May 4, 1982 - The analysis of catecholamines and metanephrines in urine samples is important for identification of pheochromocytoma. (6). 5-HIAA is of great ...
CLIN. CHEM.28/8. 1745-1748 (1982)
Use of Alumina,SephadexGlO, and Ion-ExchangeColumnsto Purify Samples for Determinationof Epinephrine,Norepinephrine,Dopamine, HomovanillicAcid, and 5-HydroxyindoleaceticAcid in Urine Ben H. C. Westerlnk,1 Fokko J. Bosker,1 and James F. O’Hanlon2 We investigated the usefulness of small Sephadex columns, prepared in Pasteur pipetteS, in purifying samples to be analyzed for catecholamines, homovanillic acid, and 5-hycfroxyindoleacetic acid. After free catecholamines in urine samples were purified on alumina followed by Sephaclex Gb, a reliable and simultaneous quantification of epinephrine, norepinephrine, and dopamine was achieved by using high-performance liquid-chromatography with electrochemical detection. Purification of urine on Bio-Rad prepacked ion-exchange columns followed by Sephadex GlO resulted in a reliable, fast (200 samples processed per week) method for determination of free catecholamines in urine. Analytical recoveries of both methods were between 80 and 95%, with a CV of about 3%. Single purification on Sephadex GlO sufficed to allow simultaneous determination of homovanillic acid and 5hydroxyindoleacetic acid in urine. Analytical recovery for this method was about 90%, with a CV of about 5%. Sephadex GlO columns, which can be re-used without regeneration for at least one year, appear to have a great potential in clinical chemistry. AddItional Keyplwases chromatography preparation .
catecholamines
#{149}
electrochemical detection column chromatography
reversed.phase sample .
Concentrations of catecholamines and related metabolites in urine may be estimated by several techniques, including classical spectrophotometric (1) or fluorometric methods (2) and, more recently, by “high-performance” liquid chromatography (HPLC)3 with ultraviolet (3) or electrochemical detection (4-6). All these techniques, however, require extensive treatment of urine, which often means two sequential purification steps. Methods such as liquid-liquid extraction, thin-layer chromatography, and adsorption on alumina are time-consuming and require careful evaluation of the analytical recovery by means of internal standards. In our laboratory we have recently developed various assays for catecholamines and related compounds from brain, based on purification of tissue extracts on small Sephadex Gb columns. In brain research Sephadex Gb has proven to be an excellent alternative to the other currently used purification techniques (7,8). In the present study we investigated whether Sephadex Gb, alone or in combination with other purification methods, can be used in routine analysis for urinary epinephrine (E), norepinephrine (NE), dopamine (DA), the DA metabolite homovanillic acid (HVA), and the indole compound 5-hy1 Pharmaceutical Laboratories, Department of Medicinal Chemistry, and 2Traffic Research Center, University of Groningen, A. Deusinglaan 2, 9713 AW, Groningen, The Netherlands. Nonstandard abbreviations: HPLC, “high-performance” liquid
chromatography; E, epinephrine; NE, norepinephrine; DA, dopamine; HVA, homovanillic acid; 5-HIAA, 5-hydroxyindoleacetic Received Feb. 26, 1982; accepted May 4, 1982.
acid.
droxyindoleacetic acid (5-HIAA). The results suggest that Sephadex GlO columns are potentially very useful in clinical chemistry.
