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Specific gas chromatographic measurement of urinary 3,4-dihydroxyphenylacetic acid
249
Clinica Chimica Acta, 59 (1975) 249-251 @ Elsevier Scientific Publishing Company, Amsterdam -
Printed in The Netherlands
SHORT COMMUNICATION CCA 6888 SPECIFIC GAS CHROMATOGRAPHIC MEASUREMENT 3,4-DIHYDROXYPHENYLACETIC ACID
OF URINARY
M.W. WEG, C.R.J. RUTHVEN, B.L. GOODWIN and M. SANDLER Bernhard Baron Memorial Research Laboratories and Institute of Obstetrics and Gynaecology, Queen Charlotte’s Maternity Hospital, Goldhawk Road, London W6 OXG (U.K.)
(Received October 14, 1974)
The endogenous excretion of 3,4_dihydroxyphenylacetic acid (DOPAC) is relatively low [l-3] and this has created problems in its assay. The determination of this major metabolite of L-DOPA and dopamine has assumed particular importance in the biochemical investigation of catecholamine-secreting tumours [4-61 and in monitoring the treatment of parkinsonism with L-DOPA [71. In our experience the estimation of DOPAC at normal urinary output concentrations by gas-liquid chromatography (GLC) of the methyl ester/trimethylsilyl (TMS) ether [8,9] has been unsatisfactory, even using support-coated open tubular (SCOT) columns. Not only does the peak occasionally coalesce with that of the hippuric acid derivative, but there is another compound in the solvent extract that has the same retention time on the stationary phase (SE 52) as the DOPAC derivative. In order to remove unwanted material, isolation of the DOPAC on alumina has therefore been substituted for solvent extraction. In addition, preparation of the pentafluoropropionyl (PFP) derivative has permitted analysis by the far more sensitive electron capture detector (ECD) in place of the flame ionisation detector (FID) used for the TMS derivative. A 10 ml portion of urine (hydrolysed at pH 1, with 0.05 ml 10% EDTA for 20 min at 100°C or unhydrolysed as appropriate) is mixed with 0.4 ml 10% EDTA (ethylenediamine tetra-acetic acid disodium salt) and 0.4 ml 10% ascorbic acid and adjusted to pH 8.4. Catechols are adsorbed on a column (i.d. 5 mm) of 1 g acid-washed alumina [lo] from which dust had been removed. This was done by repeatedly pouring a stream of dry alumina through a funnel placed 3 ft above a beaker. The fines were carried out of the beaker by air turbulence. The column was washed with 7.5 ml 0.2 M sodium acetate adjusted to pH 8.4 and containing 0.2% EDTA and 0.2% ascorbic acid. This was followed by 7.5 ml water. Catechols were then eluted with 0.5 M acetic acid (8 ml). A 20 ~1 fraction was evaporated to dryness by lyophilisation in a
250
“Reacti-Vial” of 1 ml capacity and the acids were esterified by adding 2 N HCl in dry ethanol (50 ~1) and standing at room temperature for 30 min. The ethanolic HCl was evaporated to dryness in vacua from the “Reacti-Vial”. Its plastic cap and liner were also placed under vacuum with the “Reacti-Vial” for about 20 min. Omission of this step may lead to instability of the derivative. The PFP derivative was formed by heating at 65°C for 40 min with 20 ~1 acetonitrile and 100 1.11PFP anhydride. The reagent was evaporated to dryness in a stream of nitrogen and the residue dissolved in a suitable volume (e.g. 0.1-0.5 ml) of dry ethyl acetate containing ol-lindane as internal standard. Portions of this were chromatographed at 200°C on a 12 ft column of 10% SE 54 supported on 80-100 mesh Chromosorb WHP and derivatives detected by electron capture. The procedure was calibrated with an internal standard, by adding initially 10 pg or 20 pg of pure DOPAC to a duplicate portion of one or more of the urines in the batch under test. A typical chromatogram of a urine extract is shown in Fig. 1. The DOPAC peak is one of the largest observed and very little interference from other compounds has been encountered. To obtain chromatograms which are quantitatively reproducible, it has been found necessary to prime the column with 3 ng of DOPAC derivative. This treatment increased the detector response of the DOPAC derivative relative to cu-lindane by about 10% over a period of 5 h. But more significantly, the height of the peak of the DOPAC derivative then became linear with concentration, within the working range of the method (0.005-0.07 ng per injection). Repeated injections of the same sample gave a standard deviation of + 6%. A series of urine samples assayed in duplicate gave good replication: the differ-
t 0
Fig.
5 1. Chromatogram
10 from
a typical
urinary
assay
of 3.4-dihydroxyphenylacetic
acid
(DOPAC).
