Simple gas chromatographic analysis of plasma dopa and dopamine

11

Clinica

Chimica

Acta,

74 (1977)

0 Elsevier/North-Holland

11-19 Biomedical Press

CCA 8123

SIMPLE GAS CHROMATOGRAPHIC DOPAMINE

YOSHIKUNI Department

(Received

ANALYSIS

OF PLASMA DOPA AND

MIZUNO of Neurology,

Jichi

Medical

School,

Minamikawachi,

Tochigi

32904

(Japan)

June 3rd, 1976)

Summary A method for gas chromatographic analysis of plasma dopa and dopamine is described. Deproteinized plasma was shaken with acid washed aluminium oxide at pH 8.8. Dopa and dopamine were eluted from alumina with weak acids and evaporated to dryness. The pentafluoropropionic derivative of dopamine and the trifluoroacetic derivative of dopa were made and injected into a gas chromatograph equipped with a 63Ni electron capture detector. Alpha-methyl derivatives of each substance were used as internal standards. Linearity of plasma working curves of each substance was good. This method was applied to the assay of plasma samples from Parkinsonian patients under treatment with L-dopa with or without peripheral dopa decarboxylase inhibitors. The method described is sensitive and simple enough to monitor the blood levels of dopa and dopamine of these patients.

Introduction In recent years there has been great progress in the treatment of Parkinson’s disease. L-Dopa has been found to be particularly effective against the akinesia and rigidity of Parkinson’s disease [1,2]. Recently, concomitant use of a peripheral dopa decarboxylase inhibitor has been introduced to reduce side effects of L-dopa [3,4]. However, still in the course of the treatment various kinds of untoward effects may appear and some of the patients seem to be resistant to L-dopa treatment. To solve these problems, measurement of plasma dopa and dopamine concentrations occupies an important part of the investigation. There is a need for a simple and sensitive method for the determination of plasma dopa and dopamine. Imai et al. published a sensitive gas chromatographic method for plasma catecholamines [ 5,6]. We have made some modifications to their method for the convenience of daily routine analysis. It is the purpose of this report to present our method and its clinical application.

1”

Materials and methods Reagents Dopamine . HCl, L-3,4_dihydroxyphenylalanine (L-dopa), trifluoroacetic anhydride and pentafluoropropionic anhydride were puchased from Tokyo Kasei (Tokyo); aluminium oxide (neutral, chromatography grade, activity 1) from Woelm (Eschwege). Aluminium oxide was prepared according to the method of Anton and Sayre [7] before use. Alpha-methyldopamine was kindly provided by Sterling-Winthrop Research Institute (Rensselaer, N.Y.) and alpha-methyldopa by Merk-Banyu Pharmaceutical Companies (Tokyo). Other chemicals were purchased from Wako Pure Chemical Industries (Osaka). The chemicals used were of highest purity grade available. Methanol and ethanol were distilled before use. n-Butanol was dehydrated with pieces of magnesium ribbon and then distilled. HCl-saturated butanol was prepared by introducing HCl gas from a HCI cylinder. HCl gas was washed with concentrated sulfuric acid before use. Equipment A Shimazu Model GC 4BM gas chromatograph equipped with a 63Ni electron capture detector (ECD) and fitted with a 2-m glass column (4-mm internal diameter) was used. The ECD was operated at a pulse rate of 10 kHz and pulse amplitude of 48 V. Stationary phase for the analysis of dopamine was 5% SE 54 on GasChrom Q (100-120 mesh) and the column temperature 175°C. The carrier gas was nitrogen and the flow rate 17 ml/min. Stationary phase for the analysis of dopa was 1% OV 17 on GasChrom Q (So-100 mesh) and the column temperature 153°C. The flow rate of the carrier gas was 20 ml/min. The injection port and detector temperatures were kept at 220°C. For the analysis of dopa a sample concentrating unit (Shimazu PCC-4M-2 type) was attached to the gas chromatograph. The unit is a device developed for the purpose of large volume injection. The principle of the unit is to direct the solvent gas outside the main column without losing the samples. A schematic drawing of the unit is shown in Fig. 1. The exhaust valve is opened just before the injection. Injection is made on the precolumn. Since the exhaust valve is open, flow of the carrier gas is towards the valve. Therefore most of the solvent will be led to the outside of the column. Purge gas I is to prevent diffusion of the solvent into the main

Fig.

