Microanalytical Determination of Acetylcholine, Other Choline Esters, and Choline by Pyrolysis-Gas Chromatography P. I. A. Szilagyi,’ Dennis E. Schmidt,2 and Jack Peter Green3 Cornell Unitiersity Medicul College, 1300 York Auenue, New York, N . Y . 10021 and Departments of Pharmacology, Mount Sinai School of Medicine, Fifth Avenue and 100th Street, New York, N. Y . 10029 A microanalytical technique has been described for acetylcholine and related compounds. By flashheating on a pyrolyzing ribbon, the quaternary ammonium compounds are converted to their volatile dimethylamino derivatives which are swept onto the gas chromatographic column by a nitrogen stream. The method allows the simultaneous identification and quantitative measurement in the nanogram range of several choline derivatives. These compounds can be measured in dilute solutions because the method includes concentration of the material by evaporating the solution on the pyrolyzing ribbon before pyrolysis and chromatography. MEASUREMENTS of small amounts of acetylcholine and other choline esters have required bioassay procedures which have inherent disadvantages ( I ) . Though sensitive, the methods are time-consuming, tedious, and artful. The most serious limitation is the fact that a bioassay does not distinguish acetylcholine from other choline esters or from choline. In the 1 Present address, Department of Pharmacology, Cornell University Medical College, New York, N. Y. 10021 2 Present address, Department of Pharmacology, Mount Sinai School of Medicine, New York, N. Y. 10029 3 Author to whom correspondence should be directed.
(1) V. P. Whittaker, “Handbuch der Experimentellen Pharmakologie. Cholinesterase and Anticholinesterase Agents,” G. B. Koelie, Ed., Springer-Verlag, Berlin, 1963, p 1.
absence of a specific and sensitive method for resolving and measuring choline esters, the idea that acetylcholine functions (or even exists) in the mammalian nervous system rests on bioassays that can measure only acetylcholine-like activity. The ferric hydroxamate method (2) is neither sensitive nor specific. A gas chromatographic procedure has been described, but this estimates only the acyl components of the choline esters (3). A most promising gas chromatographic method requires demethylation with sodium benzenethiolate (4). This method demands highly purified benzenethiolate, anhydrous and anoxic conditions, and a n extraction to separate the desired product from other products and excess benzenethiolate ; also the solvent does not simultaneously extract both the choline and acetylcholine products. Our initial idea was t o attempt a n on-column Hofmann degradation after preparation of the quaternary ammonium hydroxide derivative on a n anion-exchange resin. This general reaction may be shown in Reaction 1 :
(2) S . Hestrin, J. Biol. Chem., 180, 249 (1949). (3) W. B. Stavinoha and L. C . Ryan, J. Pharrnacol. Exp. Ther., 150, 231 (1965). (4) D. J. Jenden, I. Hanin, and S. I. Lamb, ANAL.CHEM., 40, 125
I
I
I
VI Q)
c
0
I
n VI L a3
m
L a,
a m
c
VI
L m
F
L m
0 V a3
2
E
U 0
Lli
0
2 4 Time (minutes)
6
Figure 1. Chromatogram of pyrolytic products of 60 ng of acetylcholine on 20 Carbowax 6000 under optimum pyrolysis conditions
z
Arrow denotes beginning of pyrolysis. Peak I had same retention time as dimethylaminoethyl
acetate
j
J
’
I
Time (minutes)
Figure 2. Chromatogram on 6 x SE-30 of pyrolytic products of 5 pg each of a mixture of acetylcholine and dimethylaminoethyl acetate (I) and of a mixture of butyrylcholine and dimethylaminoethyl butyrate (11)
Time (minutes)
Figure 3. Chromatogram on 2 0 z Carbowax of a pyrolyzed mixture containing 1 pg each of acetyl-/3-methylcholine (I), acetylcholine (11), propionylcholine (111), and butyrylcholine (IV) VOL. 40,
NO. 13, NOVEMBER 1968
0
2009
Table I.
