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).
ANALYTICAL CHEMISTRY, VOL. 44, NO. 11, SEPTEMBER 1972
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3% ov-1 COlUma
Figure 1. Gas chromatogram of a standard nitrosamine mixture that has been treated with HFBA-Py to form electron capturing derivatives. N-nitrosodiethylamine, N-nitrosopyrrolidine, and N-nitrosopiperidine have not been confirmed by mass spectral analysis; therefore, identification must be considered tentative
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EC sensitive nitramine by oxidation of N-nitrosodimethylamine; however, a difficult clean-up problem exists when the method is applied to the analysis of food extracts. The reaction of nitrosamine with anhydrides has not been reported. We observed this reaction while investigating the microbial production of amines in urine employing a gas chromatograph equipped with an EC detector and preparing heptafluorobutyric anhydride (HFBA) derivatives with pyridine (Py) as a catalyst. This observation stimulated the present study. EXPERIMENTAL
Gas Chromatography. A Barber-Colman gas chromatograph equipped with a 300-mCi tritium (EC) detector was used for routine analysis, and a flame ionization detector equipped with a 2O:l splitter was used to collect samples for low resolution mass spectrometry. The instrument employed glass columns (0.3-cm inside diameter by 7.3-m length) packed with Chromosorb W SOjlOO mesh (AWDMCS H.P.) coated with 3% OV-1 (Applied Science Laboratories). The column used for collecting was the same except that it was packed with Tabsorb (Regis Chemical Co.). The instrument was operated routinely isothermally for 5 minutes at 90 "C, then, to give a linear increase of 5 "C per minute to 220 " C ; the electrometer attenuation was 32 for the 3 z OV-1 column and 16 for the Tabsorb column; the range setting was 10, and the balance setting was lo4. For collecting samples by using the Tabsorb column, the operating parameters were the same except that the instrument was operated isothermally at 110 "C. Nitrogen was used as the carrier gas at a flow rate of 36 cc per minute for the 3% OV-1 column and 50 cc per minute for the Tabsorb column. The recorder was operated with an input signal of 1 mV and a chart speed of 30 inches per hour. Mass Spectra. The mass spectrometer was a Varian Associates M-66. It was operated at a resolution of approximately 3100; the ion source temperature was controlled at 65 "C; the direct probe inlet temperature was set at 70 "C; the electron energy was 70 eV, and the spectra were scanned from mie 10 to 500 in about 10 minutes per scan. The capillary tube was directly introduced into the ion source with a probe inserted into the direct sampling inlet port. Reagents. Trifluoroacetic anhydride (TFA), N-nitrosodimethylamine, N-nitrosodiethylamine, N-nitrosopiperidine (all Eastman grade), and 1,l-dimethylhydrazine (practical 1882
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grade) were purchased from Eastman Chemical Co.; Nnitrosopyrrolidine and HFBA, from Rare Chemical Co., and the chloroform (nanograde) and Py (spectrAR grade), from Mallinckrodt. The diethyl ether (anhydrous) was purchased from Fisher. Twenty milliliters of each new container (1 gallon) of chloroform, which was used in 20-ml volume to extract nitrosamines from biological samples, was concentrated by evaporation of solvent at room temperature to about 0.06 ml and treated with reagents as described below for sample preparation, and tested for purity by GLC analysis. When larger quantities (quantities greater than represented in Figure 1) of the HFBA-Py derivatives of N-nitrosodiethylamine and N-nitrosopyrrolidine were analyzed, contaminating components were detected by the EC detector. In addition, N-nitrosopyrrolidine contained some N-nitrosopiperidine. Procedure. N-nitrosodimethylamine (0.68 mmole), Nnitrosodiethylamine (0.922 mmole), N-nitrosopyrrolidine (75.8 pmoles), and N-nitrosopiperidine (47.6 pmoles) were diluted to 6 ml each in chloroform. One-tenth milliliter each of the diluted nitrosamines in chloroform was added to a 12- x 75-mm test tube, and the contents were evaporated to about 0.06 ml; then one drop of pyridine (Py), ca. 0.014 ml, and two drops of HFBA (ca. 7 pl) were added. The test tube was stoppered with a cork and moderately shaken to mix the contents; the reaction