1. Schematic

drawing

of the sample-concentrating

unit.

Explanation

in the

text.

13

column while the solvent is being released from the exhaust valve. Purge gas II and leak gas are to prevent the solvent gas, remaining in the column while the exhaust valve is closed, from entering the main column by diffusion and also to enhance entrance of the sample gas into the main column. The precolumn consists of a 40-cm glass column (4-mm internal diameter) packed with the same stationary phase as the main column. Subjects Blood samples were collected from eleven patients with P~ki~so~ism being treated with L-dopa. At least two samples were drawn from each patient while he was being treated with L-dopa alone and while he was being treated with Ldopa and a peripheral dopa decarboxylase inhibitor. The sample was taken into a heparinized syringe approximately 2 h after the last medication. Blood was immediately centrifuged for 5 min at 2500 X g and the plasma was kept at -25°C until assayed. Methods

Analysis of plasma dopamine 1 ml of plasma was deproteini~ed with 1 ml of 0.8 N perchlo~i& acid containing 250 ng of ~pha-m~thyldopamine as an internal standard. The mixture was centrifuged at 15 000 X g for 20 min at 4°C. The supernatant was tr~sfered~to a siliconized TO-ml glass stoppered cent~fuge tube. To the pellet 2 ml of 0.4 N perchloric acid was added and mixed well. The mixture was centrifuged again at 15 000 X g for 20 min at 4°C. Both supernatants were combined. Then 0.25 ml of 0.2 M sodium metabisulfite was added as an antioxidant and nitrogen gas was introduced for 2 min to replace oxygen in the deproteinized plasma according to the method of Imai et al. [ 5,6]. The tube was tightly capped and put into boiling water for 20 min for acid hydrolysis. After the tube had been cooled to room temperature, 0.5 ml of 0.2 M EDTA disodium salt was added and the pH of the mixture was adjusted to 6.0. Then 400 mg of acid-washed aluminium oxide was added and the pH of the mixture was adjusted to 8.8. For the adjustment of pH 3.8 N ammonia solution was used. The mixture was gently shaken 100 times and the supernatant was discarded. The alumina was washed twice with 10 ml of water. ~ate~hol~ines were eluted from alumina with 3.5 ml of 0.4 N acetic acid in methanol. The eluate was transfered to a 5-ml pea-shaped flask and evaporated to dryness at 60°C under reduced pressure. To the residue 50 ~1 of ethylacetate and 20 pl of pentafluoropropio~ic anhydride were added and the mixture was heated at 65°C for 10 min. Unreacted solvents were evaporated under a stream of dry nitrogen. The residue was dissolved in 100 ~1 of ethyl acetate and 1 ~1 was injected into the gas chromatograph.

Initial parts of the extraction procedure are the same as those for dopamine. The points of difference are as follows. As an internal standard, 1000 ng of ~pha-methyldopa were used, 800 mg of alumina were added and 3.5 ml of 0.4 N formic acid in ethanol were used to elute dopa from alumina. After the eluate had evaporated to dryness, 0.25 ml of HCl-saturated butanol was added

