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Removal of interference by antihypertensive drugs in the spectrophotometric assay of metanephrines
Clinica Chimica Acta, 169 (1987) 117-120 Elsevier
117
CCA 04009
Short communication
Removal of interference by antihypertensive drugs in the spectrophotometric assay of metanephrines Georgina A. Crawford ‘, Eileen D.M. Gallery a and Akos Z. Gyory b oDepartment of Renal Medicine, Royal North Shore Hospital and b Department of Medicine, Universicv of Sydney, New South Wales (Australia) (Received
18 March
1987: revision received 4 July 1987; accepted
Key words: Metanephrines;
Antihypertensive
drugs;
after revision 6 August
1987)
Pheochromocytoma
Introduction The spectrophotometric assay of total urinary metanephrines provides a method for detecting the presence of a pheochromocytoma without the need for specialised high pressure liquid chromatography equipment [l]. Most antihypertensive drugs do not interfere in this assay when present alone. However, we have found that certain combinations of the antihypertensive agents labetalol or oxprenolol with other drugs may give spuriously elevated results. It has been suggested that interfering material can be removed by an additional extraction step when an elevated value is obtained [2,3]. This paper presents the values measured with the extraction procedure for patients with a pheochromocytoma, normal controls and those with elevated metanephrine values due to antihypertensive drugs. Materials and methods The assay of urinary metanephrines was performed as described previously [l], except that 0.9 ml of column effluent was added to polypropylene tubes (13 ml capacity with polyethylene screw caps, Disposable Products Pty. Ltd., Adelaide, South Australia) in duplicate. Next, 22.5 ~1 of sodium metaperiodate and metabisulfite solutions were added as before and urinary metanephrines determined in the same manner as previously. Samples were not discarded after determination of absorbance (OD). For routine assays, when a patient’s value was above the upper limit of normal (6.5 pmo1/24 h), 15 ~1 of phenol red solution (0.5 pmol/l) was added to all tubes
Correspondence to: Dr. G.A. Crawford, Department Leonards, 2065 New South Wales. Australia.
0009-8981/87/$03.50
of Renal Medicine,
0 1987 Elsevier Science Publishers
Royal North
B.V. (Biomedical
Division)
Shore Hospital,
St.
118
relative to that sample (4 tubes total). The pH was adjusted to 7-8 with 7.8 mol/l phosphoric acid (20-40 /.~l), 8 ml of toluene (analytical grade) was added and the tubes capped and mixed by inversion for 10 minutes. After centrifugation at 1000 X g for 4 min, 7 ml of the upper toluene phase was transferred to similar plastic tubes containing 1.2 ml of 1 mol/l K&O,. The tubes were capped, mixed and centrifuged as before. The carbonate layer was transferred to a cuvette: the majority of the upper toluene layer was removed by suction and a portion of the lower layer transferred with a Pasteur pipette to the cuvette (capacity 0.8 ml) and the OD determined at 360 nm. Care was taken to exclude any toluene in the aqueous layer in the cuvette as this would lead to an elevated OD. The extracted urinary metanephrines (pmo1/24 h) were determined with the calculations used previously [l]. Results Toluene extraction did not significantly change the urinary metanephrine values obtained from 5 patients with a proven pheochromocytoma (Table I). In 8 patients without a pheochromocytoma whose therapy included oxprenolol (80-360 mg/day for 5 days-13 mth) or labetalol (600 mg/day for 2 days-2 mth), elevated metanephrine values were significantly reduced to within the normal range after toluene extraction. In a study of 25 healthy control subjects not taking any medication, values were slightly reduced after toluene extraction, but this did not reach statistical significance. Samples with high metanephrine values could be extracted on the same day as the original measurement as the procedure took < 3 h. However, column extracts could be frozen before extraction and kept for several weeks without loss of activity. Precision data are shown in Table II.
TABLE
I
Urinary
metanephrines
measured
with and without
Subject
Healthy controls Patients with a pheochromocytoma Hypertensive patients taking: Oxprenolol, hydralazine Oxprenolol, mefruside, verapamil Labetalol, mefruside Labetalol, captopril, atenolol. prazosin a Values shown are the range. h Difference after extraction is significant
toluene No.
