Table 11. Separation of Scandium and Zirconium Using Benzilic Acid SC203 sc203 sczo3 ZrOz ZrOz Sample added, mg found, mg recovered, % added, mg found, mg 29.05 98.7 26.31 25.45 I 29.44 27.87 94.7 26.31 29.78 I1 29.44 1.11 18.8 91.55 101.49 111 5.89 100.2 27.7 28.1 118.0 IV 117.7 101.1 9.2 8.5 119.0 V 117.7 99.7 91.55 96.07 17.61 VI 17.66 VIIQ 58.87 59.81 99.7 27.47 26.15 Mandelic acid used in place of benzilic acid.
precipitated by the addition of 100 ml of hot, 0.1M benzilic acid solution. The precipitatewas filtered through filter paper, washed several times with hot water, then dried and ignited to the oxide for weighing. The p H of the filtrate was adjusted to 2.5 and scandium determined by precipitation with benzilic acid using the procedure given previously. Several different mixtures of scandium and zirconium were analyzed and the results are listed in Table 11. One sample was analyzed using mandelic acid in place of benzilic acid and the results were comparable. The efficiency of the separation was determined using scandium-46 tracer and the study showed that the separation of scandium from zirconium using benzilic acid is quantitative.
ZrOp recovered, 96.8 113.2 110.9 101.4 92.4 104.9 95.2
DISCUSSION
The volume of the scandium benzilate precipitate is larger than that of scandium mandelate and therefore is less subject to loss during transfer. The solubility of scandium tribenzilate is about one-tenth that of scandium trimandelate ( 4 ) . This property, along with the relative ease of handling the scandium benzilate precipitate, makes benzilic acid the preferred reagent over mandelic acid for the gravimetric determination of scandium.
RECEIVED for review February 9, 1972. Accepted May 12, 1972.
Simultaneous Microestimation of Choline and Acetylcholine by Gas Chromatography D. J. Jenden, R. A. Booth, and Margaret Roch Department of Pharmacology, UCLA School of Medicine, Los Angeles, Gal$ 90024
AN ADEQUATE STUDY of acetylcholine turnover must involve concomitant measurements of both choline and acetylcholine, since the former is generally believed t o be one of the major precursors of acetylcholine and its availability under some circumstances is probably rate limiting. Many biological and chemical methods have been described for the microestimation of acetylcholine ( I ) with broad agreement regarding the tissue levels obtained. On the other hand, results obtained by different procedures for the measurement of brain levels of choline have given widely disparate results (2-4). Gas chromatographic methods have been described for the microanalysis of choline esters in tissues and perfusates (5-9). (1) D. J. Jenden and L. B. Campbell, in “Analysis of Biogenic Amines and Their Related Enzymes,” Vol. 19, D. Glick, Ed.,
Interscience, New York, N.Y., 1971. (2) J. Schuberth, B. Sparf, and A. Sundwall, in “Drugs and Cholinergic Mechanisms in the CNS,” E. Heilbronn and A. Winter, Ed., Research Institute for National Defense, Stockholm, 1970. (3) I. Hanin, R. Masserelli, and E. Costa, in “Drugs and Cholinergic Mechanisms in the CNS,” E. Heilbronn and A. Winter, Ed., Research Institute for National Defense, Stockholm, 1970. (4) D. J. Jenden, in “Drugs and Cholinergic Mechanisms in the CNS,” E. Heilbronn and A. Winter, Ed., Research Institute for National Defense, Stockholm, 1970. (5) D. E. Schmidt, P. I. A. Szilyagi, D. L. Alkon: and J. P. Green, J. Pltarmacol., 174, 337 (1970). (6) D. J. Jenden, I. Hanin, and S. I. Lamb, ANAL.CHEM.,40, 125 (1968).