mixture was permitted to sit at room temperature for 1 hour; then six drops of chloroform were added with a disposable pipet to prevent excessive loss during washing. The mixture was shaken moderately; then two drops of 4% HC1 (ca. 0.07 ml) were added and the tube contents were shaken thoroughly to remove excess Py and unreacted HFBA and permitted to stand for 3 minutes. Next, the entire contents of the test tube were drawn up into a disposable pipet; the chloroform layer was dispensed back into the test tube, and the aqueous supernatant was discarded; then two drops (ca. 0.07 ml) of a 0.1N NaOH were added; the contents of the test tube were thoroughly shaken to remove components that would interfere with subsequent GLC analysis, and the test tube was stoppered with a cork and permitted to stand for 30 minutes. Next, the entire contents of the test tube were again drawn up into a disposable pipet; the chloroform layer was dispensed into a clean dry test tube, and the aqueous supernatant discarded. The chloroform was evaporated almost to dryness (cu. 0.01 ml) with a stream of clean, dry air. One-tenth milliliter of diethyl ether was added as a final solvent for the HFBA
ANALYTICAL CHEMISTRY, VOL. 44, NO. 11, SEPTEMBER 1972
Figure 2. Mass spectra of HFBA-N-nitrosodimethylamine The fraction was collected from a gas chromatograph by using a Tabsorb column, 20 : 1 splitter, and a flame ionization detector. The structures indicated above the peaks are possible fragmentation products. m/e = mass/charge
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derivatives, the contents of the tube were shaken moderately, cooled in a refrigerator for a few minutes, and 0.2 p1 of the 100-pl sample was injected onto the gas chromatograph for analysis. After the aliquot was removed for analysis the remainder of the sample, which was contained in a 12- X 75-mm test tube, was immediately corked, taped, and stored in a freezer. Replicate analyses were good when care was taken to fill the 1-pl syringe (Hamilton) to the desired level and adequate time permitted to fill the syringe (1 minute). Three or four replicate analyses could be made from the sample without noticeable change in ether volume when the derivatives were not stored for more than a day. Hexane, ethyl acetate, benzene, xylene, and acetonitrile were tested as final solvents, but the halogenated nitrosamines were most soluble in ether as judged by the increase in the detector signal. Care was taken to prevent evaporation of the ether from the syringe before the sample was injected by first measuring the amount to be injected, then pulling the sample up into the syringe before injection. In addition, the needle was protected from hot air that escapes from the oven around the injector ports of the Model 5000 Barber-Colman Gas Chromatograph by wrapping the columns at the injector port with a few turns of asbestos. A TFA derivative of 1,l-dimethylhydrazine was prepared in the same manner as the HFBA derivative. For collecting aliquots of HFBA derivatives of N-nitrosodimethylamine for analysis by mass spectra, 40 pl of sample were injected onto the Tabsorb column, and the peak was collected into a small glass capillary that was cooled with dry ice. A portion of the collected sample was diluted in ether and analyzed by GLC by using an instrument equipped with an EC detector and a 3x OV-1 column. RESULTS AND DISCUSSION
The catalytic value of pyridine (Py) involving reactions with anhydrides and amines found in biological samples is well established (4), and the mechanism for the reaction is described (5). Although the mechanism for the reaction of HFBA-Py with nitrosamines has not been established, there is little doubt that Py is involved in the reaction. No detectable peaks were formed in the absence of Py or HFBA, even when the reaction mixture was heated for 10 minutes at 70 OC. In addition, when the amount of Py was diluted 1 :4, the amount of halogenated nitrosamine was reduced. Heating N-nitrosodimethylamine for 10 minutes at 70 OC in the presence of HFBA-Py increased the reaction rate, but a second smaller
