Detection and Confirmation of N-Nitrosodialkylamines Using Liquid

Simultaneous detection and confirmation of several N-nitrosodialkylamines are accomplished by on-line coupling of a photolysis reactor with an ...
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Anal. Chem. 1996, 68, 546-552

Detection and Confirmation of N-Nitrosodialkylamines Using Liquid Chromatography-Electrospray Ionization Coupled On-Line with a Photolysis Reactor Dietrich A. Volmer, Jack O. Lay, Jr., Stanley M. Billedeau, and David L. Vollmer*

National Center for Toxicological Research, 3900 NCTR Road, Jefferson, Arkansas 72079

N-Nitroso compounds have been found in a large variety of foods and consumer products such as meat,1 beer,2 cosmetics,3 infant pacifiers,4 and drug formulations.5 Their widespread prevalence can be attributed to the relative ease of formation and to the abundance of their amine precursors in the environment. The occurrence of N-nitroso compounds in these products is of great concern, owing to the carcinogenic properties that many of them exhibit.6 To date, the majority of the analytical methods for detection of N-nitroso compounds, hereafter termed N-nitrosodialkylamines, have employed gas chromatography (GC) or liquid chromatography (LC) in conjunction with a thermal energy analyzer (TEA),7

which relies on the pyrolytic breakdown of N-NO moieties to release the nitrosyl radical. The disadvantage of these techniques, however, is that a subsequent confirmation is needed to ensure that the method does not give rise to a false-positive response.7 Generally, it is accepted that mass spectral analysis, where at least three structurally significant ions are detected, provides unequivocal proof of N-nitrosodialkylamines in a sample.8 A combination of LC and mass spectrometry (LC-MS) offers significant analytical advantages over the aforementioned techniques. The LC-MS technique can be used to separate and detect volatile and nonvolatile N-nitrosodialkylamines without the need for a solvent cold trap. It allows for a wider choice of polar mobile phases than does LC-TEA. Moreover, the LC-MS technique allows for identification of unknown components. This is especially important for the analysis of nonvolatile N-nitrosodialkylamines, where, in contrast to volatile N-nitrosodialkylamines, information regarding their occurrence and concentration in many products is not well-known.7 There are limitations, however, to the LC-MS analysis. The two most common ionization techniques available to LC-MS, thermospray (TSP) and electrospray ionization (ESI), yield primarily molecular weight information; that is, little fragmentation is observed to confirm the structure of the analyte. Thermally induced decomposition9 and in-source collision-induced dissociation (CID)10 have been utilized as a means of producing structurally significant ions. These techniques, however, are often unreliable and can suffer from a significant loss in sensitivity.11 Alternatively, on-line photolysis can be used to induce photolytic dissociation of varying types of compounds.12-14 In these experiments, UV irradiation of the analyte in aqueous solutions generates structurally significant photolysis products and, in turn, adds a degree of selectivity to the analysis; that is, class-specific analytes undergo photolysis to give characteristic products.

* Address correspondence and reprint requests to this author at USFDA/ NCTR, HFT-233, National Center for Toxicological Research, 3900 NCTR Rd., Jefferson, AR 72079. (1) Canas, B. J.; Havery, D. C.; Joe, F. J., Jr.; Fazio, T. J. Assoc. Off. Anal. Chem. 1986, 69, 1020. (2) Billedeau, S. M.; Miller, B. M.; Thompson, H. C. J. Food Sci. 1988, 53, 1696. (3) Billedeau, S. M.; Heinze, T. M.; Wilkes, J. G.; Thompson, H. C. J. Chromatogr. A. 1994, 688, 55. (4) Billedeau, S. M.; Thompson, H. C.; Miller, B. M.; Wind, M. L. J. Assoc. Off. Anal. Chem. 1986, 69, 31. (5) Dawson, B. A.; Lawrence, R. C. J. Assoc. Off. Anal. Chem. 1987, 70, 554. (6) Magee, P. N.; Montesano, R.; Preussman, R. In Chemical Carcinogens; Searle, C. W., Ed.; ACS Monograph Series 173; American Chemical Society: Washington, DC, 1976; Chapter 11. (7) Massey, R. C. Nitrosamines 1988, 16.