and heated at 100°C for 20 min. Unreacted butanol was evaporated under reduced pressure. The residue was dissolved in 40 ~1 of ehtyl acetate and 10 1.11of trifluoroacetic anhydride was added. The mixture was kept at a room trmperature for 10 min, 150 ~1 of n-hexane were added and 1 ~1 of the mixture was injected into the gas chromatograph. The exhaust valve was opened just before irject ion and kept open for 70 seconds after injection. Results Typical chromatograms of authentic dopamine and alpha-methyldopamine and of a sample from a patient are shown in Fig. 2 and Fig. 3, respectively. Plasma standards containing known amounts of dopamine in plasma were made and assayed according to the method described. The plasmas utilized were obtained from normal persons who were not taking any medications. The results are shown in Fig. 4. There is a good linear correlation between the plasma concentration of dopamine and the ratio of dopamine to the internal standard. The ratio was calculated by cutting out the peaks of chromatogram and weighing them. The recovery of dopamine was measured by adding a known amount of dopamine to plasma and its peak weight was compared to that of a known amount of authentic dopamine. The recovery of dopamine was 69.8 f 3.4 (S.E.) %.

100

90

100

1

90

Dopomlne Standard 5% SE 54/Gaschrom Q loo-120 Column T 175’C Detector T 22O’C Carrier gas N1 0.7 kg/cm2

80

i

S-159 (HT) 5% SE 54/Goschrom Q 100-120 Column T 175’C Detector T 220°C Carrier gas N1 0.7 kg/cm*

80.

70-

70.

60-

60.

IS Dopomine

1 DoDomjne

50-

40-

:-

30-

20.

10. _

OJ

I

0

10

min

Fig.

2. Chromatogram

of standards.

Fig.

3. Chromatogram

of a sample

0

from

a patient.

10

min

15

looDow

Standard

IY~OV 171Gaschrom Column T 153°C &ector T 220°C

go-

Corner

gas

0

80 -100

Nz 0.9 kg/cm’

so-

Dopa

70-

IS

60-

I

IL v

.

100

O-’ 250

500

750 ng/mi

Fig. 4. Working less than 6.6%.

curve

Fig. 5. Chromatogram

of plasma

0

dopamine.

of standards.

I 20

< 10

The average of two independent

experiments.

L min

The S.E.M.

was

Analysis using a precolumn.

Typical chromatograms of authentic dopa and alpha-methyldopa and of a sample from a patient are shown in Fig. 5 and Fig. 6, respectively. For comparison, chromatograms of dopa and alpha-methyldopa analyzed without using the sample-concentrating unit are shown in Fig. 7. The great difference in the degree of tailing by the solvents is clearly visible. This difference is much more prominent when samples from patients are analyzed (Fig. 8). As discussed in the experimental conditions in the methods, the optimal opening period of the exhaust valve was found to be 70 seconds. If the period was too long, reduction in the peak height of the internal standard occurred and if the duration was too short, tailing from the solvents became much larger. As in the case of dopamine, plasma standards containing known amounts of dopa were made and assayed according to the methods described. The results are shown in Fig. 9. There is a good linear relationship between the plasma concentration of dopa and the ratio of dopa to the internal standard. The ratio was calculated as for dopamine by weighing the peaks of chromatogram. Recovery of dopa assayed by adding a known amount of dopa to plasma was 83.8 f 7.6 (S.E.) %. For the analysis of dopa, we tested the possibilities of using other stationary phases, i.e. 5% SE 30 on shimalite and 1% XF 1105 on Chromosorb W. In both stationary phases, separation of dopa and alpha-methyldopa was excellent, however, when plasma samples were assayed, unknown peaks overlapped with

16

100

Dopo Stan&r-d l”/.OV 17/Goschrom C ?!-‘UC Column i 153°C Detector T 22C”L Carrier qoi Yp 0.9 kg/cm’

IOC s-152 (EM)‘ Precoiumn l%OV17/Gaschrom 080-100 Column T 153°C Detector T 220°C Carrier gas NZ 0.9 kg/cm2

9c

90

80

80 70

IS 70 Dopa

60 50 50 40. 40 30. 30 20.

i lo-

-6 Fig. 6. Chromatogram

of a patient

Fit.

of standards.

7. Chromatogram

Analysis

sample.

