Identification by Selective Ion Monitoring of l-Methy1-1,2,3,4-tetrahydro-plcarboline in Human Platelets and Plasma After Ethanol Intake

Pekka Peura, Ilkka Kari and Mauno M. Airaksinent Department of Pharmacy, University of Kuopio, P.O.B. 138, 70101 Kuopio 10, Finland

l-Methyl-1,2,3,4-tetrahydro-&carboline (tetrahydroharman) has been quantified in human platelets and plasma following acute intake of ethanol using a selective ion monitoring method. It was not possible to detect this compound before ethanol intake.

INTRODUCTION

Ethyl alcohol dependence has repeatedly been connec- ted with the formation of endogenous alkaloids by condensation of acetaldehyde, the initial metabolite of ethanol, with biogenic amines such as the catecholamines to form tetrahydroisoquinolines and with indolealkylamines to form tetrahydro-p-carbo- lines.'-4 Since the condensation of tryptamine with aldehydes in uitro under physiological conditions produces tetrahydro-P-carbolines in good yield,5 the reaction may also occur in uivo, and the identification of these compounds in human and rodent tissues has been the object of several studies. 1,2,3,4-Tetrahydro-p- carboline (tetrahydronorharman, 1) has been shown to be a normal constituent in the tissues of man and and its 6-hydroxy derivative (2) has been identified in human urine and in rat brain and in rat platelets after the intake of 5-hydro~ytryptophan.~

We have adapted a gas chromatographic mass spectrometric (GCMS) method for the identification of l-methyl-1,2,3,4-tetrahydro-~-carboline (tetrahydro- harman, methtryptoline, 3) and the measurement of its concentration in human plasma and platelets by the selective ion monitoring (SIM) method before and after acute ethanol intake. 1-Methyl-THBC (3) has not been identified previously in human blood.

1 H H H 2 H H OH 3 CH3 H H 4 *H 2H H

Abbreviations: THBC = 1,2,3,4-tetrahydro-P-carboline; HFB = tetrafluorobutyryl.

t Department of Pharmacology and Toxicology.

@ Heyden & Son Ltd, 1980

EXPERIMENTAL

Chemicals and standards

All chemicals and solvents were of analytical grade. The internal standard l,l-dideutero-2,3,4-tetrahydro-p- carboline ['HJTHBC, 4j0 was prepared by a modified Fischer indole synthesis and its isotopic purity was determined to be 99% measuring the ratio between the retro Diels-Alder fragments m / z 143 (CloH9N) and m / z 145 (CIOH~D~N) . c~,cr-[~H~]Tryptamine (5) was synthesized by reduction of the appropriate amide prepared from dimethylaminomethylindole with lithium aluminium deuteride (98% deuterated, Merck AG, Darmstadt, Germany)." Its isotopic purity determined by mass spectrometry was 98%. [3H]Tryptamine was obtained from The Radiochemical Centre, Amersham, UK (sp. act. 1.5 Ci mmol-I). Reference compound 1- methyl-THBC (3) was prepared by Pictet-Sprengler condensation from tryptamine with acetaldehyde." The nuclear magnetic resonance and mass spectra were consistent with the assigned structure of the synthesized compound.

Extraction and derivatization

Platelet-rich plasma (PRP) was obtained from three healthy men, 25-34 years of age, before and after intake of ethanol (1 g kg-') during 4 h. Platelets were counted and platelets and platelet poor plasma (PPP) were separated from the samples of 2 ml.13

The platelets were broken in dilute hydro:hloric acid solution in an ultrasonic bath and 50 ng of [ H2]THBC (4) was added as an internal standard in acetone solution to the platelet samples and PPP samples. After addition of 1 ml of saturated sodium carbonate solution, the samples were extracted with 10 volumes of fresh dis- tilled aldehyde-free diethyl ether.14 For GCMS analyses the evaporated extracts were derivatized with 25 g1 of N-heptafluorobutyrylimidazole (Pierce Chemical Co., Rockford, Illinois, USA) for 1 h at 85 "C. The cooled solutions were dissolved in 1 ml of dichloromethane and

CCC-0306-O42X/ 80/0007-05 5 3 $0 1.5 0

BIOMEDICAL MASS SPECTROMETRY, VOL. 7, NOS. 11 AND 12, 1980 553

P. PEURA. I. KARI AND M. M. AIRAKSINEN

I00 200 300 400

382 367

382 367

I00 200 360 400 m/z

Figure 1. The mass spectrum of the heptafluorobutyryl deriva- tive of authentic l-methyl-l,~,3,4-tetrahydro-P-carboline (a) at 70 eV and (b) at 23 eV. (c) The mass spectrum at 23 eV of a GC peak of the same retention time as (a) from human platelet poor plasma following acute intake of ethanol.

washed with distilled water." After evaporating under nitrogen the heptafluorobutyryl (HFB)-tetrahydro-p- carboline derivatives were dissolved in 5Ogl of dichloromethane.

Instrumentation

GCMS analyses were performed on a OV-101 glass capillary column (15 m x 0.3 mm) prepared according to the method of GrobI6 with a Carlo Erba Fractovap 2150 gas chromatograph connected with a single stage glass jet separator into a JEOL JMS D 300 mass spec- trometer. The spectral data was processed with a JEOL JMA 2000 system.

