Gas Chromatography of Urinary Catecholamines

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Gas chromatographic assay of catecholamines in urine was successfully carried out by means of the following procedure. 40 ml of urine acidified to about pH 1 with 6N-HCl was boiled for 20 min. After cooling, the solution was neutralized to about pH 6.5 with 4N-NH4OH, then 2 ml of 0.2M-EDTA-Na2 and 2g of Al2O3 were added. The mixture was adjusted to about pH 8.4 with 4N-NH4OH and shaken gently for 5 min. The supernatant was discarded and the Al2O3 was transfered into a chromatographic tube. Catecholamines were eluted from the Al2O3 with 10 ml of 0.2N-CH3COOH. One ml of the eluate was evaporated to dryness under reduced pressure. The residue was treated with 2 drops of tetrahydrofuran and one drop of trifluoroacetic anhydride for 5 min at room temperature. The reaction mixture was diluted to an appropriate volume with n-hexane and was injected directly to gas chromatograph. A complete separation of three catecholamines, epinephrine, dopamine and norepinephrine, was achieved on a 2% GE-XF 1105 glass column at 175°C. Isodrin was used as an internal standard. Formation of trifluoroacetates was rapid and simple. The trifluoroacetates produced were volatile enough to permit the use of selective columns, and satisfactory responses were obtained from several ng (10-9g) of them by means of an electron capture detector. This technique was applied to determination of catecholamines in the urines of two patients (A and B) with pheochromocytoma, a patient (C) with hypertension and a normal person (D), and a tumor (T) removed from the patient B. Fig. 1 shows the chromatogram obtained from the urine of the patient B and that of the normal subject D is illustrated in Fig. 2. Gas chromatographic and fluorometric data are compared in TABLE I. If one considers these data so far, it is clear that the urinary output of norepinephrine is elevated to abnormal amounts in pheochromocytoma. Dopamine is a normal constituent of human urine and excreted in high amounts in bothof Fig. 1 and Fig. 2. In the urine of the patient C with hypertension, normal norepinephrine output is observed, indicating that the patient C has no pheochromocytoma. The gas chromatographic method has several advantages over fluorometry. Each peak indicates a reliable amount of catecholamine itself without interference of other co-existing components. Fluorometry often gives unreliable informations, since the amounts of epinephrine and norepinephrine are calculated by the use of simultaneous equations, in which dopa also interferes. An additional point of interest in gas chromatography is that the amount of each catecholamine can be estimated simultaneously on the same chromatogram. In most cases, the whole pattern of chromatogram itself may have a clinical significance without precise quantitative estimation of each catecholamine. As shown in Fig. 1 and Fig. 2, the increase in the ratio of norepinephrine to dopamine in peak height permits a rapid and simple diagnostic evaluation. Fig. 3 shows separation of the amines in the tumor and the patttern reveals norepinephrine to be the only predominant catecholamine, which suggest that this tumor produces only norepinephrine because of inability to methylate it to epinephrine. Moreover, co-existing unknown compounds, if any, may be discriminated on the same chromatogram. As observed in Fig. 1 and Fig. 2, an unknown peak X was found in a significant amount. In view of correspondence of retention times so far, it is not unreasonable to assume that the compound of peak X might be dopa itself. Dopa has not been found in urine except the cases reported in a few papers and of great interesting is the presence of considerable amounts of dopa in the urines not only of a patient with pheochromocytoma, but also of a normal person.
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Gas Chromatography of Urinary Catecholamines

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