(8) Andrezejewski, D.; Havery, D. C.; Fazio, T. J. Assoc. Off. Anal. Chem. 1981, 64, 1457. (9) Tsai, C.-P.; Sahil, A.; McGuire, J. M.; Karger, B. L.; Vouros, P. Anal. Chem. 1986, 58, 2. (10) Straub, R. E.; Voyksner, R. D. J. Am. Soc. Mass Spectrom. 1993, 4, 578. (11) Niessen, W. M. A.; van der Greef, J. Liquid Chromatography-Mass Spectrometry; Marcell-Dekker, Inc.: New York, 1992; Chapter 17. (12) Ballard, J. M.; Grinberg, N. Proceedings of the 40th ASMS Conference on Mass Spectrometry and Allied Topics; American Society for Mass Spectrometry: East Lansing, MI, 1992; p 1256. (13) Laycock, J. D.; Yost, R. A. Proceedings of the 41st ASMS Conference on Mass Spectrometry and Allied Topics; American Society for Mass Spectrometry: East Lansing, MI, 1993; p 55. (14) Asano, K. G.; Van Berkel, G. J. Proceedings of the 41st ASMS Conference on Mass Spectrometry and Allied Topics; American Society for Mass Spectrometry: East Lansing, MI, 1993; p 1068a.

Simultaneous detection and confirmation of several Nnitrosodialkylamines are accomplished by on-line coupling of a photolysis reactor with an HPLC-electrospray ionization mass spectrometer. Several parameters such as irradiation wavelength, irradiation time, mobile-phase composition, and pH, as well as different organic acid modifiers are investigated, and their impact on the detection of the N-nitrosodialkylamine-acid complex and its dissociative photolysis products is presented here. Additionally, the type of structural information obtained from the photolytic processes of N-nitrosodialkylamines is compared to that obtained by using in-source collisioninduced dissociation. To demonstrate the potential of this technique, six N-nitrosodialkylamines are studied to determine the linearity of the response, the limits of detection and confirmation, and the reproducibility. The technique’s versatility is also exhibited by utilizing negative-ion mode as a complementary means for analysis of the compounds. Finally, an illustrative application for N-nitrosodimethylamine analysis in beer is described.

546 Analytical Chemistry, Vol. 68, No. 3, February 1, 1996

This article not subject to U.S. Copyright. Published 1996 Am. Chem. Soc.

Moreover, these products of photolysis can often enhance the ESI sensitivity of the MS analysis because many of them are ionic species. The goal of this study is to utilize an on-line photolysis reactor in the LC-MS experiment (LC-hν-MS) as a means to enhance detection and allow for confirmation of N-nitrosodialkylamines. The kinetics of these photolysis reactions are largely dependent on irradiation wavelength, irradiation time, mobile-phase composition, and mobile-phase pH. Hence, these parameters are investigated to evaluate their impact on the LC-hν-MS experiment. To demonstrate the usefulness of this technique, six N-nitrosodialkylamines are studied with respect to linearity of the response, limits of detection and confirmation, and reproducibility. In addition, an LC-hν-MS method in the negative-ion mode is utilized as a complimentary means for multiresidue analysis of N-nitrosodialkylamines. Finally, an illustrative application for N-nitrosodimethylamine (NDMA) analysis in beer is described. EXPERIMENTAL SECTION Reagents. All N-nitrosodialkylamine standards (Sigma, St. Louis, MO) and trifluoroacetic acid (Sigma, St. Louis, MO), formic acid (Fluka Chemical Co., Buchs, Switzerland), and acetic acid (Fischer Scientific, Fair Lawn, NJ) were used as received. Caution! Many N-nitroso compounds are carcinogenic and should be handled with extreme care. Standards were diluted with doubledistilled water, which was purified with a Milli-Q water system (Millipore Corp., Bedford, MA). Distilled water and Optima-grade acetonitrile (Fischer Scientific, Fair Lawn, NJ) were degassed in an ultrasonic bath and continuously purged with approximately 25 mL/min of helium to keep dissolved air free of the system. All beer samples were obtained locally. Sample Extraction/Preconcentration. Two 100-mL beer samples and one 100-mL control were weighed into a 180-mL culture tube equipped with Teflon-lined screw caps. Exactly 1 mL of internal standard solution containing 200 ng/mL NDMA was added to one of the samples. Into the beer samples was added 20 mL of dichloromethane. The samples were then shaken by inverting rapidly for 1 min and then centrifuging at 500g for 10 min. The organic layer was removed using a 10-mL glass syringe fitted with a 13-cm stainless steel cannula for reaching below the aqueous layer. A second extraction was repeated with 20 mL of dichloromethane. After drying through a plug of Na2SO4, the combined 40-mL extract was collected in a 250-mL Kuderna-Danish (K-D) evaporator assembled with a 4-mL graduated K-D tube for volume measurement. A boiling chip was added to the K-D tube, which was connected to a three-ball Snyder condenser. The sample extracts were then evaporated to approximately 4 mL in a 65 °C water bath. The final concentration step was performed by a nitrogen evaporator to reduce the volume to 0.2 mL. The tubes were then stoppered and refrigerated to prevent further evaporation until analysis. Instrumentation. The eluents used in the chromatographic separation were delivered by a Constametric 4110 MS HPLC pump (Thermo Separation Products, Riveria Beach, FL) at a flow rate of 1 mL/min. For separation of the N-nitrosodialkylamine standards, a 3.9-mm × 300-mm µ-Bondapak C-18 column (Waters Associates, Inc., Milford, MA) with 5-µm particles was used. The acetonitrile-water eluent composition was linearly programmed from a 5:95 ratio held for 5 min to a 95:5 ratio over 10 min and then held for an additional 10 min at a 95:5 ratio.