Materials and Methods Standards and reagents. Materials and their sources were as follows: norepinephrine bitartrate and epinephrine bitartrate (Sigma Chemical Co., St. Louis, MO 63176), homovanillic acid and 5-hydroxyindoleacetic acid (Fluka GmbH., Buchs, CH-9470, Switzerland), dopamine HC1 (Merck GmbH., Darmstadt, D-1600, F.R.G.), Sephadex GlO (Pharmacia), aluminum oxide (alumina, N-Super I; Woelm Pharmaca GmbH, Eschwege D-3440, F.R.G.), and prepacked ion-exchange columns (filled with Bio-Rad Affi-Gel 601; Bio-Rad Laboratories GmbH., Munchen, D-8000, F.R.G., Catecholamine Kit). All other chemicals were of analyticalreagent grade and were purchased from E. Merck. All aqueous solutions were prepared from de-ionized water distilled in glass. Stock solutions of NE, E, DA, HVA, and 5-HIAA contained 100mg of analyte per liter of 30 minol/L HC1, and were stored in portions of about 1 mL at -80 #{176}C. Each week, standard solutions were freshly prepared from a portion of the stock solution by appropriate dilution with 30 mmol/L HC1. Urine collection. We collected 24-h specimens in a polyethylene container, with 500 mg of sodium metabisulfite as preservative. On completion of collection, the pH was adjusted to between 3 and 4 with concentrated formic acid. After measuring the volume of the specimen, we removed about 8 mL for analysis. This aliquot, stored at -80 #{176}C, was stable for longer than six months. Isolation procedure on aluminum oxide. Alumina was prepared according to the method of Anton and Sayre (2), modified (9) such that the suspension was very carefully mechanically (not magnetically) stirred to prevent fine separation of the Al203 particles. After centrifugation we transferred 4 mL of the 24-h aliquot to a 50-mL beaker containing 500 mg of alumina and 21 mL of 0.4 mol/L HC1. After adding 1.0 mL of a 200 g/L solution of disodium ethylenediaminetetraacetate (EDTA), we rapidly adjusted the pH (within 20-30 s) to 8.60 (±0.01) with 2.5 mol/L NaOH and kept it at this value for 5 mm (the optimal adsorption time, see ref. 9). We used a motor-driven microburet to adjust the pH. After 5 mm, the supernatant fluid was discarded; the alumina was transferred to a polyethylene screw-capped centrifuge tube and washed three times with 10 mL of water. We then added 5 mL of 32 mmol/L HC1 to the alumina, shook the suspension for 13 mm with a shaking incubator (at 33 #{176}C), and subsequently centrifuged it. The supernatant fluid, which contained the free catecholamines, was removed with a pipette and transferred to a glass tube for storage. If necessary, samples could be stored in the dark at 4#{176}C. No detectable change was observed afterstorage for as long as 48 h. Isolation procedure on Bio-Rad columns. Isolation of catecholamines on prepacked Bio-Rad ion-exchange columns (filled with Bio-Rad Affi-Gel 601) was performed as indicated .
CLINICAL CHEMISTRY,
Vol. 28, No. 8, 1982
1745
by the manufacturer: Mix 5 mL of filtered urine with 15 mL of a solution of 2.7 mmol/L EDTA and adjust the pH to 6.5 ± 0.05 with 0.5 mol/L NaOH. Apply the mixture to the ionexchange column, wash the columns twice with 5 mL of H20, and elute the free catecholamines with 8 mL of boric acid solution, 650 mmol/L. Isolation procedure on Sephadex GlO columns. Samples were 1.0 mL of 10-fold
diluted
urine
(if HVA and 5-HIAA
0*
I I
2 nA
are
to be determined) or eluates of the alumina or Bio-Rad procedure. We mixed 15 zL of a 700 mL/L perchioric acid solution
with
the 1.0-mL
sample
and applied
this to a column
