detection. The calibration curves for aliphatic aldehydes in the range 20-2500 ng were linear and the detection limits at a signal-to-noise ratio of 3 were ca. 4-100 pg injected. Aliphatic aldehydes in foods could be selectively determined by this method without any interference from coexisting substances. Overall recoveries of aldehydes added to food samples were 82-111%. Analytical results for the determination of aliphatic aldehydes in various food samples are presented.

A selective and sensitive method for the determination of glutathione (GSH) and related aminothiols such as cysteine (Cys), cysteinylglycine (CysGly) and gamma-glutamylcysteine (gamma-GluCys) by gas chromatography (GC) has been developed. GSH and related aminothiols were converted into their N,S-isopropoxycarbonyl methyl ester derivatives and measured by GC with flame photometric detection using a short capillary column (5 m x 0.53 mm i.d.) of cross-linked DB-1. The calibration curves were linear in the range 1-25 nmol for GSH and in the range 0.2-5 nmol for other aminothiols, and the detection limits of GSH, Cys, CysGly and gamma-GluCys were approximately 5, 0.2, 0.3 and 0.5 pmol per injection respectively. This method was successfully applied to blood samples without prior clean-up, and GSH and related aminothiols in these samples could be analysed without any influence from coexisting substances. Overall recoveries of GSH and other aminothiols added to blood samples were 88-107%. The analytical results of free and total blood GSH and related aminothiols in normal subjects are presented.

A sensitive and selective method for the determination of total cysteamine in urine and plasma samples by gas chromatography (GC) has been developed. After reduction of the sample with sodium borohydride, the liberated cysteamine was converted into its N,S-diisobutoxycarbonyl derivative and measured by GC with flame photometric detection using a DB-210 capillary column. The calibration curve was linear in the range 0.2-5.0 nmol, and the detection limit, at a signal-to-noise ratio of 3, was ca. 0.5 pmol injected. Using this method, total cysteamine in urine and plasma samples could be accurately and precisely determined without any interference from coexisting substances. Analytical results for the determination of total cysteamine in urine and plasma samples from normal subjects are presented.

A selective and sensitive method has been developed for the determination of sulphur amino acids by gas chromatography (GC). Sulphur amino acids were converted into their N(S)-isopropoxycarbonyl methyl ester derivatives and measured by GC with flame photometric detection using a DB-17 capillary column. The derivatives were sufficiently volatile and stable to give single symmetrical peaks. The detection limits of sulphur amino acids were ca. 0.5-1 pmol per injection, and the calibration curves were linear in the range 0.5-10 nmol for each sulphur amino acid. This method was successfully applied to small urine samples without prior clean-up, and sulphur amino acids in these samples could be analysed without any influence from coexisting substances. Overall recoveries of sulphur amino acids added to urine samples were 85-113%. The analytical results of free sulphur amino acid contents in urine samples of normal subjects are presented.

A gas chromatographic method for the determination of cysteamine and its disulphide cystamine is described. Cysteamine and cystamine are converted into N,S-diisobutoxycarbonyl and N,N-diisobutoxycarbonyl derivatives, respectively. The derivatives are analysed by gas chromatography with flame photometric detection, using a DB-210 capillary column. The calibration curves for cysteamine and cystamine in the range of 0.2-5.0 nmol are linear and sufficiently reproducible for quantitative analysis, and the detection limit is about 0.5 pmol injected. Cysteamine in mouse tissues is found in the free reduced, free oxidized and protein-bound forms. Free oxidized and protein-bound forms are reduced to free cysteamine by the use of sodium borohydride, and then derivatized. Cysteamine and cystamine in mouse tissues can be measured without any interference from coexisting substances by this method. The recoveries of cysteamine and cystamine added to the tissue samples are 91-106%, and their reproducibilities are found to be satisfactory. Analytical results for the determination of various forms of cysteamine in mouse tissues are presented.

A selective and sensitive method has been developed for the determination of secondary amines by gas chromatography (GC). After removal of primary amines by the reaction with o-phthaldialdehyde, secondary amines were converted into their N-diethylthiophosphoryl derivatives and then measured by GC with flame photometric detection using a DB-1701 capillary column. The derivatives were sufficiently volatile and stable to give single symmetrical peaks. The detection limits of secondary amines were ca. 0.05-0.2 pmol per injection. N-Methylcyclohexylamine was used as an internal standard. The calibration curves for secondary amines in the range 1-20 nmol were linear and sufficiently reproducible for quantitative determination. This method was successfully applied to small urine samples without prior clean-up. Overall recoveries of secondary amines added to urine samples were 91-105%. By using this method, secondary amines in urine samples could be analysed without any influence from primary amines and other coexisting substances. The analytical results of secondary amine content in urine samples of normal subjects are presented.

A selective and sensitive method for the determination of low molecular weight aliphatic primary amines in urine is described. These amines were converted into their benzenesulphonyl derivatives by a modified Hinsberg procedure, and measured by gas chromatography with flame photometric detection (FPD-GC) using a DB-1 capillary column. The derivatives were very stable and provided excellent FPD responses. By FPD-GC, linear calibration curves were obtained in the range 10-200 ng of methylamine, ethylamine, n-propylamine, isobutylamine and n-butylamine using tert-butylamine as an internal standard, and the detection limits of these amines were ca. 6-25 pg as the injection amount. Benzenesulphonamide derived from ammonia was converted into its N-dimethylaminomethylene derivative which has a longer retention time, and separated from benzenesulphonyl derivatives of low molecular weight primary amines on the chromatogram. The recoveries of aliphatic primary amines added to urine samples were 91-107% and the relative standard deviations were 0.2-4.5%. Analytical results of aliphatic primary amine contents in urine samples of normal subjects are presented.

A practical method for the quantitative determination of protein amino acids by gas-liquid chromatography (GLC) is described. All of the common protein amino acids except arginine can be readily converted into their N-isobutyloxycarbonyl (N-isoBOC) methyl ester derivatives by a simple procedure involving isobutyloxycarbonylation with isobutyl chloroformate in aqueous medium, followed by methylation with diazomethane. Arginine was converted into N-isoBOC ornithine methyl ester by treatment with arginase, followed by the above derivatization procedure. The resulting N-isoBOC methyl esters of the amino acids have good GLC properties. Complete resolution of the derivatives of 20 protein amino acids was achieved by using a dual-column system consisting of a 0.65% Poly-A-101A column and a 0.70% FFAP-Poly-A-101A (1:1, w/w) column. The reproducibility of response was found to be good for derivatives carried through the entire chemical and chromatographic procedure. The calibration graphs were linear and showed no statistical bias. The results of recovery experiments with synthetic mixtures containing known amounts of the amino acids were satisfactory, the recoveries ranging from 94.3 to 106.2%.