Gas Chromatographic Microestimation of Acetylcholine and Related Compounds Donald J. Jenden, Israel Hanin, and Sandra I. Lamb Department o f Pharmacology, School o f Medicine, University of California a t Los Angeles, Los Angeles, Calif. 90024
A method is described for the gas chromatographic determination of acetylcholine and other choline esters based on prior N-demethylation with sodium benzenethiolate. The technique is useful in the submicrogram range, and allows the estimation of choline and several choline esters. Demethylation of quaternary ammonium compounds with benzenethiolate may be of general value in their analysis by gas chromatography or isotopic methods.
A METHOD has been devised for the detection and estimation of very small amounts of acetylcholine and related compounds by gas chromatography. The extensive literature on tissue levels of acetylcholine has depended primarily on bioassay procedures for identification and estimation. Several methods are in routine use which allow the estimation of acetylcholine-like activity in extremely small amounts of bioassay, but the identity of the active component as acetylcholine is open to question in view of other tissue constituents which possess similar pharmacological properties. Parallel bioassays on different tissue preparations have for many years been a classical method of identifying the substance assayed ( I , 2) but their reliability has been questioned (3, 4), the quantity of material needed is much larger, and the procedures are time-consuming. A further disadvantage of bioassay becomes apparent when drugs such as atropine, anticholinesterases, or muscarinic agents are unavoidably present in the tissue extract in unknown concentration. Although their effects may theoretically be avoided by choosing a suitable test object and by appropriate controls, an additional element of uncertainty and error is introduced which may be quite large (5). A number of chemical methods have been described for estimating acetylcholine, but none of these approach bioassay in sensitivity. Most are also nonspecific, responding either to the ester or quaternary ammonium group, Gas chromatography cannot be applied directly to acetylcholine because it depends upon the compound having a significant vapor pressure; quantitative conversion of acetylcholine to a volatile compound is therefore a prerequisite for gas chromatographic analysis. Stavinoha, Ryan, and Treat (6, 7) have described a gas chromatographic procedure in which the ester group is reduced by borohydride to yield ethanol. The method now described depends upon the quantitative conversion of acetylcholine to dimethylaminoethyl acetate, a specific product sufficiently volatile to be identified and
(1) H. H. Dale and H. W. Dudley, J. Physiol. (London), 68, 97 (1929). (2) H. C. Chang and J. H. Gaddum, Zbid., 79,255 (1933). (3) E. A. Hosein, P. Proulx, and R. Ara, Biochem. J., 83, 341 (1962). (4) E. A. Hosein, P. Rambaut, J. G. Chabrol, and A. Orzeck, Arch. Biorhent. Biophys., 111, 540 (1965). ( 5 ) I. Hanin and D. J. Jenden, Experientia, 22, 537 (1966). (6) W. B. Stavinoha, L. C. Ryan, and E. L. Treat, Life Sciences, 3, 689 (1964). (7) W. B. Stavinoha and L. C. Ryan, J. Pharmacol. Exptl. Therap., 150, 231 (1965).
estimated by gas chromatography. It is applicable also to other choline esters and to choline itself, and the individual concentrations of esters may be simultaneously estimated in a complex mixture. A preliminary account of this work has already been published (8). Principle of the Method. Conversion of quaternary ammonium compounds to tertiary amines in good yield by benzenethiolate ion has been reported by Haberli (9) and by Shamma, Deno, and Remar (10). The reaction is relatively selective in removing an N-methyl group, especially when carried out at low temperatures. Although Shamma et al. warned that the reaction could not be used with esters because of attack on this group ( I I , I2), it appeared worthwhile to seek conditions in which an N-methyl group could be selectively removed from acetylcholine without concurrent ester attack. This can be achieved in anhydrous butanone at 80" C, using rigorous precautions to remove reactive impurities in the benzenethiolate reagent. The resulting dimethylaminoethyl acetate is separated by solvent extraction from excess reagent and nonbasic reaction products, and is
CH3'~ --CH2-CH,-0-C-CH3
i
+
CHzS
CH/