Scheme 1. Mechanism for Formation of Alkylidenealkylamine from Irradiated N-Nitrosodialkylamine under Acidic Conditions

The column was connected directly to a Model 7125 injection valve (Rheodyne, Cotati, CA) equipped with a 10- or 20-µL loop. A photochemical reactor (Aura Industries, Staten Island, NY) fitted with a knitted-open tubular reactor coil (0.25-mm i.d. × 15-m length) and an 8-W, 254- or 366-nm ultraviolet bulb was connected after the column, followed by a 1:4.5 split. Approximately 180 mL/min of sample and eluent were admitted into the electrospray source of a Finnigan MAT (San Jose, CA) Model TSQ 7000 mass spectrometer. Typical operating conditions for the spectrometer were as follows: capillary temperature, 240 °C; electrospray voltage, 4.5 kV; and sheath gas pressure, ∼400 kPa. RESULTS AND DISCUSSION Ultraviolet absorption of the N-nitrosodialkylamine molecule involves activation, via photolytic irradiation, from the ground state to a low-lying excited state. The resulting activated N-nitrosodialkylamine can return to the ground state by way of vibrational or rotational relaxation, or it can act as a transition state to a new molecule through associative or dissociative reactions. The type of reaction and its product yield is largely dependent on the irradiation wavelength, the irradiation time, the mobile-phase composition, and the pH.15 In most cases, the reaction involves dissociation,15 hereafter termed dissociative photolysis. N-Nitrosodialkylamines exhibit UV absorptions at ∼360 nm (n f π* transition) and at ∼230 nm (π f π* transition). They are remarkably stable compared to other nitroso derivatives because of their extensive resonance stabilization, owing to n- and π-electron interactions. Their stability is reflected in a resistance to dissociative photolysis under neutral or basic conditions.16 This behavior changes, however, under acidic conditions, where a N-nitrosodialkylamine-acid complex is thought to be the photolabile species.17 Excitation of the N-nitrosodialkylamine-acid complex (see structure a of Scheme 1) at ∼360 nm leads to dissociative photolysis to release an aminium radical (structure c) and nitric oxide, which, in aqueous solution, usually react to form protonated alkylidenealkylamine (structure d) and hyponitrous acid, respectively.18 With excitation at ∼230 nm, the N-nitrosodialkylamineacid complex (see structure a of Scheme 2) can undergo (15) Chow, Y. L.; Colon, C. J. Can. J. Chem. 1968, 46, 2827. (16) Chow, Y. L. Acc. Chem. Res. 1973, 6, 354. (17) Polo, J.; Chow, Y. L. In Environmental N-Nitroso Compounds Analysis and Formation; Walker, E. A., Bogovski, P., Griciute, L., Eds.; International Agency for Research on Cancer: Lyon, 1976; p 473.

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Scheme 2. Mechanism for Formation of the Parent Amine from Irradiated N-Nitrosodialkylamine under Acidic Conditions

Table 1. Relative Abundance Comparison of NDMA and NMOR Dissociative Photolysis Products at Different Irradiation Wavelenghthsa irradiation N-nitrosodialkylamine ion

none

366 nm

254 nm

[(CH3)(CH2)NH]+ [(CH3)(CH3)NH2]+ [M + H]+ total abundance

NDMA nd 1.1 ( 0.2 97.7 ( 0.2 10 025 480

4.4 ( 0.0 14.7 ( 0.6 70.4 ( 0.7 8 840 384

5.4 ( 0.2 74.1 ( 0.6 nd 6 318 158

[C4H7ONH]+ [C4H8ONH2]+ [M + H]+ total abundance

NMOR 1.1 ( 0.1 2.5 ( 0.1 94.3 ( 0.2 9 399 731

16.0 ( 1.2 16.0 ( 0.2 47.9 ( 1.7 11 794 332

15.1 ( 0.7 57.9 ( 0.4 nd 13 777 447

a

Data averaged from three replicate injections. nd, not determined.