Comparison of Retention Times (on Two Different Columns) of Pyrolyzates of Quaternary Ammonium Salts with Retention Times of Proposed Demethylated Products
CH3
+I
Substance pyrolyzed CH3
I
2.7s
CH3-N-CHrCH-OH
I
Retention pyrolyzate, min
c1-
CH3 B-Methvlcholine CHa
I
I
CH3-N-CHz-CH-OH
2.70
CHI 3.7a
CH~-N-CHFCHZ-OH
I
I
CH3-N-CHrCHrOH
3.7'
0.6b c1-
CHI Choline CH3
CH3
+I
I
c1-
CHI
0
+I
CHs-N-CHrCHr&C-CHa
I
CH3 Acetylcholine
CH3
/I
I CH3
Acetyl-P-methylcholine
3.8~
I
CH3
0
I
CHs-N-CHrCH-0-C-CHs
I1
3.8a
2-Dimethylaminoisopropyl acetate
CH3
It
4.7a
0
I
CHa-N-CHrCHr-C-CHa
/I
4.P 1,l b
1.16
c1-
Dimethylaminoethylacetate CH3
0
CH3
0.6*
2-Dimethylaminoethanol
0
CH~-N-CHFCH-O-C-CH~
/I
CHa-N-CHrCHrO-C-(CHzkCHs
8.Sa
II CH3 Butyr ylcholine
3.26
0
Proposed demethylated products CH3 CHI
Dimethylamhoisoprouylalcohol
+I
+I
Retention demethylated derivative, min
c1-
0
I
II
CH3-N-CHrCHrO-C-(CHz)z--CH,
8.8a 3.2b
Dimethylaminoethylbutyrate
2 0 z Carbowax 6000, conditions as in Figure 1.
* 6% SE-30, conditions as in Figure 2.
The reaction worked well for choline, yielding acetaldehyde and trimethylamine :
H ,C-!J-CH CHI OH-
x-
CH 3
0
II
2-CH z-O-C--CH
3
heat_
I+ CH3 CH 3
I
0
II
H~C-N-CH~-CH~-O-C-CH~
+ CH3X
(3)
Like the Hofmann degradation described above, this reaction, the pyrolysis of quaternary ammonium halides, had also been described by Hofmann in 1850 ( 5 , 6 )but it has not been applied to choline esters. A preliminary account of this work has been presented (7). EXPERIMENTAL
But the flash pyrolysis of acetylcholine hydroxide gave only some degradation products of choline acetate. However, the halide anion of acetylcholine (and of other choline esters and of choline) proved to be sufficiently strong nucleophiles at the high temperature of the injector port of the column to yield dimethylaminoethyl acetate from acetylcholine : 2010
ANALYTICAL CHEMISTRY
Chromatography. For most of the experiments a BarberColman Model 5000 dual column gas chromatograph equipped with a hydrogen flame ionization detector and recorder was used with a glass U-tube column (6 feet X 3.5 mm) containing 20 Carbowax 6000 on hexamethyldisilazane-treated Chromosorb W, 60/80 mesh, which was pre(5) J. H. Brewster and E. L. Eliel, Org. Reactions, 7, 142 (1953). (6) A. C. Cope and E. R. Trimbull, ibid., 11, 377 (1960). (7) P. I. A. Szilagyi, D. Schmidt, and J. P. Green, Federation Proc., 27, 471 (1968).
