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Column chromatographic separation and fluorometric estimation of metanephrine and normetanephrine in urine
CLINICA CHIMICA ACTA
453
COLUMN CHROMATOGRAPHIC MATION
OF METANEPHRINE
ZDZISLAW
KAHANE
AND
PER
SEPARATION
AND FLUOROMETRIC
AND NORMETANEPHRINE
ESTI-
IN URINE
VESTERGAARD
Research Center, Rockland State Hospital, Orangeburg, New York (U.S.A.) (Received
April ‘4,
1969)
SUMMARY
A method is proposed for the determination of urinary metanephrine (M) and normetanephrine (NM) after the chromatographic separation of the two compounds on cellulose phosphate columns. The trihydroxyindole reaction is used for the fluorometric quantitation of the compounds. Very close to 100% recovery of the metanephrines was found with this method and good separation between metanephrine and normetanephrine was consistently obtained.
We have previously published a method for the estimation of urinary normetanephrine and metanephrine based on the application of the trihydroxyindole reaction to highly purified extracts containing a mixture of both compound+. A separate estimate of each of the two compounds was obtained by performing the trihydroxyindole reaction on this extract at different pH and in different buffers. We have in our work on a number of occasions-particularly in studies of the effect of phsychophamlaceuticals on catecholamine metabolism-felt the need for a method that would include a chromatographic separation of the two compounds before estimation. We have developed such a procedure as a companion mehod to the previously published assay1 to be used when the higher specificity and accuracy and the better recovery after the chromatographic separation may justify the extra work and the additional instrumentation needed for the more complicated method. MATERIALS
Cation exchavzger P II (Cellulose phosphate, Column Chromedia, Reeve Angel, Cat. #21111). This material, suggested for the separation of catecholamines by Merrills2, was precycled through suspension in 0.5 N HCl and decantation; this procedure was repeated several times until the final supernatant remained nearly colorless and most of the fines were separated. The ion exchanger was then filtered and washed with a small amount of water on a vacuum filter. The material was resuspended in 0.5 N NH,OH and left for about 30 minutes. It was then filtered, washed with a Clin. Chim. Acta, 25 (1969) 453-458
KAHANE,VESTERGAARD
454
small amount of water and resuspended in water. The supernatant was adjusted to pH 6.2 with acetic acid. The ion exchanger was finally filtered and stored in wet form in a well closed container in the refrigerator. 0.25 A4 ami 0.05 M ammonium acetate buffers: Dissolve 0.25 and 0.05 1cI respectively of ammonium acetate and 6.0 g of urea in one liter of water. I mg of sodium fluoride should be added to these solutions as a preservative. The above buffer solutions are adjusted to pH 6.1 with ammonia or acetic acid. Chromatographic columns: have an id. of 6 mm and a length of 400 mm with a reservoir funnel attached through a standard tapered joint at the top. A teflon stopcock is used at the bottom of the column. Chromatographic pumps: We have used a Milton Roy chromatographic minipump (#2rg6-31). This is a duplex pump which makes it possible both to form the gradient and elute from the column with the same pump. METHODS
Chromatographic
separation of the metanephrines
ml of urine is hydrolyzed and the metanephrines adsorbed on a Dowex 50 W-X:! column as described previouslyl. The eluate is left overnight in a vacuum desiccator. The column is prepared in the following way: IO g of the precycled P II exchanger are suspended in 200 ml of 0.05 M ammonium acetate buffer and mixed for 30 min. A magnetic stirrer should not be used for this step, as this produces fines and greatly reduces the filtering speed. The pH of the supernatant should not rise over 6.4; otherwise the operation should be repeated. The supernatant is now discarded and the ion exchanger poured into the column and allowed to settle by gravity. For a final equilibration 20 ml of 0.05 A4 ammonium acetate buffer is pumped through the column at a flow rate of about I ml per IO min. The stopcock at the bottom should be completely open; high pressure should not be applied to the P II ion exchanger as this results in compression of the column material and brigs the filtration to a complete standstill. The pump is then turned off and the column left connected with the stopcock closed till