Analysis

lb

20

min

using a precolumn.

without

using a precolumn.

loo-

S-152 (EM) Regular anolys~s 1% OV 17/ Gaschrom Q 80 -100 Column T 153°C Detector T220’C Carrier gas N2 1.0 kg /cm*

go-

60.

50-

400.8

30-

0.6

.

20-

lo-

b

lb

2b

min

0.5

1.0

O-

Fig. 8. Chromatogram Fig. 9. Working than 6.8%.

curve

of a patient of plasma

sample. dopa.

Analysis

without

The average

3.0

2.0 a/m1

using a precolumn.

of 4 independent

experiments.

The S.E.M.

was less

17

the peak of either dopa or alpha-methyldopa and they could not be used. To eliminate tailing from solvents, we attempted to remove unreacted solvents under a stream of dry nitrogen. However, peak heights diminished significantly, indicating partial hydrolysis having occurred during this procedure. Pentafluoropropionic derivatives of dopa and alpha-methyldopa were more stable and it was possible to remove unreacted solvents under a stream of dry nitrogen without significant loss of peak heights, however, the base line of the chromatogram rose up gradually during multiple injections so it also could not be used for our purpose. We found that the sample-concentrating unit could be utilized to remove most of the tailing from solvents. Results obtained by the analyses of samples from patients are shown in Table I. All of the patients were treated with L-dopa alone initially and then switched to combined treatment with L-dopa and a peripheral dopa decarboxylase inhibitor. Either MK-486 * or Ro4-4602 ** was used as an inhibitor. TABLE

I

PLASMA

DOPA

AND

DOPAMINE

Case

L-Dopa

LEVELS

IN PARKINSONIAN

PATIENTS

alone

Dopa

Dopa

Dopamine

Dopal

dosage

Cog/ml)

(nglml)

Dopamine

(mg/d) H.M.

53

3000

52

1600

350 -

752

K.T. T.T.

66

2400

166

1800

H.T.

27

750

799

623

1.28

K.M.

43

1200

307

815

0.38

0.47

_

-

0.09

W.M.

65

1800

294

464

0.63

E.M.

64

2400

1309

2100

0.62

N.T.

60

2000

1366

N.H.

47

2 400

342

1043

T.M.

36

1800

1107

469

2.36

W. K.

59

2400

430

0.64

L-Dopa

240

276

5.69 0.33

+ inhibitor

Dopa

Inhibitor

dosage

dosage

(mgld)

(mg/d)

H.M.

53

600

MK

60

K.T.

52

300

MK

30

T.T.

66

700

MK

70

H.T.

27

600

MK

60

*

Dopa

Dopamine

Dopal

(nglml)

(nglml)

Dopamine

1464

404

3.62

670

0.86

577 88

51

1.73

941

309

3.05 4.26

K.M.

43

800

MK

80

1600

376

W.M.

65

500

MK

50

4836

556

8.70

E.M.

64

700

MK

70

1414

552

2.56

60

4.46

N.T.

60

600

MK

1391

300

N.H.

47

600

R 150

938

368

2.55

T.M.

36

600

R 150

731

294

2.49

W.K.

59

600

R 150

* MK,

MK-486;

* MK-486: ^n..+i^^l

R.

6692

16.36

409

Ro4-4602.

