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Determination of N-Nitrosodimethylamine in Dimethylamine by Gas Chromatography with Nitrogen Selective Detection and Nitrosamine Selective Detection David Marc Parees Air Products and Chemicals, Box 538, Allentown, Pennsylvania 18 105
The separation of N-nitrosodimethylamine from dimethylamine using a silica gel column chromatography procedure which permits trace analysis of the nitrosamine In the amine matrix using a gas chromatographic nitrogen selective detector (NPD) procedure is described. Recovery percentages are over 90 % for nitrosamine levels as low as 4 ppm and 60-80 % for levels around 0.1-0.2 ppm. Preclsion at the lowest level was * I S % . Confirmation of the nitrosamine identity was provided by examination on gas chromatographic columns of different characteristics, gas chromatography/mass spectometry, Thermal Energy Analysis (TEA) and photodecomposition studies. Conflrmatlon of the quantitative GC/NPD work was provided by GCITEA. The presence of dimethylnitramlne, and its detection using GC/TEA, are also discussed.
The toxic and carcinogenic nature of most nitrosamines is well documented from the studies concerning N-nitrosodimethylamine (1, 2) to the extensive review article of Druckrey et al. (3)and the recent summary by Douglass et al. (4). Many investigators have studied various food stuffs (5-9), clinical specimens (1&13),and air (14-19) and water (17,201 samples in efforts to determine the distribution of nitrosamines in the diet and environment of humans, particularly those residing in localities with high cancer incidence (21). Within the past few years, the development of a nitrosamine selective detector, the Thermal Energy Analyzer (TEA, Thermo Electron Corporation, Waltham, Mass.) (22), has enabled Fine and his co-workers to assay, rapidly and sensitively, various types of air (16-18) and water (17,20) samples and commercial products (23, 24) for nitrosamine content. Pesticides have been analyzed (25) because many contain a dialkylamine moiety. The nitrosamine concentrations detected were attributed t o the nitrosation precursors from reaction chemistry by-products or storage corrosion inhibitors. Spiegelhalder et al. (26) published a brief study of various commercial amines in which a TEA was used. Anhydrous dimethylamine was not investigated. Although the selectivity of the TEA allows direct analysis of amines for nitrosamine content, the detector is not in widespread use. Similarly, high resolution mass spectral specific ion monitoring could be used to analyze for Nnitrosodimethylamine (NDMA) in dimethylamine (DMA), but the use of equipment of sufficient sensitivity and resolution is not widespread. Use of the nitrogen selective gas chromatography detector for trace nitrosamine analysis has been demonstrated to be effective (6, 7, 27-31). However, in order to analyze dimethylamine for traces of NDMA, prior isolation of the nitrosamine is necessary. The isolation of nitrosamines from complex matrices has been investigated and the use of extractions, distillations, liquid/solid chromatography and thin-layer chromatography is widespread (32, 33). Various procedures were investigated for isolation of NDMA from dimethylamine. The most successful, producing good
recoveries a t levels as low as 0.075 ppm, was based upon silica gel column chromatography.