dissociative photolysis to yield the parent amine (structure f) and nitrous acid, where it is believed that water and a proton act in a concerted manner to cleave the N-N bond.19 Effect of Different Irradiation Wavelengths. Because the yield of dissociative photolysis products for N-nitrosodialkylamines is dependent on the wavelength of excitation, irradiation at different wavelengths is investigated. Two N-nitrosodialkylamines, NDMA and N-nitrosomorpholine (NMOR), are chosen as model compounds for these studies because their photolysis reactions and the resulting products have been well-characterized (see, for example, refs 15-17). In consecutive experiments, the relative abundance of both N-nitrosodialkylamine-acid complexes, written as [M + H]+, decreases from no irradiation, to 366-nm irradiation, and to 254nm radiation, where no acid complex is detected (see Table 1). Conversely, the relative abundance of the dissociative photolysis products, [R(R′HC)NH]+ and [R(R′H2C)NH2]+, increases from no irradiation, to 366-nm irradiation, and to 254-nm radiation. The results can be directly correlated to the molar absorptivity, am, for N-nitrosodialkylamines at low-wavelength irradiation (254 nm, am = 5500 for NDMA) compared to that at high-wavelength irradiation (366 nm, am = 110 for NDMA).20 That is, a larger molar absorptivity yields a greater relative abundance of dissociative photolysis products upon irradiation. Careful examination of the data also reveals an interesting detail with regard to the relative abundance ratios for the (18) Lau, M. P.; Cessna, A. J.; Yip, R. W.; Chow, Y. L. J. Am. Chem. Soc. 1971, 93, 3808. (19) Chow, Y. L.; Lau, M. P.; Perry, R. A.; Tam, J. N. S. Can. J. Chem. 1972, 50, 1044.

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alkylidenealkylamine (d) and the parent amine (f) dissociative products. For NDMA, the d/f ratio is 0.3 at 366-nm irradiation (n f π* excitation) and 0.1 at 254-nm irradiation (π f π* excitation). The same trend is observed for NMOR; that is, the d/f ratio is 1.0 and 0.3 for 366 and 254 nm, respectively. These ratios are consistent with previous findings which showed that photolytic dissociation by way of n f π* excitation primarily yields alkylidenealkylamine products18 and that dissociation by way of π f π* excitation generally gives parent amine products.19 Relying on the earlier criteria, where at least three structurally significant ions are needed for mass spectral confirmation, it follows that 366-nm irradiation, which yields the unreacted N-nitrosodialkylamine-acid complex and two dissociative photolysis products, is the wavelength of choice. The structures of these three ions are verified on the basis of comparisons of their tandem (MS/MS) mass spectra with those from model compounds. That is, the MS/MS spectra of nonirradiated and 366nm irradiated samples for the acid complex of NMOR are the same. Similarly, the MS/MS spectra of the NMOR photolysis products (protonated alkylidenealkylamine and the protonated parent amine) and those from protonated morpholine and its protonated alkylidenealkylamine analog are identical. Effect of Irradiation Residence Time. From the comparison of irradiation at different wavelengths (Table 1), it is clear that 366-nm excitation is the most suitable wavelength for obtaining molecular weight and structural class information. It is not obvious, however, if this irradiation time gives the optimum conditions for formation of the photolysis products. This parameter is also important because of the flow rate restrictions, owing to the chromatographic separation and because of the flow rate dependence on the ESI sensitivity. The flow rate through the on-line photolysis reactor is varied, effectively changing the irradiation residence time. The relative abundance of the acid complexes of NDMA and NMOR decreases exponentially with the residence time, while the dissociative photolysis products increase (see Figure 1). A log plot of the data is linear (NDMA, correlation coefficient, r2 ) 0.98; NMOR, r2 ) 0.99) and is consistent with previous reports,17 indicating that photolysis of N-nitrosodialkylamines follows first-order kinetics. The relative rate of the dissociative photolysis is compound specific (i.e., the relative rate is different for each N-nitrosodialkylamine). The relative rate for NDMA and NMOR is approximately 89 and 71 au, respectively, which can be directly attributed to their differences in molar absorptivity at 366 nm. That is, NDMA has a larger molar absorptivity at 366 nm than does NMOR, and hence, NDMA dissociates at a greater relative rate. These results demonstrate that optimum signals (i.e., greatest ESI sensitivity and at least 10% relative abundance for dissociative photolysis products) are obtained for the N-nitrosodialkylamineacid complexes and their photolysis products when their irradiation residence time in the photolysis reactor is 45-75 s. This residence time corresponds approximately to a 1 mL/min flow rate through a 15-m length knitted-open tubular reactor of 0.25mm id. A shorter reactor with a larger inner diameter may be used to achieve the same residence time, but this may increase band broadening in the LC-hν-MS experiment. Comparison of On-Line Photolysis and In-Source CID. The ability of in-source CID to obtain structurally informative ions (20) Sadtler Handbook of Ultraviolet Spectra; Simons, W. W., Ed.; Sadtler Research Laboratories: Philadelphia, 1979; p 300.

