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Measurement of 3-methoxy-4-hydroxyphenylacetic acid (HVA) in plasma by high-performance liquid chromatography with electrochemical detector (HPLC-EC)
ANALYTICAL
135,326-33 1 (1983)
BIOCHEMISTRY
Measurement of 3-Methoxy-4-hydroxyphenylacetic Acid (HVA) in Plasma by High-Performance Liquid Chromatography with Electrochemical Detector (HPLC-EC) J . I . JAVAID,*”
T. S. LIU,* J. W. MAAS,?
AND J. M. DAVIS*
*Illinois State Psychiatric Institute, 1601 West Taylor Street, Chicago, Illinois 60612; and iDepartment of Psychiatry, University of Texas at San Antonio, Health Science Center, 7703 Floyd Curl Drive, San Antonio, Texas 78284 Received May 6, 1983 A simple and sensitive high-performance liquid chromatography method with electrochemical detector is described for the determination of free 3-methoxy-4-hydroxyphenylacetic acid (HVA) in human plasma. The method does not involve any extraction, is specific and reproducible, and has the potential to measure serotonin (5HT) and 5hydroxyindoleacetic acid (5-HIAA) simultaneously. The plasma concentration of free HVA in eight normal, healthy adult volunteers was 10.9 k 4.6 (mean + SD). In a preliminary study, in one schizophrenic patient the plasma HVA increased twofold after neuroleptic treatment.
3-Methoxy-4-hydroxyphenylacetic acid (homovanillic acid, HVA)* is one of the main metabolites of dopamine in primates (l-3). In animal studies, the measurement of HVA in the brain has been shown to reflect the functional activity of dopaminergic neurons (4-6). Several investigators have confirmed the presence of HVA in plasma from animals and humans (7-9). Although the contribution of brain HVA to plasma HVA has not been established, it has been shown that in experimental animals the treatments which change brain HVA, also result in parallel changes in plasma HVA (10). This suggests that plasma, an easily available body fluid, may serve as an indicator of the brain dopaminergic systems. In urine and cerebrospinal fluid (CSF), HVA has been measured by spectrophotometry (11,12), fluorometry (13-15), and gas ’ To whom all correspondence should be addressed. 2 Abbreviations used: HVA, 3-methoxy-4-hydroxyphenylacetic acid (homovanillic acid); CSF, cerebrospinal fluid; EC, electrochemical; 5-HT, serotonin; 5-HIAA, 5hydroxyindoleacetic acid; VA, vanillic acid; CV, coefficient of variation; CC-MS, gas chromatography-mass spectrometry.
All
rights
0 of
1983
by
reproduction
MATERIALS
Academic in any
Press. form
Inc. reserved.
AND
METHODS
The instrument used was a Perkin-Elmer Series 212 liquid chromatograph (Perkin-El326
0003-2697183 $3.00 Copyright
chromatography ( 16- 18). However, spectrophotometric and fluorometric methods are not specific and lack the sensitivity to measure low concentrations of HVA present in plasma. Gas chromatography with electron-capture detection has been used to determine the HVA in human plasma (19,20). However, these methods involve extensive sample preparation and derivatization and the reported values are much higher than those measured with more specific mass spectrometric detection methods (2 l-23). Recently, subnanogram concentrations of HVA in discrete areas of animal brains have been measured (24-26) by high-performance liquid chromatography (HPLC). Here, we describe an HPLC method with electrochemical (EC) detector for HVA determination in plasma. The method is simple, reproducible and requires minimum sample preparation and has the potential to also measure serotonin (5-HT) and %hydroxyindoleacetic acid (5-H&A) simultaneously in the same run.