the next day when the small amount of buffer on top of the column is removed by a syringe. The eluate from the Dowex column is taken from the desiccator and after adjustment to pH 6.1 poured through the reservoir funnel attached to the top of the column and the beaker washed with small amounts of 0.05 M ammonium acetate buffer. The stopcock at the bottom of the column is opened and the eluate passes under gravity. The funnel is now disconnected and 20 ml of the acetate buffer is pumped through the column at a speed not exceeding I ml per IO min. The chromatography is accomplished with a gradient and for this purpose a duplex pump is used: one for the formation of the gradient and one for the elution of the column. The gradient is formed through the addition of the 0.25 M ammonium acetate buffer to the 0.05 M buffer, both previously adjusted to pH 6.1. Of the weak buffer 50 ml are placed in an Erlenmeyer flask with an outlet at the bottom, connected with the intake of the eluting pump; to this buffer are added with the second pump 50 ml of the stronger buffer at a flow rate equal to the speed of the elution of the column. A thorough mixing of both buffers should be accomplished through the use of 25
Clin.Chim. Acta,
25 (1969) 453-458
METANEPHRINE
AND NORMETANEPHRINE
455
IN URINE
a magnetic stirrer. The wash with 20 ml 0.05 M buffer and the first 25 ml of gradient are discarded and the following eluate collected in 2 ml fractions. There are at this point two ways to proceed with the analysis. The trihydroxyindole reaction can be applied to each chromatographic fraction or it is possible to locate the metanephrine and normetanephrine fractions by examining their native fluorescence and then to apply the trihydroxyindole method to the pooled fractions containing each of these compounds. Locating the metanephrine and normetanephrine fractions by native juorescence The native fluorescence can be used for the separation of fractions containing normetanephrine from those of metanephrine but is not useful for the quantitation of these compounds on account of the presence of other contaminating fluorescent material. The fractions were diluted with 2 ml water and the native fluorescence measured with the Farrand spectrofluorometer; the wavelengths were adjusted before reading to 285 m/c for excitation and 330 m,u for fluorescence which correspond with the maxima for pure metanephrine and normetanephrine; a Corning filter #o-54 was inserted on the fluorescence side of the instrument for better separation from the exciting light. The first continual rise indicates the start of the metanephrine peak. The values peak and then decline, and the last fraction with the lowest reading is added to metanephrine. The readings for the next fractions show a second peak corresponding to the normetanephrine and fall to blank values at the end of this peak. The tubes are separated into fractions according to the native fluorescence and the metanephrine and normetanephrine tube areas pooled giving volumes varying from 20 to 32 ml for each of these compounds. Fluorometric quantitation of the metanephrines The quantitation of both metanephrines is performed on the basis of the trihydroxyindole method at pH 7 using the procedure as given in our previous publication1 with the following modifications: Standard solutions are diluted with 0.05 M buffer to contain 0.2 pg/ml of M respective NM. 2 ml sample and standard are taken for the assay, diluted with 4 ml borate buffer and adjusted to pH 7. From there on the procedure is as previously described. When the method has been used after separation and pooling of fractions according to native fluorescence the normetanephrine fraction has been checked for metanephrine by performing also the lutine reaction at pH 2.85 as previously described’. We have in all determinations found metanephrine missing from the normetanephrine fraction indicating that the separation of metanephrine and normetanephrine by native fluorescence is a valid procedure. Internal standards have been used throughout. There seems however to be little need for them, since they came out almost identical with external standards in all experiments. In case the trihydroxyindole method is applied to every fraction separately, 4 ml of borate buffer are added and otherwise the procedure is as described above. RESULTS
A complete chromatographic separation of metanephrine and normetanephrine is accomplished with the proposed technique as shown in Fig. I. It can be seen that Clin. Chim.