EXPERIMENTAL Reagents. Standard nitrosamine solutions were obtained from Nanogens, Inc. (Watsonville, Calif.). Air Products and Chemicals,Inc. (Trexlertown, Pa.) supplied the anhydrous dimethylamine. HzO2(50%)was obtained from Fisher Scientific Company and trifluoroacetic anhydride from Aldrich Chemical Co., Inc. Matheson, Coleman and Bell (grade 12, 28/200 mesh) silica gel was used. Burdick and Jackson (Muskegon, Mich.) high purity distilled in glass dichloromethane (DCM), isooctane, and ethyl acetate were used. Isolation Methods. Water-jacketed columns (25-mm i.d.1 packed with 35 g of 25% H 2 0 deactivated silica gel were used. The silica gel was applied as a slurry in DCM and the same solvent was used to elute the nitrosamine. Dimethylamine (typically 5 g) was poured onto the head of the column. A polyethylene-lined cap was sufficient to hold the amine in a vial, without boiling, for weighing. After addition to the water-cooled column, adsorption on the silica gel prevented extensive evaporation of the DMA. The amount of amine was determined by the weight difference of the vial in which it was contained. The elution rate was held to 0.4 mL/min for the first 30 mL (after the amine was drained into the column bed), and about 2.4 mL/min thereafter. The fraction from 30-230 mL was collected for analysis. For trace analysis of nitrosamines, only high purity solvents were used. Typically, 200 mL of solvent was concentrated to about 2 mL using a Kuderna-Danish evaporator and the possible presence of interfering species was checked using both a nitrogen selective detector and the TEA. For analysis of samples, final volumes of 2-10 mL were typical. Especially for volumes as low as 2 mL, it is advisable to add a keeper such as 1.0 mL of isooctane t o the concentrator. Quantitation requires weighing of the concentrate, and accounting for the two-solvent system to determine the final volume when a keeper is present. In this work it was assumed that the isooctance was recovered quantitatively in the evaporator. The Kuderna-Danish evaporator consisted of a three-bulb Snyder column, 500-mL flask and 4-mL conical, graduated receiver. It was operated at 54-56 "C in a warm water bath. Recovery of NDMA in DCM was shown to be quantitative at 10 pg/FL levels in the concentrate. As an alternate to the silica gel column separation procedure, dimethylamine in DCM was reacted at 0 "C with a slight excess of 6 N HC1. The organic layer was separated and the aqueous phase was extracted with DCM. The combined organic phases were dried over Na2S04and concentrated for analysis. Instrumentation. Gas chromatography was carried out on a Perkin-Elmer 3920B instrument equipped with a nitrogen selective detector, a Hewlett-Packard5840A instrument equipped similarly, and a Perkin-Elmer3920 interfaced to a Thermal Energy Analyzer. The gas chromatograph/massspectrometerwas a Perkin-Elmer 270 instrument. Typical resolution was approximately 500. The electron impact source was operated at 80 eV. Some manufacturers advise against use of halogenated solvents such as DCM with their nitrogen/phosphorus selective detectors. When the Perkin-Elmer detector was used, a heated 4-port valve was installed to vent the solvent. Analysis and vent columns were identical in all respects. Each consisted of a pair of columns (2
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and 15 feet long, 0.125 inch X 0.020 inch wall, grade 316 SS) packed with 10% Carbowax 20M on Chromosorb W/AW/DMCS (80/100).
The arrangement described above was not necessary with the Hewlett-Packard detector. However, a few injections of a standard nitrosamine in DCM were typically made before analysis was begun each day because of the need for the detector to stabilize under the effect of a halogenated solvent. The following gas chromatographic columns were used in this work. (A) 6 feet X 0.125 inch (0.016-inch wall, grade 304 SS), 60/80 mesh Chromosorb 103. (B) 15 feet X 0.125 inch (0.020-inch wall, grade 316 SS), 10% Carbowax 20M on Chromosorb W/AW/DMCS (80/100). (C) 8 feet X 0.125 inch (0.016-inch wall, grade 304 SS), 10% Carbowax 20M on Gas Chrom Q (80/100). (D) 5 feet X 0.125 inch (0.016-inch wall, grade 304 SS), 10% Carbowax 20M on Chromosorb W/AW/DMCS (80/ 1001, used for GC/MS work. G€/TEA Interface. The interface between the Perkin-Elmer 3920 and the TEA was constructed from,a 25-cm length of 0.0625-inch narrow bore stainless steel tubing which was sheathed in a 20-cm length of 0.125-inch copper tubing. The interface is heated by a flexible heating tape (VWR Scientific, catalog number 3373379, Briscoe Manufacturing Company, Columbus, Ohio 43216) for use with conducting surfaces. Electrical power was supplied by a Powerstat variable autotransformer (Superior Electric Company, Bristol, Conn.). Three thermocouples were placed along the length of the interface under asbestos tape insulation in contact with the copper tubing and were connected to the gas chromatograph temperature meter. Finally, the interface was wrapped in aluminum foil. Temperatures to at least 280 "C have been maintained. Care was exercised in constructing the interface to allow enough length and flexibility to insert wrenches between the gas chromatograph and the Thermal Energy Analyzer to complete the interfacing operation. The interface was routed through a hole drilled in the left side of the chromatograph to join with the TEA which has its GC inlet on the right side. Thermo Electron now supplies detectors with left-sided inputs. GC/TEA Analysis. The Thermal Energy Analyzer was used in conjunction with a double cold trap fabricated from a &foot length of 0.25-inch 0.d. stainless steel bent into a "W' shape. The fmt leg was kept at -78 "C (solid COz/isopropanol) and the second a t -160 "C (liquid nitrogen/2-methylbutane). A 4 inch X 0.25 inch copper tube filled with 60/80 mesh Tenax GC resin was put between the cold traps and the detection cell. T o inject DMA directly for GC/TEA analysis, samples were chilled to -14 "C in a freezer along with the syringe. An aluminum block 1 inch square by 3.125 inches long was drilled to accept the syringe barrel, with a machined slot between the bore and one side for reading volumes. It was chilled with the syringe. Used as a syringe holder, it helped to keep the volatile sample in the syringe. While useful for this work, 10-pL Hamilton syringes had a tendency to develop cracks in the barrels with this procedure, as the syringe instructions warn, and a certain amount of breakage must be accepted. If injected volumes are kept at 15 pL or above, use of a Series A-2 25-pL syringe with valve (Supelco, Inc., Supelco Park, Bellefonte, Pa., 16825) is acceptable. The dead volume of the valve may cause precision problems with smaller injections of DMA. However, the Thermal Energy Analyzer easily handled the larger injections. Alternatively, water or ethyl acetate was used to make DMA solutions for injection. Photolability Confirmation. The mercury vapor ultraviolet lamp (Hanovia, Inc.) drew about 500 W. Samples were put in a quartz vessel 10 cm from the lamp. Irradiation times of 4-8 min were sufficient to destroy NDMA in DCM. Exposure in bright sunlight was also used. Nitramine Synthesis. The procedure of Sen (34) was modified as follows: 400 pL of 50% HzOZwas added slowly to 500 pL of trifluoroacetic anhydride in a small vial. To this was added 5 p L of 100 pg NDMA/mL benzene (Nanogens). After standing overnight, the solution was poured over 2 g crushed ice and 3 mL 20% K&03 was added. The solution was extracted with 2 X 10 mL of DCM and dried over 1g of NaZSO4.The DCM extract was then filtered and the NaZSO4was washed with 2 X
TIME ( M I N U T L S l
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Figure 1. Dichloromethane extract of industrial dimethylamine with HCI. GC:Hewlett-Packard 5840A. Nitrogen selective detection: Column A.
3 mLlmin H,, 50 mLlmin air, 35 mL/min He; peak at 4.25 min coincides with NDMA. Temperatures: injector and detector: 200 "C; column: 190 "C
0
30
60
90
120
150
I80
210
240
210
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Figure 2. Silica gel separation of NDMA from dimethylamine. Plots show elution of the nitrosamine (GClNPD detection). (A) 25 g 20% H,O deactivated silica gel. (B) 30 g 50% H,O deactivated silica gel.
(C) 35 g 25% H,O deactivated sillca gel. Break points in the lines represent mid-points of the collected aliquots 5 mL DCM. The combined DCM portions were then analyzed.