VI
Time (minutes)
x
Figure 4. Chromatogram on 20 Carbowax of a pyrolyzed mixture containing 1 pg each of p-methylcholine, produced by hydrolyzing acetyl-p-methylcholine (I) and choline (11)
1
P u L
=k
2
0
4
6
Time (minutes)
Figure 5. Chromatogram on 2 0 x Carbowax of a pyrolyzed mixture of acetyl-/3 methylcholine (I), acetylcholine (II), and butyrylcholine (111) After hydrolysis of mixture, material resulted with retention time of dimethylaminoisopropyl alcohol (IV) and dimethylaminoethanol (V), which are products of demethylation of p-methylcholine and choline, respectively. Chromatograms of the hydrolyzate in admixture with authentic alcohols showed an increase in the size of peaks VI and VII
conditioned at 190 "C in a stream of nitrogen for 24 hours. The temperatures were: flash heater 240 "C, column 140 "C, detector 310 "C. Nitrogen flow rate was 40 ml/minute (30 psi). Air pressure was 40 psi and hydrogen pressure was adjusted to give maximum detector sensitivity. In some experiments (Table I and Figure 2) a Victoreen Model 4000 gas chromatograph was used. This too was equipped with a hydrogen flame ionization detector and a recorder. The columns were coiled stainless steel tubes (6 inch) containing 6 x SE-30 on Anakrom ABS, feet X 9O/lOO mesh, preconditioned at 250 "C in a stream of nitrogen for 6 hours. The temperatures were: flash heater 270 "C, column 100 "C, detector 310 "C. The nitrogen flow rate was 45 ml/minute, the air flow rate was 1000 ml/minute, and the hydrogen flow rate was 40 ml/minute. The peak areas were integrated by a Nester/Faust Model 1502 Summatic Integrator equipped with an automatic base line compensator. The areas of the curves were also calculated by multiplying height by the width at half-height. Pyrolysis. The derivatives of the various esters and alcohols were prepared on a nickel ribbon in the BarberColman Model 5180 pyrolyzer. Pyrolyzer body temperature was 165 "C. An aqueous solution of the substances, 1 to 20 p l , was placed on the pyrolyzer ribbon and the water was allowed to evaporate at 165 "C. The pyrolyzer chamber was closed and purged for 2 minutes to remove air. The pyrolyzer setting was then placed in the analyze position, the temperature of the pyrolyzer was set, and pyrolysis was carried out for 30 seconds. The volatile products of pyrolysis are automatically introduced onto the column. Alkaline Hydrolysis. The esters were hydrolyzed by heating them in 0.1N NaOH for 60 minutes in a boiling water bath, after which the pH of the solution was adjusted to 4.5 with HCI.
RESULTS AND DISCUSSION
Optimum Pyrolysis Conditions. Formation of the pyrolytic product from acetylcholine chloride (peak I, Figure 1) required a temperature of at least 540 "C; the product was detectable at temperatures up to 900 "C. Optimum conditions were found to be 730 "C for 30 seconds, and these conditions were routinely used. Both the iodide and chloride salts of acetylcholine yielded products with the same retention time, and ecluimoles of the two salts gave peaks of equal area. The peak showed little tailing. The material (s) of short retention time (e.g., Figure 1) was not identified. From work described below, it can be presumed that methyl chloride is one of these substances, Identification of Products. The retention times of the pyrolysis products were identical to the retention times of the corresponding authentic N,N-dimethyl derivatives (Table I). Equimolar quantities of acetylcholine and N,N-dimethylaminoethyl acetate were, separately and together, pyrolyzed and chromatographed. The peak area resulting from a mixture containing 6.7 nanomoles each of acetylcholine and dimethylaminoethyl acetate was 2.951, almost exactly equal to the sum of the areas of the individual compounds which were, respectively, 1.548 and 1.459. Analogously, the peak area resulting from a mixture containing 10.4 nanomoles each of choline and dimethylaminoethanol was 1.778, almost exactly equal to the sum of the areas of the individual compounds which were, respectively, 0.992 and 0.810. On the column containing SE-30, pyrolysis of a mixture of 5 pg each of acetylcholine and dimethylaminoethyl acetate gave one peak (I in Figure 2). Analogously, 5 pg each of butyrylVOL. 40, NO. 1 3 , NOVEMBER 1968
* 201 1
"7-
?OK
4.0
3000
3.0
2
-
a 0
N
E
E 2000
?.O -u
-2 L
0
L a
--
a
m
c
IO00
1.0
Acetylcholine lng)