estimated by gas chromatography. Hexyldimethylamine is used as an internal chromatographic standard which is carried through the entire procedure. EXPERIMENTAL
Purification of Solvents. Water was distilled from glass and boiled before use. Butanone (J. T. Baker, AR) was purified and dried by slow distillation from Linde, Type 4A Molecular Sieve, using a 60-cm glass helixes fractionating column. Chloroform (J. T. Baker, Spectroquality) was shaken with an equal volume of 2N ",OH until both phases were clear (2-4 hours), washed twice with 2N H2S04 and three times with glass distilled water. I t was stored with 1% absolute ethanol in a dark bottle. Pentane (M.C.B.Spectroquality) was treated as chloroform and stored in pure form, All organic solvents were stored in tinted, groundglass-stoppered vessels and kept in a cool, dark place. Preparation of Sodium Benzenethiolate. Thiophenol (Aldrich T3280-8) (83 grams, 0.75 mole) and 20 grams (0.50 mole) of sodium hydroxide were warmed in 100 ml of an(8) D. J. Jenden, S. I. Lamb, and I. Hanin, Federation Proc., 26(2) No. 226 (1967). (9) J. Haberli, Ph.D. Thesis. Univ. Microfilms #62-5746. Brown Univ., 1960. (10) M. Shamma. N. C. Deno. and J. F. Remar. Tetrahedron Letters, 13, 1375 (1966). (11) W. R. Vaughan and J. B. Baumann, J. Org. Chem., 27, 739 (1962). (12) J. C. Sheehan and G. D. Daves, Jr., Zhid., 29, 2006 (1964). VOL 40, NO. 1, JANUARY 1968
0
125
LI
I
0
5
-d
I
10
15
20
25
M I N'u T E s
Figure 1. Products of reaction between acetylcholine chloride (0.1M) and sodium benzenethiolate (0.2M) in anhydrous butanone Conditions: column: S.S., 6 foot, l/&ch 0.d.; packed with Polypak 1 (80/120). col. temp. = 182" C; inj. port = 233" C; det. = 227" C; NZ[carrier] 20 ml/min; air = 490 ml/ min; HZ30 rnl/min. hydrous ethanol until dissolution occurred. Toluene (700 ml) was added and the mixture was distilled slowly at atmospheric pressure; the product crystallized out as the ethanol and water were distilled. A total of 600 ml more of toluene was added in aliquots of 100 ml to maintain the volume of the boiling mixture at 500 to 600 ml. The product was then filtered (Whatman No. 42) under dry nitrogen and washed with boiling toluene. The two steps following were necessary to obtain a quantitative yield of dimethylaminoethyl acetate from acetylcholine at high dilutions. The product from the above reaction was added to 200 ml of absolute ethanol and 5 ml of ethyl acetate, shaken in an atmosphere of nitrogen until dissolved, and allowed to stand at room temperature for 60 hours. It was then filtered and the solvents were removed by distillation as before. The benzenethiolate salt was recovered by filtration under dry nitrogen and washed with boiling toluene. I t was immediately placed in a vented desiccator (Aquasorb, Mallinckrodt) over PzOs (Granusic, Baker) together with 100-200 grams of dry ice, and left for 65 hours. The product was then rapidly transferred to individually sealed vials for storage. For the demethylation reaction, a solution containing 6 mg/ml (50 mM) sodium benzenethiolate in butanone was freshly prepared each day and stored under nitrogen until
used. 126
ANALYTICAL CHEMISTRY
Preparation of Hexyldimethylamine (HDA). Bromohexane (K&K Labs, Inc.) (0.2 mole) was added slowly to a 25% methanolic solution of dimethylamine (0.4 mole) at 0" C, the mixture was allowed to warm spontaneously to 35" C and was left at room temperature for 60 hours. After boiling for 30 minutes, 250 ml of water and 8 grams of NaOH were added, and the solution was extracted twice with 50 ml of diethyl ether, which was then dried with anhydrous MgSOa. The product was precipitated as the hydrochloride by adding an excess of dry ethereal hydrogen chloride, and recrystallized from ethanollethyl acetate. Melting point of the product was 169'-171 " C. Procedure for Demethylation Reaction. The sample to be tested containing 0.5-25 nmoles of acetylcholine and a precisely known amount (-10 nmoles) of hexyldimethylamine hydrochloride is placed in a conical centrifuge tube (Corning No. 8122) fitted with a Teflon-lined screw cap, and evaporated to dryness. The sodium benzenethiolate reagent (0.5 ml) is added and air is displaced from the remainder of the tube with a gentle stream of dry nitrogen. The tube is then tightly capped and placed in a water bath at 80" C for 30 minutes, with shaking every 5 minutes. Procedure for Extraction of Dimethylaminoethyl Acetate. The following procedure was evolved to