LIQUID
CHROMATOGRAPHY
OF
3-METHOXY4HYDROXYPHENYLACETIC
mer, Norwalk, Conn.) equipped with dualpiston pump and precision flow control and a Rheodyne Model 7 125 (Bheodyne, Berkeley, Calif.) injection valve. The amperometric EC detector Model LC-4A (Bioanalytical Systems Inc., West Lafayette, Ind.) was used at a potential of 0.95 V vs Ag/AgCl with a glassy carbon electrode. A stainless-steel column (4.5 X 250 mm) packed with octylsilane C-8 bonded to silica (Analytical C8, 10 pm, Perkin-Elmer) was used for separation. The mobile phase was 0.1 M sodium acetate, pH 4.5, containing 1 mM EDTA and 10% methanol (v/v). The flow rate was maintained at 1.0 ml/min. The HVA, vanillic acid (VA) and 5-HIAA were all obtained from Sigma Chemical Company, St. Louis, MO., while 5-HT was from Calbiochem-Behring Corp., San Diego, Calif. HPLC-grade methanol was purchased from Burdick and Jackson Labs, Inc., Muskegon, Mich. Deionized glass distilled water was used for the buffers. All other reagents used were of analytical grade. Mobile phase was filtered and degassed before use. The standard stock solutions were made in 0.4 N perchloric acid to give final concentrations of 1 mg/ml and kept frozen for up to 1 month. Working standard solutions were made by diluting the stock solution with 0.4 N perchloric acid to give 1- 10 pg/ml concentrations. Working solutions were made once a week and kept refrigerated. Sample preparation. Initial attempts to extract HVA from 2-4 ml of plasma for HPLC analysis were unsuccessful. Although a variety of procedures were tried, either the extraction was poor or the resulting HVA peak was masked by a larger peak just prior to HVA. To circumvent these problems, deproteinized plasma was directly used for analysis. To 1 ml of ice-cold plasma samples, 100 ng of VA was added as internal standard. The proteins were precipitated by the addition of 35 ~1 of concentrated perchloric acid (0.4 N, final concentration). The sample was thoroughly mixed and centrifuged for 15 min at 14,000 rpm. The clear, supematant was transferred to clean
ACID
327
glass vials and 50- 100 ~1 of the samples was directly injected. With each experiment a standard curve was generated by adding varying concentrations of HVA (5-20 ng) to the plasma and analyzing the samples as described. The HVA concentrations were calculated from the standard curve as well as from the ratios of peak heights of HVA and VA. Animal study. In one experiment rats were injected with 5 mg/kg of fluphenazine and decapitated 1.5 h later, along with saline injected controls. The caudate was dissected and extracted with 10 vol of 0.4 N perchloric acid (w/v) and 50- 100 ~1 of the extract were directly injected for HPLC analysis. Patient sample. In one schizophrenic patient, plasma HVA was determined on three consecutive days after the patient was free of all medication for 2 weeks and also after day 4 of treatment with haloperidol(20 mg/day). RESULTS
After experimenting with different mobile phases, 0.1 M sodium acetate, pH 4.5, containing 1 mM EDTA and 10% methanol was used for the separation of 5-HT, 5-HIAA, HVA, and VA. The use of methanol reduced the elution time and resulted in sharper peaks. Figure 1 shows the detector response for 5HT, 5-HIAA, HVA, and VA at different oxidation potentials across a glassy carbon electrode and a Ag/AgCl electrode. The 5-HT and 5-HIAA were completely oxidized at a potential of 0.7 V. Since the present method was being optimized for HVA, however, a potential of 0.95 V was used in all experiments. The linearity of the detector response with varying concentrations of 5-HT, 5-HIAA, and HVA is shown in Fig. 2. The response was linear whether area under the curve or peak height was used for the plots. Figure 3A shows the separation of a mixture of 5-HIAA, HVA, and VA standards. The peaks were symmetrical and elution of all compounds was achieved in I5 min. Figure 3B shows the chromatogram from a human plasma sample after
JAVAID
0.5
0.6
0.7 OXIDATION
0.8
0.9
POTENTIAL
I .o (‘4)
FIG. 1. Voltogram for 5-HT (0), 5-HIAA (m), HVA (O), and VA (0) with glassy carbon electrode and Ag/ AgCl electrode.
deproteinization and Fig. 3C is a chromatogram of a CSF sample. The catecholamines, NE and DA and the metabolites MHPG, DO-
0
0.25
ET AL.