25 5 5 1 1 1I
p < 0.001.
extraction Urinary metanephrines (pmo1/24 h)
a
Without extraction
With extraction
1.1- 6.1 22.6-84.0
1.6- 4.5 27.8-85.0
7.5-12.0
2.3-
4.5 ’
119 TABLE Precision
II data for metanephrines
measured
Without
extraction
Mean ( n mo1/24
with and without
extraction
With extraction cv
h)
toluene
(W)
Mean (pmo1/24
cv h)
(%E)
Inter-assay (3 urines examined over 10 days)
2.4 6.3 19.5
16.4 9.5 12.9
1.6 4.2 17.9
29.4 26.8 25.2
Intra-assay (3 urines examined 10 times in 1 day)
1.5 6.3 16.5
8.9 10.1 7.4
0.9 4.1 19.3
23.4 17.0 11.3
After extraction to 0.233 indicating in the process.
of the standard on 8 separate occasions, a mean OD of 0.323 fell that on each occasion 28% f 4.2% of the original sample was lost
Discussion Toluene extraction of vanillin formed in the assay of metanephrines removes interference which may occur when oxprenolol or labetalol are used in conjunction with other drugs. The method can be easily added on to the routine”assay of metanephrines whenever a high value is obtained. The use of recovery samples removes the effect of interference by most antihypertensive drugs, particularly when only one drug is present [l]. However, in some cases where drugs or their metabolites are similar to vanillin, an additional extraction procedure is necessary. In this study, a combination of drugs rather than a single antihypertensive drug appeared to show this type of interference. Vanillin itself, for example from dietary sources, does not interfere as it is not retained by the cation exchange columns used in the assay. The large CV values of the method are due to the coupling of the toluene extraction procedure (with CV 3.9913%) to the spectrophotometric assay of metanephrines (with CV 7.4-16.4s). The variation of the extraction procedure is most likely due to the double solvent extraction step which is necessary to remove all the drug metabolite interfering compounds. *Despite the high CV of this method, the difference between those with a pheochromocytoma and those without could readily be identified. Furthermore, in all cases examined, high values due to drug interference returned to within the normal range after extraction. References 1 Crawford GA, Gallery EDM, Gyory AZ. An improved spectrophotometric assay of urinary metanephrines. Clin Chim Acta 1986;157:121-126. 2 Ruthven RJ, Sandler M. The estimation of 4-hydroxy-3-methyl-phenylgycol and total metadrenalines in human urine. Clin Chim Acta 1965;12:318-324. 3 Lax P, Ruthven CRJ, Sandler M. Labetalol and urinary metadrenalines. Br Med J 1979;1:953.
117
CCA 04009
Short communication
Removal of interference by antihypertensive drugs in the spectrophotometric assay of metanephrines Georgina A. Crawford ‘, Eileen D.M. Gallery a and Akos Z. Gyory b oDepartment of Renal Medicine, Royal North Shore Hospital and b Department of Medicine, Universicv of Sydney, New South Wales (Australia) (Received
18 March
1987: revision received 4 July 1987; accepted
Key words: Metanephrines;
Antihypertensive
drugs;
after revision 6 August
1987)
Pheochromocytoma
Introduction The spectrophotometric assay of total urinary metanephrines provides a method for detecting the presence of a pheochromocytoma without the need for specialised high pressure liquid chromatography equipment [l]. Most antihypertensive drugs do not interfere in this assay when present alone. However, we have found that certain combinations of the antihypertensive agents labetalol or oxprenolol with other drugs may give spuriously elevated results. It has been suggested that interfering material can be removed by an additional extraction step when an elevated value is obtained [2,3]. This paper presents the values measured with the extraction procedure for patients with a pheochromocytoma, normal controls and those with elevated metanephrine values due to antihypertensive drugs. Materials and methods The assay of urinary metanephrines was performed as described previously [l], except that 0.9 ml of column effluent was added to polypropylene tubes (13 ml capacity with polyethylene screw caps, Disposable Products Pty. Ltd., Adelaide, South Australia) in duplicate. Next, 22.5 ~1 of sodium metaperiodate and metabisulfite solutions were added as before and urinary metanephrines determined in the same manner as previously. Samples were not discarded after determination of absorbance (OD). For routine assays, when a patient’s value was above the upper limit of normal (6.5 pmo1/24 h), 15 ~1 of phenol red solution (0.5 pmol/l) was added to all tubes
Correspondence to: Dr. G.A. Crawford, Department Leonards, 2065 New South Wales. Australia.
0009-8981/87/$03.50
of Renal Medicine,
0 1987 Elsevier Science Publishers
Royal North
B.V. (Biomedical
Division)
Shore Hospital,
St.