remove excess benzenethiolate and undesired reaction products, and to concentrate dimethylaminoethyl acetate into a minimum volume, while retaining a high and consistent yield. After completion of the reaction the tube is cooled and opened. After adding 0.1 ml of aqueous citric acid (0.5M) and 2 ml of pentane, the contents are shaken vigorously and centrifuged (International Clinical Centrifuge Model CL) to achieve complete phase separation. The upper organic layer is discarded and the aqueous phase is twice washed with 1 ml of pentane. The remaining traces of pentane are removed by evaporation with a gentle stream of dry nitrogen. To the aqueous residue, 50 p1 of chloroform and 0.1 ml of ammonium citrate (2M)/ammonium hydroxide (7.5M) are added, shaken vigorously, and centrifuged. An aliquot of -5 pl of the lower (organic) phase is injected into the gas chromatograph. Gas Chromatography. An F & M 5750A dual-column gas chromatograph equipped with flame ionization detectors was employed, using a silanized glass column (6 feet X 1/4 inch) containing 80/120 mesh Polypak 1 (F & M), coated with 1 (w/w) phenyldiethanolamine succinate (PDEAS) (Analabs, Inc.). Injections were made with a Hamilton No. 701-N 10-pl syringe. Chromatographic runs were performed isothermally, and the column temperature was maintained at 180" C. Injection port and flame detector temperatures were 204' and 200" C, respectively. Nitrogen was used as carrier gas, at a rate of 45 ml/minute (80 psi). Air flow was maintained at 280 mliminute (30 psi), and hydrogen at 30 ml/minute (26 psi). All three gases were separately passed through special drying tubes containing Molecular Sieve (Linde, Type 5A) before coming in contact with the chromatographic apparatus. Peaks were recorded OR a Varian Associates Recorder, Model G-2000. These conditions were used throughout this work unless otherwise stated. RESULTS AND DISCUSSION Gas chromatography of a reaction mixture initially containing acetylcholine chloride (0.1 M> and sodium benzenethiolate (0.2M) in acetone, butanone, or dimethylformamide revealed that after brief refluxing two volatile products appeared (Figure 1). Dimethylaminoethyl acetate was isolated from the mixture as the hydrochloride, and positively identified by mixed melting point with the authentic compound (129"-131" C). Methyl phenyl sulfide was isolated from the
'""1 Figure 2. N-Demethylation of acetylcholine chloride (100 p M ) by sodium benzenethiolate (50 p M ) in butanone at 80" C Each point was obtained on a separate reaction tube, the contents of which were extracted for dimethylaminoethyl acetate as described in the text. Correction has been made for extraction losses
/o
D
80
E 0 60
-
40-
P
8
mixture by distillation and its identity confirmed by a comparison of its infrared spectrum with that of the authentic compound. Retention times of these two compounds were identical with those of the two chromatographic peaks appearing during the reaction; the peak heights indicated that the reaction proceeded to completion within the limits of experimental measurement. This was confirmed by experiments in which hexyldimethylamine was included as an internal standard. In subsequent experiments the concentrations of the reactants were reduced until acetylcholine chloride was in the range 0-10 p M and sodium benzenethiolate was 50 mM. Quantitative recoveries were obtained provided that the benzenethiolate reagent was exposed to ethyl acetate and to carbon dioxide as described above. If these steps were omitted, excellent yields were obtained at high concentrations ( > l o mM) but poor yields (0-70z)in the micromolar range. These results are attributed to removal by these steps of trace amounts of more reactive bases-e.g., alkoxides and hydroxide-which attack the ester group. The time course of the reaction is shown in Figure 2. It is complete in 20 minutes, and more prolonged reaction causes only an extremely slow decline in yield. The extraction procedure was evolved to concentrate dimethylaminoethyl acetate and separate it from other nonbasic components which might interfere chromatographically
R E20 ACTION
10
0-
TIME 30 [ MINUTES)40
50
I
60