Acta,
25 (1969)
453-458
KAHANE, VESTERGAARD
456
NM
200 t 175.
h loo5075. * oJo50. B ” 0.25.
I 0
5
IO
20
I
Frachn
30
,’ 5
35
40
5
0
10
15
20
Fraction
No
25
30
35
40
No.
Fig. I. Separation of a metanephrine and normetanephrine standard (left) and normetanephrine and methanephrine in a pooled urine elutate (right). The trihydroxyindole reaction was performed on each fraction.
the separation is as effective in a urinary extract from pooled urine as for standards. It should be emphasized, as can be seen in the differences in elution volume in the two runs, that the elution volume varies from column to column. We have not been able to pack these columns so that we could obtain equal elution volumes. We have always however been able to obtain a clear separation. It is therefore necessary for each column to separate into metanephrine and normetanephrine fractions using the native fluorescence as a guide before performing the trihydroxyindole reaction on the pooled fraction or to perform the trihydroxyindole method fraction by fraction. In our experiment with 5 Ftg of each standard we obtained a recovery of 4.87 for metanephrine and 4.93 for normetanephrine. In order to compare the chromatographic method with our previous’ procedure an experiment was designed in which IO urine samples were analyzed with both methods. Recovery experiments were performed adding before hydrolysis 3 big or TABLE
I
RECOVERY A
WHEN -_
5 ,Ug AMOUNTSOF
OF 3 ,AUgAND
COMPARISON
BETWEEN
PERFORMED
ON
VALUES THE
SAME
#
NM
Recovery
WITH
THE
AND
PROPOSED
METANEPHRINE METHOD
ADDED
AND
THE
TO
PREVIOUS1
URINE
.__
Method
Recov-
M
of3rug erY in % oy 5 Pg NM added
Recovery of3iug or 5 fig M added
G
NM
Old A&hod ____.. NM in % of chrom. meth.
e+,Y in %
M in “/ of chrom. meth.
I
5.32
2.83
94
4.72
2.54
84
4.83
91
3.88
82
2
5.35
3.07
IO2
4.20
2.90
96 95 103 95 9I 108 90
86 97 II3 80 90
3.48 2.44 3.12 4.84 2.46 4.50 IO.70
83 106 76 Io5 92 ‘03 IO9
3.35
124
102
5.44
Io6
3 4 : ; 9 IO
2.18 13.30 8.35 5.04 2.13 8.35 3.36
2.87 5.20 4-75 5.16 4.42 5.00 5.74
6.10
5.00
Average Recovery Clin.
Chim.
Acta,
‘03
4.33 9.80 2.70
2.89 5.15 4.75 4.56 4.52 5.40 5.00
IO0
4.60 2.10 15.00 7.50 4.03 2.46 7.20 3.06
100
5.14
4.84
96
6.20
96 IO4 95 Io3 IO0 88
98.5
25 (1969) 453-458
AND
METHOD
URINES
Chromatographic Samplr
NORMETANEPHRINE
OBTAINED
2.31 4.ro 2.68 4.60
95.6
II5 86 91
METANEPHRINEANDNORMETANEPHRINE IN URINE
457
5 pg of metanephrine and normetanephrine to separate samples of urine which were then taken through the full procedure. It can be seen from the results listed in Table I that the recovery in the chromatographic method is very close to IOO”/~. DISCUSSION We feel that the proposed method offers clear advantages when compared with the previous’ assay. It has better recovery of normetanephrine. Average recovery in the older method was for 12 analyses 89.5%‘. The similar figure for IO analyses for the new method is 98.5%. The difference in recovery is due to some loss of normetanephrine on the Florex used in the old assay. There is also higher consistency in recovery for both metanephrines. In the non-chromatographic method recoveries for 12 determinations varied between 62.5% and 122~/~for normetanephrine and between 64% and 114% for metanephrine. In contrast similar values for the chromatographic method in IO determinations are: for normetanephrine 88°/o-~030/oand for metanephrine 84%-108%. The more inconsistent results in the old method are probably due to the fact that any error in the quantitation of metanephrine is automatically reflected in the determination of normetanephrine for which the values are obtained through subtraction from the total, whereas after chromatographic separation both compounds are determined independently. That this is not a too serious weakness in the old method is shown by good agreement between “new” and “old” methods demontsrated in Table I. Overall the easier and faster older method would seem quite adequate for general use whereas the more complicated chromatographic separation should be reserved for situations where the highest accuracy is of prime importance. An advantageof the proposed method compared with the previous is that the two compounds are physically separated. This