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Quantitative determination of homovanillic acid in serum by mass fragmentography
EUROPEAN JOURNAL OF PHARMACOLOGY24 (1973) 37-42. NORTH-HOLLANDPUBLISHINGCOMPANY
QUANTITATIVE DETERMINATION OF HOMOVANILLIC A C I D I N S E R U M BY MASS F R A G M E N T O G R A P H Y Erik ~.NGGARD, Birgitta SJOQUIST, Bengt FYRO and GtSran SEDVALL Department of Pharmacology, Karolinska Institutet, S 104 01 Stockholm 60. Sweden
Accepted 22 May 1973
Received 25 May 1972
E. ANGGARD, B. SJOQUIST, B. FYRO and G. SEDVALL, Quantitative determination of homovanillic acid in serum by mass fragmentography, European J. Pharmacol. 24 (1973) 37-42. The analysis of homovanillic acid (HVA) in serum of humans and goats is described for the first time. The method uses gas chromatographic separation and mass spectrometric detection with the deuterated methyl ester of HVA as an internal standard. Simultaneous measurement of the protium and deuterium forms was performed with a multiple ion detector focused at the molecular ions of the methyl ester heptaflurobutyryl derivatives. The method has high specificity, a precision of +10% (S.E.) and a sensitivity which determines normal levels (100 pmole/ml) at 10 times above the background. The serum level in man was increased several fold foUowingphysical exercise. In goats probenecid elevated the level of HVA, whereas nialamide had the opposite effect. Dopamine metabolism Gas chromatography
Homovanillic acid analysis Mass spectrometry
1. Introduction Fluorimetric and gas chromatographic methods for measuring homovanillic acid (HVA) have been applied to brain, cerebrospinal fluid (CSF) and urine (Sharman, 1963; And6n et al., 1968; Sato, 1965). Analysis in blood has however not been possible due to low levels and interference by background material (Werdinius, 1967a). We now report the application of a new technique for the quantitative analysis of HVA in serum from humans and goats. The method uses gas chromatography-mass spectrometry with a deuterated internal standard and simultaneous measurement of the protium and deuterium forms by means of an accelerating voltage unit (Samuelsson et al., 1970; Gaffney et al., 1971; Sjt~quist et al., 1971; Sjoquist and Angg~Ird, 1972).
2. Materials and methods 2.1. Materials
The deuterated methyl ester of HVA was prepared by dissolving 200 mg HVA in 5 ml CDaOD and ad-
Probenecid Nialamide
cling 0.25 ml of acetyl chloride to the solution. The reaction mixture was left over night and then evaporated. The residue was dissolved in 1 ml H20 pH 7 - 8 and extracted with 3 X 2 ml ethyl acetate. During evaporation the product crystallized ( 1 9 0 ~ . 42-43°C). The product gave a single peak on GLC after treatment with heptafluorobutyric anhydride (HFBA) in ethyl acetate (Angg~rd and Sedvall, 1969). The mass spectrum showed a molecular ion at m/e 395 and prominent ions at M+ - 6 2 and M+ - 1 9 7 , probably due to elimination of O O ~1 II C-OCD 3 and C3F7-C (cf. SjOquist and ~ngghrd, 1972). 7 .3 H-HVA (specific activity 1.05 Ci/mmole) was a custom synthesis by the New England Nuclear Corp., Boston, Mass. The radiopurity was checked by thin layer chromatography on silica gel (solvent system toluene : HOAc : H20, 70 : 36 : 1.5) of the free acid and the methyl ester and radio gas chromatography of the methyl ester heptafluorobutyryl (HBF) derivative. The radioactive compound cochromatographed with inactive HVA. The radiopurity was assessed to be in excess of 99%.
38
E. A'nggard et aL, Homovanillic acid in serum
The ion exchange resin Dowex IX-2 (CI-, 2 0 0 - 4 0 0 mesh) was washed 3 times with distilled water prior to use. Diazomethane was prepared from p-toluene sulfonylmethylnitrosamide (Fluka AG, St. Gallen, Switzerland). The heptafluorobutyric anhydride was purchased from Pierce Chemical Co. The compound was distilled before use and kept at-20 ° . Serum from humans or goats was isolated from clotted blood by centrifugation at 2000 X g.
PEAK
HEIGHT
RATIO .10 .09 .08
.07 .06 .05
.04
2.2. Instrumental conditions
.03 .02.
