ANALYTICAL
BIOCHEMISTRY
94, 358-359 (1979)
An Improved Gas-Liquid Chromatographic Method Analysis of Trimethylamine in Urine PETER
for
SCHLESINGER
Genetics Research Unit, Royal Children’s Hospital Research Foundation, Parkville, Victoria, 3052, Australia
Flemington Road,
Received October 4, 1978 An established gas-liquid chromatographic method for analysis of trimethylamine in urine was substantially improved by the use of an exchangeable lithium hydroxide, kieselguhr precolumn. Biological samples were acidified immediately upon collection and were analyzed by direct injection onto the precolumn. Using this technique, a normal urinary level of trimethylamine was found to be 5.0 + 3.1 ymol/mmol creatinine in a group of 17 adults.
The gas-liquid chromatography (glc) method of Marks et al. (1,2) for measuring trimethylamine (TMA)’ in urine had been found satisfactory in our laboratory, however, when acidified biological fluids were directly injected the column very quickly deteriorated. Thus, at times, after only 30 injections of 2-~1 doses, nonlinear response, excessive adsorption characteristics such as “ghosting” and “tailing” of peaks and generally irreproducible results were obtained. A substantial lengthening of column life, for up to 1000 injected doses, was achieved by use of a short exchangeable lithium hydroxide, chromosorb W precolumn. A normal range for urinary TMA was measured, using the precolumn. METHOD
All materials were readily available commercially. Trimethylammonium chloride was obtained from Fluka and desiccated before being used to prepare reference solutions. Purity of FFAP was important. A modification of the glc method of Marks et al. (1,2) was used, technical details being summarized below. Chromatograph: Pack’ Abbreviation
used: TMA, trimethylamine.
0003-2697/79/060358-02$02.00/O Copynghr 0 1979 by Academic Press, Inc. All rights of reproduction in any form reserved.
358
ard-Becker Model 419 with a Model 706 flame ionization detector; integrator: Spectra-physics system 1; recorder: Rikadenki Model 8341; columns: single, 6 ft x 4 mm internal diameter, glass main column packed with 10% FFAP, 5% potassium hydroxide on Chromosorb W 801100 mesh linked by a Swagelock connector with two GFF 4 Graphlok ferrules to a 12 cm x 4 mm internal diameter glass precolumn packed with 20% lithium hydroxide on Chromosorb W. In preparing the main column alcohol was used to achieve KOH penetration into the FFAP as well as onto the Chromosorb. Amplification/sensitivity: lop3 V, lo-” A full-scale deflection. Temperature: injector 13O”C, detector 2OO”C, column 68°C isothermal. Gas flows: nitrogen 50 mllmin, air 300 ml/min, hydrogen 40 mhmin. A standard curve of the ratio of peak area TMA/peak area acetone (internal standard, 10 ppm) versus concentration of reference TMA was graphed on each day of analysis. Immediate upon collection, urine samples were acidified with hydrochloric acid (equivalent to 0.1 moYliter) and stored frozen in bottles with tight Teflon-lined closures. Urinary creatinine was measured calorimetrically (3).
GLC ANALYSIS
359
OF TRIMETHYLAMINE
RESULTS
A straight line standard curve was obtained between 3 ppm TMA (lower limit of detectability) and 200 ppm TMA, the range used to analyze urine samples or their dilutions, when 20% lithium hydroxide Chromosorb W precolumns were used. When similar potassium hydroxide precolumns were tried, nonlinearity was observed below 10 ppm TMA and with sodium hydroxide below 50 ppm. The precision of the method with the lithium hydroxide precolumn was estimated by the coefficient of variation calculated from 14 intraexperiment, triplicate samples as 7%. The day-to-day reproducibility expressed as the coefficient of variation obtained using a 50 ppm TMA solution interexperimentally was 8.5%. Improved recovery, as measured by the ratio of the area of TMA peak to acetone peak, was demonstrated by lower ratios being obtained when lithium hydroxide precolumns were replaced by glass-wool precolumns. Other amines which were detected by direct injection of acidified, aqueous reference solutions were the primary, secondary, and tertiary amines up to butylamine in the homologous series as well as piperidine. None interfered with TMA. Ammonia had a very low detector response. Urines from 17 presumably healthy adults
were analyzed and found to contain 5 .O + 3.1 pmol TMA/mmol creatinine (mean 2 SD). DISCUSSION
The technique presented incorporates the general advantages of the well-established methods for analysis of aqueous solutions of amines injected directly onto columns where alkali is used to maintain the volatile free bases and to saturate excessive column adsorption sites. It has the special advantages of providing an exchangeable precolumn where the debris in the samples of urine or other biological fluid may be deposited, thereby substantially lengthening the life of the main column, and allowing amine hydrochlorides to be converted, with minimal loss, to volatile free bases, within the precolumn. Of the materials tried, 20% lithium hydroxide on Chromosorb W was found best for the precolumn. ACKNOWLEDGMENTS Thanks are extended to Mr. A. Grimes for excellent technical assistance and to Professor Danks and Dr. R. G. H. Cotton for very helpful discussion.
REFERENCES 1. Marks, R., Greaves, M. W., Prottey, C., and Hartop, P. J. (1977) Bn’f. J. Dermar. 96, 399. 2. Marks, R., Greaves, M. W., Danks, D. M., Plummer, V. (1976)Brir.J. Dermat. %(Suppl. la), 11. 3. Edwards, K. D. G., and Whyte, H. M. (1959) Aust. Ann. Med. 8, 218.
