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A microquantitative technique for the detection of some biologically important amines by gas-liquid chromatography
SHORT COMMUNICATIONS
A microquantitative biologically important
193
technique for the detection of some amines by gas-liquid chromatography*
The possibility of applying the methods of gas chromatography toward the analysis of biologically important amines, has recently been explored. FALES AND PISANO~ demonstrated but the low sensitivity
that they could separate free amines on a siloxane polymer, of detection generally limited analysis to a I”/$ amine solution.
BROCHMANN-HANSSEN AND SVENDSON~ noted with ketone solvents to give sharp symmetrical was accomplished,
that a number of amines condensed peaks. Separations of various amines
but here too o.~--I~/~ solutions
were required.
The recently
pre-
pared3 silyl derivatives of several amines afforded somewhat incomplete separation and a generally low order of sensitivity. All of the above methods lack the necessary sensitivity for a quantitative assay of an amine such as normetanephrine, less than I mg of which is excreted in human urine in a 24-h period4p5. Since only micro-liter quantities of the amine solution is normally delivered to the detector, one must be able to detect nanogram The high sensitivity
quantities of the amine. of the electron capture detector for electronegative groups was noted by LANDOWNE AND LIPSKY 637. Their observations were confirmed in this laboratory during chromatographic studies of numerous derivatives of various primary and secondary amines. It was finally determined that heptafluorobutyric anhydride could yield amine derivatives with sufficient sensitivity as well as ease of synthesis and separation to suggest clinical usefulness in the study of amine excretion of human subjects. This paper will describe the synthesis and separation of the Nheptafluorobutyramide derivatives of five amines: normetanephrine, tyramine p-phenylethylamine, ,!?-hydroxyphenylethylamine and tryptamine. The amines were obtained from the California Corporation for Biochemical Research and heptafluorobutyric anhydride (HFB) was obtained from the Chemicals Procurement Corporation, College Point, New York. To a I5-ml ground glass test tube, IOO mg of normetanephrine. HCl, 4 ml glacial acetic acid and I ml HFB was added. The tube was stoppered and heated on a steam bath for 3 h. At the end of the reaction period, the mixture was concentrated by evaporation under a stream of air, poured into goml of ice water and refrigerated until crystallization was complete. The precipitate was collected on a Buchner funnel, washed several times with water and dried in a desiccator. The above procedure yields crystalline material for normetanephrine, tyramine and ,6&phenylethylamine. Tryptamine was allowed to react at room temperature, since heating resulted in a breakdown of the amine and a lowered yield. It was found that fi-hydroxyphenylethylamine was destroyed when exposed to glacial acetic acid and HFB. The substitution of N,N-dimethylformamide as solvent resulted in a good yield of the p-hydroxyphenylethylamine derivative, which was recrystallized from ethanol. N-heptafluorobutyramide derivatives could not be prepared from the secondary amines metanephrine and epinephrine, or from the easily oxidizable catechols such as 3-hydroxytyramine and norepinephrine. * Supported by grants from the United States Public Health Service, National stitute (Grant Number HE-06546) and the New York Heart Association. Clin. Chim. A&,
Heart In-
IO (1964)
rgprgg
I94
SHORT
COMMUNICATIONS
Standard solutions of the amine derivatives in a concentration of IO ,ug/ml were prepared in ethyl acetate or acetone. Chromatography was accomplished using the Packard Gas Chromatograph, Model 750 B. Separations were effected on a 6 ft x 4 mm OD coiled glass column packed with 5% XE-60 coated on Anakrom ABS IOO-IIO mesh 5% silanized (Analabs). The column was cured for one week at 220’ prior to use. These derivatives proved unstable to high column temperature and chromatography was therefore carried out at 150’. The nitrogen gas flow was adjusted to 60 ml/min. Q :“I $HIlSfl,rnl W
TIME (MINUTES)
Fig. I. Separation b-Phenylethylamine
of 2 ,ul of a model solution of the N-heptafluorobutyramide
derivatives
of:
(15 pg/ml) ; B-Hydroxy-phenylethylamine (2.5 pg/ml) ; Normetanephrine ..n,ml\. ..“,rnl\ /e pm&, ..~/rnl\~ I JI UllllllL Ira ,’ J p5, ‘L”, , Tr.rn+~minn A’Jy’*o”“L’c /n \L.Jc /.46,“,‘,. \J ““, , TT,mm;nn