Table 2. Relative Abundance Comparison of NDMA and NMOR Dissociative Photolysis Products with 366-nm Irradiation at Different Formic Acid Concentrationsa pH N-nitrosodialkylamine ion

2.5

2.75

3.0

[(CH3)(CH2)NH]+ [(CH3)(CH3)NH2]+ [M + H]+ total abundance

NDMA 5.1 ( 0.1 9.6 ( 0.4 76.6 ( 0.1 4 626 190

3.5 ( 0.5 11.5 ( 1.0 76.9 ( 1.4 6 747 479

3.9 ( 0.3 21.7 ( 2.8 60.6 ( 3.8 4 156 316

[C4H7ONH]+ [C4H8ONH2]+ [M + H]+ total abundance

NMOR 11.6 ( 0.6 14.2 ( 0.6 53.8 ( 0.6 7 069 912

8.9 ( 0.6 22.6 ( 0.5 46.0 ( 1.1 10 000 000

10.4 ( 0.4 30.1 ( 1.2 29.0 ( 0.4 9 836 237

a

Figure 1. Kinetic plot of relative abundance of NDMA-acid complex, [M + H]+, and that of its dissociative photolysis products, [(CH3)2NH2]+ and [(CH3)(CH2)NH]+.

has been demonstrated for several types of compounds.21 Generally, in-source CID is produced in the electrospray transport region by a potential difference between the heated capillary and the first skimmer. As a result, internal energy can be added to the ions in the transport region to induce the bond cleavage necessary for structural confirmation. In these experiments, a comparison is done concerning the type and the amount of information obtained from dissociative photolysis processes to that from insource CID. Both techniques offer a means for producing structurally significant ions. For on-line photolysis, the dissociative photolysis process is class-specific; that is, the primary mode of dissociation for N-nitrosodialkylamine-acid complexes is formation of alkylidenealkylamine and parent amines. For in-source CID, the fragmentation patterns are more complicated, and they are not class-specific. For example, the CID spectra for protonated NDMA give three main peaks, whereas the CID spectra for protonated NMOR give four main peaks (see Table 2). These differences can be attributed to a greater number of fragmentation possibilities for NMOR compared to those for NDMA. Moreover, the structure of the fragment ions is not always clear. Upon collision, protonated NDMA dissociates to produce an ion at m/z 43, which can correspond to [CHNO]+ or [C2H5N]+. Likewise, protonated NMOR fragments to give an ion at m/z 57, which can be [CHN2O]+, [C2H3NO]+, or [C3H7N]+. There are also distinct differences between on-line photolysis and in-source CID with respect to the total abundance of signal. To produce CID spectra needed for structure confirmation, the capillary-skimmer potential difference is increased to amounts which cause a 20-60% loss in total ion signal for NDMA and a 10-70% loss in total ion signal for NMOR. These relatively high losses in total ion signal are probably a result of scattering, of which low-mass ions are more susceptible than high-mass ions. Of course, MS/MS-type experiments offer another means of (21) Voyksner, R. D.; Keever, J. In Analysis of Pesticides in Ground and Surface Water II, Vol. 12; Stan, H.-J., Ed.; Springer: Berlin, 1995; p 109.