A LKB 9000 mass spectrometer was used. The instrument was equipped with a 6 foot 1% SE-30 column. The usual column temperature was 170°, flash heater 210 ° and ion source 290 °. The energy of the electrons was kept at 22.5 eV and the trap current was 60/aA. The column support was coated with the liquid phase using the fluidization technique. The column was conditioned at maximum temperature for 2 days.
2. 3. Procedure 5 ml of serum was combined with 5 ml of distilled water and 0.05 /ICi (22 pmole) of 3 H-HVA and the pH adjusted to 7.5. The sample was applied to a column of Dowex (7 X 40 mm). After washing with 10 ml of H20, HVA and related compounds were eluted with 10 ml of 0.05 M HC1 in 50% ethanol (Korf et al., 1971). The eluate was neutralized with dilute NaOH and evaporated to about 3 ml in vacuo. 5 ml of 5 M NaC1 was added to the residue and the pH was adjusted to pH 2 - 3 . HVA was extracted twice with 10 ml of ethyl ether. The ether phase was dried by freezing out water at - 7 0 ° C followed by filtration. HVA-CD3, 5.23 nmole was added and the radioactivity was counted on an aliquot (for determination of recovery). The organic solvent was evaporated in vacuo and reconstituted in 50 /21 of ethyl acetate. Diazomethane in ether was added, the content was mixed and the diazomethane was removed by a stream of nitrogen within 1 min. The dry residue was dissolved in 50/al of ethyl acetate and 50/21 of HFBA. After 30 min at room temperature, the reagent and solvent were removed under reduced pressure. The final sample was dissolved in 50 /al of ethyl
.01 . . . . . . . . . . .01 .02 J~, .04 D6 911, ,O'r J~l D9
WEIGHT
RATIO
JO H~tt - C H | * HFll MVA-¢O~ - H F I
Fig. 1. Standard curve showing linearity between the peak height ratio determination of HVA-CH 3-HFB and the internal standard HVA-CD 3-HFB. acetate containing 2-5% of HFBA and 1 /al was injected into the GLC-MS system. The instrument was focused alternately on m/e 392 and 395, representing the molecular ion and base peak of the protium and deuterium derivative respectively. The peak heights were measured manually and the ratios of protium to deuterium forms were plotted on a standard curve (fig. 1) prepared by injecting standards containing known proportions of the protium and deuterium derivatives. Corrections were made for losses in the extraction step in the determination of the concentration in plasma. The recovery of 3 H-HVA through the isolation procedure was 65 -+ 4% (S.D.;n = 10). The standard deviation of the determination of the peak height ratios was found to be 2% after repeated injections (n = 5) of the same sample when the ratio of the protium to deuterium derivative was 0.05. The background peak height ratio of the internal standard at 392 and 395 was 0.003, due to small amounts of protium derivative present. This value was subtracted in the calculation of the final result.
2. 4. Applications o f the method Female goats weighing about 35 kg were injected i.v. with probenecid/100 mg/kg (Benemid ®, Astra, Stidert•lje, Sweden) or nialamide 25 mg/kg
E. .;fnggdrd et aL, Homovanillic acid in serum
39
(Niamid ® Pfizer, USA). Both drugs were dissolved in saline. Blood samples were drawn from the jugular vein of goats and from the antecubital vein of 11 fasting healthy female and male subjects at 8 am. In a female subject blood samples were drawn before, during and after 30 min of physical exercise on a bicycle ergometer. Probenecid in serum was determined by the method of Perel et al. (1970).
3. Results The precision of the method was determined from a pool of serum obtained from healthy blood donors. The mean level of 18 determinations was 160+16 pmole/ml. The recovery was studied by adding 147 or 294 pmole/ml of HVA to samples of serum. The amounts recovered were found to be 105-+2% (n = 5) and 92+4% (n = 4) respectively. The specificity of the mass spectrometric detection is high since only compounds having the same
t I I
30
I
t I I
I t
I I
I f
I t
'
//
!
,o/
-
" ,
I]
~
.
,f
I!
I
/ \
l
,
2
3
//\/ "~--'~.
-.----.
i
w
4
5
I~IN
Fig. 2. Multiple ion mass fragmentography of a serum sample containing 10 pmole/ml of HVA. The relative intensity of ions occurring at m/e 195 ( e - - - e ) , 198 ( o - - - o ) , 333 (X--×), 395 (e ;) and 392 (a o) were recorded by running a full mass spectrum at the indicated intervals. Column (6 foot) 1% SE-30, temp. 160°. The energy of the electrons was 22.5 eV, the trap current 60 t~A and the gain setting at 6.