will make it possible to use the method for isotope work where the older method cannot be applied. It will also preclude some of the ambiguity of the method when it is used under conditions where drugs are given and it is unknown whether drug metabolites might interfere with the assay. The chances of separating the metanephrines from possible interfering metabolites would seem to be better when a chromatographic separation of the individual metanephrines is performed. The validity of the separation of the fractions by native fluorescence has been checked by reading all normetanephrine fractions at pH 2.85 for the presence of metanephrine. In no case have measurable amounts of metanephrine been found. It involves considerably more work to perform the trihydroxyindole reaction on each fraction cut. There may be situations where this may be preferable, particularly if there are difficulties in identifying the two fractions by native fluorescence alone. This has not occurred in our material of normal controls and patients treated with various tranquilizers and anti-depressants but might be found in other conditions. The fact that the trihydroxyindole reaction products give a fluorescence approximately IOO times as strong as the native fluorescence and that this reaction is specific for the catecholamines makes the unequivocal separation into metanephrine and normetanephrine peaks easier if the trihydroxyindole reaction is performed on each fraction. It would seem possible that the performance of the trihydroxyindole reaction as Clin. Chim. Ada,
25
(1969) 453-458
KAHANE,
458
VESTERGAARD
performed in this method can be automated by modification of published methodP6 and the fraction by fraction determination using this reaction might then become the preferable way of performing the analysis. Two other procedures have been published for the estimation of urinary metanephrine and normetanephrine after column chromatographic separation. One, the method of Haggendala, uses the eluate from an Amberlite column directly for fluorometry. According to Randrup’ such eluates are frequently colored and have high blanks. The other is the method of Taniguchi et al. 8. This method has been criticized by Weil-Malherbe and Smiths, in their survey of fluorometric methods for the determination of metanephrine and normetanephrine, as having low sensitivity and giving high blanks besides giving very unstable fluorophores. Also Weil-Malherbe and Smith claim they could not reproduce the chromatographic separation. The current method has high sensitivity, low blank readings and gives stable fluorophores. It has consistently given complete separation between the two compounds. ACKNOWLEDGEMENT
This work was supported Center Grant MH 07292.
by General
Support
Grant
FRO5651
and Research
REFERENCES I 2. KAHANE AND P. VESTERGAARD, J. Lab. Gin. Med., 70 (1967) 333. 2 R. J. MERRILLS, in L. T. SKEGGS (Ed.), Automation in Analytical Chemistry, Mediad, New York, 1966. p. 390. 3 R. J. MERRILLS, Anal Biochem., 6 (1963) 272. 4 R. L. ROBINSON AND D. T. WATTS, Clin. Chem., II (1965) 986-997. 5 V. FIORICA, Clin. Chim. Acta, 12 (1965) IgI--197. 6 J. HAGGENDAL, Acta, Physiol. Sand., 56 (1962) 258-266. 7 A. RANDRUP, Clin. Chim. Acta, 6 (1961) 584-586. 8 K. TANIGUCHI, Y. KAKIMOTO AND M. D. ARMSTRONG, J. Lab. Clin. Med., 64 (1964) 469. g H. WEIL-MALHERBE AND E. R. B. SMITH, in G. H. ACHESON (Ed.), Second Symposium on Catecholamines, Williams & Wilkins Company, Baltimore, 1966, p. 331.. C&z. Chim. Acta,
25 (1969) 453-458
453
COLUMN CHROMATOGRAPHIC MATION
OF METANEPHRINE
ZDZISLAW
KAHANE
AND
PER
SEPARATION
AND FLUOROMETRIC
AND NORMETANEPHRINE
ESTI-
IN URINE
VESTERGAARD
Research Center, Rockland State Hospital, Orangeburg, New York (U.S.A.) (Received
April ‘4,
1969)
SUMMARY
A method is proposed for the determination of urinary metanephrine (M) and normetanephrine (NM) after the chromatographic separation of the two compounds on cellulose phosphate columns. The trihydroxyindole reaction is used for the fluorometric quantitation of the compounds. Very close to 100% recovery of the metanephrines was found with this method and good separation between metanephrine and normetanephrine was consistently obtained.