PAC, and VMA eluted earlier in the chromatogram and did not interfere with HVA analysis. The reproducibility of the method was determined by analyzing four aliquots of a pooled plasma sample. The mean concentration of HVA in plasma was 14.3 rig/ml with a coefficient of variation (CV) of 8.7%. In another reproducibility experiment five plasma samples were split into duplicate aliquots and were coded before analysis. The average CV for these five duplicate samples was 6.8 + 2.5% (SEM). The standard HVA added to plasma coeluted with the plasma HVA peak and there was a linear relationship between the amount added and increase in peak height, with -x intercept as the endogenous HVA concentration. The specificity of the HVA peak was further confirmed by the analysis of rat caudates. Only the peak corresponding to HVA was increased (from 114 to 798 rig/g) after fluphenazine treatment (5 mg/kg) while there was no effect on the concentration of 5-HT and 5-HIAA. To determine the stability of HVA, freshly drawn pooled plasma was frozen as such and in HCl (final pH 1). The samples were analyzed the same day and after weeks 1, 2, 4, and 8. There were no differences in plasma
0.50
0.75
I .o
t&INJECTION
FIG. 2. Linearity of the detector response. Three to five samples for each concentration were injected. The results are mean + SEM for S-HT (6), S-HIAA (9 ), and HVA (0 ).
LIQUID
CHROMATOGRAPHY
OF 3-METHOXY-4-HYDROXYPHENYLACETIC
ACID
329
I ,;
: Z -- Ic
i
TIME
(min.)
FIG. 3. Typical HPLC-EC’chromatograms for: (A) A standard mixture containing I .O ng of 5-HT, 1.O ng of 5-HIAA, 0.5 ng of HVA, and 5.0 ng of VA in a 50-~1 volume. (B) A plasma sample from a patient to which VA (as an internal standard) was added. The sample was prepared as described under Materials and Methods and 50 pl was injected. (C) A CSF sample from the same patient. VA was added as an internal standard and after protein precipitation as described under Materials and Methods, 50 ~1 of the sample was injected. The arrow indicates the origin of the sample injected.
HVA up to week 2, whether the plasma was kept frozen as such or in the presence of HCl. However, samples from weeks 4 and 8 resulted in an extra peak and HVA values were 50% lower in both samples stored as such and in HCl.
The method described here was used to determine HVA concentrations in the plasma of normal individuals. These results are shown in Table 1, and are compared with previously reported levels in Table 2. Although the values as determined by the present HPLC method
330
JAVAID TABLE
CONCENTRATIONS
ET AL.
1
TABLE 3
OF FREE HVA IN PLASMA HEALTHY CONTROLS
FROM
CONCENTRATION SCHIZOPHRENIC
OF FREE HVA PATIENT
AFTER NEUROLEPTIC
Sample
Sex
HVA (rig/ml)
1 2 3 4 5 6 7 8
M F F F M F M M
12.5 15.4 5.8 3.0 7.0 16.0 12.9 14.7
Study Day’
R?SD=
10.9 + 4.6
1 1 2 3 7
CSF Plasma Plasma Plasma Plasma
IN ONE
BEFORE
AND
TREATMENT
HVA (&ml) 18.2 8.4 12.0 11.0 22.5
’ The patient was free of all medications for 2 weeks before entering the study. Day 7 of the study represents 4th day of the treatment with haloperidol (20 mg/day).