Safety. Since N-nitrosodimethylamine is a confirmed animal carcinogen, all procedures were conducted in ventilation hoods and all instruments were located in or vented to hoods. All solutions were double bottled and isolated in storage. Standards were kept in a refrigerator. Amber bottles were used for the inside bottles (Erno Products Company, Philadelphia, Pa). Latex gloves were used for all critical operations and were discarded immediately after each procedure. All glassware contacting nitrosamine-containing solutions were rinsed in a solution of 1:l concentrated hydrochloric acid/glacial acetic acid before general cleaning.
RESULTS AND DISCUSSION Analysis of the DCM extract of industrial product dimethylamine which had been neutralized with acid was not possible using a nitrogen selective detector owing t o the presence of many trace impurities which were extracted in the organic phase. Typical nitrogen-containing impurities possible in industrial amine products include imines, nitriles, and amides besides other amines. One impurity preceded and almost completely masked the NDMA peak. See Figure 1. Figure 2 shows the results of experiments conducted to determine the optimum activity and amount of silica gel for the isolation procedure. Dimethylamine spiked with NDMA was eluted from each column and collected in 30-mL fractions. The spiked amount (250 pg) was great enough to allow direct determination of the nitrosamine in each fraction without concentration. While dimethylamine is held up indefinitely on all three experimental columns, other trace nitrogen-
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li,
U I
0
0 2 4 5 8 0 2 4 5 8 TIME IMlNUTESl
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Figure 3. Injection of concentrated silica gel column isolated fraction from DMA into GC/NPD. Peak at 4.39 min coincides with NDMA. Column and conditions as in Figure 1
containing components do elute in the NDMA fractions. One species which eluted somewhat before the nitrosamine on the 15-foot Carbowax 20M column also eluted within the first 30 mL on the silica gel columns. Therefore, a column containing 35 g of 25% water deactivated silica gel was chosen over the one containing 25 g of 20% water deactivated silica gel. This held up the nitrosamine sufficiently to allow elution of the other component, so that it was not present in the nitrosamine fraction. Figure 3 is a typical GC/NPD chromatogram of the nitrosamine-containing fraction from the silica gel column, after concentration. NDMA elutes at about 4.4 min under these conditions on Column A. This identification was confirmed by retention time comparisons with standards on this column and Column B. It was also confirmed by the Thermal Energy Analyzer. Other solutions were also analyzed using selective ion monitoring GC/MS. The molecular ion ( m / e 74) and the base peak ( m / e42) were monitored (35). Solutions suspected of containing NDMA were also exposed in a quartz vessel to sunlight. The photolability of N-nitroso compound allows facile identification of possible nitrosamines (15, 36, 37). Figure 4 shows GC/TEA chromatograms of a silica gel-isolated NDMA solution from a production DMA sample before and after exposure to sunlight. The NDMA peak disappeared (there appears to be a trace amount of another nitrosamine, possibly N-nitrosomethylethylamine), but an unidentified species which eluted approximately where N-nitrosodi-npropylamine elutes did not degrade, indicating that it is not a simple dialkyl nitrosamine. Subsequently, in related work, a species eluting at this time was identified by GC/MS as dimethylnitramine (N-nitrodimethylamine, NDMAO). Dimethylnitramine had the same retention time as the unknown. Fine et al. (17) described an unknown which could be NDMAO. I t appears that NO2 is converted to NO in the TEA furnace, possibly through reaction with carbonaceous pyrolysis residues. This has not been investigated. Examination of NDMAO under the UV lamp showed that it decomposed readily under these conditions and, therefore, could represent a significant source of false positive GC/TEA “identifications” of “nitrosamines” if a strong enough UV source is used to perform a photolability check. It appears to have the same molar response as NDMA, as might be expected if the TEA produces NO from NDMAO as efficiently as from NDMA. The conversion of nitrosamines to their
Figure 4. Solution containing NDMA before and after exposure to sunlight. GClTEA Detection: Solution is silica gel isolated eluent from DMA sample. The unknown is probably dimethylnitramine. See text. (A) Before exposure: 5.0 pL injected. (6)After exposure: 4.0 pL injected. Column: C. Column temperature: 160 O C ; injection port and interface: 200 O C . Argon carrier gas: 32 mL/min; traps: -78 O C and -160 O C cold traps and Tenax trap in use. Attenuation: X8, 5-V recorder span. Detection cell pressure: 1.0 Torr; oxygen flow rate: 15 mL/min
Table I. Recovery of Spiked NDMA from Dimethylamine on 35 g 25% H,O Deactivated Silica Gel
a
NDMA spike
% recoveredn
75 PPb 150 ppb 4.15 ppm 11.5 ppm
60-80 88-9 3 93-95 9 2-9 4
DMA sample data shown in Table 11.