Data averaged from three replicate injections.

improvement compared to in-source CID; that is, the signal-tonoise in the MS/MS experiment will improve upon reduction of the chemical noise in the low-mass region. For on-line photolysis, a 10-15% loss in total ion signal is observed for NDMA, whereas a 20-30% gain in total ion signal is seen with NMOR (see Table 1). The enhanced ESI sensitivity for irradiated NMOR is presumably related to the greater ability of its photolysis products to form ions in solution.22 Conclusively, on-line photolysis can be regarded as an alternative, and in some cases superior, means of producing structurally ions in the LCMS experiment. Effect of Solvent Composition and Solvent pH. N-Nitrosodialkylamines readily undergo photolysis in the presence of acid solutions. The type of photolysis and its kinetics, however, are largely dependent on the solvent. Photolysis in acidic methanol solutions principally yields additive products.19 Conversely, photolysis in acidic aqueous solutions predominantly gives dissociative products rather than associative products.19 Thus, to promote photolytic dissociation of N-nitrosodialkylamines, water is determined to be the primary solvent for these experiments. Efficient chromatographic separations, however, require the addition of an organic modifier to the mobile phase which may affect the kinetics of photolysis. To determine if the type of photolysis and its kinetics are influenced by the addition of an organic modifier, the solvent composition is investigated. For these experiments, acetonitrile is used as the organic modifier in the composition because of its better separation efficiencies for N-nitrosodialkylamines.23 Moreover, acetonitrile possesses a low surface tension over a wide solvent-composition range,24 which should, in turn, yield better ESI sensitivities.25 The relative abundance of the acid complexes of NDMA and NMOR decreases as the acetonitrile composition increases, while the relative abundance of the dissociative photolysis products increases (see Figure 2). That is, as the acetonitrile composition increases, the rate of photolysis increases. No associative products are detected in these experiments, although noncovalent (as determined by MS/MS experiments) solvent adducts (e.g., [M + CH3CN + H]+ ) are observed at high acetonitrile compositions. (22) Quirke, J. M. E.; Adams, C. L.; Van Berkel, G. J. Anal. Chem. 1994, 66, 1302. (23) Righezza, M.; Murello, M. H.; Siouffi, A. M. J. Chromatogr. 1987, 410, 145. (24) CRC Handbook of Chemistry and Physics, 65th ed.; CRC Press, Inc.: Boca Raton, FL, 1985; p F-31. (25) Kerbarle, P.; Tang, L. Anal. Chem. 1993, 65, 972A.

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Table 3. Partial CID Spectra of NDMA and NMOR as a Function of Capillary Voltagea m/z

0V

33 43 58 [M + H]+ total

nd nd nd 100 4 435 426

45 57 73 87 [M + H]+ total

nd nd nd nd 100 6 081 345

a

10 V

20 V

30 V

40 V

NDMA nd 4.3 ( 0.9 nd 4.2 ( 0.5 nd 7.1 ( 0.2 100 84.3 ( 0.6 9 051 495 7 476 223

48.8 ( 3.1 17.8 ( 1.1 6.9 ( 0.7 26.5 ( 0.2 3 433 955

70.4 ( 2.0 23.0 ( 1.4 nd 6.6 ( 0.8 1 044 410

NMOR nd 2.6 ( 0.1 nd 1.4 ( 0.1 nd 1.9 ( 0.1 0.2 ( 0.0 7.0 ( 0.5 99.7 ( 0.0 87.0 ( 0.4 18 000 000 16 000 000

28.9 ( 1.8 8.3 ( 0.4 6.8 ( 0.1 10.7 ( 0.5 45.3 ( 1.7 6 029 156

74.1 ( 1.5 9.8 ( 0.7 3.0 ( 0.3 3.5 ( 0.2 9.7 ( 0.6 1 862 612

Data averaged from three replicate injections. nd, not determined.

Figure 2. Plot of relative abundance of NMOR-acid complex, [M + H]+, and that of its dissociative photolysis products, [(C4H8O)NH2]+ and [(C4H7O)NH]+, as a function of acetonitrile composition in water.