We have previously published a method for the estimation of urinary normetanephrine and metanephrine based on the application of the trihydroxyindole reaction to highly purified extracts containing a mixture of both compound+. A separate estimate of each of the two compounds was obtained by performing the trihydroxyindole reaction on this extract at different pH and in different buffers. We have in our work on a number of occasions-particularly in studies of the effect of phsychophamlaceuticals on catecholamine metabolism-felt the need for a method that would include a chromatographic separation of the two compounds before estimation. We have developed such a procedure as a companion mehod to the previously published assay1 to be used when the higher specificity and accuracy and the better recovery after the chromatographic separation may justify the extra work and the additional instrumentation needed for the more complicated method. MATERIALS
Cation exchavzger P II (Cellulose phosphate, Column Chromedia, Reeve Angel, Cat. #21111). This material, suggested for the separation of catecholamines by Merrills2, was precycled through suspension in 0.5 N HCl and decantation; this procedure was repeated several times until the final supernatant remained nearly colorless and most of the fines were separated. The ion exchanger was then filtered and washed with a small amount of water on a vacuum filter. The material was resuspended in 0.5 N NH,OH and left for about 30 minutes. It was then filtered, washed with a Clin. Chim. Acta, 25 (1969) 453-458
KAHANE,VESTERGAARD
454
small amount of water and resuspended in water. The supernatant was adjusted to pH 6.2 with acetic acid. The ion exchanger was finally filtered and stored in wet form in a well closed container in the refrigerator. 0.25 A4 ami 0.05 M ammonium acetate buffers: Dissolve 0.25 and 0.05 1cI respectively of ammonium acetate and 6.0 g of urea in one liter of water. I mg of sodium fluoride should be added to these solutions as a preservative. The above buffer solutions are adjusted to pH 6.1 with ammonia or acetic acid. Chromatographic columns: have an id. of 6 mm and a length of 400 mm with a reservoir funnel attached through a standard tapered joint at the top. A teflon stopcock is used at the bottom of the column. Chromatographic pumps: We have used a Milton Roy chromatographic minipump (#2rg6-31). This is a duplex pump which makes it possible both to form the gradient and elute from the column with the same pump. METHODS
Chromatographic
separation of the metanephrines
ml of urine is hydrolyzed and the metanephrines adsorbed on a Dowex 50 W-X:! column as described previouslyl. The eluate is left overnight in a vacuum desiccator. The column is prepared in the following way: IO g of the precycled P II exchanger are suspended in 200 ml of 0.05 M ammonium acetate buffer and mixed for 30 min. A magnetic stirrer should not be used for this step, as this produces fines and greatly reduces the filtering speed. The pH of the supernatant should not rise over 6.4; otherwise the operation should be repeated. The supernatant is now discarded and the ion exchanger poured into the column and allowed to settle by gravity. For a final equilibration 20 ml of 0.05 A4 ammonium acetate buffer is pumped through the column at a flow rate of about I ml per IO min. The stopcock at the bottom should be completely open; high pressure should not be applied to the P II ion exchanger as this results in compression of the column material and brigs the filtration to a complete standstill. The pump is then turned off and the column left connected with the stopcock closed till the next day when the small amount of buffer on top of the column is removed by a syringe. The eluate from the Dowex column is taken from the desiccator and after adjustment to pH 6.1 poured through the reservoir funnel attached to the top of the column and the beaker washed with small amounts of 0.05 M ammonium acetate buffer. The stopcock at the bottom of the column is opened and the eluate passes under gravity. The funnel is now