are lower than those determined by gas chro- 3. There was a twofold increase in free HVA matography with electron-capture detection, after neuroleptic treatment. they are similar to those determined by more specific GC-MS methods (Table 2). The apDISCUSSION plicability of the described procedure was also established by analyzing five plasma samples In this report we have described a simple and one CSF sample from one schizophrenic and reliable HPLC method for the determipatient. The patient was admitted to the Re- nation of HVA in human plasma. The method search Ward, University of Texas hospital, San has the potential for the simultaneous deterAntonio, Texas. After 2 weeks of washout pe- mination of 5-HT and 5-HIAA along with riod, plasma samples were collected for 3 con- HVA. Unlike previously described gas chrosecutive days. During this baseline period, a matograph and mass spectrometric methods, CSF sample was also obtained by lumber the HPLC procedure described here does not puncture. One plasma sample was collected require any extensive extraction procedure or after 4 days of treatment with haloperidol. derivatization. The HVA, 5-HT, and 5-HIAA The samples were shipped frozen and analyzed from deproteinized plasma are well resolved in Chicago. These results are shown in Table from the other components with the described analytical system in a reasonably short time. It has been shown that turnover rates of TABLE 2 neurotransmitter amines are correlated with functional activity of amine-containing neuHVA CONCENTRATIONS IN HUMAN PLASMA REPORTED BY DIFFERENT METHODS rons. The measurement of amine metabolites in rat brain or human urine and CSF has been HVA used as an index of aminergic activity. ReRef. Method (@ml) f SD N Range cently, Swann et al. (27) and Kendler et al. (19) GC-ECD” 62.0 2 45.0 (28) have reported that in man and rats, treat(20) GC-ECD’ 89.9 f 51.3 ment with peripheral monoamine oxidase in(21) GC-MSb 10.2 f 4.96 5 4-19 hibitor debrisoquin significantly decreased (22) GC-MS* 10.5 f 1.3 (2% GC-MSb l1.3f 4.2 23 2.4-18.4 plasma HVA. As suggested by these authors, This study HPLC 10.9 f 4.6 8 3-16 HVA determination in plasma after debrisoquin treatment provides a tool for determining L1Gas chromatography with electroncapture detector. central dopamine activity. Both of these studb Gas chromatography-mass spectrometry.
LIQUID
CHROMATOGRAPHY
OF 3-METHOXY-4-HYDROXYPHENYLACETIC
ies had used the GC-MS method for HVA determination. The method described in this paper would be useful for such future research, since many plasma samples can be analyzed in a short time. Although the plasma sample after deproteinization is directly injected, we have not observed any deterioration in column performance over a 3-month period. Instead, with more regular use the analytical system appears to be more stable.
ACID
331
10. Bacopoulos, N. G., Hattox, S. E., and Roth, R. G. (1979) Eur. J. Pharmacol. 56, 225. 11. Ruthven, C. R. J., and SandIer, M. ( 1966) Clin. Chim. Acta 14, 411. 12. Knight, J. A., and Haymond, R. E. ( 1977) Clin. Chem. 23, 2007.
13. Anden, N. E., Roos, B. E., and Werdinius, B. (1963) Life Sci. 3, 149. 14. Korf, J., Ottema, S., and Van du Veen, I. (197 1) Anal. Biochem. 40, 187. 15. Prockop, L., Fahn, S., and Barbour, P. (1974) Brain Rex 80, 435. 16. Sjoquist, B., and Anggard, E. (1972) Anal. Chem. 44, 2297.
REFERENCES 1. Shaw, K. N. F., McMillan, A., and Armstrong, M. D. (1957) J. Biol. Chem. 226, 255. 2. Goldstein, M., Friedhoff, A. J., and Simmons, C. (1959) Biochim. Biophys. Acta 33, 572. 3. Maas, J. W., Hattox, S. E., Marin, D. M., and Landis, D. H. (1979) J. Neurochem. 32, 839. 4. Bunney, B. S., Walters, J. R., Roth, R. H., and Aghajanian, G. K. (1973) J. Pharmacol. Exp. Ther. 185, 560. 5. Roth, R. H., Munin, L. C., and Walter, J. R. (1976) Eur. J. Pharmacol. 36, 163. 6. Westerink, B. H. C., and Korf, J. (1976) Eur. J. Pharmacol. 37, 249, 7. Sjoquist, B., Lindstrom, B., and Anggard, E. (1973) Life Sci. 13, 1655. 8. Maas, J. W., Hattox, S. E., and Landis, H. (1979) Biochem. Pharmacol. 28, 3153. 9. Muskiet, F. A. J., Nagel, G. T., and Wolthers, B. G. (1980) Anal. Biochem. 109, 130.