peak eluting not far after the major peak was detected. Heating of the compound under the above conditions did not produce any more of the major compound than did permitting the reaction mixture to stand for 1 hour at room temperature. Derivatives were formed that were highly sensitive to the EC detector. With a 0.2-ml injection of sample, the detector gave a full-scale response to 22.6 nmoles of N-nitrosodimethylamine, 30.7 nmoles of N-nitrosodiethylamine, 1.5 nmoles of N-nitrosopiperidine, and full-scale deflection with 2.5 nmoles of N-nitrosopyrrolidine (Figure 1). Electron capturing derivatives were also formed with N-nitrosodimethylamine and N-nitrosodiethylamine by using TFA. The derivatives formed with TFA-Py had higher boiling points (as determined by their elution on 3x OV-1 columns) than the HFBA-Py derivatives. N-nitrosopyrrolidine and N-nitrosopiperidine were not tested except with HFBA-Py. To test the possibility that N-nitrosodimethylamine was reduced in the process of the reaction to the corresponding hydrazine before derivatization and a hydrazide is formed with the anhydride and hydrazine, 1,l-dimethylhydrazine was treated with HFBA-Py and analyzed by GLC. An electron capturing compound was detected that eluted much later than did the HFBA derivatives of N-nitrosodimethylamine, thus indicating another type of derivate, possibly an ester. The 1,l-dimethylhydrazine, prior to derivatization, was non-electron capturing. Moreover, the electron capturing compound was not formed in the absence of Py. In analyzing urine samples, we found that a more desirable method for forming the HFBA derivatives of N-nitrosodiethylamine in the presence of other amines was to dilute the Py 1 :4 in chloroform,use one drop of the diluent (ca. 0.01 ml), and let the reaction proceed in the mixture of HFBA-Py for 3 minutes instead of 1 hour. Otherwise, the derivative was prepared in the same manner described here. The advantage of the modified procedure was that there was less Py, which is difficult to evaporate, and less time was required for preparing derivatives. The modified procedure was reproducible, its only disadvantage being a slight loss in the amount of HFBA derivative formed from the nitrosamine. Low resolution mass spectral analysis of the collected sample of N-nitrosodimethylamine did not give a parent ion peak (Figure 2); however, the parent peak is often very weak in aliphatic fluorinated compounds (6). Elemental compositions of major peaks were not determined, but structural as-
(4) J. B. Brooks and W. E. C. Moore, Can. J. Microbiol., 15, 1433
(1969). ( 5 ) L. F. Fieser and Mary Fieser, “Introduction to Organic Chemistry,” D. C. Heath and Co., Boston, 1957, p 148.
(6) R. M. Silverstein and G. C . Bassler, “Spectrometric Identification of Organic Compounds,” second ed., John Wiley and Sons, New York, N.Y., 1967, pp 27-31.
ANALYTICAL CHEMISTRY, VOL. 44, NO. 11, SEPTEMBER 1972
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signment for the peaks may be inferred from the literature presented. The most characteristic peak for polyfluorinated compounds is m / e 69 and is due to CF3+ (6). This and other prominent peaks commonly associated with fluorinated hydrocarbons are present (Figure 2, m / e 119 and 169). Fragments reported to be associated with N-nitrosodimethylamine ( 7 ) were also present: m / e 27, 28, 29, 41, 42, 43, 53, 5 5 , 58, and 59 (Figure 2). Peak m / e 42 has been reported (8) to be representative of N-nitrosodimethylamines in the nonderivatized compound and is due to the H2C=N+=CH2 fragment. Fragment m / e = 44,assigned the structure CHaN+-CH3, could be the result of homolytic cleavage of the molecular ion 0
It
CHI
\
O-C-R N++N
/ CH3
/
. \
O-C-R
ll
0 to yield the fragment H3C-N+-CH3 shown in Figure 2 or N20+. The procedure is well documented (9). Although we have tentatively assigned to the peak at m / e 43 H3CN2,it is equally plausible the peak might be CHsNCH2. Although mass spectral data of halogenated compounds leave much to be desired from a standpoint of clearly revealing the actual molecular weight and structure of the derivatized N-nitrosodimethylamine, the presence of fluorine and nitrosamine fragments are clearly indicated, and they are evidence that halogenation of the nitrosamine did occur. N-nitrosodiethylamine, N-nitrosopyrrolidine, and N-nitrosopiperidine (7) T. Fazio, J. H. Damico, J. W. Howard, R. H. White, and J. 0. Watts, J . A g r . Food Chem., 19,250 (1971). (8) H. Budzikiewicz, C. Djerassi, and D. H. Williams, “Mass Spectrometry of Organic Compounds,” Holden and Day, San Francisco, 1967, p 523. (9) J. Collins, Bull. SOC.Roy. Sci. Liege, 23, 201 (1954).
