BIOCHEMICAL

MEDICINE

12, 55-65 (1975)

Tissue

Catecholamines

Tetrahydroisoquinoline A Gas

Electron G.

BIGDELI

Potential

Alkaloid

Chromatographic

with MOSTAFA

and

Metabolites:

Assay

Capture

Method

Detection1

AND MICHAEL

A. COLLINS"

Department of Biochemistry and Biophysics, Loyola University of Chicago, Stritch School of Medicine, Maywood, Illinois 60153 Received June 4, 1974

Tetrahydroisoquinoline (TIQ) alkaloids have been suggested as biologically active metabolites of catecholamine (CA) transmitters in certain pathological or drug-related states. The alkaloids are postulated to form in viva from the bimolecular cyclization of epinephrine (E), norepinephrine (NE), or dopamine (DA) with ethanol (EtOH)-derived acetaldehyde (AcH) (1,2), and CA-derived aldehydes (3) during alcoholism and with formaldehyde during methanol toxicity (4). 3-Carboxylated and I-benzyl TIQs have been considered as active metabolites of the CA precursor drug, L-DOPA, during chemotherapy for Parkinson’s disease (5). Since AcH-derived TIQ formation has yet to be demonstrated in tissues during EtOH metabolism, however, it seems necessary to develop a highly sensitive method for the determination of potential TIQs concomitant with parent CAs. With the possible exception of more costiy mass fragmentography (6), gas chromatography (GC) using electron capture (EC) is the most selective method available for the analysis of picogram (pg) quantities of CA-related compounds. In electron capture gas chromatography (EC/GC), CA functional groups are generally derivatized with halogen-containing acyl substituents. Numerous derivatization studies have been done on the CAs (7-12), but at the outset of this research, these procedures had not been examined with CA-derived TIQs. While this study was in progress, several complete CA assay procedures involving EC/GC were published (13- 15), and DA-derived TIQs were detected by EC/GC and mass fragmentography in the urine of Parkinson patients (16). ’ A preliminary report of a portion of this work has appeared: M. A. Collins, M. G. Bigdeli, and F. Kemozek, Pharmacologist 13, 309 (1971). * To whom correspondence is addressed. 55 Copyright All rights

@ 1975 by Academic Press. Inc. Printed of reproduction in any form reserved.

in the United

States.

56

BIGDELI

AND

COLLINS

RI R,

I

R3

R4

-H

-OH

-OH

-OH

II

-H

-OH

-OCH,

-OH

III

-H

-OH

-OH

-OCH,

Ip

-H

-OH

-0

P

-H

-H

-OH

-OH

‘3LI

-CH3

-H

-OH

-OH

-DHB

-II

-OH

VII FIG.

R2

- CH2-O-

OH

1. Crystalline TIQ substrates used in study. DHB = 3,4-dihydroxybenzyl.

We report here the results of fluoroacylation procedures with CAs and representative TIQs (Fig. l), including several newly synthesized 4hydroxy-TIQs which are the postulated in vivo cyclization products of E and NE with aldehydes (1). Recoveries were determined using various alumina extraction procedures. The possibility of “direct derivatization” of CA or TIQ in the aluminum oxide (A&O&bound state in the absence of aqueous acid was explored and was found not feasible. A simple and quantitative method for the determination of endogenous CAs and their potential TIQ products is presented which involves tissue homogenization in HCIOB, A&O, extraction, aqueous HCl elution, formation of heptatIuorobutyry1 (HFB)-derivatives in acetonitrile, and EC/GC on 5% GE XF-1105 columns. Determination of CA levels in rat brain and adrenal glands are made with this method. MATERIALS AND METHODS

Results were obtained with a Varian 2100 biomedical gas chromatograph equipped with a ‘j3Ni EC detector (direct current mode) and injection port extenders. Six foot by 2 mm i.d. glass columns were packed with 3-5% OV-17, SE-30, SE-54, XE-60, or GE XF-1105 on SO/l00 mesh Gas Chrom Q and conditioned 24 hr at the respective maximum temperatures. Sources of column packings were Applied Science and Varian.