IO i t Ix
Fig. 3. Mass fragmentographic determination of HVA in human serum. The instrument was focussed on m/e 395 (HVA-CD3-HFB, broken line) and on m/e 392 (HVACH 3-HFB, solid line), gain setting at 11. retention time as H V A - M e - H F B and having as high intensity fragments at m/e values of the molecular ions of H V A - C H a - H F B (m/e = 392) and H V A C D 3 - H F B (m/e = 395) respectively will disturbe the analysis. In fig. 2 is shown the analysis of serum sample using multiple ion detection of the ions 195, 198, 333, 392 and 395 by repetitive scanning. A peak height ratio determination between 195 and 198 is clearly unsuitable since the reagent gives a highbackground at 198. A focussing on m/e 333 is not practical since it is formed by elimination of the carbomethoxy group (m/e = 59) and thus is the same for both H V A - C H a - H F B and H V A - C D 3 - H F B . The molecular ions proved to be satisfactory since they occur in the high mass end, where background is low, and since they constitute the base peak in the methyl esters of the H V A - H F B derivatives (Angg~rd and Sedvall, 1969). The tracing from a mass fragmento-' graphic analysis of HVA in human plasma is shown in fig. 3. The dominating peaks are those due to H V A CH 3 - H F B and H V A - C D a - H F B respectively. The possibility that dihydroxyphenylacetic acid (DOPAC) could contribute to the yields of HVA found in the blood samples was studied. Additions of 1190 pmole of DOPAC per ml serum gave the same level of HVA (141 pmole/ml -+ 1.9%; n = 4) as those of the controls (144 pmole/ml -+ 12.9 %; n = 4).
E. ,~'nggard et aL, Homovanillic acid in serum
40
I00 -
Table 1
E
Subject
Sex
A.B. E.A. R.R. M.E. B.J. B.B. U.J. C.D. U.N. S.S. H.P.
f f m m f f f f f m f
Age
Weight (k_g)
o
HVA (pmole/ml) Duplicates Mean
E o.
50. 1"
I1: I,iJ tD
Z T l
l
1
4
6
HOURS
NIALAMIOE
25rng/kg
i.v.
Fig. 4. The effect of nialamide on the HVA level in the serum of a goat.
21 37 27 29 26 42 38 21 26 26 38
44 63 66 68 69 60 63 70 55 72 51
63 68 74 76 81 93 93 94 124 154 155
Mean S.D.
The sensitivity of the method is high. Normal levels of serum HVA in man and in the goat peak height ratios which were 10 times above the background (fig. 3). Since the background is determined partly by the amount of protium in the deuterated and tritiated internal standards, it is possible to increase the sensitivity further by adding less of these labelled internal standards. Treatment of goats with nialamide, a monoamine oxidase inhibitor, markedly reduced the level of HVA in serum (fig. 4), whereas administration of probenecid increased several fold the level of HVA in serum
+ 4
600
500. IOOmg / k9
i.v.
400
200.
E o
E
*6 E I00
200 t~ 1.) IM Z i,i 113 O
"1-
" ~,
I
IZ LIJ O')
PROBENECID
I I
O.
2
65 71 75 77 81 94 94 97 126 159 160 100 -+32
The serum levels of HVA in 11 healthy subjects are shown in table 1. The interindividual variation was considerable, ranging from 65 to 160 pmole/ml, while the standard deviation for the duplicates was only -+4 pmole/ml. The effect of physical exercise on the levels of HVA in human serum is shown in fig. 6. The HVA concentrations increased about 3-fold following the start of the exercise. The HVA level declined to normal levels within 180min after exercise was stopped.
(fig. 5).
PROBENECID
66 74 75 78 81 95 95 99 128 166 165
;o
.ou.s
Fig. 5. The effect of probenecid on the serum level of HVA in a goat.