17. Watson, E., Wilk, S., and Roboz, J. (1974) Anal. Biochem. 59, 44 1. 18. Pearson, J. D. M., and Sharman, D. F. (1975) Brit. J. Pharmacol. 53, 143. 19. Markianos, E., and Ruther, E. (1976) J. Clin. Chem. Clin. Biochem. 14, 437. 20. Markianos, E., and Beckmann, H. (1976) J. Neural Transm. 39, 79. 21. Takahaski, S., Hoshioka, M., Hoshiue, S., and Tamura, Z. (1978) J. Chromatogr. 145, 1. 22. Swann, A. C., Maas, J. W., Hattox, S. E., and Landis, H. (1980) Life Sci. 27, 1857. 23. Muskiet, F. A. J., Nagel, G. T., and Wolthers, B. G. ( 1980) Anal. Biochem. 109, 130. 24. Hefti, F. (1979) L@ Sci. 25, 775. 25. Kempf, E., and Mandel, P. (1981) Anal. Biochem. 112, 223.
26. Sperk, G. (1982) J. Neurochem. 38, 840. 27. Swann, A. C., Maas, J. W., Hattox, S. E., and Landis, H. (1980) Life Sci. 27, 1857. 28. Kendler, K. S., Heninger, G. R., and Roth, R. H. (1981) Eur. J. Pharmacol. 71, 321.
135,326-33 1 (1983)
BIOCHEMISTRY
Measurement of 3-Methoxy-4-hydroxyphenylacetic Acid (HVA) in Plasma by High-Performance Liquid Chromatography with Electrochemical Detector (HPLC-EC) J . I . JAVAID,*”
T. S. LIU,* J. W. MAAS,?
AND J. M. DAVIS*
*Illinois State Psychiatric Institute, 1601 West Taylor Street, Chicago, Illinois 60612; and iDepartment of Psychiatry, University of Texas at San Antonio, Health Science Center, 7703 Floyd Curl Drive, San Antonio, Texas 78284 Received May 6, 1983 A simple and sensitive high-performance liquid chromatography method with electrochemical detector is described for the determination of free 3-methoxy-4-hydroxyphenylacetic acid (HVA) in human plasma. The method does not involve any extraction, is specific and reproducible, and has the potential to measure serotonin (5HT) and 5hydroxyindoleacetic acid (5-HIAA) simultaneously. The plasma concentration of free HVA in eight normal, healthy adult volunteers was 10.9 k 4.6 (mean + SD). In a preliminary study, in one schizophrenic patient the plasma HVA increased twofold after neuroleptic treatment.
3-Methoxy-4-hydroxyphenylacetic acid (homovanillic acid, HVA)* is one of the main metabolites of dopamine in primates (l-3). In animal studies, the measurement of HVA in the brain has been shown to reflect the functional activity of dopaminergic neurons (4-6). Several investigators have confirmed the presence of HVA in plasma from animals and humans (7-9). Although the contribution of brain HVA to plasma HVA has not been established, it has been shown that in experimental animals the treatments which change brain HVA, also result in parallel changes in plasma HVA (10). This suggests that plasma, an easily available body fluid, may serve as an indicator of the brain dopaminergic systems. In urine and cerebrospinal fluid (CSF), HVA has been measured by spectrophotometry (11,12), fluorometry (13-15), and gas ’ To whom all correspondence should be addressed. 2 Abbreviations used: HVA, 3-methoxy-4-hydroxyphenylacetic acid (homovanillic acid); CSF, cerebrospinal fluid; EC, electrochemical; 5-HT, serotonin; 5-HIAA, 5hydroxyindoleacetic acid; VA, vanillic acid; CV, coefficient of variation; CC-MS, gas chromatography-mass spectrometry.