One of these may be simply modified t o measure free choline ( I , 3, 4). However, the original procedure cannot be employed for the simultaneous estimation of both, because the choline modification requires conditions under which its acetate ester is unstable. In this report a procedure is described for the derivatization of choline and acetylcholine, which allows their simultaneous measurement without mutual interference. EXPERIMENTAL
The basic procedure is similar to that described earlier (7, 8) except that: a) silver p-toluenesulfonate in acetonitrile is used in place of a suspension of Biorex 9 ion exchange resin t o remove Reineckate ion; b) choline is converted to propionylcholine by reaction with propionyl chloride in acetonitrile solution; Ag+ serves as a catalyst for the acylation; c) dimethylaminoethanol may be removed from the sample if necessary by extraction of the dry residue with pentane containing trimethylamine ; and d) pivaloylcholine is used as a n internal standard in place of hexyltrimethylammonium. (7) I. Hanin and D. J. Jenden, Biochem. Pharmacol., 18, 837 (1969). (8) D. J. Jenden, L. B. Campbell, and M. Roch, Afial. Biochem., 35, 209 (1970). (9) W. B. Stavinoha and L. C. Ryan, J. Pharmacol., 150, 231 (1965).
ANALYTICAL CHEMISTRY, VOL. 44, NO. 11, SEPTEMBER 1972
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15
0 W
I-
1
05
w D
0
Figure 1. Typical chromatogram of brain extract using the conditions described in the text The three peaks correspond to acetylcholine as dimethylaminoethyl acetate, RT 6.6 min; choline as dimethylaminoethyl propionate, RT 9.5 min; and pivaloyl choline as dimethylaminoethyl pivalate, RT 12.2 min. Total quantities of acetylcholine and choline were 15.1 and 54.4 nmoles/gram, respectively
Reagents. Preparation of pivaloylcholine: choline chloride (Mann Research Laboratories) (100 mmole :14.0 grams) was dissolved in 50 ml of cold chloroform, and pivaloyl chloride (Aldrich) (200 mmole :24.1 gram) slowly added with stirring. After 4-hr refluxing, the mixture was cooled and 200 ml of ether was added. The crystalline precipitate was filtered, washed with ether, and dissolved in 75 ml of ice cold absolute ethanol. Perchloric acid (23 grams, 60%) was slowly added with cooling, and after 1 hr the crystalline precipitate was filtered. The product was twice recrystallized from absolute ethanol to give a final yield of 25.7 grams (89 %) pivaloylcholine perchlorate, mp 146.5-147.5 "C. Anal. Calcd: C, 41.74; H, 7.71, C1, 12.32. Found: C, 41.69; H, 7.77; C1, 12.27. Silver p-toluenesulfonate (Eastman) is made up in acetonitrile (Matheson) at a concentration of 5mM. This must be kept dry but is stable at room temperature for at least one month. Pentane (Matheson) and propionyl chloride (Analabs, Aldrich, or J. T. Baker) were used without further purification. Three different samples of Eastman propionyl chloride were unacceptable because of low recoveries or complete disappearance of the product and internal standard; the reasons for this were not further investigated. A solution of trimethylamine (-0.1M) in pentane was prepared by transferring 0.22 ml of liquid trimethylamine to a chilled 25-ml volumetric flask and making up to volume with pentane at 0 "C. All other reagents were prepared as described in earlier publications ( I , 7, 8). Procedure. The initial processing of the sample has been previously described in detail (1, 7). Brain or other tissues 1880
are rapidly frozen by immersion in liquid nitrogen, powdered in a cold stainless steel mortar, and homogenized in 85% acetone, 15 % 1 N aqueous formic acid. Pivaloylcholine (50 m o l e s ) is added as an internal standard, and the homogenate is allowed to stand at 0 "C for 30 min before centrifuging for 20 min at 15,000 rpm (Sorvall Model RC-2). Acetone is removed by extraction with diethyl ether and residual organic solvent evaporated with a stream of dry nitrogen. Quaternary ammonium compounds are separated from the sample by Reineckate precipitation, and the residue is dried under vacuum. Silver p-toluenesulfonate (5mM) in acetonitrile (0.5 ml) is added and triturated with the precipitate. After centrifuging in a clinical centrifuge (International Equipment Company, Model CL), the supernatant is transferred to a second tube, and 50 p1 of propionyl chloride