118
relative to that sample (4 tubes total). The pH was adjusted to 7-8 with 7.8 mol/l phosphoric acid (20-40 /.~l), 8 ml of toluene (analytical grade) was added and the tubes capped and mixed by inversion for 10 minutes. After centrifugation at 1000 X g for 4 min, 7 ml of the upper toluene phase was transferred to similar plastic tubes containing 1.2 ml of 1 mol/l K&O,. The tubes were capped, mixed and centrifuged as before. The carbonate layer was transferred to a cuvette: the majority of the upper toluene layer was removed by suction and a portion of the lower layer transferred with a Pasteur pipette to the cuvette (capacity 0.8 ml) and the OD determined at 360 nm. Care was taken to exclude any toluene in the aqueous layer in the cuvette as this would lead to an elevated OD. The extracted urinary metanephrines (pmo1/24 h) were determined with the calculations used previously [l]. Results Toluene extraction did not significantly change the urinary metanephrine values obtained from 5 patients with a proven pheochromocytoma (Table I). In 8 patients without a pheochromocytoma whose therapy included oxprenolol (80-360 mg/day for 5 days-13 mth) or labetalol (600 mg/day for 2 days-2 mth), elevated metanephrine values were significantly reduced to within the normal range after toluene extraction. In a study of 25 healthy control subjects not taking any medication, values were slightly reduced after toluene extraction, but this did not reach statistical significance. Samples with high metanephrine values could be extracted on the same day as the original measurement as the procedure took < 3 h. However, column extracts could be frozen before extraction and kept for several weeks without loss of activity. Precision data are shown in Table II.
TABLE
I
Urinary
metanephrines
measured
with and without
Subject
Healthy controls Patients with a pheochromocytoma Hypertensive patients taking: Oxprenolol, hydralazine Oxprenolol, mefruside, verapamil Labetalol, mefruside Labetalol, captopril, atenolol. prazosin a Values shown are the range. h Difference after extraction is significant
toluene No.
25 5 5 1 1 1I
p < 0.001.
extraction Urinary metanephrines (pmo1/24 h)
a
Without extraction
With extraction
1.1- 6.1 22.6-84.0
1.6- 4.5 27.8-85.0
7.5-12.0
2.3-
4.5 ’
119 TABLE Precision
II data for metanephrines
measured
Without
extraction
Mean ( n mo1/24
with and without
extraction
With extraction cv
h)
toluene
(W)
Mean (pmo1/24
cv h)
(%E)
Inter-assay (3 urines examined over 10 days)
2.4 6.3 19.5
16.4 9.5 12.9
1.6 4.2 17.9
29.4 26.8 25.2
Intra-assay (3 urines examined 10 times in 1 day)
1.5 6.3 16.5
8.9 10.1 7.4
0.9 4.1 19.3
23.4 17.0 11.3
After extraction to 0.233 indicating in the process.
of the standard on 8 separate occasions, a mean OD of 0.323 fell that on each occasion 28% f 4.2% of the original sample was lost
Discussion Toluene extraction of vanillin formed in the assay of metanephrines removes interference which may occur when oxprenolol or labetalol are used in conjunction with other drugs. The method can be easily added on to the routine”assay of metanephrines whenever a high value is obtained. The use of recovery samples removes the effect of interference by most antihypertensive drugs, particularly when only one drug is present [l]. However, in some cases where drugs or their metabolites are similar to vanillin, an additional extraction procedure is necessary. In this study, a combination of drugs rather than a single antihypertensive drug appeared to show this type of interference. Vanillin itself, for example from dietary sources, does not interfere as it is not retained by the cation exchange columns used in the assay. The large CV values of the method are due to the coupling of the toluene extraction procedure (with CV 3.9913%) to the spectrophotometric assay of metanephrines (with CV 7.4-16.4s). The variation of the extraction procedure is most likely due to the double solvent extraction step which is necessary to remove all the drug metabolite interfering compounds. *Despite the high CV of this method, the difference between those with a pheochromocytoma and those without could readily be identified. Furthermore, in all cases examined, high values due to drug interference returned to within the normal range after extraction. References 1 Crawford GA, Gallery EDM, Gyory AZ. An improved spectrophotometric assay of urinary metanephrines. Clin Chim Acta 1986;157:121-126. 2 Ruthven RJ, Sandler M. The estimation of 4-hydroxy-3-methyl-phenylgycol and total metadrenalines in human urine. Clin Chim Acta 1965;12:318-324. 3 Lax P, Ruthven CRJ, Sandler M. Labetalol and urinary metadrenalines. Br Med J 1979;1:953.