It is reasonable to surmise that the increased rate of dissociative photolysis (as a function of solvent strength) is related to pKa of the solvent composition. It is well-known that the pKa increases (acid concentration decreases) in nonaqueous solvents.26 Further, in lower concentrations of acid (pH > 3), the presence of hydrogen bonding between the N-nitrosodialkylamine and the acid (see structure a of Scheme 1)27 promotes the photolysis of Nnitrosodialkylamines.27 Conversely, in higher concentrations of acid (pH < 3), the rate of photolysis for protonated N-nitrosodialkylamines markedly decreases.28 Thus, as the acetonitrile composition increases, the probability of forming an N-nitrosodialkylamine-acid complex rather than a protonated N-nitrosodialkylamine increases. To confirm that concentration of acid plays a role in the kinetics of photolysis, the reactions are investigated at different pH values in pure water. In consecutive experiments, the relative abundance of the N-nitrosodialkylamine-acid complexes decreases as the pH increases from 2.5 to 3.0 (see Table 3). Conversely, the relative abundance of the dissociation products increases as a function of pH. Similar trends are observed when comparing the same concentrations of acetic acid (pKa ) 4.75), formic acid (pKa ) 3.75), and trifluoroacetic acid (pKa ) 0.2) in pure water. That is, the rate of photolysis decreases as the strength of the acid increases. The results demonstrate that the type of photolysis is independent of the acetonitrile-water solvent composition (i.e., only two dissociative photolysis products are observed in varying compositions of acetonitrile and water). The kinetics of the photolysis, however, is dependent on the solvent composition and the solvent pH. This, in turn, may have an effect on the detection and confirmation limits of the N-nitrosodialkylamines that elute under high acetonitrile compositions in the LC-hν-MS experiment. (26) For examples, see: Dean, J. A. Lange’s Handbook of Chemistry, 13th ed.; McGraw-Hill Book Co.: New York 1985; pp 5-69. (27) For discussion of pH dependence and modes of N-nitrosodialkylamineacid interactions, see: Layne, W. S.; Jaffe, H. H.; Zimmer, H. J. Am. Chem. Soc. 1963, 85, 1815. (28) Lau, M. P. Ph.D. Dissertation, Simon Fraser University, Burnaby, BC, Canada, 1970.

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Figure 3. Extracted ion current profile obtained, in the full-scan mode, from a mixture of six N-nitrosodialkylamines at a concentration of ∼110 ng for each compound. See Experimental Section for LChν-MS conditions.

Linearity, Limits of Detection and Confirmation, and Reproducibility of LC-hν-MS. To evaluate the LC-hν-MS method, the linearity, limits of detection and confirmation, and reproducibility are measured for NDMA, N-nitrosodiethylamine (NDEA), N-nitrosoethylbutylamine (NEBA), NMOR, N-nitrosopiperdine (NPIP), and N-nitrosopyrrolidine (NPYR). External standard calibration curves are determined from the individual peak areas of the N-nitrosodialkylamine-acid complex generated from the LC-hν-MS ion trace (see Figure 3 for a typical LC-hν-MS ion trace). The calibration curves for the six N-nitrosodialkylamines are linear from 6 to 800 ng (see Table 4). The full scan limits of detection (LODs) of the LC-hν-MS technique, as estimated by a signal-to-noise ratio of 3:1 from the low end of the calibration curve, are 2-6 ng for the six N-nitrosodialkylamine-acid complexes (see Table 4). The LODs

Table 4. Linear Regression Analysis with Full-Scan Limits of Detectiona and Confirmationa for Six N-Nitrosodialkylaminesb N-nitrosodialkylamine slope/106 y-intercept/105 NDMA NMOR NPYR NDEA NPIP NEBA

6.47 0.97 8.26 7.1 17.99 3.13

0.29 0.33 0.71 0.25 0.86 0.1

r2 0.999 0.999 0.999 0.997 0.998 0.997

LOD (ng) LOC (ng) 2 2 4 6 4 6

5 4 7 24 11 30

a Estimated by signal-to-noise ratio of 3:1 from the low end of the calibration curve. Data from three or four replicate injections of at least five different concentrations. b Listed in order of increasing retention times.