GC ASSAY OF CATECHOLAMINES

AND

ISOQUINOLINES

57

Derivatizing reagents and solvents (acetonitrile, dimethylformamide) were purchased from Pierce Chemical and stored at -20°C when not in use. Ethyl acetate (EtOAc Eastman analytical grade) was batch-distilled before use. E, NE, and DA were obtained as hydrochloride (HCl) salts from Regis Chemical. 14C-DA (56 mC/mm sp act) and 14C-E (50 mC/mm sp act) were purchased as bitartrate salts from AmershamlSearIe. Seven crystalline 1,2,3,4-TIQ substrates were used in this study (Fig. 1). The HCI salts of four 4-hydroxylated alkaloids, 4,6,7-trihydroxy-TIQ (I), 4,7-dihydroxy-6-methoxy-TIQ (II), 4,6-dihydroxy-7methoxy-TIQ (III), and 4-hydroxy-6,7-methylenedioxy-TIQ (IV), were synthesized in this laboratory by reported procedures (17), with our modifications (18). 6,7-Dihydroxy-TIQ hydrobromide (HBr) (V) was prepared by Bischler-Napieralski cyclization (19) and demethylation with refluxing HBr. 1-Methyl-6,7-dihydroxy-TIQ HBr (VI; Salsolinol HBr) was purchased from Aldrich Chemical. Tetrahydropapaveroline HBr (VII) was a gift from Wellcome Research. The l-methyl-4,6,7trihydroxy-TIQs in Fig. 2 were used as obtained from pH 6 aqueous reactions of NE or E with AcH (1). Derivative preparation. One milligram of the amine salt dissolved in 0.1 ml of derivatization solvent in a 5-ml screw-capped vial was allowed to react for varying lengths of time with 0.2 ml of the fluoroacyl reagent. The solution was dried with a nitrogen (N,) stream, stored at -20°C and dissolved in the appropriate volume of EtOAc prior to GC analysis. Throughout this study 1 ~1 portions were injected into the chromatograph.

5

hi,*

“‘1 1

DA OR SALSoLlNoL’ I PITT.: 4 x I OP,

RETENTIOM

TIME

(min)

FIG. 2. Gas chromatogram of the HFB-derivatives of 50 pg each of the CAs and three lmethyl-TIQ products. EC/GC conditions: 5% GE XF-I 105 on 80/100 Gas Chrom Q; Column T = 175°C; NZ = 40 mUmin; Att. = 2 x 10-l” AFS except where noted.

58

BIGDELI

AND

COLLINS

Calibration curves were prepared by appropriately diluting standard solutions of the HFB-derivative with EtOAc and plotting resultant peak heights vs amine salt in pg. Minimum detectable quantities (MDQ), pg substrate with a peak height equal to twice the baseline noise level, were obtained directly from calibration curves. Aluminim oxide (A&O,) extraction procedure for crystalline substrates: 100 mg A&O3 (Woelm Neutral Activity-Grade l), activated according to Shellenburger and Gordon (20), was added to a solution of 1 mg CA or TIQ salt or 14C-CA in 2 ml 0.1 N HClO,, or to the supernatant obtained from a 10 min centrifugation (2000 rpm) of a rat brain which had been previously homogenized in 0.1 N HCIO, with added CA. The pH of the acid solution was adjusted to 7.8-8.0 by dropwise addition of 2.0 N NaOH followed by 0.2 N NaOH. While still at room temperature the mixture was mechanically shaken 10 min and centrifuged (2000 rpm) with a clinical centrifuge. The Al,O, was washed with three 5-ml portions of distilled water. Ten milliliters 0.1 N HCI was added to the A&O, and the mixture was shaken 10 min. After 3 min centrifugation in a clinical centrifuge, the acid supernatant was lyophylized to dryness. The residue remaining was treated with HFB-anhydride/acetonitrile (0.2 ml/O.1 ml) for 30 min and prepared for EC/GC analysis as described previously. CA in tissues. Single whole brains or pairs of adrenal glands from 350 g male Sprague-Dawley rats were rapidly removed, weighed, and homogenized 5 min in the cold in a 10 ml conical homogenizing tube containing 2 ml of a mixture of 1 N HClO,, 5 mu sodium metabisulfite, and approximately 5,000 cpm 14C-DA (brain) or 14C-E (adrenals). These amounts of isotopes, which were insufficient to contribute to the quantitative EC/GC of endogenous CAs, were prepurified on short A&O, columns prior to use (22). Ten percent of the homogenate was taken for counting by scintillation spectroscopy. The remaining 90% was centrifuged (2000 rpm) for 10 min at 4°C and the supernatant was transferred to a 15-ml screw cap vial. The precipitate was resuspended in 2 ml fresh ice-cold homogenizing medium (less the 14C-isotope), briefly recentrifuged, and the supernatant pooled with the original supernatant. The pH of the combined supernatants was carefully brought to 4 with 2 N NaOH, 100 mg activated A&O, was added, and the pH was further adjusted to 7.8-8.0 with 0.2 N NaOH. The mixture was mechanically shaken 10 min and the supernatant was decanted and discarded. The Al,O, was washed with three 5-ml portions of H,O and the catechols were then eluted from A&O3 precipitate by shaking 10 min with 2 ml 0.1 N HCl. The HCl supernatant was decanted and lyophylized. The residue after lyophylization was treated with HFB-anhydridelacetonitrile and 30

GC

ASSAY

OF

min later the reaction were added and 50% 14C-CA by scintillation luted with EtOAc if calibration curves for CA analyses.