E. Jfnggt2rd et aL, Homovanillic acid in serum
200
IOO
300
" • 2 00 ,oo
J m
p.Ys,c.L
ExE.c,s~
Fig. 6. The effect of physical exercise (bicycle ergometer) on the serum levels of HVA in a female human subject. 4. Discussion
The use of internal standards is widely practised in quantitative gas chromatographic analysis. The ideal internal standard should have physical and chemical properties closely related to the compound to be analyzed in order to give the same yields through the purification and derivatization procedures, but it should be distinguished in the final GLC-analysis. Using the mass spectrometer as detector for the GLCeffluents through focusing at single ions, it could be predicted that labelling with stable isotopes would provide suitable internal standards, since they have a small difference in molecular weight but are otherwise identical. With the development of techniques for the alternate or simultaneous focusing of up to 4 ions (Sweeley et al., 1966; Hammar and Hessling, 1971) deuterium-labelled internal standards have been used for the analysis of prostaglandin El in serum (Samuelsson, Hamberg and Sweeley, 1970), 5hydroxyindolacetic acid in CSF (Bertilsson et al., 1972), nortriptyline in plasma (Gaffney et al., 1971) and HVA in CSF (Sjtiquist and Angg~rd, 1972). In the present paper we extend the mass fra~nento-
41
graphic technique for the measurement of HVA in CSF to applications on serum from humans and goats. The precision was found to be +10% (S.D.). At present the main sources of error probably lie in the focusing. Small drifts of magnetic current may move the peak slightly off the slit thus influencing the protium to deuterium ratio. In the future a computer may aid in the focusing, thus improving the accuracy and precision of the procedure. Following the administration of nialamide, a potent MAO-inhibitor, the enzymatic formation of HVA from dopamine should be blocked (Bliss and Ailon, 1971). Probenecid inhibits transport of organic acids like HVA from the cerebrospinal fluid and the kidney (Werdinius, 1967b; Neff et al., 1967). The demonstration that HVA levels in serum are reduced by nialamide and increased by probenecid provides pharmacological evidence for the specificity of our method and indicates its applicability for pharmacological and physiological studies. With the present method, HVA levels in serum of fasting healthy humans were found to be about 100 pmoles/ml. During physical exercise a marked elevation of the levels was found. This could be expected since physical work has been shown to cause increased levels of catecholamines in serum and urine (Haggendal et al., 1970). Previously available methods have not permitted the determination of HVA in serum (Werdinius, 1967a). Together with new procedures{or measuring HVA in human cerebrospinal fluid (Sjt~quist et al., 1971; Sjt~quist and Angg~rd, 1972) and urine (Sjtiquist et al., 1971; Daily and )~ngg~ird, 1973) the present method provides means for obtaining an increased knowledge about the brain dopamine system in man.
Acknowledgements
This study was supported by grants from the Swedish Medical Research Council No. 40X-3560 and from the Swedish Delegation for Applied Defense Research No 452. The authors are indebted to Dr. Rolf SjiSstrOmfor determination of probenecid in serum and to Barbro Beck, Berit Holmberg, Befit Johansson and Margareta S~ker for skilful technical assistance.
42
E. .~ngg~rd et al., Homovanillic acid in serum
References And~n, N.-E., B.-E. Roos and B. Werdinius, 1968, On the occurrence of homovaniUic acid in brain and cerebrospinal fluid and its determination by a fluorimetric method, Life Sci. 7,448. Angg~ird, E. and G. Sedvall, 1969, Gas chromatography of eatecholamine metabolites using electron capture detection and mass spectrometry, Anal. Chem. 41, 1250. Bertilsson, L., A.J. Atkinson Jr., J.R. Althaus, A. H~irfast and B. Holmstedt, 1972, Quantitative determination of 5-hydroxyindole-3-acetic acid in cerebrospinal fluid by gas chromatography-mass spectrometry, Anal. Chem. 44, 1434. Bliss, E. and J. Ailon, 1971, Relationship of stress and activity to brain dopamine and homovanillic acid, Life Sci. 10, 1161. Dailey, J.W. and E. Angg~rd, 1973, Gas chromatographic determination of homovaniUic acid in human and rat urine, Biochem. Pharmacol. 22, in press. Gaffney, T.E., C.G. Hammar, B. Holmstedt and R.E. McMahon, 1971, Ion specific detection of internal standards labelled with stable isotopes, Anal.Chem. 43, 307. Hammar, C.