corresponding nitramines followed by confirmatory analysis appears to be an excellent means of substantiating the identity of chromatographic peaks (38). The cumulative evidence indicated that the NDMA peak was correctly assigned. The UV lamp was used to irradiate samples to help to distinguish nitrosamines and nitramines from other nitrogen- (or phosphorus-) containing compounds detected by the GC/NPD system. A GC/NPD chromatogram containing numerous components, even after using the silica gel isolation procedure on this particular DMA sample, is shown in Figure 5A. After 8 min of irradiation, Figure 5B shows that the suspected NDMA peak is almost gone. (Compare peak at 3.86 min before irradiation with peak 3.88 min after irradiation.) The relative sizes of other peaks also change upon irradiation. Most evident is the appearance of the large peak a t 5.67 min in the post-irradiation injection. The base line is also considerably more level. Both of these changes are due to the solvent, dichloromethane, or impurities in it. Figures 6A and 6B demonstrate this solvent reaction, showing chromatograms before and after irradiation. Use of a lower wattage lamp would allow nitrosamines and nitramines to be readily distinguished (as sunlight was used for this purpose) and, possibly, the solvent species may not appear. Lamps used to view TLC plate spots are suitable for this purpose. Dimethylamine was spiked at low levels with NDMA and the recoveries were determined by gas chromatography. Table I summarizes the results. At the spike level, 75 ppb, the recovery was 6G80%. This includes all steps in the isolation,
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B
Table 11. Analysis for N-Nitrosodimethylamine in Anhydrous Dimethylamine GC/NPD GC/TEA GC/TEA results, silica results, silica results, DMA gel isolated gel isolated analyzed material, ppm material, ppm directly, ppm o.090a o.loc 0.137d 0.090 0.124 0.080 0.11 0.11 0.128 0.080
0.11
0.078
0.075
av: 0.088 f 0.013 av: 0.097 + 0.015 0.130 t 0.007 assuming 70% recoveryb 0.13 t 5% 0.126 = 0.138 = 0.13 ? 15% 0.14 t 15% a Each number represents 1 silica gel procedure and 3-6 GC injections. Spike work is discussed in the text with results shown in Table I. Each number represents 3-6 iniections. Each number remesents 1 iniection.