Figure 6. Relationship beween peak area on 2 0 z Carbowax and amount of pyrolyzed acetylcholine in range 2 to 14 ng 0 =
Integrator response
A = Manually calculated peak area
choline and dimethylaminoethyl butyrate gave one peak (I1 in Figure 2). O n this column, the products tailed. Resolution of Isomers and Homologs of Acetylcholine. Figure 3 shows the chromatogram resulting from pyrolysis of a mixture containing 1 pg each of acetyl-0-methylcholine (peak I), acetylcholine (peak II), propionylcholine (peak 111), and butyrylcholine (peak IV). In addition there are five early peaks. Two of the early peaks are methyl iodide and methyl chloride resulting from the pyrolysis of the iodide of acetylcholine and the chlorides of the other esters. The other early peaks remain unidentified. It is possible that when large amounts of material are pyrolyzed (4 pg in the experiment shown in Figure 3), fragmentation products are detectable that are not observed when small amounts of material are pyrolyzed (60 ng in the experiment shown in Figure 1). Methacrylylcholine had a retention time of 9.2 minutes in this system. The products of choline and /3-methylcholine are
3My)
0 0
20
60 40 Acetylcholine lngl
80
1
1
Figure 7. Relationship between peak area on 20% Carbowax and amount of pyrolyzed acetylcholine in range 20 to 100 ng 0 =
A
=
Integrator response Manually calculated peak area
also resolved but with tailing (Figure 4). Choline and acetylP-methylcholine had retention times too similar (Table I) to be resolved, but these can be detected after alkaline hydrolysis (Figure 4). Pyrolysis-Chromatography after Hydrolysis. Pyrolysis of the products formed by alkaline hydrolysis of a mixture containing 1 pg each of acetyl-P-methylcholine, acetylcholine, and butyrylcholine yielded material with the retention times of the products produced by pyrolyzing P-methylcholine and choline (Figure 5 ) . When the hydrolyzate was pyrolyzed and chromatographed in mixture with dimethylaminoethanol and P-dimethylaminoisopropyl alcohol, the peak areas increased (Figure 5).
r
4 Figure 8. Relationship between peak area on 2 0 z Carbowax and amount of pyrolyzed choline in range 60 to 190 ng 0 = Choline lag1
2012
ANALYllCAL CHEMISTRY
Integrator response
A = Manually calculated peak area
Pyrolysis-chromatography of 10-12 mole) of acetylcholine resulted in an identifiable peak which was only of qualitative value, but the peak resulting from 2 ng was measurable and integrable. The relationship between peak area and amount of acetylcholine is shown in the ranges 2 to 14 ng of acetylcholine. (Figure 6) and 20 to 100 ng of acetylcholine (Figure 7). Figure 6 was obtained at an attenuation setting of 1 on the electrometer, Figure 7 at a setting of 10; there is no linearity between the two attenuation settings with the Barber-Colman instrument used for these measurements. Figure 8 shows the relationship between peak area and amount of choline from 60 to 190 ng. As with the measurements of acetylcholine (Figures 6 and 7), areas obtained by either integration or manual estimation, though plotted on different scales, were linearly related to mass of the compound. The sensitivity of the method is greater than indicated by these figures. For in the method, the solvent containing the quaternary ammonium compound is evaporated on the Quantitative Measurements.
1 ng (7
x
pyrolyzing ribbon before pyrolysis, and, hence, dilute solutions of the compounds can be concentrated before pyrolysis by pipetting and evaporating small aliquots. The sensitivity of the method appears to be limited only by the patience of the examiner. Solutions containing 100 ng of acetylcholine in 0.5 ml have been concentrated and pyrolyzed, with the acetylcholine readily measured. As with other methods (e.g., ref. 4), the application of this method to tissue extracts will require purification steps, for direct pyrolysis of tissue extracts will result in products that interfere with the determination of choline esters and choline. Purification steps are being devised. RECEIVED for review May 6, 1968. Accepted July 11, 1968. Work supported by grants from the U. S. Public Health Service (GM-14278-02 and 5-Sol-FR-05396-6) and the National Science Foundation (GB-6248). P. I. A. Szilagyi and D. E. Schmidt were supported by a training grant from the U. S. Public Health Service (5T1 GM99).