packed with Sephadex Gb. Sephadex Gb columns (5 X 70 mm) were prepared in long (7 cm) Pasteur pipettes as described previously (7,8). Numerous columns (at least 80) can be handled in one run by using automated pipettes. Before use we washed the packed columns with 3.0 mL of 20 mmol/L ammonia and 3.0 mL of 10 mmol/L formic acid. After samples had passed through the columns, 2.5 mL of 10 mmol/L formic acid was added. The free catecholamines were then eluted with another 2.5 mL of 10 mmol/L formic acid. To determine HVA and 5-HIAA, we applied diluted urine samples to the Sephadex columns and washed with 3.0 mL of 10 mmol/L formic acid followed by 1.5 mL of 5 mmol/L phosphate buffer (Na2HPO4. 2H20). HVA and 5-HIAA were subsequently eluted with 2.0 mL of 20 mmol/L ammonia. The fraction containing HVA and 5-HIAA was acidified with 50 tL of 6 mol/L formic acid. If Sephadex eluates had to be preserved until the next day, they were stored at 4 #{176}C. The columns are stored in 20 mmol/L ammonia. Chromatography. We used a liquid chromatograph (Model 3500 B; Spectra-Physics, Inc., Berkeley, CA) equipped with a Cb8 reversed-phase column (Nucleosil 5 C 18; MachereyNagel, DUren, F.R.G.), in conjunction with a rotating disc electrochemical detector (8). The potential setting was 450 mV (for the catecholamines) or 700 mV (for HVA and 5HIAA). Two types of mobile phases were investigated. The first was a phosphate-citrate buffer (prepared from 0.1 mol/L citric acid and 0.2 mol/L Na2HPO4- 2H20); the second, 0.1 mol/L trichloroacetic acid, adjusted to the appropriate pH (see Results) with solid sodium acetate, contained 0.1 mmol of EDTA, and 60-b 50 mL of methanol per liter. We filtered the solutions (0.5-tim pore size Millipore filter) before assay, and used pure standard solutions for calibration.
Results Analysis of catecholamines after one pretreatment step. When alumina was used as the only purification step, DA could
be quantitated
from the chromatogram,
but NE and E
were seriously overlapped by interfering peaks. The chromatogram was further complicated by late elution of various compounds with relatively long retention times (up to 20 mm). Single purification on Sephadex GlO was similarly insufficient. Use of the Bio-Rad ion-exchange column produced a
much cleaner chromatogram but with a broad negative front; a compound occasionally co-eluted with E, limiting the usefulness of this method as a routine procedure. We therefore concluded that a single purification step was not reliable for the determination of the free catecholamines. In contrast, HVA and 5-HIAA were sufficiently purified on Sephadex to proceed forthwith to HPLC Purification on alumina
Sephadex
Gb.
analysis (see below). followed by purification
The combination
GlO purification
resulted
free catecholamines
in a much
better
on
and Sephadex
separation
of the
The use of a citrate-phosphate buffer (studied pH range: 3.0-6.5), however, resulted in a chromatogram in which E was seriously overlapped
by several
and interfering
of alumina
compounds,
compounds.
whereas
very near to the void volume of the column. 1746
CLINICAL CHEMISTRY,
Vol. 28, No. 8, 1982
NE was eluted
Heeding
the
I 0
I 2
I 4
I B
0
I 10
mm .
Fig. 1. Typical chromatogram of catecholamines in a urine sample purified on alumina, then on Sephadex GlO Flow rate 1.0 mL/min, mobile phase: trichloroacetic acid, 0.1 mol/L. pH 4.0. and methanol, 80 mL/L; detector potential 500 mV. NA, norepinephrmne; A.
epmnephrmne. indicates change in recorder sensitivity (10), that amines
are much material when trichioracetic acid is present in the eluent, we included trichloracetic acid in the mobile phase, adjusted to pH 4.0 ± 0.1 (studied pH range: 3-6.5), and 60-80 mL of methanol per liter; separation between catecholamines and interfering compounds was complete. A typical chromatogram (Figure 1) illustrates the suggestion of Asmus and Freed better retained on reversed-phase
efficiency used.
and
specificity
of the
chromatographic
system
To optimize the isolation of the catecholamines from alumina, we investigated several factors that influenced paralytical recovery. First, we found that adjustment of the pH to 8.6 during the adsorption phase apparently should be carried out as quickly as possible (within 20-30 s). Second, when the alumina suspension is stirred, one should be careful not to divide the A1203 particles too finely; we therefore stir with a
piece of a paperclip
wrapped
in Parafilm.