is added. The tube is capped and left at room temperature for 5 min, then evaporated to dryness in a stream of dry nitrogen. The remainder of the procedure is identical to that described earlier ( I , 7), and may be scaled down if necessary for greater sensitivity (8). Quantitative N-demethylation is achieved by reacting with sodium benzenethiolate and benzenethiol in anhydrous butanone at 80 "C for 45 min, and the tertiary amines are recovered and concentrated by partition between aqueous acid and pentane, followed by extraction from basic aqueous solution into chloroform or dichloromethane. Gas chromatographic analysis utilizes a 3-m x 2-mm (i.d.) column containing Gas Chrom Q coated with 5 % OV 101 and 5 % dodecyldimethylenetriamine succinamide, a basic polymer recently described for the analysis of aminoesters (IO), at a temperature of 125 "C. For some purposes, the small quantities of endogenous dimethylaminoethanol in brain (11) may be significant. They may be removed by washing the precipitate with 0.1M trimethylamine in pentane after evaporation of the propionyl chloride. No significant loss of quaternary ammonium compounds occurs under these conditions. The pentane washes are discarded and the residue is dried before proceeding to the dimethylation reaction. RESULTS AND DISCUSSION The procedure described gives quantitative recovery of choline as dimethylaminoethyl propionate, with a linear relation between the mole ratio to the internal standard (pivaloylcholine) and the peak height ratio over the range 0.01-10. The standard deviation for individual analyses corresponded to a peak height ratio of 0.026 or 0.19 nmole injected. The peak height ratio for the demethylation product of acetylcholine did not change significantly when the propionylation step was included, and showed no significant regression on the quantity of choline added. The standard deviation for the peak height ratio was 0.039 or 0.20 nmole injected. Dimethylaminoethanol has been reported to be a normal constituent of mammalian tissues ( I I , 1 2 ) , and the estimation of choline after N-demethylation is therefore subject to error because choline and dimethylaminoethanol would be indistinguishable. The quantity of the latter in brain is small [< 2 nmoles/gram ( I ] ) ] , and could for most purposes be ignored compared to choline. Substantial losses of dimethylaminoethanol also occur in the precipitation step and subsequent exchange. Finally, it was found that after acylation > 98 of the tertiary amine could be removed by extracting the solid residue twice with pentane (200 p1) containing tri(10) D. J. Jenden, M. Roch, and R. Booth, J . Chromatogr. Sci., 10, 151 (1972). (11) C . G. Honneger and R. Honneger, Nature, 184, 530 (1959). (12) F. Bremer and D. M. Greenberg, Biochim. Biophys. Acta, 35, 287 (1959).
ANALYTICAL CHEMISTRY, VOL. 44, NO. 11, SEPTEMBER 1972
methylamine (O.lM), without significant alteration of the recovery of choline. Trimethylamine is included in this step because dimethylaminoethyl propionate is present in the residue at this stage as the chloride or tosylate salt, and can only be extracted into a nonpolar solvent in the presence of an excess of base. The choline content of mouse brain was 57.4 rt 3.3 nmoles/ gram while the acetylcholine content was 14.7 =I= 0.7 nmoles/ gram. A representative chromatogram from a brain extract is shown in Figure 1. No significant interference was found in brain extracts from components not identifiable as choline and acetylcholine. A pooled mouse brain homogenate was divided into 12 aliquots of which 6 each were processed with and without the pentane/trimethylamine step. Results per aliquot were 18.9 f 0.7 and 18.9 rt 0.5 nmoles for choline and 6.03 =t0.23 and 6.23 i 0.04 nmoles for acetylcholine. Values obtained after pentane/trimethylamine extraction were not significantly different, nor would the reported dimethylaminoethanol content (see above) be detectable as an increment in the choline value. Experiments were conducted in which a pooled homogenate of mouse brain was divided into two parts, to one of which choline was added. Replicate estimations of choline and acetylcholine were made on each. The choline content of the unspiked samples was 18.5 f 0.3 nmoles, and those to which 40 nmoles each had been added gave a value of 59.5 f 1.0 nmoles, corresponding to a recovery of the added choline of 102.5 f 2 . 