Aliquots of the derivatized samples ( 2 PI) were injected with a splitless technique (30 s) into the column. High purity helium was used as carrier gas at a flow rate of 25 ml min-' The temperature of the injection port

( a 1

I I ' 1 1 I I

8 9 10 8 9 10 Time (rnin)

Figure 2. Selected ion recordings of the ions at m/z 382 and 367 for the heptafluorobutyryl derivative of (a) pure l-methyl-1,2,3,4- tetrahydro-p-carboline, and (b) material extracted from platelet poor plasma after intake of ethanol.

was 250 "C and GC oven was temperature programmed as follows: 1 min at 60 "C, 60-170 "C at the rate of 39"Cmin-l, 170-230°C at the rate of 7"Cmin-'. Under these conditions the retention times of HFB-1- methyl-THBC and HFB-['H2]THBC were approximately 9.1 min and 10.0 rnin, respectively. The separator and the ion source temperatures of the mass spectrometer were 260 "C and 265 "C, respectively. The ionization current was adjusted to 300 PA. The mass spectra were measured and the SIM of ions at m/z 382.1 370.1 and 367.1 of the effluent were recorded at elec- tron energy of 23 eV and 70 eV with the resolution set at 500.

RESULTS AND DISCUSSION

1 -Methyl-THBC (3) was identified using GCMS from the samples and solutions containing the reference compounds after derivatization with N-heptafluorobu- tyrylimidazole. The identification was based on the observation of identical ions with the same retention times as the reference compounds. The fragmentation of HFB-1-methyl-THBC was very slight. When an elec- tron energy of 23 eV was used, the molecular ion at m / z 382 was the base peak. A major fragment appeared at m/z 367. It was due to P-fission of the methyl sidechain at C-1. These were the only peaks in the spectrum with relative intensities over 20%. The 70 eV spectrum was quite similar; however, the peak at m/z 367 was the base peak (Fig. 1.).

Table 1. The concentrations (ng mi-') of l-methyl-1,2,3,4- tetrahydro-p -carboline in platelet poor plasma (PPP) and platelets of three persons following acute intake of ethanol

Time (hl after the completion of drinking

1 Before

Person drinking 0 20

1 PPP 0 1.2 19.1 1.4 plateletsa 0 1.1 1 .o 1.1

2 PPP 0 0.5 0.7 2.2 plateletsa 0 1 .o 1.2 0.8

3 PPP 0 3.1 7 .o 0.8 platelets' 0 0.7 0.7 1.5

a ng per platelets obtained from 1 ml platelet rich plasma.

554 BIOMEDICAL MASS SPECTROMETRY, VOL. 7, NOS. 11 AND 12, 1980 0 Heyden & Son Ltd, 1980

IDENTIFICATION OF l-METHYL-l,2,3,4-TETRAHYDRO- 0-CARBOLINE IN HUMAN PLASMA

I I I I I I 1 I I I I , I I I I l l l l f 1 1 1 1 1

10 P4 100 P4 I n4 I-Methyl-THBC

Figure 3. Standard curve for the determination of l-methyl- 1,2,3,4-tetrahydro-p-carboline (1-methyl-THBC) by selective ion monitoring using 1 ng of 1 ,1-[*H2]-2,3,4-trihydro-p-carboline as internal standard.

~~ ,a - [~H~]Tryp tamine and [3H]tryptamine were added to some of the samples as an internal control for a possible in uitro formation of 1-methyl-THBC (3) in the condensation of tryptamine with acetaldehyde. The product of this reaction, l-meth~l-3,3-[~H~]THBC, was not found in samples. Radioactivity of a l-methyl- THBC spot on a thin-layer chromatogram RP-8 F 254, Merck AG, Darmstadt, Germany, solvent system n - butanol+acetic acid + water, 4 : 1 : 1) was measured with a thin-layer scanner (Berthold 2LB 2723) and no activity was found.

The heights of the two most intense ions at m / z 382.1 and 367.1 were used in the quantitation of l-methyl- THBC (3). [*H2]THBC (4) with an intense ion at m/z 370 (100%) was used as internal standard. The concen- trations were calculated on the basis of ions at m / z 382 and 367 (Fig. 2). The use of the intensity ratio of m / z 382/370 at 23 eV gave the greatest instrumental pre- cision, and thus it was used in the quantitation (Table 1). The accuracy of the method was not checked. The calibration curve prepared from pure reference solutions was linear over the concentration range of

50-5OOOpg (Fig. 3). Recovery of extraction and derivatization was found to be 7 5 4 5 % .

On a short glass capillary with a thin film of methyl phenyl silicone (OV-17) we were also able to chromatograph 1-methyl-THBC (3) without derivatization, but when this column was connected to the mass spectrometer the full aromatizing of l-methyl- THBC (3) took place at the hot separator temperature which must be used, producing the mass spectrum of 1-methyl-p-carboline (unpublished results). 1 -Methyl- P-carboline has been identified recently in the rat arcuate nucleus and in human platelets by GCMS on packed column without deri~atization.’~

1-Methyl-THBC (3) was present in measurable concentrations both in platelets and PPP both during intoxication (0 and 1 h after the end of the drinking) and during the hangover (20 h sample) (Table 1). The reason for the variability in the plasma concentrations of 1- methyl-THBC (3), particularly one high value, is not known, but a possible dependence of its synthesis on the availability of tryptamine may partly explain it.

THBC (1) has been shown to increase voluntary ethanol consumption in rats when given repeatedly into brain ventricles.18 THBC (1) and 1-methyl-THBC (3) may be also more effective in elevating membrane fluidity than ethanol itself.” Those findings and our preliminary results provide a basis for further study on this subject. At present it is not possible to make generalized conclusions from the results as the experi- ments were made with material from only three subjects. It remains to study whether 1-methyl-THBC (3) has any significance for the symptoms of ethanol intoxication and hangover and what, if any, is its role in ethanol dependence.

Acknowledgements

This work is supported in part by The Finnish Pharmaceutical Society and Sigrid Jusklius Foundation.

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Received 13 June 1980

@ Heyden & Son Ltd, 1980

Paper presented a t the Third International Symposium on Quan- titative Mass Spectrometry in Life Sciences, Gent, Belgium, June 1980

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