disconnected and 20 ml of the acetate buffer is pumped through the column at a speed not exceeding I ml per IO min. The chromatography is accomplished with a gradient and for this purpose a duplex pump is used: one for the formation of the gradient and one for the elution of the column. The gradient is formed through the addition of the 0.25 M ammonium acetate buffer to the 0.05 M buffer, both previously adjusted to pH 6.1. Of the weak buffer 50 ml are placed in an Erlenmeyer flask with an outlet at the bottom, connected with the intake of the eluting pump; to this buffer are added with the second pump 50 ml of the stronger buffer at a flow rate equal to the speed of the elution of the column. A thorough mixing of both buffers should be accomplished through the use of 25
Clin.Chim. Acta,
25 (1969) 453-458
METANEPHRINE
AND NORMETANEPHRINE
455
IN URINE
a magnetic stirrer. The wash with 20 ml 0.05 M buffer and the first 25 ml of gradient are discarded and the following eluate collected in 2 ml fractions. There are at this point two ways to proceed with the analysis. The trihydroxyindole reaction can be applied to each chromatographic fraction or it is possible to locate the metanephrine and normetanephrine fractions by examining their native fluorescence and then to apply the trihydroxyindole method to the pooled fractions containing each of these compounds. Locating the metanephrine and normetanephrine fractions by native juorescence The native fluorescence can be used for the separation of fractions containing normetanephrine from those of metanephrine but is not useful for the quantitation of these compounds on account of the presence of other contaminating fluorescent material. The fractions were diluted with 2 ml water and the native fluorescence measured with the Farrand spectrofluorometer; the wavelengths were adjusted before reading to 285 m/c for excitation and 330 m,u for fluorescence which correspond with the maxima for pure metanephrine and normetanephrine; a Corning filter #o-54 was inserted on the fluorescence side of the instrument for better separation from the exciting light. The first continual rise indicates the start of the metanephrine peak. The values peak and then decline, and the last fraction with the lowest reading is added to metanephrine. The readings for the next fractions show a second peak corresponding to the normetanephrine and fall to blank values at the end of this peak. The tubes are separated into fractions according to the native fluorescence and the metanephrine and normetanephrine tube areas pooled giving volumes varying from 20 to 32 ml for each of these compounds. Fluorometric quantitation of the metanephrines The quantitation of both metanephrines is performed on the basis of the trihydroxyindole method at pH 7 using the procedure as given in our previous publication1 with the following modifications: Standard solutions are diluted with 0.05 M buffer to contain 0.2 pg/ml of M respective NM. 2 ml sample and standard are taken for the assay, diluted with 4 ml borate buffer and adjusted to pH 7. From there on the procedure is as previously described. When the method has been used after separation and pooling of fractions according to native fluorescence the normetanephrine fraction has been checked for metanephrine by performing also the lutine reaction at pH 2.85 as previously described’. We have in all determinations found metanephrine missing from the normetanephrine fraction indicating that the separation of metanephrine and normetanephrine by native fluorescence is a valid procedure. Internal standards have been used throughout. There seems however to be little need for them, since they came out almost identical with external standards in all experiments. In case the trihydroxyindole method is applied to every fraction separately, 4 ml of borate buffer are added and otherwise the procedure is as described above. RESULTS
A complete chromatographic separation of metanephrine and normetanephrine is accomplished with the proposed technique as shown in Fig. I. It can be seen that Clin. Chim.