-G. and R. Hessling, 1971, A novel peak matching technique by means of a new and combined multiple ion detector-peak marcher device: elemental analyses of compounds in submicrogram quantities without prior isolation, Anal. Chem. 43, 298. H~iggendal, J., L.H. Hartley and B. Saltin, 1970, Arterial noradrenaline concentration during exercise in relation to the relative levels, Scand. J. Clin. Lab. Invest. 26, No. 4,337. Korf, J., B.-E. Roos and B. Werdinius, 1971, Fluorometric determination of homovanillic acid in tissues, using an ion exchange separation and mixed solvent elution, Acta Chem. Scand. 26,333. Neff, N.M., T.N. Tozer and B.B. Brodie, 1967, Application of steady-state kinetics to studies of the transfer of 5-hy-
droxyindoleacetic acid from brain to plasma, J. Pharmacol. Exptl. Therap. 158, 214. Perel, J.M., R.F. Cunningham, H.M. Fales and P.G. Dayton, 1970, Identification and renal excretion of probenecid metabolites in man, Life Sci. 9, 1337. Samuelsson, B., M. Hamberg and C.C. Sweeley, 1970, Quantitative gas chromatography of prostaglandin E1 at the nanogram level: Use of deuterated carrier and multipleion analyser, Anal. Biochem. 38, 301. Sato, T.L., 1965, The quantitative determination of 3methoxy-4-hydroxyphenylacetic acid (homovaniliic acid) in urine, J. Lab. Clin. Med. 66, 507. Sharman, D.F., 1963, Fluorimetric method for the estimation of 4-hydroxy-3-methoxyphenylacetic acid (homovaniUic acid) and its identification in brain tissue, Brit. J. Pharmacol. Chemotherap. 20, 204. Sweeley, C.C., W.H. Elliot, J. Tories and R. Ryhage, 1966, Mass spectrometric determination of unresolved components in gas chromatographic effluents, Anal. Chem. 38, 1549. Sjtiquist, B. and E. J~nggard, 1972, Determination of homovanillic acid in cerebrospinal fluid by gas chromatography with electron capture or mass spectrometric detection, Anal. Chem. 44, 2297. Sjtiquist, B., J.W. Dailey and E. J~ngg~rd, 1971, New gas chromatographic methods for the determination of homovanillic acid in urine and cerebrospinal fluid, Acta Pharmacol. Toxicol. 29, Suppl. 4, 55. Werdinius, B., 1967a, Elimination of 3,4-dihydroxyphenylacetic acid from the blood, Acta Pharmacol. Toxicol. 25, 9. Werdinius, B., 1967b, Effect of probenecid on the levels of monoamine metabolites in the rat brain, Acta Pharmacol. Toxicol. 25, 18.
QUANTITATIVE DETERMINATION OF HOMOVANILLIC A C I D I N S E R U M BY MASS F R A G M E N T O G R A P H Y Erik ~.NGGARD, Birgitta SJOQUIST, Bengt FYRO and GtSran SEDVALL Department of Pharmacology, Karolinska Institutet, S 104 01 Stockholm 60. Sweden
Accepted 22 May 1973
Received 25 May 1972
E. ANGGARD, B. SJOQUIST, B. FYRO and G. SEDVALL, Quantitative determination of homovanillic acid in serum by mass fragmentography, European J. Pharmacol. 24 (1973) 37-42. The analysis of homovanillic acid (HVA) in serum of humans and goats is described for the first time. The method uses gas chromatographic separation and mass spectrometric detection with the deuterated methyl ester of HVA as an internal standard. Simultaneous measurement of the protium and deuterium forms was performed with a multiple ion detector focused at the molecular ions of the methyl ester heptaflurobutyryl derivatives. The method has high specificity, a precision of +10% (S.E.) and a sensitivity which determines normal levels (100 pmole/ml) at 10 times above the background. The serum level in man was increased several fold foUowingphysical exercise. In goats probenecid elevated the level of HVA, whereas nialamide had the opposite effect. Dopamine metabolism Gas chromatography
Homovanillic acid analysis Mass spectrometry
1. Introduction Fluorimetric and gas chromatographic methods for measuring homovanillic acid (HVA) have been applied to brain, cerebrospinal fluid (CSF) and urine (Sharman, 1963; And6n et al., 1968; Sato, 1965). Analysis in blood has however not been possible due to low levels and interference by background material (Werdinius, 1967a). We now report the application of a new technique for the quantitative analysis of HVA in serum from humans and goats. The method uses gas chromatography-mass spectrometry with a deuterated internal standard and simultaneous measurement of the protium and deuterium forms by means of an accelerating voltage unit (Samuelsson et al., 1970; Gaffney et al., 1971; Sjt~quist et al., 1971; Sjoquist and Angg~Ird, 1972).