alpha-L-hydrazinomethyldopa, Pr. ‘F,.lr.,n

which

was

kindly

provided

by

the

Merk-Banyu

Pharma-

While patients were being treated with L-dopa alone, plasma level of dopa was relatively low and that of dopamine high, therefore, the ratio of dopa to dopamine was small (below 1.0 in most of the patients). On the other hand, when the patients were treated with L -dopa and a peripheral dopa clecarboxylase inhibitor, plasma dopa level was higher and dopamine level lower than those while being treated with L-dopa alone. Therefore, the ratio of dopa to dopamine increased significantly (above 2.0 in most of the cases). Discussion Plasma dopa and dopamine could be assayed fluorimetrically. However, the presence of a large amount of dopa interferes with the analyses of dopamine and other catecholamines. Therefore, preliminary separation with column choromatography is necessary [8,9], which is cumbersome for routine clinical use. Other problems of fluorimetric assay of catecholamines include high tissue blank, instability of fluorophores, quenching and critical pH adjustment. In the present method dopa and dopamine do not interfere with each other because the pentafluoro-derivative of unesterified dopa takes the form of an oxazolone appearing in the early part of the chromatogram and does not overlap with the peaks of dopamine or alpha-methyldopamine [6]. On the other hand, in the process of esterification, catecholamines are destroyed and they do not interfere with the analysis of dopa. We have made some modifications to the original method described by Imai et al. [ 5,6]. They used XF 1105 as a stationary phase which gave an excellent separation of catecholamines, and utilized isodrin or dieldrin as an external standard. In their method, a parallel assay of recovery was necessary for each sample to obtain plasma concentration. We utilized alpha-methyldopamine and alpha-methyldopa as internal standards and obtained plasma working curves which showed a linear relationship between the plasma concentration and the ratio of dopamine or dopa to the respective internal standard. Therefore, we could eliminate parallel assay of recovery. This is a great advantage when many clinical samples must be handled. Because of this modification, we had to use other stationary phases, i.e. SE 54 for dopamine and OV 17 for dopa. Our method could probably be extended into the analysis of plasma norepinephrine and epinephrine. However, concentrations of those catecholamines are much lower, therefore some modifications are probably necessary, which is under investigation. The sample-concentrating unit, first developed by Imai and Tamura [lo], enabled injection of large samples, up to 50 ~1. This unit can be utilized to reduce tailing from solvents as we showed in the analysis of dopa. We applied our method to the analysis of plasma dopa and dopamine concentrations in patients with Parkinsonism being treated with L-dopa with or without a peripheral dopa decarboxylase inhibitor. It was found that most of the concentrations of our specimens fell into the range where a linear relationship existed between the peak weight and plasma concentration. However, at times much higher concentrations were encountered where another injection with a smaller amount was necessary. As shown in Table I, with the use of a peripheral dopa decarboxylase inhibitor with L-dopa, a significant increase of plasma dopa concentration and a significant decrease of plasma dopamine con-

!. 9

centration were noted, indicating that conversion of dopa to dopamine was effectively inhibited. When the ratio of dopa to dopamine was calculated, this difference was more prominent. The dosage of dopa could be reduced to one third to one fourth by the combined treatment. The use of a peripheral dopa decarboxylase inhibitor is a great advantage for those Parkinsonian patients who cannot tolerate large amount of dopa, because most of the peripheral side effects of dopa seem to be due to large amount of dopamine in the plasma. Detailed clinical reports on these patients will appear elsewhere. There was no linear correlation between the plasma dopa and dopamine concentrations and the daily dopa dosage. This is probably due to several factors, such as individual differences in the rate of absorption from the gastrointestinal tract or in the rate of conversion from dopa to dopamine or to other metabolites such as 3-0methyldopa. Another factor is the difference of the interval between the last dosage and the sampling of the blood. Although we made a great effort to draw blood approximately 2 h after the last dosage, many of the patients were outpatients and at times the interval was greater than 2 h. Although peripheral side effects of dopa were significantly reduced by a concomitant use of a peripheral dopa decarboxylase inhibitor, central side effects such as dyskinesia and mental symptoms could not be reduced with the inhibitor and seem even more prominent during combined treatment [ 111. Orthostatic hypotension also is not infrequent. To investigate these problems and to offer better treatment of Parkinsonism, analyses of plasma dopa and catecholamines occupy an important part. We will extend our study to include other patients and will make an effort to analyze other catecholamines in the plasma. Acknowledgement We are grateful for the kind advice and helpful criticisms of Dr. Zenzo Tamura, Professor and Chairman, and Dr. Kazuhiro Imai at the Department of Analytical Chemistry, University of Tokyo. Also, we thank Miss Kumiko Hamano for her technical assistance. References 1

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Vol. York

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in the