Generation of Nitrosamines for Gas Chromatographic Analysis via Direct Injection D. J. Freed* and A. M. Mujsce Bell Laboratories, 600 Mountain Avenue, Murray Hili, New Jersey 07974
Techniques for in situ generation of nitrosamines for chromatographic analysis are descrlbed. Using amine salt precursors, reaction on potasslum nitrite precolumns affords high (greater than 95%) and reproduclble yields of the corresponding nitrosamines in the 10- to 100-ng range.
Although the estimation of nitrosamines is an extremely important subject, in which much interest has been generated, the extraordinary toxicity of these compounds renders the preparation of standards extremely hazardous (1-3). Recently, we have demonstrated that it is possible to generate precisely controlled amounts of toxic or hazardous materials directly in the injection port of a gas chromatograph (4).Application of this in situ method circumvents the necessity for storage and manipulation of these potent carcinogens and facilitates the preparation of accurate (*3%) trace level comparison standards (in the 10- to 100-ng range). This report describes techniques and methods for generating submicrogram amounts of nitrosamines for gas chromatographic analysis.
EXPERIMENTAL Instrumentation. A Varian MAT 112 Gas Chromatograph-Mass Spectrometer was used for all investigations. Chromatograms were recorded by use of a second ionizer operated at 20 eV with detection by means of a Faraday cup and dc amplifier. The column used for this work was 2 m by 3.2 mm (stainless steel) packed with Chromosorb 103 (100-120 mesh). Injections were made with Hamilton 7101 syringes which had been gravimetrically calibrated with mercury. These were found to be reproducible to f1.8% for a 0.5-pL injection. Materials. Morpholine, pyrrolidine, piperidine, and diethylamine were purchased from Aldrich, Inc., and redistilled before use. Dimethylamine (gaseous) was purchased from Linde, Inc., and used directly. Standard solutions of the above (in the range 0.1 to 1.0 mg/mL) were prepared by dissolution in water and titration with 0.1 M HC1. Diethyl nitrosamine was prepared in small amounts (ca. 5 mg) using glove box techniques (5). Standards were prepared from the above material using a sealed ampoule technique (6). Precolumns were constructed from Teflon tubing (8 cm by 6 mm o.d.), packed with 1g of ACS Reagent Grade KN02, plugged with glass wool at each end, and preconditioned at 200 "C for 1 2 h before use.
RESULTS AND DISCUSSION The generation of nitrosamines is easily accomplished by direct injection of the corresponding amine salt onto a suitably prepared precolumn of KN02. Solutions of the desired amine were prepared by dilution of a freshly prepared and standardized master solution with 1M sulfuric acid. These solutions gradually decreased in strength (after two weeks 90% of the original amine was still present) and therefore, for highest accuracy, periodic restandardization by titration was employed. Attempts to use the amines as their hydrochloride salts led to poor reproducibility. We attribute this to the volatilization of the amine hydrochloride which results in incomplete reaction with the KN02. Table I lists nitrosamines prepared by this technique together with absolute yields 1544
ANALYTICAL CHEMISTRY, VOL. 49, NO. 11, SEPTEMBER 1977
Table I. Yields and Conditions for Generation of Nitrosoaminesa Amount generated, Compound nmol Yield t ' ~min , N-Nitrosodiethylamine 0.86 95 i 3% 3.7 0.66 9 4 + 3% 0.44 94i 3% 0.22 9 4 + 3% N-Nitrosodimethylamine b 1.5 N- Nitrosopyrrolidine b 11 N- Nitrosopiperidine b 12.6 N-Nitrosomorpholine b 10.6 The column was operated at 220 "C with a helium flow rate of 20 mL/min. The injector port temperature was 200 "C. See text. obtained for diethyl nitrosamine generation. These latter were obtained as follows. Aqueous solutions of chromatographically pure diethyl nitrosamine were prepared in the range 10 to 100 F g / d by gravimetry and suitable dilution. A calibration plot of total integrated ion current in the nitrosamine peak was obtained for amounts from 10 to 100 ng. Aliquots of the diluted diethylamine sulfate solution were then injected and the integrated total ion currents under the generated nitrosamine peak were also plotted on the same graph after correction by the gravimetric factor for the reaction stoichiometry (for the case of diethyl nitrosamine this is 0.716 for the reaction Etz" + HONO EhNNO + HzO). These curves are shown in Figure la. In order to ensure that the nitrosamine did not react with KN02, amounts from 5 to 50 ng were injected onto both packed and empty precolumns. No differences greater than 2% in the measured peak areas could be seen (cf. Figure la). The yields exhibited little dependence on either injector temperature (in the range 180 to 250 "C)or on flow rates (in the range 10 to 30 mL/min). Therefore these parameters were chosen so as to optimize column efficiencies. The efficiency of nitrosamine generation depends strongly on the acid concentration of the precursor amine solution. A plot of relative yields as a function of sulfuric acid concentration is shown in Figure lb. As expected, increasing acid concentrations favor increased yields (i.e., by ensuring an adequate generation of nitrous acid); however, too strong an acid concentration actually lowers the yields. This is due to the reverse reaction of the nitrosamine with acid to regenerate the original amine salt. For this work, an optimum acid concentration was found to be 1M. At this concentration the absolute yield of diethyl nitrosamine was high and reproducible (95 f 3% in the range from 10 to 100 ng). Precolumns were found to be stable for approximately 30 injections and, as also expected, lifetimes decreased drastically at higher acid concentrations. Although a mass spectrometer was used as a detector, it was not feasible to use single ion monitoring at the parent peak for high sensitivity detection of the nitrosamines. A previous -+
to ensure that the method results in nitrosamine production by operating the source at 60 O C . However, because of the low volatility of the nitrosamines, condensation in the source quickly resulted in high backgrounds a t this temperature. Therefore measurements were made at a source temperature of 200 "C. The resulting mass spectra were those of the amine portion of the nitrosamine, although they appeared at the GC retention time of the authentic nitrosamine. In this work, other nitrosamines have not been synthesized directly for absolute yield studies. However, comparison of the measured ion currents from the respective nitrosamines generated in situ with that of diethylamine (for which the sensitivities should be similar) suggests that the efficiency of generation for all amines tested should be well over 90%.