All
rights
0 of
1983
by
reproduction
MATERIALS
Academic in any
Press. form
Inc. reserved.
AND
METHODS
The instrument used was a Perkin-Elmer Series 212 liquid chromatograph (Perkin-El326
0003-2697183 $3.00 Copyright
chromatography ( 16- 18). However, spectrophotometric and fluorometric methods are not specific and lack the sensitivity to measure low concentrations of HVA present in plasma. Gas chromatography with electron-capture detection has been used to determine the HVA in human plasma (19,20). However, these methods involve extensive sample preparation and derivatization and the reported values are much higher than those measured with more specific mass spectrometric detection methods (2 l-23). Recently, subnanogram concentrations of HVA in discrete areas of animal brains have been measured (24-26) by high-performance liquid chromatography (HPLC). Here, we describe an HPLC method with electrochemical (EC) detector for HVA determination in plasma. The method is simple, reproducible and requires minimum sample preparation and has the potential to also measure serotonin (5-HT) and %hydroxyindoleacetic acid (5-H&A) simultaneously in the same run.
LIQUID
CHROMATOGRAPHY
OF
3-METHOXY4HYDROXYPHENYLACETIC
mer, Norwalk, Conn.) equipped with dualpiston pump and precision flow control and a Rheodyne Model 7 125 (Bheodyne, Berkeley, Calif.) injection valve. The amperometric EC detector Model LC-4A (Bioanalytical Systems Inc., West Lafayette, Ind.) was used at a potential of 0.95 V vs Ag/AgCl with a glassy carbon electrode. A stainless-steel column (4.5 X 250 mm) packed with octylsilane C-8 bonded to silica (Analytical C8, 10 pm, Perkin-Elmer) was used for separation. The mobile phase was 0.1 M sodium acetate, pH 4.5, containing 1 mM EDTA and 10% methanol (v/v). The flow rate was maintained at 1.0 ml/min. The HVA, vanillic acid (VA) and 5-HIAA were all obtained from Sigma Chemical Company, St. Louis, MO., while 5-HT was from Calbiochem-Behring Corp., San Diego, Calif. HPLC-grade methanol was purchased from Burdick and Jackson Labs, Inc., Muskegon, Mich. Deionized glass distilled water was used for the buffers. All other reagents used were of analytical grade. Mobile phase was filtered and degassed before use. The standard stock solutions were made in 0.4 N perchloric acid to give final concentrations of 1 mg/ml and kept frozen for up to 1 month. Working standard solutions were made by diluting the stock solution with 0.4 N perchloric acid to give 1- 10 pg/ml concentrations. Working solutions were made once a week and kept refrigerated. Sample preparation. Initial attempts to extract HVA from 2-4 ml of plasma for HPLC analysis were unsuccessful. Although a variety of procedures were tried, either the extraction was poor or the resulting HVA peak was masked by a larger peak just prior to HVA. To circumvent these problems, deproteinized plasma was directly used for analysis. To 1 ml of ice-cold plasma samples, 100 ng of VA was added as internal standard. The proteins were precipitated by the addition of 35 ~1 of concentrated perchloric acid (0.4 N, final concentration). The sample was thoroughly mixed and centrifuged for 15 min at 14,000 rpm. The clear, supematant was transferred to clean
ACID
327
glass vials and 50- 100 ~1 of the samples was directly injected. With each experiment a standard curve was generated by adding varying concentrations of HVA (5-20 ng) to the plasma and analyzing the samples as described. The HVA concentrations were calculated from the standard curve as well as from the ratios of peak heights of HVA and VA. Animal study. In one experiment rats were injected with 5 mg/kg of fluphenazine and decapitated 1.5 h later, along with saline injected controls. The caudate was dissected and extracted with 10 vol of 0.4 N perchloric acid (w/v) and 50- 100 ~1 of the extract were directly injected for HPLC analysis. Patient sample. In one schizophrenic patient, plasma HVA was determined on three consecutive days after the patient was free of all medication for 2 weeks and also after day 4 of treatment with haloperidol(20 mg/day). RESULTS
