Determination of Xylitol in Human Urine by Gas-Liquid Chromatography Lynda R. Horn, Lawrence J. Machlin, and James G. Hamilton, Department of Biochemical Nutrition, Roche Research Center, Hoffmann-La Roche, Inc., Nutley, New Jersey 07110
Abstract A rapid and specific method for the quantitative determination of xylitol in human urine has been developed. The method consists of the gas-liquid chromatographic analysis of the acetate ester derivative of the alditol in deionized urine using dulcitol as an internal standard. As little as 20 ng xylitol can be detected. At concentrations ranging from 25 to 400/xg/ml urine, the accuracy is + 4 . 0 % .
Introduction Xylitol is a sweet tasting pentitol which is a normal metabolite of the glucuronic acid cycle. Current methodology for the quantitative analysis of xylitol in urine is not satisfactory. Measurement by enzymatic oxidation to L-xylulose is not sensitive enough to analyze trace amounts of xylitol due to the unfavorable equilibrium. The Km for xylitol has been reported (1) to be 25.4 mM, or 3.86 mg/ml. The authors' requirements for the analysis of much less than 1 mg xylitol per ml urine led to a search for a more sensitive method. Thin layer and paper chromatography produced poor separations (2). Robyt (3) was able to achieve better separation of polyols. However, the procedure was time consuming and quantitation was still a problem. A report (4) on the gas-liquid chromatographic (GLC) analysis of xylitol described neither the specificity for pentitols nor quantitation. Sawardeker, et al. (5) achieved quantitation of xylitol on a 3% ECNSS-M column, but the sensitivity was only 10 mg/ml with rather long retention times. Sjostrom and Juslin (6) observed the gas chromatographic separation of pentitol acetates on a column of Silicone oil XF-1150 and EGS, but also reported sensitivity no greater than 10 mg/ml. In this study, the development of a quantitative gas chromatographic method for the detection of xylitol in human urine is reported. The present method distinguishes between xylitol and other pentitols, as well as xylulose, glucose, and dulcitol.
Experimental Preparation of Urine for Pentitol Analysis Fresh urine was obtained from a Caucasian male who fasted for 12 hours prior to the analysis. Aliquots of 1 ml each were mixed with aqueous solutions of xylitol and dulcitol. These samples were applied to ion exchange columns in Pasteur pipettes fitted with glass wool plugs. The resins used were the H+ form of Ag 5OW-X8 (BioRad, Richmond, California) and Dowex 1-X2 (BioRad), which had been converted to the OH' form with 1 N NaOH. The column was filled to a height of 2 cm with the cationic exchange resin, then the same amount of anionic resin was added. After washing the column with water
the urine mixtures were applied. Nonionic species were collected by elution with 5 ml water and then were lyophilized. Aqueous standard solutions of 25 to 400 jig pentitol and dulcitol were lyophylized without prior treatment. Acetate esters were prepared by reacting the lyophilized powders with 1 ml acetic anhydride in 0.5 ml pyridine in screwcapped tubes for 1 hour at 70°C. Following evaporation to dryness under nitrogen at 50°C, acetate esters were dissolved in 1 ml chloroform for injection into the gas chromatograph. Gas Chromatographic Apparatus and Conditions for Detection of Polyols The gas chromatograph used was a Hewlett-Packard (Avondale, Pennsylvania) research chromatograph, Model 7620A, equipped with a hydrogen flame ionization detector and a Hewlett-Packard integrator, Model 338OA. The chromatograph was fitted with a 6 ft x 2 mm glass column, which was packed using suction and gentle tapping with 3% ECNSS-M (a polymer consisting of an ethylene succinate structure combined with cyanoethyl silicone) on 100/120 mesh Gas-Chrom Q (Applied Science Laboratories, State College, Pennsylvania), and plugged with silanized glass wool. A Hamilton syringe of 10 \x\ capacity was employed for 2 y.\ injections. The analyses were run isothermally at a column temperature of 170°C. The injector temperature was 230°C and the detector temperature was 250°C. Helium was used as carrier gas with a flow rate of 50 ml/min. Results and Discussion The linearity of the detector response for xylitol can be seen from a plot of xylitol concentration versus the ratio of the area of xylitol to the area of dulcitol (Figure 1). The detector response, K, was calculated from the slope of the curve: K=
area of xylitol pentaacetate/area of dulcitol hexaacetate concentration of xylitol/concentration of dulcitol
K = 0.95 ±0.02 Concentrations of xylitol in urine were then calculated from the peak areas for xylitol pentaacetate by the following equation: Concentration of xylitol = area of xylitol pentaacetate X concentration of dulcitol area of dulcitol hexaacetate X K That the peak obtained at a retention time of 6.% min was
Reproduction (photocopying) of editorial content of this journal is prohibited without publisher's permission.