or contaminate the column because of nonvolatility. In particular it was clearly undesirable to inject relatively large quantities of sodium benzenethiolate. The procedure provides a high, uniform recovery of dimethylaminoethyl acetate which can be further increased at the expense of diluting the product by increasing the volume of chloroform in the final step (Table I). The recovery of hexyldimethylamine is quantitative; benzenethiol and methyl phenyl sulfide are completely removed. It is essential for good extraction that the chloroform be washed according to the procedure described above. If this is omitted, recovery of both hexyldimethylamine and dimethylaminoethyl acetate may be grossly reduced, particularly the former. When unwashed chloroform was used, the net recovery fell as the volume of chloroform was increased,
Table I. Recovery of Dimethylaminoethyl Acetate as a Function of the Volume of Chloroform Used in the Final Extraction Step Vol. CHCI,, p l 30 50 80
Recovery, 88.5 93.7 96.9
1 Figure 3. N-Demethylation products of acetylcholine and two of its analogs I. Hexyldimethylamine, 25 nmoles; 11. dimethylaminoethyl acetate, 50 nmoles; 111. dimethylaminopropyl acetate, 50 nmoles; IV. dimethylaminobutyl acetate, 50 nmoles. For conditions, see text
0
5
10
15
20
25
30
MINUTES
VOL 40, NO. 1, JANUARY 1968
0
127
Table 11. Linear Dependence of Ratio of Chromatographic Peak Heights for Dimethylaminoethyl Acetate and Hexyldimethylamine on the Mole Ratio of Which the Reaction Was Run A constant quantity (6.25 nmoles) of hexyldimethylamine was used throughout
Mole ratio acetylcholine/ hexyldimethylamine 0.0 0.4 0.8 1.2 1.6 2.0
Peak height ratio O.Oo0 0.164 0.455 0.630 0.840 1.010
presumably indicating the presence of impurity in the chloroform which reacted with the amines and prevented their extraction. Table I1 shows typical calibration data relating the peak height ratio for dimethylaminoethylacetate/hexyldimethylamine as a function of the mole ratio on which the reaction was run. The total quantity of hexyldimethylamine was constant at 6.25 nmoles, and the amounts of acetylcholine estimated were therefore 0-12.5 nmoles. The detection limit for dimethylaminoethyl acetate under the chromatographic conditions used was about 0.02 nmole; if one fourth (-10 p1) of the chloroform phase contains this amount, the detection limit for acetylcholine would be 0.08 nmole or 14 ng. Reproducibility of the entire reaction, extraction and chromatography sequence is indicated by a coefficient of variation of 3.9% in the peak height ratio starting with a 2 :1 mole ratio in 18 successive experiments run over a period of a month. The procedure can be used without modification for the simultaneous estimation of acetylcholine, propionylcholine, and butyrylcholine (Figure 3). However, the demethylation product of choline, dimethylaminoethanol, is poorly extracted into the chloroform phase under the conditions described above; satisfactory extraction can be obtained by using 400 p1 of a potassium citrate (2M) plus potassium hydroxide (2M) solution in place of the ammonium citrate/ammonium hydroxide buffer, and increasing the volume of chloroform to 300 pl. Under these conditions acetylcholine is rapidly hydrolyzed and total choline is estimated (Figure 4). The results presented confirm the reports of Haberli (9) and Shamma et af. (IO)that the benzenethiolate is a mild and highly specific reagent for the removal of a methyl group from quaternary ammonium compounds. In spite of published evidence of attack on esters by thiolates, the reaction has been shown to proceed without significant concurrent attack on a labile neighboring ester group, provided suitable precautions
128
ANALYTICAL CHEMISTRY
V
h I
0
5
I
I
#
10 MINUTES
1s
20
Figure 4. N-Demethylation product of 500 nmoles of choline (I) in presence of 360 nmoles of hexyldimethylamine (11). Conditions: col. 160" C; inj. port. 194" C; det. 192" C. Column and packing as specified in text are taken. In addition, the reaction can be demonstrated in a concentration range low enough to be useful for analytical procedures. It seems likely that demethylation of quaternary ammonium compounds by benzenethiolate may prove to be a preparatory reaction of general utility for their analysis by gas chromatography. It may also prove to be of value in isotopic analysis of tertiary amines containing an N-methyl group, which can be labeled by quaternization with isotopically labeled methyl iodide, followed by benzenethiolate demethylation.
RECEIVED for review August 31, 1967. Accepted November 13, 1967. Work supported by USPHS Grants NB-03007 and GRSG 1 SOL FR-05354, and conducted during tenure of a USPHS Fellowship (GM 22,905) by Israel Hanin.