After experimenting with different mobile phases, 0.1 M sodium acetate, pH 4.5, containing 1 mM EDTA and 10% methanol was used for the separation of 5-HT, 5-HIAA, HVA, and VA. The use of methanol reduced the elution time and resulted in sharper peaks. Figure 1 shows the detector response for 5HT, 5-HIAA, HVA, and VA at different oxidation potentials across a glassy carbon electrode and a Ag/AgCl electrode. The 5-HT and 5-HIAA were completely oxidized at a potential of 0.7 V. Since the present method was being optimized for HVA, however, a potential of 0.95 V was used in all experiments. The linearity of the detector response with varying concentrations of 5-HT, 5-HIAA, and HVA is shown in Fig. 2. The response was linear whether area under the curve or peak height was used for the plots. Figure 3A shows the separation of a mixture of 5-HIAA, HVA, and VA standards. The peaks were symmetrical and elution of all compounds was achieved in I5 min. Figure 3B shows the chromatogram from a human plasma sample after
JAVAID
0.5
0.6
0.7 OXIDATION
0.8
0.9
POTENTIAL
I .o (‘4)
FIG. 1. Voltogram for 5-HT (0), 5-HIAA (m), HVA (O), and VA (0) with glassy carbon electrode and Ag/ AgCl electrode.
deproteinization and Fig. 3C is a chromatogram of a CSF sample. The catecholamines, NE and DA and the metabolites MHPG, DO-
0
0.25
ET AL.
PAC, and VMA eluted earlier in the chromatogram and did not interfere with HVA analysis. The reproducibility of the method was determined by analyzing four aliquots of a pooled plasma sample. The mean concentration of HVA in plasma was 14.3 rig/ml with a coefficient of variation (CV) of 8.7%. In another reproducibility experiment five plasma samples were split into duplicate aliquots and were coded before analysis. The average CV for these five duplicate samples was 6.8 + 2.5% (SEM). The standard HVA added to plasma coeluted with the plasma HVA peak and there was a linear relationship between the amount added and increase in peak height, with -x intercept as the endogenous HVA concentration. The specificity of the HVA peak was further confirmed by the analysis of rat caudates. Only the peak corresponding to HVA was increased (from 114 to 798 rig/g) after fluphenazine treatment (5 mg/kg) while there was no effect on the concentration of 5-HT and 5-HIAA. To determine the stability of HVA, freshly drawn pooled plasma was frozen as such and in HCl (final pH 1). The samples were analyzed the same day and after weeks 1, 2, 4, and 8. There were no differences in plasma
0.50
0.75
I .o
t&INJECTION
FIG. 2. Linearity of the detector response. Three to five samples for each concentration were injected. The results are mean + SEM for S-HT (6), S-HIAA (9 ), and HVA (0 ).
LIQUID
CHROMATOGRAPHY
OF 3-METHOXY-4-HYDROXYPHENYLACETIC
ACID
329
I ,;
: Z -- Ic
i
TIME
(min.)
FIG. 3. Typical HPLC-EC’chromatograms for: (A) A standard mixture containing I .O ng of 5-HT, 1.O ng of 5-HIAA, 0.5 ng of HVA, and 5.0 ng of VA in a 50-~1 volume. (B) A plasma sample from a patient to which VA (as an internal standard) was added. The sample was prepared as described under Materials and Methods and 50 pl was injected. (C) A CSF sample from the same patient. VA was added as an internal standard and after protein precipitation as described under Materials and Methods, 50 ~1 of the sample was injected. The arrow indicates the origin of the sample injected.