Third, for optimal
elution of the catecholamines from alumina, both the volume and molarity of HC1 should be carefully controlled. A volume of 5 mL (range tested, 2-8 mL) and a concentration of 32 mmol/L (range tested, 25-75 mmol/L) produced optimal recovery. Finally we studied the shaking time (optimal, 13 mm; range tested, 10-20 mm) and the temperature (optimal, 33 #{176}C; range tested, 18-35 #{176}C) during the elution phase. Table 1 shows recoveries for 20,40, and 100 ng of the catecholamines
added to 1 mL of water. Analytical recoveries of pure standards from the Sephadex Gb column varied between 95 and 100% (mean ± SD) for NE and DA, and were about 90% for E (cf. 7,8).
The recoveries
(mean
± SD) of 50 ng of NE, 20 ng
Table 1. Recovery of Pure Catecholamines Treated with the Alumina Procedure Amount added,
20
40 100
ng
% recovery Noroplnephrlne 98.5
± 1.2
99.2 ± 1.1 98.7 ± 1.5
(mean ± SO, n Eplnephrlne 99.0
± 0.2
99.5 ± 0.4 99.4 ± 0.8
=
4)
Dopamine 986
± 0.7
98.2 ± 1.3 98.7 ± 0.6
L
NA
H 5HlAA
0*
LL
.1..1 OVA
I
0
I
I
4
I
I
I
I
12
I lB
mu +
Fig. 3. Typical chromatogram of HVA and 5-HIAA in a urine sample purified on Sephadex GlO Fig. 2. Typical chromatogram of catecholamines in a urine sample purified on Blo-Rad ion-exchange column, then on Sephadex GlO Flow rate 1.0 mL/min; mobIle phase: trichloroacetic acid, 0.1 mol/L, pH 4.0, and methanol,
60 mLIL; detector potential
500
my. Abbreviations as in Fig.
of E, and 200 ng of DA added to b mL of urine and processed through the alumina as well as the Sephadex Gb were (n = 12 each): NE, 92.5 ± 2.6%; E, 79.3 ± 5.2%; and DA, 89.0 ± 1.8%. Values for recovery by the alumina procedure were corrected for the amount of liquid trapped in the alumina after centrifugation: 0.49 (SD 0.01) g/500 mg of alumina (n = 20). Mean (±SD) 24-h values, corrected for the recoveries, in urine obtained from healthy men were: NE, 41.5 ± 11.0 g (n = 48); E, b2.b ± 4.5 ig (n = 47); and DA, 224.7 ± 60.4 ig (n = 48). These data are in good agreement with the literature (5, 11, 12). Purification on Bio-Rad ion-exchange column followed by Sephadex Gb. This combination of columns resulted in an
excellent separation of the catecholamines from interfering compounds (Figure 2). In fact, we noted no other peaks, and the chromatograms of routine urine samples could not be distinguished from those for pure standards. The analytical recovery was assessed by assaying urine supplemented with various amounts of NE, E, and DA; the results are given in Table 2. Because no interfering compounds were present, we increased the proportion of methanol to 80 mL/L, to shorten the time of analysis (about 8 mm) during routine use. Determination Sephadex Gb.