I
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Figure 5. GC/NPD chromatograms of silica gel isolated fraction from DMA containing NDMA. (A) Before irradiation. Peak at 3.86 min coincides with NDMA. 4.8 I.LL injected. (B) After 8-min UV irradiation under lamp. 5.25pL injected. Instrument: Hewlett-Packard 5840A. Column: C. Conditions: Column temp.: 145 "C;injector temp.: 200 "C; detector temp.: 250 "C. Flow rates: Helium: 45 mL/min; hydrogen: 3 mLlmin; air: 50 mL/min. Attenuation: X 4 A
Flgure 6. G U N P D chromatograms of dichloromethanebefore and after UV irradiation. (A) Before irradiation. 5.0 pL injected. (B) After 6min UV irradiation. 5.6 pL injected. See Figure 5 for chromatographic conditions concentration, and chromatography. These data indicated that dimethylamine, which has a recovered concentration of 0.088 f 0.013 ppm NDMA by GC/NPD, needs to have a recovery factor applied. Assuming a recovery of about 7 0 % , the actual concentration would be approximately 0.13 ppm. Because of the selectivity of the GC/TEA system, dimethylamine can be analyzed directly to determine the NDMA concentration. The TEA result for this sample, 0.13 ppm, verified the GC/NPD work. The GC/TEA chroma-
0
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Figure 7. GC/TEA of NDMA. (A) 5.25pL of 98.3 pg/FL NDMA in DCM. (B) 3.0 pL of DMA. Column: 6. Conditions as in Figure 4 tograms for both standard and sample are free of extraneous peaks. See Figure 7 . Table I1 summarizes these GC results. The percentage recovered a t the spike level of 0.075 ppm varied more than was expected. The results are based on five analyses at this level. The Thermal Energy Analyzer results indicated that the recovered percentage of NDMA was about 62-77% of that present in the dimethylamine. One of the silica gel isolated concentrates was rechromatographed on an identical silica gel column and the concentrated eluent was reanalyzed. The recovery percentage was about 85%. Although concentration of the nitrosamine in dichloromethane under the stated conditions was quantitative, attempts a t preconcentration of the nitrosamine in dimethylamine to adjust the concentration for maximum recovery using the silica gel column procedure were unsuccessful. Ambient temperature distillation of DMA (boiling point ca. 7 "C) led to a loss of about half of the NDMA (boiling point ca. 150 "C) in a sample when the initial concentration of the nitrosamine was 0.05 ppm. Although the addition of dichloromethane to DMA causes problems because of formation of the amine hydrochloride, even under refrigerated conditions (NMR and elemental analysis confirmed this observation), the use of other solvents as a distillation keeper may prove useful in circumventing the codistillation problem. Results to date show that if the solvent blank is kept low (under 0.1 ppb), levels of NDMA in DMA can be analyzed below a level of 0.2 ppm with good recoveries, selectivity, and precision using the silica gel separation technique and a nitrogen selective detector. Detection limits for the two nitrogen selective detectors used were comparable to the Thermal Energy Analyzer; all were about 30-40 pg NDMA under the chromatographic and instrumental conditions used.
ACKNOWLEDGMENT
I would like to thank Thomas Boyle for technical assistance and J. P. Casey, R. E. Mayo, L. B. Tepper, and W. J. Zubyk for helpful ideas. I also had valuable discussions with W.
:+?ALYTICAL CHEMISTRY, VOL. 51, NO. 11, SEPTEMBER 1979
Fiddler of the U S . Department of Agriculture, L. K. Keefer of the National Cancer Institute, and D. H. Fine, T. Y. Fan, and R. Ross of the Thermo Electron Corporation.