HVA up to week 2, whether the plasma was kept frozen as such or in the presence of HCl. However, samples from weeks 4 and 8 resulted in an extra peak and HVA values were 50% lower in both samples stored as such and in HCl.
The method described here was used to determine HVA concentrations in the plasma of normal individuals. These results are shown in Table 1, and are compared with previously reported levels in Table 2. Although the values as determined by the present HPLC method
330
JAVAID TABLE
CONCENTRATIONS
ET AL.
1
TABLE 3
OF FREE HVA IN PLASMA HEALTHY CONTROLS
FROM
CONCENTRATION SCHIZOPHRENIC
OF FREE HVA PATIENT
AFTER NEUROLEPTIC
Sample
Sex
HVA (rig/ml)
1 2 3 4 5 6 7 8
M F F F M F M M
12.5 15.4 5.8 3.0 7.0 16.0 12.9 14.7
Study Day’
R?SD=
10.9 + 4.6
1 1 2 3 7
CSF Plasma Plasma Plasma Plasma
IN ONE
BEFORE
AND
TREATMENT
HVA (&ml) 18.2 8.4 12.0 11.0 22.5
’ The patient was free of all medications for 2 weeks before entering the study. Day 7 of the study represents 4th day of the treatment with haloperidol (20 mg/day).
are lower than those determined by gas chro- 3. There was a twofold increase in free HVA matography with electron-capture detection, after neuroleptic treatment. they are similar to those determined by more specific GC-MS methods (Table 2). The apDISCUSSION plicability of the described procedure was also established by analyzing five plasma samples In this report we have described a simple and one CSF sample from one schizophrenic and reliable HPLC method for the determipatient. The patient was admitted to the Re- nation of HVA in human plasma. The method search Ward, University of Texas hospital, San has the potential for the simultaneous deterAntonio, Texas. After 2 weeks of washout pe- mination of 5-HT and 5-HIAA along with riod, plasma samples were collected for 3 con- HVA. Unlike previously described gas chrosecutive days. During this baseline period, a matograph and mass spectrometric methods, CSF sample was also obtained by lumber the HPLC procedure described here does not puncture. One plasma sample was collected require any extensive extraction procedure or after 4 days of treatment with haloperidol. derivatization. The HVA, 5-HT, and 5-HIAA The samples were shipped frozen and analyzed from deproteinized plasma are well resolved in Chicago. These results are shown in Table from the other components with the described analytical system in a reasonably short time. It has been shown that turnover rates of TABLE 2 neurotransmitter amines are correlated with functional activity of amine-containing neuHVA CONCENTRATIONS IN HUMAN PLASMA REPORTED BY DIFFERENT METHODS rons. The measurement of amine metabolites in rat brain or human urine and CSF has been HVA used as an index of aminergic activity. ReRef. Method (@ml) f SD N Range cently, Swann et al. (27) and Kendler et al. (19) GC-ECD” 62.0 2 45.0 (28) have reported that in man and rats, treat(20) GC-ECD’ 89.9 f 51.3 ment with peripheral monoamine oxidase in(21) GC-MSb 10.2 f 4.96 5 4-19 hibitor debrisoquin significantly decreased (22) GC-MS* 10.5 f 1.3 (2% GC-MSb l1.3f 4.2 23 2.4-18.4 plasma HVA. As suggested by these authors, This study HPLC 10.9 f 4.6 8 3-16 HVA determination in plasma after debrisoquin treatment provides a tool for determining L1Gas chromatography with electroncapture detector. central dopamine activity. Both of these studb Gas chromatography-mass spectrometry.
LIQUID
CHROMATOGRAPHY
OF 3-METHOXY-4-HYDROXYPHENYLACETIC
ies had used the GC-MS method for HVA determination. The method described in this paper would be useful for such future research, since many plasma samples can be analyzed in a short time. Although the plasma sample after deproteinization is directly injected, we have not observed any deterioration in column performance over a 3-month period. Instead, with more regular use the analytical system appears to be more stable.
ACID
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