7 z . Corresponding values of the acetylcholine content were 5.05 =t0.19 and 5.15 f 0.14 nmoles, which do not differ significantly. Choline therefore can be recovered quantitatively from a brain homogenate, and does not interfere with the estimation of acetylcholine when present in 10-fold excess. A similar series of experiments was conducted in which brain homogenates were spiked with acetylcholine (10 nmoles). The recovery of added acetylcholine was 9.02 nmoles, or 90.2 + 1.9 %. When this procedure was used, commercial samples of acetylcholine were found to contain up to about 2 % of choline,
which is not easily removed by recrystallization. It may readily be acetylated by dissolving in an excess of 5 0 z acetyl chloride in methylene chloride, legving at room temperature for 10 min, and evaporating to dryness immediately before use. After this procedure less than 0.1 mole ratio of choline was found. Earlier reports of the gas chromatographic estimation of choline after acylation and N-demethylation involved the use of Biorex 9 ion exchange resin suspended in methanol to remove Reineckate (3, 4). This yields high and variable results, because a substance is eluted from the resin by methanol which is chromatographically indistinguishable from dimethylaminoethanol either as such or after acylation. Although this problem could theoretically be circumvented by exhaustive washing of the resin immediately before use, the silver p-toluenesulfonate reagent is obviously preferable and is stable for long periods of time. The Reineckates of both acetylcholine and choline are soluble in acetonitrile, while silver Reineckate forms an extremely insoluble and compact precipitate, allowing a higher recovery from this step. Silver p-toluenesulfonate serves another purpose, since the silver ions effectively catalyze the subsequent acylation by propionyl chloride. In the absence of silver p-toluenesulfonate, quantitative acylation in acetonitrile requires 10-20 min at 80 OC; it occurs at room temperature in 5 min with 5mM Ag+ present in the same solvent. Although the subsequent evaporation leaves silver ptoluenesulfonate in the analytical sample, this does not interfere with the demethylation reaction since the benzenethiolate used is present in large excess. A precipitate remains in the subsequent solvent extraction but does not interfere. If it is found to be troublesome in the final extraction step, it may be dissolved by including 0.1M KCN in the ammonium hydroxide/citrate buffer in this step (6).
z
RECEIVED for review Feburary 16, 1972. Accepted May 25, 1972. This investigation was supported by USPHS Grant No. MH 17691.
Reaction of Nitrosamine with Fluorinated Anhydrides and Pyridine to Form Electron Capturing Derivatives John B. Brooks, Cynthia C. Alley, and Roy Jones' Center for Disease Control, Health Services and Mental Health Administration, Public Health Service, US.Department of Health, Education, and Wevare, Atlanta, Ga. 30333 THESTUDY OF NITROSAMINE chemistry has not been extensive ( I ) , and there is still much to be learned concerning reactions of this group of compounds. Of great value would be an electron capturing derivative that would permit a practical approach to the analysis of minute quantities of nitrosamines in biological samples by gas-liquid chromatography (GLC). Nitrosamines are highly toxic, carcinogenic compounds that 1 Present address, Chemistry Department, Emory University, Atlanta, Ga. 30322.
(1) W. J. Serfontein and P. Hurter, Nature, 209, 1238 (1966).
some microorganisms produce in vitro. Some investigators have speculated that some organisms produce nitrosamines in vivo, possibly during infection (2). Electron capturing techniques are desirable because of the extreme sensitivity (usually picomole) of electron capture (EC) detectors to these compounds and because many other compounds present in biological samples are much less sensitive to this detector. Nitrosamines tested in this study, as such, were not sensitive to this detector. Other workers (3) have reported formation of an (2) G. Hawksworth and M. J. Hill, Biochem. J., 122, 28 P (1970). (3) N. P. Sen, J. Chromatogr., 51, 301 (1970).
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