BIOCHEMISTRY
94, 358-359 (1979)
An Improved Gas-Liquid Chromatographic Method Analysis of Trimethylamine in Urine PETER
for
SCHLESINGER
Genetics Research Unit, Royal Children’s Hospital Research Foundation, Parkville, Victoria, 3052, Australia
Flemington Road,
Received October 4, 1978 An established gas-liquid chromatographic method for analysis of trimethylamine in urine was substantially improved by the use of an exchangeable lithium hydroxide, kieselguhr precolumn. Biological samples were acidified immediately upon collection and were analyzed by direct injection onto the precolumn. Using this technique, a normal urinary level of trimethylamine was found to be 5.0 + 3.1 ymol/mmol creatinine in a group of 17 adults.
The gas-liquid chromatography (glc) method of Marks et al. (1,2) for measuring trimethylamine (TMA)’ in urine had been found satisfactory in our laboratory, however, when acidified biological fluids were directly injected the column very quickly deteriorated. Thus, at times, after only 30 injections of 2-~1 doses, nonlinear response, excessive adsorption characteristics such as “ghosting” and “tailing” of peaks and generally irreproducible results were obtained. A substantial lengthening of column life, for up to 1000 injected doses, was achieved by use of a short exchangeable lithium hydroxide, chromosorb W precolumn. A normal range for urinary TMA was measured, using the precolumn. METHOD
All materials were readily available commercially. Trimethylammonium chloride was obtained from Fluka and desiccated before being used to prepare reference solutions. Purity of FFAP was important. A modification of the glc method of Marks et al. (1,2) was used, technical details being summarized below. Chromatograph: Pack’ Abbreviation
used: TMA, trimethylamine.
0003-2697/79/060358-02$02.00/O Copynghr 0 1979 by Academic Press, Inc. All rights of reproduction in any form reserved.
358
ard-Becker Model 419 with a Model 706 flame ionization detector; integrator: Spectra-physics system 1; recorder: Rikadenki Model 8341; columns: single, 6 ft x 4 mm internal diameter, glass main column packed with 10% FFAP, 5% potassium hydroxide on Chromosorb W 801100 mesh linked by a Swagelock connector with two GFF 4 Graphlok ferrules to a 12 cm x 4 mm internal diameter glass precolumn packed with 20% lithium hydroxide on Chromosorb W. In preparing the main column alcohol was used to achieve KOH penetration into the FFAP as well as onto the Chromosorb. Amplification/sensitivity: lop3 V, lo-” A full-scale deflection. Temperature: injector 13O”C, detector 2OO”C, column 68°C isothermal. Gas flows: nitrogen 50 mllmin, air 300 ml/min, hydrogen 40 mhmin. A standard curve of the ratio of peak area TMA/peak area acetone (internal standard, 10 ppm) versus concentration of reference TMA was graphed on each day of analysis. Immediate upon collection, urine samples were acidified with hydrochloric acid (equivalent to 0.1 moYliter) and stored frozen in bottles with tight Teflon-lined closures. Urinary creatinine was measured calorimetrically (3).
GLC ANALYSIS
359
OF TRIMETHYLAMINE
RESULTS
A straight line standard curve was obtained between 3 ppm TMA (lower limit of detectability) and 200 ppm TMA, the range used to analyze urine samples or their dilutions, when 20% lithium hydroxide Chromosorb W precolumns were used. When similar potassium hydroxide precolumns were tried, nonlinearity was observed below 10 ppm TMA and with sodium hydroxide below 50 ppm. The precision of the method with the lithium hydroxide precolumn was estimated by the coefficient of variation calculated from 14 intraexperiment, triplicate samples as 7%. The day-to-day reproducibility expressed as the coefficient of variation obtained using a 50 ppm TMA solution interexperimentally was 8.5%. Improved recovery, as measured by the ratio of the area of TMA peak to acetone peak, was demonstrated by lower ratios being obtained when lithium hydroxide precolumns were replaced by glass-wool precolumns. Other amines which were detected by direct injection of acidified, aqueous reference solutions were the primary, secondary, and tertiary amines up to butylamine in the homologous series as well as piperidine. None interfered with TMA. Ammonia had a very low detector response. Urines from 17 presumably healthy adults
were analyzed and found to contain 5 .O + 3.1 pmol TMA/mmol creatinine (mean 2 SD). DISCUSSION
The technique presented incorporates the general advantages of the well-established methods for analysis of aqueous solutions of amines injected directly onto columns where alkali is used to maintain the volatile free bases and to saturate excessive column adsorption sites. It has the special advantages of providing an exchangeable precolumn where the debris in the samples of urine or other biological fluid may be deposited, thereby substantially lengthening the life of the main column, and allowing amine hydrochlorides to be converted, with minimal loss, to volatile free bases, within the precolumn. Of the materials tried, 20% lithium hydroxide on Chromosorb W was found best for the precolumn. ACKNOWLEDGMENTS Thanks are extended to Mr. A. Grimes for excellent technical assistance and to Professor Danks and Dr. R. G. H. Cotton for very helpful discussion.
REFERENCES 1. Marks, R., Greaves, M. W., Prottey, C., and Hartop, P. J. (1977) Bn’f. J. Dermar. 96, 399. 2. Marks, R., Greaves, M. W., Danks, D. M., Plummer, V. (1976)Brir.J. Dermat. %(Suppl. la), 11. 3. Edwards, K. D. G., and Whyte, H. M. (1959) Aust. Ann. Med. 8, 218.