LITERATURE CITED (1) J. M. Barnes and P. N. Magee, Er. J . Ind. Med., 11, 167-74 (1954). (2) P. N. Magee and J. M. Barnes, Er. J . Cancer, 10, 114-22 (1956). (3) H. Druckefy,R. Preussman, S. Ivankovic, and D. Schmahi, 2.Krebsfwsch. Klin. Onkol., 69, 103-201 (1967). (4) M. L. Dougiass, B. L. Kobacoff, G. A. Anderson, and M. C. Cheng. J . SOC. Cosmet. Chem., 29, 581-606 (1978). (5) D. H. Fine. D. P. Rounbehler, and P. E. Oettinger, Anal. Chim. Acta, 7 8 , 383-9 (1975). (6) A. E. Wasserman, W. Fiddler, R. C. Doerr, S. F. Osman, and C. J. Dwley, Food Cosmet. Toxicol., 10, 681-4 (1972). (7) T. Fazio, J. N. Damico, J. W. Howard, R. H. White, and J. 0. Watts, J . Auric. Food Chem.. 19. 250-53 119711. (8) R r A l Scanlan, Crit.' Re;. Food Techno/.,5, 357-402 (1975). (9) N. P. Sen, D. C. Smith, L. Schwinghamer, and B. Howsam. Can. Inst. Food Technol., 3(2), 66-9 (1970). (IO) G. Schwuing and D. Ziebarlh, in "Environmental N-Nitroso Compounds, Analysis and Formation", E. A. Walker, P. Bogovski, and Griciute, Ed., Proceedings of Conference at Tallinn, Estonian SSR, October 1-3, 1975, International Agency for Research on Cancer Scientific Publication 14, Lyon. France, 1976, pp 269-77. (11) J. Sander and G. Waly, Ref. IO, pp 291-300. (12) R. M. Hicks, T. A. Gough, and C. L. Waiters, in "Environmental Aspects of N-Nitroso Compounds", E. A. Walker, M. Castegnaro, L. Griciute, and R. E. Lyle, Ed., Proceedings of Conference at Durham, N.H., August 22-24. 1977, International Agency for Research on Cancer Scientific Publication 19, Lyon, France, 1978, pp 465-73. (13) L. Lakritz, A. E. Wasserman, Gates, and A. M. Spinelii, Ref. 12, pp 425-29. (14) E. D. Peliizzari, J. E. Bunch, J. T. Bursey, and R. E. Berkley, Anal. Len., 9(61. 579-94 11976). (15) K: Bretschneider and J. Matz, Ref. IO, pp 395-9. (16) D. H. Fine, D. P. Rounbehier, N. M. Beicher, and S. S. Ipstein, Ref. 10, pp 401-8. (17) D. H. Fine, D. P. Rounbehler, A. Rounbehler, A. Silvergleid, E. Sawicki, K. Krost, and G. A. DeMarrais, Environ. Sci. Technoi., 11, 581-4 (1977). (18) D. H. Fine, D. P. Rounbehier, E. D. Pellizzari J. E. Bunch, R. W. Berkiey, J. McCrae, J. T. Bursey, E. Sawicki, K. Krost, and G. A. DeMarrais, Buil. Environ. Contam. Toxicoi., 15, 739-46 (1976).
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D. H. Fine, D. P. Rounbehier, E. Sawicki, K. Krost, and G. A. DeMarrais Anal. Len., 9 , 595-604 (1976). D. H. Fine, D. P. Rounbehler. F. Huffman, A. W. Garrison, N. L. Wolfe, and S. S. Epstein, Bull. Environ. Contam. Toxicol., 14, 404-8 (1975). P. Bogovski, in "N-Nitroso Compounds, Analysis and Formation", P. Bogovski, R. Preussman and E. A. Wakec. Ed., R o c d i n g s of Conference at Heidelberg, Federal Republic of Germany, October 13-15, 1971, International Agency for Research on Cancer Scientific Publication 3, Lyon, France, 1972, pp 1-5. D. H. Fine, D. Lieb. and F. Rufeh, J . Chromatogr.. 107, 351-7 (1975). T. Y . Fan, J. Morrison, D. P. Rounbehler, R. Ross, D. H. Fine, W. Miles, and N. P. Sen, Science, 196, 70-71 (1977). R. D. Ross, D. H. Fine, D. P. Rounbehler, T. Y. Fan, A. Siberglei. L. Song, and J. Morrison, presented at the 173rd ACS National Meeting, New Orleans, La., 1977. Fed. Regist., 42(152), August 8 , 1977, pp 40009-.40015. B. Spiegelhalder, G. Eisenbrand, and R. Preussmann, Angew. Chem., I n t . Ed. Engl., 17. 367-8 (1978). D. C. Havery and T. Fazio. J . Assoc. Off. Anal. Chem., 60, 517-19 (1977). N. T. Crosby, J. K. Foreman, J. F. Palframan and R. Sawyer, Ref. 21, pp 38-42. L. Hedler, H. Kaunitz, P. Marquardt. H. Faies, and R. E. Johnson, Ref. 21, pp 71-3. J. W. Howard, T. Fazio, and J. 0. Watts, J . Assoc. Off. Anal. Chem., 53, 269-74 (1970). W. Fiddler, R. C. Doerr, J. R. Ertel, and A. W. Wasserman, J . Assoc. Off. Anal. Chem., 54, 1160-63 (1971). A. E. Wasserman, Ref. 21, pp 10-15. G. Eisenbrand, in "N-Nboso Compounds in the Environment", P. Bogovski and F. A. Walker, Ed., Proceedings of a Conference at Lyon, France, October 17-20. 1973, International Agency for Research on Cancer Scientific Publication 9, Lyon, France, 1974, pp 6-11. N. P. Sen, J . Chromatogr., 51, 301-4 (1970). W. Lijinsky, W. H. Chris%, and W. T. Rainey. Jr., "Mass Spectra of NN&w Compounds", Oak Ridge National Laboratory, Oak Ridge, Tennessee (1973). J. Polo and Y. L. Chow, Ref. IO, pp 473-86. R. C. Doerr and W. Fiddler, J . Chromatogr., 140, 284-7 (1977). G. M. Telling, J . Chromatogr., 73, 79-87 (1972).