Acta,
25 (1969)
453-458
KAHANE, VESTERGAARD
456
NM
200 t 175.
h loo5075. * oJo50. B ” 0.25.
I 0
5
IO
20
I
Frachn
30
,’ 5
35
40
5
0
10
15
20
Fraction
No
25
30
35
40
No.
Fig. I. Separation of a metanephrine and normetanephrine standard (left) and normetanephrine and methanephrine in a pooled urine elutate (right). The trihydroxyindole reaction was performed on each fraction.
the separation is as effective in a urinary extract from pooled urine as for standards. It should be emphasized, as can be seen in the differences in elution volume in the two runs, that the elution volume varies from column to column. We have not been able to pack these columns so that we could obtain equal elution volumes. We have always however been able to obtain a clear separation. It is therefore necessary for each column to separate into metanephrine and normetanephrine fractions using the native fluorescence as a guide before performing the trihydroxyindole reaction on the pooled fraction or to perform the trihydroxyindole method fraction by fraction. In our experiment with 5 Ftg of each standard we obtained a recovery of 4.87 for metanephrine and 4.93 for normetanephrine. In order to compare the chromatographic method with our previous’ procedure an experiment was designed in which IO urine samples were analyzed with both methods. Recovery experiments were performed adding before hydrolysis 3 big or TABLE
I
RECOVERY A
WHEN -_
5 ,Ug AMOUNTSOF
OF 3 ,AUgAND
COMPARISON
BETWEEN
PERFORMED
ON
VALUES THE
SAME
#
NM
Recovery
WITH
THE
AND
PROPOSED
METANEPHRINE METHOD
ADDED
AND
THE
TO
PREVIOUS1
URINE
.__
Method
Recov-
M
of3rug erY in % oy 5 Pg NM added
Recovery of3iug or 5 fig M added
G
NM
Old A&hod ____.. NM in % of chrom. meth.
e+,Y in %
M in “/ of chrom. meth.
I
5.32
2.83
94
4.72
2.54
84
4.83
91
3.88
82
2
5.35
3.07
IO2
4.20
2.90
96 95 103 95 9I 108 90
86 97 II3 80 90
3.48 2.44 3.12 4.84 2.46 4.50 IO.70
83 106 76 Io5 92 ‘03 IO9
3.35
124
102
5.44
Io6
3 4 : ; 9 IO
2.18 13.30 8.35 5.04 2.13 8.35 3.36
2.87 5.20 4-75 5.16 4.42 5.00 5.74
6.10
5.00
Average Recovery Clin.
Chim.
Acta,
‘03
4.33 9.80 2.70
2.89 5.15 4.75 4.56 4.52 5.40 5.00
IO0
4.60 2.10 15.00 7.50 4.03 2.46 7.20 3.06
100
5.14
4.84
96
6.20
96 IO4 95 Io3 IO0 88
98.5
25 (1969) 453-458
AND
METHOD
URINES
Chromatographic Samplr
NORMETANEPHRINE
OBTAINED
2.31 4.ro 2.68 4.60
95.6
II5 86 91
METANEPHRINEANDNORMETANEPHRINE IN URINE
457
5 pg of metanephrine and normetanephrine to separate samples of urine which were then taken through the full procedure. It can be seen from the results listed in Table I that the recovery in the chromatographic method is very close to IOO”/~. DISCUSSION We feel that the proposed method offers clear advantages when compared with the previous’ assay. It has better recovery of normetanephrine. Average recovery in the older method was for 12 analyses 89.5%‘. The similar figure for IO analyses for the new method is 98.5%. The difference in recovery is due to some loss of normetanephrine on the Florex used in the old assay. There is also higher consistency in recovery for both metanephrines. In the non-chromatographic method recoveries for 12 determinations varied between 62.5% and 122~/~for normetanephrine and between 64% and 114% for metanephrine. In contrast similar values for the chromatographic method in IO determinations are: for normetanephrine 88°/o-~030/oand for metanephrine 84%-108%. The more inconsistent results in the old method are probably due to the fact that any error in the quantitation of metanephrine is automatically reflected in the determination of normetanephrine for which the values are obtained through subtraction from the total, whereas after chromatographic separation both compounds are determined independently. That this is not a too serious weakness in the old method is shown by good agreement between “new” and “old” methods demontsrated in Table I. Overall the