TABLE I Amine derivative
I. p-Phenylethylamine /3-Hydroxy-phenylethylamine 3. Normetanephrine 4. Tyramine 5. Tryptamine 2.
Retention time min 1.P
I.9 7.8 13.8 23.4
The retention times of the five derivatives are listed in Table I. Fig. I shows the separation of 2 pl of a model solution composed of these compounds in varying concentrations. The normetanephrine derivative yields an initial sharp peak at 7.8 min, and a late diffuse peak at 41.1 min. Normetanephrine may be quantitated with respect to the first peak since its area is linearly related to the amount of this derivative delivered to the column. The other four derivatives gave single peaks and excellent separations. That &phenylethylamine and tryptamine possessing but one reactive group, form a derivative with HFB suggests that this reaction takes place at the side chain amino group forming the N-heptafluorobutyramide. Ester formation by reaction of HFB with hydroxyl groups is also possible but it is not known whether this takes place with normetanephrine. This method appears to offer the basis for a quantitativedetermination of normetanephrine and other amines of clinical importance in human urine. Clin. China.Acta, 10 (1964) 19x-195
195
SHORT COMMUNICATIONS
The authors wish to thank Mr. GEORGE KRAMER of the Packard Corporation, Medicine
Inc.
and Dr.
M. GOLDSTEIN of the New
for their cooperation
and assistance
York
University
Instrument College
of
in this investigation. SHERWIN WILK
Department of Medicine, The Mount Sinai Hospital, New York, N.Y. Department of Psychiatry, Boston University, School of Medicine,
STANLEY E. GITLOW MORTON J. FRANKLIN HERMAN E. CARR
Boston, Mass. (U.S.A.) 1 H. M. FALES AND J. J. PISANO, Anal. Biochem., 3 (1962) 337. p E. BROCHMANN-HANSSEN AND A. B. SVENDSON, J. Pharm. Sci., 51 (1962) 938. s N. P. SEN AND P. L. MCGREER, Biochem. Biophys. Res. Commun., 13 (1963) 390. 4 J. J. PISANO, Clin. Chim. Ada, 5 (1960) 406. 5 S. BRUNJES, D. WYBENGO AND V. J. JOHNS JR., Clin. Chem., IO (1~64) I. 8 R. A. LANDOWNE AND S. R. LIPSKY, Nature, Igg (1963) 141. 7 R. A. LANDOWNE AND S. E. LIPSKY, Anal. Chem., 35 (1963) 532.
Received March rqth, 1963
BRIEF
TECHNICAL
Clin. C&L Acta, IO (1964) 193-195
NOTE
Recovery of solutions after calorimetry
using a flow-through
cuvette
When large numbers of coloured solutions have to be read manually, the use of a calorimeter fitted with a flow-through cuvette saves time. In general, it is unnecessary to recover the solution after taking a reading. However, when the same solution has to be re-read after an interval of time or at a different wave length or for some experimental purpose, it may be desirable to recover the solution. A simple device for doing this is shown in Fig. I. The device consists of a rubber bung held in a clip on a stand. Two plastic tubes pass through the rubber bung. The inlet tube is thin (e.g. internal diameter I-Z mm) and connects to the outlet of the flow-through cuvette. The outlet tube is wider (e.g. internal diameter 2-3 mm) and is connected to a water pump or other source of suction. The diameter of the rubber bung is such that it just fits the test-tube which contains the coloured solution. The tube is held in the stand a few inches above the level of the cuvette. The solution to be read in the calorimeter is poured into the cuvette and the suction turned on. At this stage there must be no test-tube fitted to the rubber bung. Because the tube is held above the level of the cuvette, the coloured solution rises only a small way up the inlet tube. After the reading has been taken, the test-tube is fitted onto the bung and the suction carries the solution back into the test-tube. The test-tube is detached and the next solution read. The device was designed for use with a home-made flow-through cuvette fitted C&n. Ckim. Acta, IO (1964) 195-196
A microquantitative biologically important
193
technique for the detection of some amines by gas-liquid chromatography*
The possibility of applying the methods of gas chromatography toward the analysis of biologically important amines, has recently been explored. FALES AND PISANO~ demonstrated but the low sensitivity
that they could separate free amines on a siloxane polymer, of detection generally limited analysis to a I”/$ amine solution.