2. Materials and methods 2.1. Materials
The deuterated methyl ester of HVA was prepared by dissolving 200 mg HVA in 5 ml CDaOD and ad-
Probenecid Nialamide
cling 0.25 ml of acetyl chloride to the solution. The reaction mixture was left over night and then evaporated. The residue was dissolved in 1 ml H20 pH 7 - 8 and extracted with 3 X 2 ml ethyl acetate. During evaporation the product crystallized ( 1 9 0 ~ . 42-43°C). The product gave a single peak on GLC after treatment with heptafluorobutyric anhydride (HFBA) in ethyl acetate (Angg~rd and Sedvall, 1969). The mass spectrum showed a molecular ion at m/e 395 and prominent ions at M+ - 6 2 and M+ - 1 9 7 , probably due to elimination of O O ~1 II C-OCD 3 and C3F7-C (cf. SjOquist and ~ngghrd, 1972). 7 .3 H-HVA (specific activity 1.05 Ci/mmole) was a custom synthesis by the New England Nuclear Corp., Boston, Mass. The radiopurity was checked by thin layer chromatography on silica gel (solvent system toluene : HOAc : H20, 70 : 36 : 1.5) of the free acid and the methyl ester and radio gas chromatography of the methyl ester heptafluorobutyryl (HBF) derivative. The radioactive compound cochromatographed with inactive HVA. The radiopurity was assessed to be in excess of 99%.
38
E. A'nggard et aL, Homovanillic acid in serum
The ion exchange resin Dowex IX-2 (CI-, 2 0 0 - 4 0 0 mesh) was washed 3 times with distilled water prior to use. Diazomethane was prepared from p-toluene sulfonylmethylnitrosamide (Fluka AG, St. Gallen, Switzerland). The heptafluorobutyric anhydride was purchased from Pierce Chemical Co. The compound was distilled before use and kept at-20 ° . Serum from humans or goats was isolated from clotted blood by centrifugation at 2000 X g.
PEAK
HEIGHT
RATIO .10 .09 .08
.07 .06 .05
.04
2.2. Instrumental conditions
.03 .02.
A LKB 9000 mass spectrometer was used. The instrument was equipped with a 6 foot 1% SE-30 column. The usual column temperature was 170°, flash heater 210 ° and ion source 290 °. The energy of the electrons was kept at 22.5 eV and the trap current was 60/aA. The column support was coated with the liquid phase using the fluidization technique. The column was conditioned at maximum temperature for 2 days.
2. 3. Procedure 5 ml of serum was combined with 5 ml of distilled water and 0.05 /ICi (22 pmole) of 3 H-HVA and the pH adjusted to 7.5. The sample was applied to a column of Dowex (7 X 40 mm). After washing with 10 ml of H20, HVA and related compounds were eluted with 10 ml of 0.05 M HC1 in 50% ethanol (Korf et al., 1971). The eluate was neutralized with dilute NaOH and evaporated to about 3 ml in vacuo. 5 ml of 5 M NaC1 was added to the residue and the pH was adjusted to pH 2 - 3 . HVA was extracted twice with 10 ml of ethyl ether. The ether phase was dried by freezing out water at - 7 0 ° C followed by filtration. HVA-CD3, 5.23 nmole was added and the radioactivity was counted on an aliquot (for determination of recovery). The organic solvent was evaporated in vacuo and reconstituted in 50 /21 of ethyl acetate. Diazomethane in ether was added, the content was mixed and the diazomethane was removed by a stream of nitrogen within 1 min. The dry residue was dissolved in 50/al of ethyl acetate and 50/21 of HFBA. After 30 min at room temperature, the reagent and solvent were removed under reduced pressure. The final sample was dissolved in 50 /al of ethyl
.01 . . . . . . . . . . .01 .02 J~, .04 D6 911, ,O'r J~l D9
WEIGHT
RATIO
JO H~tt - C H | * HFll MVA-¢O~ - H F I
Fig. 1. Standard curve showing linearity between the peak height ratio determination of HVA-CH 3-HFB and the internal standard HVA-CD 3-HFB. acetate containing 2-5% of HFBA and 1 /al was injected into the GLC-MS system. The instrument was focused alternately on m/e 392 and 395, representing the molecular ion and base peak of the protium and deuterium derivative respectively. The peak heights were measured manually and the ratios of protium to deuterium forms were plotted on a standard curve (fig. 1) prepared by injecting standards containing known proportions of the protium and deuterium derivatives. Corrections were made for losses in the extraction step in the determination of the concentration in plasma. The recovery of 3 H-HVA through the isolation procedure was 65 -+ 4% (S.D.;n = 10). The standard deviation of the determination of the peak height ratios was found to be 2% after repeated injections (n = 5) of the same sample when the ratio of the protium to deuterium derivative was 0.05. The background peak height ratio of the internal standard at 392 and 395 was 0.003, due to small amounts of protium derivative present. This value was subtracted in the calculation of the final result.