of HVA
and
5-HIAA
after
isolation
on
Flow rate 1.0 mL/mln; mobile phase: trichloroacetlc acid, 0.1 mol/L, pH 4.0, and methanol, 150 mi/L; detector potential 700 mV
to 150 mL/L) HVA and 5-HIAA were reliably quantified. A typical chromatogram is shown in Figure 3. The potential setting of the detector was 700 mV. The analytical recovery of 5-HIAA and HVA added to urine is shown in Table 3. Mean (±SD) excretion of HVA and 5-HIAA by healthy men was: HVA, 3.9 ± 1.1 mg/24 h (n = 6); 5-HIAA, 3.4 ± 0.9 mg/24 h (n = 6). These values are in good agreement with those previously published (13-16). anol increased
Discussion The analysis of catecholamines and metanephrines in urine samples is important for identification of pheochromocytoma (6). 5-HIAA is of great importance for diagnosis of carcinoid tumors (15, 16) and catecholamine excretion during stress is a subject of an increasing interest (e.g., 17, 18). A rapid and reliable method of analysis for monoamines and related compounds is therefore desirable. HPLC in combination with amperometric detection is now widely used in analysis for these compounds. The purification of urine samples, however, is a matter that deserves further attention. In previous reports we emphasized
(7, 8) the usefulness
of small
Sephadex
GlO
columns
as a means for rapid and miniaturized purification of catecholamines and related metabolites from tissue ex-
Table 3. Recovery of HVA and 5-HIAA Added to Urine and Purified on Sephadex GlO
Specimens of 10-fold diluted urine were purified on Sephadex GlO columns. The fraction containing HVA and 5-HIAA was injected into the chromatograph. With a mobile phase similar to that used in the assay of catecholamines (0.1 mol/L trichloracetic acid, pH 4.0, and the meth-
Amount added,
pg
HVA
5-HIAA
1.9 2.8
2.7 4.4
% recovery
(mean
HVA
± SD,
n
=
4)
5-HIAA
92.3 ± 6.3 83.3 ± 4.4
99.0 ± 5.5 90.7 ± 2.7
Table 2. Recovery of Catecholamines Added to Urine and Carried through Bio-Rad ion-Exchange and Sephadex GlO Purification Amount added,
% recovery (mean
no
± SD.
n
=
4
NE
E
OA
NE
E
DA
30 60
10
300 400 500
97.5 ± 1.0
89.5 ± 2.7 91.2 ± 5.3
80.6 ± 1.5
93.7 ± 3.2
97.2 ± 2.6
90.4 ± 5.4
83.0 ± 2.3
90
20 30
80.7 ± 0.8
CLINICALCHEMISTRY,Vol. 28, No. 8, 1982 1747
tracts,
because
of ion-pairs
of the specific
with perchlorate,
adsorption of amines, in the form to the Sephadex GlO resin
(8).
Here, we investigated
whether this purification procedure to determinations from urine samples. of urine on Sephadex GbO was sufficiently effective to allow determination of HVA and 5-HIAA, for the catecholamines a second purification step was necessary. Purification on alumina + Sephadex Gb or on BioRad ion-exchange columns + Sephadex GlO completely also could be applied Although purification
separated
the
catecholamines
from the interfering comvarious factors (see Results) we wereable to optimize the isolation of free catecholamines
pounds. By carefully controlling
from alumina, and obtained recoveries of about 90%. This method with alumina is reliable and cheap but time consuming, and no more than 10 samples can be processed in a day. On the other hand, the combination of Sephadex Gb and Bio-Rad ion-exchange columns resulted in a very rapid, simple, and reliable purification procedure. Apart from the compounds described here, the Sephadex Gb and Bio-Rad column may also be used for the separation and purification of metanephrines (unpublished data). The disadvantage of the Bio-Rad disposable columns is their high cost, but we routinely re-use the material several times without change in the recoveries. The high recoveries and precision mean that the assays can be performed without using internal standards. Efficient separation between catecholamines requires less than 10 mm, so that a large number of samples can be processed (about 200 per week). Including trichloroacetic acid in the eluent is an attractive alternative to the currently used alkylsulfonate-containing mobile phases (4, 6), which reportedly cause column instability (19). The properties of trichioroacetic acid as an ion-pairing reagent for high-efficiency separations of various amines in reversedphase chromatography have recently been described by Asmus and Freed (10). In summary, small Sephadex Gb columns are a useful, versatile, and rapid tool for purification of catecholamines, HVA, and 5-HIAA (and probably metanephrines) from urine samples. The Sephadex Gb columns are easy to handle, do not need regeneration, and can be used for at least a year without change in the recoveries of the various compounds.