251
ence between pairs, expressed as a percentage of the mean for the pair, gave a standard deviation for the overall method of 8.6%. The recovery of DOPAC by this technique was over 90%. To test whether the lyophilisation step in the procedure was essential, a set of replicate portions of column eluates was lyophilised and another set evaporated in a stream of nitrogen. The latter showed a thin crust of aluminium acetate on the inside of the vial which was insoluble in ethanolic HCl, and might have been expected to trap DOPAC. However, this fear was unfounded, as no significant differences were observed between the sets on assay. When applied to human urine, this method detected the presence of 0.33-1.85 mg of free DOPAC in 24 h urine collections from 12 subjects with a mean + SD. of 0.88 f 0.32 mg per 24 h. The range of total DOPAC output was 1.12-2.95 mg, with a mean f S.D. of 1.79 + 0.63 mg per 24 h. The proportion of conjugated DOPAC was 47 + 20% (mean + S.D.). These results are similar to those obtained by other workers using an entirely different technique [l-3]. References 1
U.S.
2
H. Weil-Malherbe
Y. Euler,
3
E.R.B.
4
L.B.
Smith
5
A.H. C.R.J.
7
D.B.
8
F. Karoum,
9
Anton.
M.
D.F.
Ph.D.
Thesis,
CO. C.R.J.
C.R.J.
Ruthven
Uppsal., 74
Chim.
Acta,
(1964)
379
and C.M.
Ruthven
Williams,
of London. and
M. Sandier,
and M.
Med. Med..
43
Ruthven
and
Sot. Clin.
Clin.
University
C.R.J.
Ruthven Anah.
Sayre
Acta
J. Lab.
Medicine,
Grew,
C.R.J.
F. Karoum, Wong,
Jacoby,
F. Karoum,
K.P.
F. Lishajko,
van Buren.
H. Weil-Malherbe.
G.A.
Ruthven, Caine,
and
and J.M. and
Page and
6
10
I. Floding
and
Sandier,
35
64
(1969)
(1971)
Am. 1965.
42
221
(1967)
and
M. Sandier.
Br. J. Pharmacol.,
Clin.
Acta.
Chim.
20
(1968)
M. Sandier,
Clin.
Chim.
Clin.
Acta.
47
Chim.
217
505
J. Med.. pp.
(1959) 305
Acta,
(1973)
1469
242 37
(1969)
59
427 24 215
(1969)
341
Clinica Chimica Acta, 59 (1975) 249-251 @ Elsevier Scientific Publishing Company, Amsterdam -
Printed in The Netherlands
SHORT COMMUNICATION CCA 6888 SPECIFIC GAS CHROMATOGRAPHIC MEASUREMENT 3,4-DIHYDROXYPHENYLACETIC ACID
OF URINARY
M.W. WEG, C.R.J. RUTHVEN, B.L. GOODWIN and M. SANDLER Bernhard Baron Memorial Research Laboratories and Institute of Obstetrics and Gynaecology, Queen Charlotte’s Maternity Hospital, Goldhawk Road, London W6 OXG (U.K.)
(Received October 14, 1974)
The endogenous excretion of 3,4_dihydroxyphenylacetic acid (DOPAC) is relatively low [l-3] and this has created problems in its assay. The determination of this major metabolite of L-DOPA and dopamine has assumed particular importance in the biochemical investigation of catecholamine-secreting tumours [4-61 and in monitoring the treatment of parkinsonism with L-DOPA [71. In our experience the estimation of DOPAC at normal urinary output concentrations by gas-liquid chromatography (GLC) of the methyl ester/trimethylsilyl (TMS) ether [8,9] has been unsatisfactory, even using support-coated open tubular (SCOT) columns. Not only does the peak occasionally coalesce with that of the hippuric acid derivative, but there is another compound in the solvent extract that has the same retention time on the stationary phase (SE 52) as the DOPAC derivative. In order to remove unwanted material, isolation of the DOPAC on alumina has therefore been substituted for solvent extraction. In addition, preparation of the pentafluoropropionyl (PFP) derivative has permitted analysis by the far more sensitive electron capture detector (ECD) in place of the flame ionisation detector (FID) used for the TMS derivative. A 10 ml portion of urine (hydrolysed at pH 1, with 0.05 ml 10% EDTA for 20 min at 100°C or unhydrolysed as appropriate) is mixed with 0.4 ml 10% EDTA (ethylenediamine tetra-acetic acid disodium salt) and 0.4 ml 10% ascorbic acid and adjusted to pH 8.4. Catechols are adsorbed on a column (i.d. 5 mm) of 1 g acid-washed alumina [lo] from which dust had been removed. This was done by repeatedly pouring a stream of dry alumina through a funnel placed 3 ft above a beaker. The fines were carried out of the beaker by air turbulence. The column was washed with 7.5 ml 0.2 M sodium acetate adjusted to pH 8.4 and containing 0.2% EDTA and 0.2% ascorbic acid. This was followed by 7.5 ml water. Catechols were then eluted with 0.5 M acetic acid (8 ml). A 20 ~1 fraction was evaporated to dryness by lyophilisation in a