2. 4. Applications o f the method Female goats weighing about 35 kg were injected i.v. with probenecid/100 mg/kg (Benemid ®, Astra, Stidert•lje, Sweden) or nialamide 25 mg/kg
E. .;fnggdrd et aL, Homovanillic acid in serum
39
(Niamid ® Pfizer, USA). Both drugs were dissolved in saline. Blood samples were drawn from the jugular vein of goats and from the antecubital vein of 11 fasting healthy female and male subjects at 8 am. In a female subject blood samples were drawn before, during and after 30 min of physical exercise on a bicycle ergometer. Probenecid in serum was determined by the method of Perel et al. (1970).
3. Results The precision of the method was determined from a pool of serum obtained from healthy blood donors. The mean level of 18 determinations was 160+16 pmole/ml. The recovery was studied by adding 147 or 294 pmole/ml of HVA to samples of serum. The amounts recovered were found to be 105-+2% (n = 5) and 92+4% (n = 4) respectively. The specificity of the mass spectrometric detection is high since only compounds having the same
t I I
30
I
t I I
I t
I I
I f
I t
'
//
!
,o/
-
" ,
I]
~
.
,f
I!
I
/ \
l
,
2
3
//\/ "~--'~.
-.----.
i
w
4
5
I~IN
Fig. 2. Multiple ion mass fragmentography of a serum sample containing 10 pmole/ml of HVA. The relative intensity of ions occurring at m/e 195 ( e - - - e ) , 198 ( o - - - o ) , 333 (X--×), 395 (e ;) and 392 (a o) were recorded by running a full mass spectrum at the indicated intervals. Column (6 foot) 1% SE-30, temp. 160°. The energy of the electrons was 22.5 eV, the trap current 60 t~A and the gain setting at 6.
IO i t Ix
Fig. 3. Mass fragmentographic determination of HVA in human serum. The instrument was focussed on m/e 395 (HVA-CD3-HFB, broken line) and on m/e 392 (HVACH 3-HFB, solid line), gain setting at 11. retention time as H V A - M e - H F B and having as high intensity fragments at m/e values of the molecular ions of H V A - C H a - H F B (m/e = 392) and H V A C D 3 - H F B (m/e = 395) respectively will disturbe the analysis. In fig. 2 is shown the analysis of serum sample using multiple ion detection of the ions 195, 198, 333, 392 and 395 by repetitive scanning. A peak height ratio determination between 195 and 198 is clearly unsuitable since the reagent gives a highbackground at 198. A focussing on m/e 333 is not practical since it is formed by elimination of the carbomethoxy group (m/e = 59) and thus is the same for both H V A - C H a - H F B and H V A - C D 3 - H F B . The molecular ions proved to be satisfactory since they occur in the high mass end, where background is low, and since they constitute the base peak in the methyl esters of the H V A - H F B derivatives (Angg~rd and Sedvall, 1969). The tracing from a mass fragmento-' graphic analysis of HVA in human plasma is shown in fig. 3. The dominating peaks are those due to H V A CH 3 - H F B and H V A - C D a - H F B respectively. The possibility that dihydroxyphenylacetic acid (DOPAC) could contribute to the yields of HVA found in the blood samples was studied. Additions of 1190 pmole of DOPAC per ml serum gave the same level of HVA (141 pmole/ml -+ 1.9%; n = 4) as those of the controls (144 pmole/ml -+ 12.9 %; n = 4).
E. ,~'nggard et aL, Homovanillic acid in serum
40
I00 -
Table 1
E
Subject
Sex
A.B. E.A. R.R. M.E. B.J. B.B. U.J. C.D. U.N. S.S. H.P.
f f m m f f f f f m f
Age
Weight (k_g)
o
HVA (pmole/ml) Duplicates Mean
E o.
50. 1"
I1: I,iJ tD
Z T l
l
1
4
6
HOURS
NIALAMIOE
25rng/kg
i.v.
Fig. 4. The effect of nialamide on the HVA level in the serum of a goat.
21 37 27 29 26 42 38 21 26 26 38
44 63 66 68 69 60 63 70 55 72 51
63 68 74 76 81 93 93 94 124 154 155
Mean S.D.