BROCHMANN-HANSSEN AND SVENDSON~ noted with ketone solvents to give sharp symmetrical was accomplished,
that a number of amines condensed peaks. Separations of various amines
but here too o.~--I~/~ solutions
were required.
The recently
pre-
pared3 silyl derivatives of several amines afforded somewhat incomplete separation and a generally low order of sensitivity. All of the above methods lack the necessary sensitivity for a quantitative assay of an amine such as normetanephrine, less than I mg of which is excreted in human urine in a 24-h period4p5. Since only micro-liter quantities of the amine solution is normally delivered to the detector, one must be able to detect nanogram The high sensitivity
quantities of the amine. of the electron capture detector for electronegative groups was noted by LANDOWNE AND LIPSKY 637. Their observations were confirmed in this laboratory during chromatographic studies of numerous derivatives of various primary and secondary amines. It was finally determined that heptafluorobutyric anhydride could yield amine derivatives with sufficient sensitivity as well as ease of synthesis and separation to suggest clinical usefulness in the study of amine excretion of human subjects. This paper will describe the synthesis and separation of the Nheptafluorobutyramide derivatives of five amines: normetanephrine, tyramine p-phenylethylamine, ,!?-hydroxyphenylethylamine and tryptamine. The amines were obtained from the California Corporation for Biochemical Research and heptafluorobutyric anhydride (HFB) was obtained from the Chemicals Procurement Corporation, College Point, New York. To a I5-ml ground glass test tube, IOO mg of normetanephrine. HCl, 4 ml glacial acetic acid and I ml HFB was added. The tube was stoppered and heated on a steam bath for 3 h. At the end of the reaction period, the mixture was concentrated by evaporation under a stream of air, poured into goml of ice water and refrigerated until crystallization was complete. The precipitate was collected on a Buchner funnel, washed several times with water and dried in a desiccator. The above procedure yields crystalline material for normetanephrine, tyramine and ,6&phenylethylamine. Tryptamine was allowed to react at room temperature, since heating resulted in a breakdown of the amine and a lowered yield. It was found that fi-hydroxyphenylethylamine was destroyed when exposed to glacial acetic acid and HFB. The substitution of N,N-dimethylformamide as solvent resulted in a good yield of the p-hydroxyphenylethylamine derivative, which was recrystallized from ethanol. N-heptafluorobutyramide derivatives could not be prepared from the secondary amines metanephrine and epinephrine, or from the easily oxidizable catechols such as 3-hydroxytyramine and norepinephrine. * Supported by grants from the United States Public Health Service, National stitute (Grant Number HE-06546) and the New York Heart Association. Clin. Chim. A&,
Heart In-
IO (1964)
rgprgg
I94
SHORT
COMMUNICATIONS
Standard solutions of the amine derivatives in a concentration of IO ,ug/ml were prepared in ethyl acetate or acetone. Chromatography was accomplished using the Packard Gas Chromatograph, Model 750 B. Separations were effected on a 6 ft x 4 mm OD coiled glass column packed with 5% XE-60 coated on Anakrom ABS IOO-IIO mesh 5% silanized (Analabs). The column was cured for one week at 220’ prior to use. These derivatives proved unstable to high column temperature and chromatography was therefore carried out at 150’. The nitrogen gas flow was adjusted to 60 ml/min. Q :“I $HIlSfl,rnl W
TIME (MINUTES)
Fig. I. Separation b-Phenylethylamine
of 2 ,ul of a model solution of the N-heptafluorobutyramide
derivatives
of:
(15 pg/ml) ; B-Hydroxy-phenylethylamine (2.5 pg/ml) ; Normetanephrine ..n,ml\. ..“,rnl\ /e pm&, ..~/rnl\~ I JI UllllllL Ira ,’ J p5, ‘L”, , Tr.rn+~minn A’Jy’*o”“L’c /n \L.Jc /.46,“,‘,. \J ““, , TT,mm;nn