538-SEPTEMBER 1979
JOURNAL OF CHROMATOGRAPHIC SCIENCE-VOL. 17
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Abstract A rapid and specific method for the quantitative determination of xylitol in human urine has been developed. The method consists of the gas-liquid chromatographic analysis of the acetate ester derivative of the alditol in deionized urine using dulcitol as an internal standard. As little as 20 ng xylitol can be detected. At concentrations ranging from 25 to 400/xg/ml urine, the accuracy is + 4 . 0 % .
Introduction Xylitol is a sweet tasting pentitol which is a normal metabolite of the glucuronic acid cycle. Current methodology for the quantitative analysis of xylitol in urine is not satisfactory. Measurement by enzymatic oxidation to L-xylulose is not sensitive enough to analyze trace amounts of xylitol due to the unfavorable equilibrium. The Km for xylitol has been reported (1) to be 25.4 mM, or 3.86 mg/ml. The authors' requirements for the analysis of much less than 1 mg xylitol per ml urine led to a search for a more sensitive method. Thin layer and paper chromatography produced poor separations (2). Robyt (3) was able to achieve better separation of polyols. However, the procedure was time consuming and quantitation was still a problem. A report (4) on the gas-liquid chromatographic (GLC) analysis of xylitol described neither the specificity for pentitols nor quantitation. Sawardeker, et al. (5) achieved quantitation of xylitol on a 3% ECNSS-M column, but the sensitivity was only 10 mg/ml with rather long retention times. Sjostrom and Juslin (6) observed the gas chromatographic separation of pentitol acetates on a column of Silicone oil XF-1150 and EGS, but also reported sensitivity no greater than 10 mg/ml. In this study, the development of a quantitative gas chromatographic method for the detection of xylitol in human urine is reported. The present method distinguishes between xylitol and other pentitols, as well as xylulose, glucose, and dulcitol.
Experimental Preparation of Urine for Pentitol Analysis Fresh urine was obtained from a Caucasian male who fasted for 12 hours prior to the analysis. Aliquots of 1 ml each were mixed with aqueous solutions of xylitol and dulcitol. These samples were applied to ion exchange columns in Pasteur pipettes fitted with glass wool plugs. The resins used were the H+ form of Ag 5OW-X8 (BioRad, Richmond, California) and Dowex 1-X2 (BioRad), which had been converted to the OH' form with 1 N NaOH. The column was filled to a height of 2 cm with the cationic exchange resin, then the same amount of anionic resin was added. After washing the column with water
the urine mixtures were applied. Nonionic species were collected by elution with 5 ml water and then were lyophilized. Aqueous standard solutions of 25 to 400 jig pentitol and dulcitol were lyophylized without prior treatment. Acetate esters were prepared by reacting the lyophilized powders with 1 ml acetic anhydride in 0.5 ml pyridine in screwcapped tubes for 1 hour at 70°C. Following evaporation to dryness under nitrogen at 50°C, acetate esters were dissolved in 1 ml chloroform for injection into the gas chromatograph. Gas Chromatographic Apparatus and Conditions for Detection of Polyols The gas chromatograph used was a Hewlett-Packard (Avondale, Pennsylvania) research chromatograph, Model 7620A, equipped with a hydrogen flame ionization detector and a Hewlett-Packard integrator, Model 338OA. The chromatograph was fitted with a 6 ft x 2 mm glass column, which was packed using suction and gentle tapping with 3% ECNSS-M (a polymer consisting of an ethylene succinate structure combined with cyanoethyl silicone) on 100/120 mesh Gas-Chrom Q (Applied Science Laboratories, State College, Pennsylvania), and plugged with silanized glass wool. A Hamilton syringe of 10 \x\ capacity was employed for 2 y.\ injections. The analyses were run isothermally at a column temperature of 170°C. The injector temperature was 230°C and the detector temperature was 250°C. Helium was used as carrier gas with a flow rate of 50 ml/min. Results and Discussion The linearity of the detector response for xylitol can be seen from a plot of xylitol concentration versus the ratio of the area of xylitol to the area of dulcitol (Figure 1). The detector response, K, was calculated from the slope of the curve: K=
area of xylitol pentaacetate/area of dulcitol hexaacetate concentration of xylitol/concentration of dulcitol
K = 0.95 ±0.02 Concentrations of xylitol in urine were then calculated from the peak areas for xylitol pentaacetate by the following equation: Concentration of xylitol = area of xylitol pentaacetate X concentration of dulcitol area of dulcitol hexaacetate X K That the peak obtained at a retention time of 6.% min was
Reproduction (photocopying) of editorial content of this journal is prohibited without publisher's permission.
538-SEPTEMBER 1979
JOURNAL OF CHROMATOGRAPHIC SCIENCE-VOL. 17
CD
8 «>!