250
“Reacti-Vial” of 1 ml capacity and the acids were esterified by adding 2 N HCl in dry ethanol (50 ~1) and standing at room temperature for 30 min. The ethanolic HCl was evaporated to dryness in vacua from the “Reacti-Vial”. Its plastic cap and liner were also placed under vacuum with the “Reacti-Vial” for about 20 min. Omission of this step may lead to instability of the derivative. The PFP derivative was formed by heating at 65°C for 40 min with 20 ~1 acetonitrile and 100 1.11PFP anhydride. The reagent was evaporated to dryness in a stream of nitrogen and the residue dissolved in a suitable volume (e.g. 0.1-0.5 ml) of dry ethyl acetate containing ol-lindane as internal standard. Portions of this were chromatographed at 200°C on a 12 ft column of 10% SE 54 supported on 80-100 mesh Chromosorb WHP and derivatives detected by electron capture. The procedure was calibrated with an internal standard, by adding initially 10 pg or 20 pg of pure DOPAC to a duplicate portion of one or more of the urines in the batch under test. A typical chromatogram of a urine extract is shown in Fig. 1. The DOPAC peak is one of the largest observed and very little interference from other compounds has been encountered. To obtain chromatograms which are quantitatively reproducible, it has been found necessary to prime the column with 3 ng of DOPAC derivative. This treatment increased the detector response of the DOPAC derivative relative to cu-lindane by about 10% over a period of 5 h. But more significantly, the height of the peak of the DOPAC derivative then became linear with concentration, within the working range of the method (0.005-0.07 ng per injection). Repeated injections of the same sample gave a standard deviation of + 6%. A series of urine samples assayed in duplicate gave good replication: the differ-
t 0
Fig.
5 1. Chromatogram
10 from
a typical
urinary
assay
of 3.4-dihydroxyphenylacetic
acid
(DOPAC).
251
ence between pairs, expressed as a percentage of the mean for the pair, gave a standard deviation for the overall method of 8.6%. The recovery of DOPAC by this technique was over 90%. To test whether the lyophilisation step in the procedure was essential, a set of replicate portions of column eluates was lyophilised and another set evaporated in a stream of nitrogen. The latter showed a thin crust of aluminium acetate on the inside of the vial which was insoluble in ethanolic HCl, and might have been expected to trap DOPAC. However, this fear was unfounded, as no significant differences were observed between the sets on assay. When applied to human urine, this method detected the presence of 0.33-1.85 mg of free DOPAC in 24 h urine collections from 12 subjects with a mean + SD. of 0.88 f 0.32 mg per 24 h. The range of total DOPAC output was 1.12-2.95 mg, with a mean f S.D. of 1.79 + 0.63 mg per 24 h. The proportion of conjugated DOPAC was 47 + 20% (mean + S.D.). These results are similar to those obtained by other workers using an entirely different technique [l-3]. References 1
U.S.
2
H. Weil-Malherbe
Y. Euler,
3
E.R.B.
4
L.B.
Smith
5
A.H. C.R.J.
7
D.B.
8
F. Karoum,
9
Anton.
M.
D.F.
Ph.D.
Thesis,
CO. C.R.J.
C.R.J.
Ruthven
Uppsal., 74
Chim.
Acta,
(1964)
379
and C.M.
Ruthven
Williams,
of London. and
M. Sandier,
and M.
Med. Med..
43
Ruthven
and
Sot. Clin.
Clin.
University
C.R.J.
Ruthven Anah.
Sayre
Acta
J. Lab.
Medicine,
Grew,
C.R.J.
F. Karoum, Wong,
Jacoby,
F. Karoum,
K.P.
F. Lishajko,
van Buren.
H. Weil-Malherbe.
G.A.
Ruthven, Caine,
and
and J.M. and
Page and
6
10
I. Floding
and
Sandier,
35
64
(1969)
(1971)
Am. 1965.
42
221
(1967)
and
M. Sandier.
Br. J. Pharmacol.,
Clin.
Acta.
Chim.
20
(1968)
M. Sandier,
Clin.
Chim.
Clin.
Acta.
47
Chim.
217
505
J. Med.. pp.
(1959) 305
Acta,
(1973)
1469
242 37
(1969)
59
427 24 215
(1969)
341