TABLE I Amine derivative
I. p-Phenylethylamine /3-Hydroxy-phenylethylamine 3. Normetanephrine 4. Tyramine 5. Tryptamine 2.
Retention time min 1.P
I.9 7.8 13.8 23.4
The retention times of the five derivatives are listed in Table I. Fig. I shows the separation of 2 pl of a model solution composed of these compounds in varying concentrations. The normetanephrine derivative yields an initial sharp peak at 7.8 min, and a late diffuse peak at 41.1 min. Normetanephrine may be quantitated with respect to the first peak since its area is linearly related to the amount of this derivative delivered to the column. The other four derivatives gave single peaks and excellent separations. That &phenylethylamine and tryptamine possessing but one reactive group, form a derivative with HFB suggests that this reaction takes place at the side chain amino group forming the N-heptafluorobutyramide. Ester formation by reaction of HFB with hydroxyl groups is also possible but it is not known whether this takes place with normetanephrine. This method appears to offer the basis for a quantitativedetermination of normetanephrine and other amines of clinical importance in human urine. Clin. China.Acta, 10 (1964) 19x-195
195
SHORT COMMUNICATIONS
The authors wish to thank Mr. GEORGE KRAMER of the Packard Corporation, Medicine
Inc.
and Dr.
M. GOLDSTEIN of the New
for their cooperation
and assistance
York
University
Instrument College
of
in this investigation. SHERWIN WILK
Department of Medicine, The Mount Sinai Hospital, New York, N.Y. Department of Psychiatry, Boston University, School of Medicine,
STANLEY E. GITLOW MORTON J. FRANKLIN HERMAN E. CARR
Boston, Mass. (U.S.A.) 1 H. M. FALES AND J. J. PISANO, Anal. Biochem., 3 (1962) 337. p E. BROCHMANN-HANSSEN AND A. B. SVENDSON, J. Pharm. Sci., 51 (1962) 938. s N. P. SEN AND P. L. MCGREER, Biochem. Biophys. Res. Commun., 13 (1963) 390. 4 J. J. PISANO, Clin. Chim. Ada, 5 (1960) 406. 5 S. BRUNJES, D. WYBENGO AND V. J. JOHNS JR., Clin. Chem., IO (1~64) I. 8 R. A. LANDOWNE AND S. R. LIPSKY, Nature, Igg (1963) 141. 7 R. A. LANDOWNE AND S. E. LIPSKY, Anal. Chem., 35 (1963) 532.
Received March rqth, 1963
BRIEF
TECHNICAL
Clin. C&L Acta, IO (1964) 193-195
NOTE
Recovery of solutions after calorimetry
using a flow-through
cuvette
When large numbers of coloured solutions have to be read manually, the use of a calorimeter fitted with a flow-through cuvette saves time. In general, it is unnecessary to recover the solution after taking a reading. However, when the same solution has to be re-read after an interval of time or at a different wave length or for some experimental purpose, it may be desirable to recover the solution. A simple device for doing this is shown in Fig. I. The device consists of a rubber bung held in a clip on a stand. Two plastic tubes pass through the rubber bung. The inlet tube is thin (e.g. internal diameter I-Z mm) and connects to the outlet of the flow-through cuvette. The outlet tube is wider (e.g. internal diameter 2-3 mm) and is connected to a water pump or other source of suction. The diameter of the rubber bung is such that it just fits the test-tube which contains the coloured solution. The tube is held in the stand a few inches above the level of the cuvette. The solution to be read in the calorimeter is poured into the cuvette and the suction turned on. At this stage there must be no test-tube fitted to the rubber bung. Because the tube is held above the level of the cuvette, the coloured solution rises only a small way up the inlet tube. After the reading has been taken, the test-tube is fitted onto the bung and the suction carries the solution back into the test-tube. The test-tube is detached and the next solution read. The device was designed for use with a home-made flow-through cuvette fitted C&n. Ckim. Acta, IO (1964) 195-196