easier and faster older method would seem quite adequate for general use whereas the more complicated chromatographic separation should be reserved for situations where the highest accuracy is of prime importance. An advantageof the proposed method compared with the previous is that the two compounds are physically separated. This will make it possible to use the method for isotope work where the older method cannot be applied. It will also preclude some of the ambiguity of the method when it is used under conditions where drugs are given and it is unknown whether drug metabolites might interfere with the assay. The chances of separating the metanephrines from possible interfering metabolites would seem to be better when a chromatographic separation of the individual metanephrines is performed. The validity of the separation of the fractions by native fluorescence has been checked by reading all normetanephrine fractions at pH 2.85 for the presence of metanephrine. In no case have measurable amounts of metanephrine been found. It involves considerably more work to perform the trihydroxyindole reaction on each fraction cut. There may be situations where this may be preferable, particularly if there are difficulties in identifying the two fractions by native fluorescence alone. This has not occurred in our material of normal controls and patients treated with various tranquilizers and anti-depressants but might be found in other conditions. The fact that the trihydroxyindole reaction products give a fluorescence approximately IOO times as strong as the native fluorescence and that this reaction is specific for the catecholamines makes the unequivocal separation into metanephrine and normetanephrine peaks easier if the trihydroxyindole reaction is performed on each fraction. It would seem possible that the performance of the trihydroxyindole reaction as Clin. Chim. Ada,
25
(1969) 453-458
KAHANE,
458
VESTERGAARD
performed in this method can be automated by modification of published methodP6 and the fraction by fraction determination using this reaction might then become the preferable way of performing the analysis. Two other procedures have been published for the estimation of urinary metanephrine and normetanephrine after column chromatographic separation. One, the method of Haggendala, uses the eluate from an Amberlite column directly for fluorometry. According to Randrup’ such eluates are frequently colored and have high blanks. The other is the method of Taniguchi et al. 8. This method has been criticized by Weil-Malherbe and Smiths, in their survey of fluorometric methods for the determination of metanephrine and normetanephrine, as having low sensitivity and giving high blanks besides giving very unstable fluorophores. Also Weil-Malherbe and Smith claim they could not reproduce the chromatographic separation. The current method has high sensitivity, low blank readings and gives stable fluorophores. It has consistently given complete separation between the two compounds. ACKNOWLEDGEMENT
This work was supported Center Grant MH 07292.
by General
Support
Grant
FRO5651
and Research
REFERENCES I 2. KAHANE AND P. VESTERGAARD, J. Lab. Gin. Med., 70 (1967) 333. 2 R. J. MERRILLS, in L. T. SKEGGS (Ed.), Automation in Analytical Chemistry, Mediad, New York, 1966. p. 390. 3 R. J. MERRILLS, Anal Biochem., 6 (1963) 272. 4 R. L. ROBINSON AND D. T. WATTS, Clin. Chem., II (1965) 986-997. 5 V. FIORICA, Clin. Chim. Acta, 12 (1965) IgI--197. 6 J. HAGGENDAL, Acta, Physiol. Sand., 56 (1962) 258-266. 7 A. RANDRUP, Clin. Chim. Acta, 6 (1961) 584-586. 8 K. TANIGUCHI, Y. KAKIMOTO AND M. D. ARMSTRONG, J. Lab. Clin. Med., 64 (1964) 469. g H. WEIL-MALHERBE AND E. R. B. SMITH, in G. H. ACHESON (Ed.), Second Symposium on Catecholamines, Williams & Wilkins Company, Baltimore, 1966, p. 331.. C&z. Chim. Acta,
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