CATECHOLAMINES

AND

ISOQUINOLINES

59

was evaporated with N,. Two milliliters EtOAc was taken for determination of % recovery of spectroscopy. The remaining solution was dinecessary and examined by EC/GC. External the CAs were determined the same day as tissue RESULTS

Of several fluoracylation procedures examined with crystalline substrates, only HFB-anhydride in acentonitrile, 30-60 min reaction times at room temperature, produced a single derivative for each TIQ and CA. HFB-derivative decomposition was evident with longer reaction times, and dimethylformamide was found to be less suitable than acetonitrile as a solvent. While TFA-anhydride in EtOAc (7) or in acetonitrile (15) or HFB-anhydride in EtOAc (12) produced single derivatives for the CAs and the DA-related TIQs (substrates V-VIII, Fig. l), these methods tended to yield multiple peaks on 3% OV- 17 for the 4-hydroxy-TIQs related to the p-hydroxy-CAs. The TMSi-imidazole/HFB-imidazole method (9) likewise gave multiple 4-hydroxy-TIQ derivatives with relatively poor EC responses, and HFB-imidazole alone in EtOAc produced multiple derivatives for all substrates. The HFB-derivatives of E and NE (obtained from HFBanhydride/acetonitrile treatment of the CAs), not well resolved on the usual “CA” columns of 3-5% SE-30, OV-17, XE-60, or SE-54, were readily separated from one another as well as from HFB-DA on 5% GEXF- 1105 (Fig. 2). In addition, with the exception of DA and its AcH cyclization product, salsolinol (compound VI in Fig. I), this liquid phase separated the HFB-derivatives of the TIQs derived from NE/AcH and ElAcH cyclizations (Fig. 2). Linear calibration curves and minimum detectable quantities (MDQs) were obtained for the HFB-derivatives of two representative TIQs and the CAs (Fig. 3). Salsolinol was extremely sensitive to the EC detector as the HFB-derivative, with a MDQ of 0.2-0.5 pg at the conditions shown in Fig. 3. HFB-DA was nearly as sensitive, its MDQ being l-3 pg. The MDQ for the HFB-derivative of 4,6,7-trihydroxy-TIQ (I, Fig. 1) was 40-50 pg, and NE and E (HFB-derivatives) were intermediate between this TIQ and DA. Not shown in Fig. 3 is the calibration curve for the HFB-derivative of tetrahydropapaveroline (VII), 1-(3’4’-dihydroxybenzyl)-6,7-dihydroxyTIQ, an alkaloid of interest in alcoholism and Parkinson’s disease studies (3,16,29). In contrast to salsolinol, tetrahydropapaveroline as the HFB-derivative was remarkably insensitive to EC detection, since

60

BIGDELI

AND COLLINS 0 Sol%llnol 0 0 0 A

AmIne

Dopomine COPI Norepmephrlne INE) Epmephrme IEI 4.6.7.trlhydroxy-TIO

salt

Ipg)

3. EC/CC calibration curves for three CAs, 4,6,7-trihydroxy-TIQ (I) and salsolinol (VI), following derivatization with HFB-anhydridelacetonitrile. GC conditions are the same as Fig. 2. FIG.

TABLE

1

% RECOVERIES (MEAN 2 SEM) OF 14C-CA FROM AQUEOUS MEDIA FOLLOWING ALUMINA= EXTRACTION, LYOPHYLIZATION, AND DERIVATIZATION WITH HFB-ANHYDRIDEIACETONITRILE

Procedure 14C-CA in 1 N HCIO,: 1. Extraction with A&O,, elution with 0.1 N HCl 2. Extraction with A&OS, elution with 0.2 N HOAc or 0.05 N HClOa 3. Extraction with freshly prepared . Al(OH)B, eluhon wtth 0.1 N HCI 4. “Direct Derivatization” of A1203bound, lyophylized CA (no acid elution) 5. EtOAc extraction

%-DA

“C-NE

14C-E

58.9 k 2.8 (4)*

87.2 f 1.0(4)

-

c 10 (2)

-