Gas-Solid Chromatographic Studies of Cyclic Hydrocarbons on Salt-ModifiedAluminas and Porous Silica Beads David J. Brookman and Donald T. Sawyer Department of Chemistry, University of California, Ricerside, Calif. 92502
The differential enthalpies, entropies, and free energies of adsorption for a series of cyclic C,, C,, C,, and Cs alkenes, polyalkenes, and alkanes on saltmodified porous silica beads and aluminas have been determined using elution gas chromatography. The results established that the degree of specific interaction by the pi-electron systems is highly dependent on geometric and steric factors. This permits gassolid chromatography to be used as an effective tool for the determination of molecular structures of cyclic unsaturated hydrocarbons. The data confirm that 1,3,5,7-~yclooctatetraeneand 1,3,5-cycloheptatriene are both in the tub configuration when adsorbed, and that conjugated cyclic dienes cannot interact as effectively as the nonconjugated isomers capable of having the two pi bonds in a plane.
PREVIOUS WORK has shown that physical adsorption at the interface between a gas and a salt-modified alumina adsorbent can be expressed as a summation of nonspecific and specific interactions. Gas chromatographic studies (I) of selected model compounds have allowed isolation of the specific and the nonspecific functional group contributions to adsorption at these surfaces such that prediction of retention volumes is possible (2). Specific interactions include those due to unsaturation in the adsorbate molecule and to perturbations of aromatic ring electron density caused by substituent groups (3). Because specific and nonspecific interactions contribute in a linear fashion to the free energy of adsorption, and because the chromatographically measured isosteric adsorption enthalpies for each of the tested model compounds is invariant over at least a 75 "C operating range, the nonspecific and specific interaction contributions to the observed enthalpy (1) D. J. Brookman and D. T. Sawyer, ANAL.CHEM.,40, 106 (1968). (2) D. T. Sawyer and D. J. Brookman, ibid., p 1847. (3) D. J. Brookman and D. T. Sawyer, ibid., p 1368.
and entropy of adsorption can be isolated. This practice permits the prediction of the retention volume for any compound from its structure within the operable temperature range of the index system so long as the various enthalpy and entropy contributions, and hence the resulting free energy, remain additive. An interesting point in the tabulation of specific interaction contributions is the large differences observed for olefinic bonds in various environments. Nonconjugated, conjugated, and aromatic pi-electron systems have significantly different specific enthalpies and entropies. Isolation of these specific quantities makes possible ascertaining how a given molecular pi-electron system interacts with an adsorbent and therefore provides data about the molecular configuration and its orientation with respect to the adsorptive surface. Extensive data are available concerning the structure of the saturated and unsaturated cyclic hydrocarbons from cyclopentane through cyclooctatetraene (COT). With the exception of benzene, studies of the adsorption for most of these compounds have been limited to graphitized carbon (4). Because the adsorption of COT and its related compounds (as well as of the C7, C6, and C5 cyclic hydrocarbons) has not been studied by gas-solid chromatography, a detailed investigation of the retention of these compounds in relation to their known structures is of interest. The structure of COT has been a subject of controversy because spectroscopic data support both crown and tub forms (5-8). Later work tends to support the latter confor(4) A. V. Kiselev, Q u a r f .Reu., 15, 99 (1961). ( 5 ) E. R. Lippincott and R. C . Lord,J. Amer. Chem. Soc., 73,3889 (1951). (6) E. R. Lippincott, R. C. Lord, and R. S . McDonald, ibid., p 3770. (7) W. B. Person, G. C . Pimentel, and K. S. Pitzer, ibid., 74, 3437 (1952). (8) T. L. Karle, J . Chem. Phys., 20, 65 (1952). VOL. 40, NO. 13, NOVEMBER 1968
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