b
a
I
I
.20
.40 .60 .SO nanornoles i n j e c t e d
1
-1
0
10 g k
-2
-3
LITERATURE CITED
-4
2 s 04
Figure 1. (a) Calibration curve for generation of diethyl nitrosamine. Generated standard (- - -). Gravimetrically prepared standard (-). Gravimetric standard on unpacked precolumn (b) Yiekl vs. solution pH for 50 ng of generated diethyl nitrosamine (-e-).
report (7) demonstrates, and we have confirmed, that at ion source temperatures much above 100 "C thermal decomposition is evident, resulting in loss of NO and much variability in the parent peak intensity. During our work, it was possible
(1) G. Hawksworth and M. J. Hill, Biochem. J., 122, 28 (1970). (2) N. P. Sen, J . Chromatogr., 51, 301 (1970). (3) D. H. Fine, D. Lieb, and F. Rufeh, J . Chromatogr., 82, 291 (1973). (4) D. J. Freed and A. M. Mujsce, Anal. Chem., 49, 139 (1977). (5) A. I. Vogel, "A Textbook of Practical Organic Chemistry", 3rd ed. John Wiley & Sons, New York, N.Y., 1966, p 426. (6) S. Siggia, "Quantitative Organic Analysis via Functional Groups", John Wlley and Sons, New York, N.Y., 1963, Chap. 26. (7) G. Schroll, R. G. Cooks,P. Klemmenses, and S A .Lawesson, Ark. Kemi, 28, 413 (1967).
RECEIVED for review April 18,1977. Accepted June 13,1977.
Determination of Intact Oxazepam by Electron Capture Gas Chromatography after an Extractive Alkylation Reaction
'
Jorgen Vessman, * Margareta Johansson, Per Magnusson, and Signhild Stromberg AB KABI, Research Department, Analytical Chemistry, Fack,
S-172 87 Stockholm, Sweden
Oxazepam was converted to an N,,03-dlmethyl derivative in an extractive alkyiatlon reaction. The derivative was quantltated by electron capture gas chromatography using iorazepam as an Internal standard. Concentrations down to 1 ng/mL could be determlned. At the 25 ng/mL level 98.4 f 3.2 % were recovered. Serum samples taken after adminlstratlon of diazepam or clorarepate contalned measurable concentrations of oxazepam after 2 to 4 h. The dialkyl derivatives of the two benrodlareplnes, especially that of lorazepam, are converted to Isomeric derlvatlves with hlgh concentratlons of the quaternary ammonium hydroxide In the organic phase. The condltions which glve quantltatlve formation without Isomerization are discussed.
The 1,Cbenzodiazepines are a widely used class of drugs. The compounds have been assayed in biological fluids by various techniques, but gas chromatography (1) and polarography (2) are the principal ones. The bioanalytical field has been reviewed (3). The gas chromatographic techniques used in the early sixties were based on the product of hydrolysis, e.g. aminochlorobenzophenone (4).The sensitivity of electron capture detection was good, but the selectivity could be doubtful in some cases because of the interferences of metabolites yielding 'Present address AB HAESSLE, Fack, S-43120 Molndal, Sweden.
the same hydrolysis product. Direct determinations for diazepam and medazepam were introduced by de Silva (5). Oxazepam cannot be determined directly as it undergoes ring contraction when injected into the gas chromatographic column. The product of the rearrangement, a quinazoline derivative, has a shorter retention time than that of the unchanged benzodiazepine, but the gas chromatographic properties of the compound make analysis difficult below about 50 ng/mL. The structure of oxazepam favors ring contraction. However, alkylation at the N1 position or absence of the 3-hydroxy group or derivatization of this group will give thermally stable derivatives (6). The present paper describes a procedure for dialkylation of oxazepam by an extractive alkylation procedure and its subsequent determination by electron capture gas chromatography.
EXPERIMENTAL Apparatus. A Varian Model 1400 gas chromatograph with a scandium type electron capture detector was used with a 1.5 m X 1.8 mm glass column, filled with 3 % OV-225 on Chromosorb G (100-120 mesh, acid washed and silanized). The column temperature was kept at 265 "C after conditioning with gas flow for 2 h at 290 "C. The detector temperature was 300 "C. The nitrogen flow rate was 30 mL/min. The mass spectra with electron impact ionization were run in an LKB 9000 gas chromatograph mass spectrometer with an ionization energy of 70 eV. The gas chromatographic column was as indicated above, but used with a helium flow of 20 mL/min at 250 "C. The mass spectra with chemical ionization were run in an LKB 2091 instrument with ANALYTICAL CHEMISTRY, VOL. 4 9 , NO. 11, SEPTEMBER 1977
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