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A selective and sensitive gas chromatographic method for the analysis of lipoic acid in biological samples has been developed. After base hydrolysis of the sample, the liberated lipoic acid was converted into its S,S-diethoxycarbonyl methyl ester derivative and measured by gas chromatography using a DB-210 capillary column and a flame photometric detector. The calibration curve was linear in the range 20-500 ng, and the detection limit was ca. 50 pg injected. The best hydrolysis conditions for the biological samples were obtained by using 2 M potassium hydroxide containing 4% bovine serum albumin at 110 degrees C for 3 h. Using this method, lipoic acid in the hydrolysate could be selectively determined without any interference from matrix substances. Analytical results for the determination of lipoic acid in the mouse tissue and bacterial cell samples are presented.

The occurrence of O-phosphoserine (P-Ser), O-phosphothreonine (P-Thr) and O-phosphotyrosine (P-Tyr) in the hydrolysate of human urine was demonstrated. Urine samples were separated into non-adsorbed and adsorbed fractions by DEAE-cellulose column chromatography. After acid and base hydrolyses of these fractions, the O-phosphoamino acids in the hydrolysates were converted into their N-isobutoxycarbonyl methyl ester derivatives and identified by gas chromatography-mass spectrometry. By gas chromatography with flame photometric detection, the urinary contents of P-Ser, P-Thr and P-Tyr were estimated to be 945 +/- 3, 109 +/- 3 and 2.9 +/- 0.2 ng/ml, respectively.

A selective and sensitive method for the determination of urinary beta-phenylethylamine (PEA) by gas chromatography (GC) has been developed. After extraction of the urine sample with n-pentane, PEA was converted into its N-benzenesulphonamide derivative and then determined by GC with flame photometric detection using a DB-1301 capillary column. By using this method, nanogram amounts of PEA in urine could be accurately and precisely determined without any influence from coexisting substances. Analytical results for the determination of PEA in urine samples from normal subjects are presented.

A gas chromatographic method was developed for the determination of monoacetylputrescine, monoacetylcadaverine, N1-acetylspermidine and N8-acetylspermidine in human urine. The amines were isolated from urine by silica gel column chromatography. 1,10-Diaminodecane was used as internal standard. The amines were reacted with ethyl chloroformate in aqueous medium to four ethyloxycarbonyl derivatives prior to application to gas chromatography using a flame ionization detector. Separation and determination of the derivatives were carried out on a Uniport HP column (1.0 m) impregnated with 0.5% SP-1000 under temperature-programmed conditions. The monoacetylpolyamines could be measured accurately at the nanomole level. The method was used for the determination of the monoacetylpolyamines in urine of healthy volunteers. The values obtained were in the range of the published data.

Application of the gas--liquid chromatographic method previously reported by us was made to the analysis of the 22 amino acids including asparagine and glutamine in serum. The method permitted that aqueous serum samples obtained after deproteinization with perchloric acid were directly subjected to derivatization without any further clean-up procedure such as ion-exchange chromatography. The N-ethyloxycarbonyl methyl esters, which were prepared in the same manner as the N-isobutyloxycarbonyl methyl esters, were introduced for the determination of leucine, isoleucine, arginine and tyrosine. Both derivatives were prepared by two-step procedures involving alkyloxycarbonylation in aqueous media and esterification with diazomethane, and simultaneously analyzed by using the dual set of columns with the same thermal conditions. The advantages of this method are that the sample pretreatment and derivatization are very simple and rapid, and that both asparagine and glutamine along with other amino acids in serum can be determined.

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%.

A method has been developed for the determination of taurine by gas-liquid chromatography. The method involves the conversion of taurine into its N-isobutyloxycarbonyl di-n-butylamide derivative and chromatography on a 1.5% OV-17 column. The derivative can be prepared in quantitative yield, having good chromatographic properties. The calibration curve for taurine in the range 5-500 nmol was linear and sufficiently reproducible for quantitative determination. Complex biological material such as urine can be analysed accurately and precisely by this method without prior clean-up of the sample.