RECEIVED for review July 25,1978. Accepted May 10,1979.
Fluorometric Determination of Pyruvic Acid and a-Ketoglutaric Acid by High Performance Liquid Chromatography Hiroshi Nakamura' and Zenzo Tamura Department of Analytical Chemistry, Faculty of Pharmaceutical Sciences, University of Tokyo, 7-3- 1, Hongo, Bunkyo-ku, Tokyo 1 13, Japan
A HPLC method has been developed for the fluorometric determination of pyruvic acid (PA) and a-ketoglutaric acid (a-KG). They were separated by anion-exchange chromatography, reacted with N'-methylnicotinamidechloride (NMN) In the presence of alkali, and heated after the acidification with formic acid to produce fluorophores. By using the optimized conditions for chromatography and the post-column derivatlzatlon, 15 pmol of PA and 75 pmol of a-KG could be determined. The relative standard deviations of the method were 3.82% and 3.32% for the analyses of 500 pmol each of PA and a-KG, respectively.
Numerous methods have been reported for the analysis of a-keto acids based on diverse principles. Among them the colorimetric method using 2,4-dinitrophenylhydrazine( I , Z ) , UV absorptiometric (3-5) and the fluorometric (6, 7 ) methods coupled with enzymic reactions are most widely used. However, these spectrophotometric methods do not permit the simultaneous analysis of coexisting a-keto acids. Gasliquid chromatography (8) and gas chromatography-mass spectrometry (8)are suitable for this purpose, but they require 0003-2700/79/0351-1679$01.00/0
in general cumbersome pretreatment of samples to prepare volatile derivatives and inherit instability of derivatives at low concentrations. The application of high performance liquid chromatography (HPLC) to the analysis of 0-keto acids is a recent development. Hayashi et al. (9) reported a HPLC-UV method for the assay of phenylpyruvic acid which involved the pre-column derivatization with naphthalene-2,3-diamine to 3-benzyl2-hydroxybenzoquinoxaline. Terada et al. (IO) determined pyruvic acid (PA) and a-ketoglutaric acid (a-KG) in serum by separating their 2,4-dinitrophenylhydrazonesfollowed by UV detection. Liao et al. (11) reported a HPLC-UV method for the analysis of urinary PA and a-KG by utilizing the reaction of a-dioxo compounds with o-phenylenediamine to form quinoxalones. On the other hand, fluorescence detection coupled with HPLC separation may provide a more specific and sensitive method to analyze a-keto acids. However, in spite of the presence of several fluorometric methods for the analysis of a-keto acids (12-I4), the attempt of such a HPLC-fluorescence detection is rare. Quite recently, Grushka et al. (15) reported the derivatization of a-keto acids with 4-bromomethyl-7-methoxycoumarin and successive reversed-phase HPLC separation of the fluorescent derivatives. 62 1979 American Chemical Society