References 1. Von Euler, U. S., and Haxnberg, U., Colorimetric determination of noradrenaline and adrenaline. Acta Physiol. Scand. 19, 74-84 (1949).
P. T., Determination of catecholamines in urine by reverse-phase liquid chromatography with electrochemical detection. Anal. Chem. 49, 2109-2111 (1977). 5. Moyer, T. P., and Jiang, N.-S., Optimized isocratic conditions for 4. Riggin, R. M., and Kissinger,
analysis of catecholamines by high-performance reversed-phase paired-ion chromatography with amperometric detection. J. Chromatogr. 153,365-372 (1978). 6. Moyer, T. P., Jiang, N.-S., Tyce, G. M., and Sheps, S. G., Analysis for urinary catecholamines by liquid chromatography with amperometric detection: Methodology and clinical interpretation of results. Clin. Chem. 25, 256-263 (1979). 7. Westerink, B. H. C., and Korf, J., Rapid concurrent automatic fluorimetric assay of noradrenaline, dopamine, 3,4-dihydroxyphenylacetic acid, homovanillic acid and 3-methoxytyrammne in milligram amounts of nervous tissue after isolation on Sephadex G-b0. J. Neurochem. 29, 697-706 (1977). 8. Westerink, B. H. C., and Mulder, T. B. A., Determination of picomole amounts of dopamine, noradrenaline, 3,4-dihydroxypheny-
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(1981).
9. O’Hanlon, J. F.,
Campuzano,
H. C., and Horvath, S. M., Fluoro-
metric
assay for subnanogram concentrations of adrenaline and noradrenaline in plasma. Anal. Biochem. 34,568-581 (1970). 10. Asmus, P. A., and Freed, C. R., Reversed-phase high-performance
chromatography of catecholamines and their congeners with simple acids as ion-pairing reagents. J. Chromatogr. 169,303-311 (1979). 11. Crout, J. R., Catecholamines in urine. Stand. Methods Clin. Chem. 3,62 (1961). 12. Nagatsu, T., Biochemistry of Catecholamines: The Biochemical Method, University Park Press, Baltimore, MD, 1973, p 236. 13. Stott, A. W., Lindsay Smith, J. R., Hanson, P., and Robinson, R, A simple chromatographic procedure for the concurrent estimation
of urinary 4-hydroxy-3-methoxymandelic acid and homovanillic acid using a scanning technique. Clin. Chim. Acta 63,7-12 (1975). 14. Yoehida, A., Yoshioka, M., Yamazaki, T., et al., Urinary levels of vanilmandelic
acid and homovanillic
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by high speed
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17. Frankenhaeuser, M., Psychoneuroendocrine sex differences in adaptation to the psychosocial environment. In Clinical Psychoneuroendocrinology in Reproduction, L. Carenza and P. Pancheri, Eds., Academic Press, New York, NY, 1978, pp 215-223.
18. Rauste-von Wright, M., von Wright, J., and Frankenhaeuser, M.,
2. Anton, A. H., and Sayre, D. F., A study of the factors affecting the
Relationships
aluminum oxide-trihydroxyindole procedure for the analysis of catecholamines. J. Pharmacol. Exp. Ther. 138, 360-375 (1962). 3. Moln#{225}r, I., and HorvSth, C., Reverse-phase chromatography of polar biological substances: Separation of catechol compounds by high-performance liquid chromatography. Clin. Chem. 22, 1497-1502 (1976).
during adolescence and catecholamine excretion during achievement
1748 CLINICALCHEMISTRY, Vol. 28,No.8, 1982
between
sex-related
psychological
characteristics
stress. Psychophysiology 18,362-370. 19. Scratchley, G. A., Masoud, A. N., Stohs, S. J., and Wingard, D. W., High performance liquid chromatographic separation and detection of catecholamines and related compounds. J. Chromatogr. 169, 313-319 (1979).