were not collected and analyzed by mass spectra; therefore the labeling of these compounds (Figure 1) must be considered tentative; however, the retention times observed for the HFBA derivatives of these amines (Figure 1) seem reasonable on the basis of the known differences in boiling points of the non-derivatized compounds. The response of the EC detector to the fluorinated nitrosamine was about 6 thousand times the response of the flame ionization detector, as indicated by analysis of N-nitrosodimethylamine with both detectors on the same instrument with the same column. To obtain 2 1 3 of a full-scale response from the flame detector, one millimole of sample was derivatized, and a 2-111 injection of the sample (100 pl total) was used at an attenuation of 8. All other parameters were the same as for the analysis with the EC detector. Procedures reported (7) for detection of known underivatized N-nitrosodimethylamine with a modified thermionic detector-mass spectrometer combination required that a minimum of 50 ng/5 p1 injection of sample be present before identification could be made, Based on calculations made from data obtained by using HFBA-Py derivatives of N-nitrosodimethylamine, an EC detector, and 0 . 2 4 injection of sample, the minimum amount of N-nitrosodimethylamine detectable (about scale) with a 5-111 injection of sample should be about 17 ng. In addition, the use of HFBA-Py derivatives and a 3 z OV-1 column permits analysis of other much higher boiling nitrosamines. The results reported here indicate that some of the biologically important nitrosamines form electron capturing derivatives when reacted with fluorinated anhydrides in the presence of pyridine. Data presented elsewhere (IO) show that these derivatives have been used successfullyto analyze nitrosamines in urine samples.
RECEIVED for review January 21, 1972. Accepted May 12, 1972. Use of trade names is for identification only and does not constitute endorsement by the Public Health Service or by the U. S. Department of Health, Education, and Welfare. (10) J. B. Brooks, W. B. Cherry, L. Thacker, and C. C. Alley, J. Inf. Dis., in press.
Electrolytic Sample Preparation and Its Application to Atomic Absorption Rapid Determination of Magnesium in Cast Iron A. H. Jones and W. D. France, Jr. Chemistry Department, Research Laboratories, General Motors Corporation, Warren, Mich. 48090
ELECTROLYTIC CONSTANT CURRENT techniques have been used for many years in corrosion research, in the associated areas of metallographic polishing and etching ( I ) , and only to a limited extent in analytical chemistry (2, 3). Because such proce(1) S. W. Dean, Jr., W. D. France, Jr., and S. J. Ketcham, in
“Handbook on Corrosion Testing and Evaluation,” W. H. Ailor, Ed., John Wiley & Sons, New York, N.Y., 1971, p 171. (2) Silvio Barabas and Sydney G. Lea, ANAL.CHEM.,37, 1132 (1965). (3) Abraham Aladjem, ibid., 41,989 (1969). 1884
dures can accelerate metal dissolution in a corrosive environment, they represent one means of rapidly preparing solutions of metal samples for instrumental analysis; by comparison, conventional procedures can be time-consuming. For example, the ASTM method (4) for determining magnesium in cast iron by atomic absorption includes the preparation of (4) “1971 Annual Book of ASTM Standards,” Part 32, American
Society for Testing and Materials, 1961 Race Street, Philadelphia, Pa., p 878.
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