The sensitivity of the method is high. Normal levels of serum HVA in man and in the goat peak height ratios which were 10 times above the background (fig. 3). Since the background is determined partly by the amount of protium in the deuterated and tritiated internal standards, it is possible to increase the sensitivity further by adding less of these labelled internal standards. Treatment of goats with nialamide, a monoamine oxidase inhibitor, markedly reduced the level of HVA in serum (fig. 4), whereas administration of probenecid increased several fold the level of HVA in serum
+ 4
600
500. IOOmg / k9
i.v.
400
200.
E o
E
*6 E I00
200 t~ 1.) IM Z i,i 113 O
"1-
" ~,
I
IZ LIJ O')
PROBENECID
I I
O.
2
65 71 75 77 81 94 94 97 126 159 160 100 -+32
The serum levels of HVA in 11 healthy subjects are shown in table 1. The interindividual variation was considerable, ranging from 65 to 160 pmole/ml, while the standard deviation for the duplicates was only -+4 pmole/ml. The effect of physical exercise on the levels of HVA in human serum is shown in fig. 6. The HVA concentrations increased about 3-fold following the start of the exercise. The HVA level declined to normal levels within 180min after exercise was stopped.
(fig. 5).
PROBENECID
66 74 75 78 81 95 95 99 128 166 165
;o
.ou.s
Fig. 5. The effect of probenecid on the serum level of HVA in a goat.
E. Jfnggt2rd et aL, Homovanillic acid in serum
200
IOO
300
" • 2 00 ,oo
J m
p.Ys,c.L
ExE.c,s~
Fig. 6. The effect of physical exercise (bicycle ergometer) on the serum levels of HVA in a female human subject. 4. Discussion
The use of internal standards is widely practised in quantitative gas chromatographic analysis. The ideal internal standard should have physical and chemical properties closely related to the compound to be analyzed in order to give the same yields through the purification and derivatization procedures, but it should be distinguished in the final GLC-analysis. Using the mass spectrometer as detector for the GLCeffluents through focusing at single ions, it could be predicted that labelling with stable isotopes would provide suitable internal standards, since they have a small difference in molecular weight but are otherwise identical. With the development of techniques for the alternate or simultaneous focusing of up to 4 ions (Sweeley et al., 1966; Hammar and Hessling, 1971) deuterium-labelled internal standards have been used for the analysis of prostaglandin El in serum (Samuelsson, Hamberg and Sweeley, 1970), 5hydroxyindolacetic acid in CSF (Bertilsson et al., 1972), nortriptyline in plasma (Gaffney et al., 1971) and HVA in CSF (Sjtiquist and Angg~rd, 1972). In the present paper we extend the mass fra~nento-
41
graphic technique for the measurement of HVA in CSF to applications on serum from humans and goats. The precision was found to be +10% (S.D.). At present the main sources of error probably lie in the focusing. Small drifts of magnetic current may move the peak slightly off the slit thus influencing the protium to deuterium ratio. In the future a computer may aid in the focusing, thus improving the accuracy and precision of the procedure. Following the administration of nialamide, a potent MAO-inhibitor, the enzymatic formation of HVA from dopamine should be blocked (Bliss and Ailon, 1971). Probenecid inhibits transport of organic acids like HVA from the cerebrospinal fluid and the kidney (Werdinius, 1967b; Neff et al., 1967). The demonstration that HVA levels in serum are reduced by nialamide and increased by probenecid provides pharmacological evidence for the specificity of our method and indicates its applicability for pharmacological and physiological studies. With the present method, HVA levels in serum of fasting healthy humans were found to be about 100 pmoles/ml. During physical exercise a marked elevation of the levels was found. This could be expected since physical work has been shown to cause increased levels of catecholamines in serum and urine (Haggendal et al., 1970). Previously available methods have not permitted the determination of HVA in serum (Werdinius, 1967a). Together with new procedures{or measuring HVA in human cerebrospinal fluid (Sjt~quist et al., 1971; Sjt~quist and Angg~rd, 1972) and urine (Sjtiquist et al., 1971; Daily and )~ngg~ird, 1973) the present method provides means for obtaining an increased knowledge about the brain dopamine system in man.
Acknowledgements
This study was supported by grants from the Swedish Medical Research Council No. 40X-3560 and from the Swedish Delegation for Applied Defense Research No 452. The authors are indebted to Dr. Rolf SjiSstrOmfor determination of probenecid in serum and to Barbro Beck, Berit Holmberg, Befit Johansson and Margareta S~ker for skilful technical assistance.
42
E. .~ngg~rd et al., Homovanillic acid in serum
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