for the acid complexes are slightly higher for the early-eluting N-nitrosodialkylamines, and they are probably related to the higher concentration of acid at a lower acetonitrile mobile-phase composition than that for the late eluting compounds (see earlier discussion of solvent composition). Because of the lower abundance of the photolysis products relative to the N-nitrosodialkylamine-acid complex, the limits of confirmation (LOCs), as determined by a signal-to-noise ratio of at least 3:1 for photolysis products and the N-nitrosodialkylamine-acid complex, increase by nearly an order of magnitude (see Table 4). The LOCs also vary slightly for each N-nitrosodialkylamine and can be related to the acid concentration of the mobile phase and to their differing molar absorptivities (see earlier discussion of different irradiation wavelength). Under the current conditions of the LC-hν-MS experiment, the LOD can be lowered to 440 pg for NDMA with selected-ion monitoring (SIM), where only the NDMA-acid complex is detected. The LOCs for the two primary photolysis products and NDMA-acid complex is 4 ng with SIM (see Figure 4). The LODs and LOCs are subject to change, however, depending on the mobile-phase composition for a given experiment. For example, at high acetonitrile compositions (i.e., low concentration of acid), the abundance of the NDMA-acid complex decreases, and, in turn, the LOC decreases relative to the LOD. In a regulatory environment, the ability of the LC-hν-MS experiment to provide stable and reproducible ion currents over an extended period of time is of great importance. Hence, the reproducibility is investigated by observing, under identical conditions, the short-term and long-term standard deviations of the abundance of the photolysis products relative to that of the NDMA-acid complex. Although the replicate injections (see Tables 1-3) usually give relative standard deviations of less than 10%, the intra- and interday experiments often result in deviations exceeding 20%. The large deviations for the intra- and interday experiments may be attributed to variances in back pressure from LC pumps to the knitted-coil reactor. The back pressure deviates over extended periods of time and subsequently changes the flowrate (i.e., irradiation time).29 Utilization of pulse-free LC pumping system should overcome these problems.30 Formation of NO2- and NO3- from N-Nitrosodialkylamines. The capacity to form positive or negative ions makes ESI well suited for a wide range of compounds. Subsequently, ESI is a versatile technique for investigation of reactions that simultaneously form positively and negatively charged products. Such is the case for the photolysis of N-nitrosodialkylamines.

Figure 4. Extracted ion current profile, in the SIM mode, of NDMAacid complex (m/z 75) and that of its dissociative photolysis products, [(CH3)2NH2]+ (m/z 46) and [(CH3)(CH2)NH]+ (m/z 44). See Experimental Section for LC-hν-MS conditions.

Upon excitation at ∼360 nm, formation of nitric oxide occurs for all N-nitrosodialkylamines (see Scheme 1), but not for C-nitrosodialkylamines.31 The nitric oxide radical is rapidly oxidized to nitrogen dioxide by oxygen, followed by combination with another nitric oxide or nitric dioxide to give dinitrogen trioxide or dinitrogen tetraoxide, respectively (see Scheme 3, eqs 1-3).32 Hydrolysis of either of these intermediates yields the nitrite ion (see Scheme 3, eq 4).33 Formation of the nitrate ion, however, has also been observed in reactions of dialkylamines with dinitrogen tetraoxide (see Scheme 3, eq 5).34 Scheme 3. Mechanism for Formation of Nitrite Ion and Nitrate Ion from Irradiated N-Nitrosodialkylamines NO• + 1/2O2 f NO2•

(1)

NO2• + NO• f N2O3

(2)

NO2• + NO2• f N2O4

(3)

hydrolysis

N2O3 or N2O4 98 NO2-

(4)

N2O4 + 2R(R′CH2C)NH f [R(R′CHC)NH2]+NO3- + R(R′CH2C)N-NO (5) The identical photolytic formation of two product ions, nitrite and nitrate, from each N-nitrosodialkylamine allows for use of SIM as a negative-ion detection method in the LC-hν-MS experiment. In the negative ion mode, the nitrate ion (m/z 62) is more abundant than the nitrite (m/z 46) for each compound (see Figure 5). These results are reflective of thermodynamic measurements, which indicate that the nitrate ion is more stable in the aqueous solution phase (∆Hf,aq(NO3-) ) -207.5 kJ/mol; ∆Hf,aq(NO2-) ) Analytical Chemistry, Vol. 68, No. 3, February 1, 1996

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Figure 5. Extracted ion current profile, in the SIM mode, of nitrite ion (m/z 46) and of nitrate ion (m/z 62) obtained from a mixture of six N-nitrosodialkylamines at a concentration of ∼130 ng of each compound. See Experimental Section for LC-hν-MS conditions.

-104.6 kJ/mol)35 and in the gas phase (∆Hf,g(NO3-) ) -307 kJ/ mol; ∆Hf,g(NO2-) ) -189 kJ/mol).36 Although formation of the nitrate or nitrite ion does not fit the earlier criteria for detection and confirmation of N-nitrosodialkylamines, it demonstrates the versatility of the LC-hν-MS technique as a multiresidue method that gives an identical response for a specific class of compounds. Under present conditions (low pH), however, the negative ESI response for the LC-hν-MS method does not give adequate limits of detection. Current efforts are underway to investigate the aforementioned parameters and their effect on formation of nitrate and nitrite.37 NDMA in Beer. In conjunction with the LC-hν-MS method, the SIM approach is chosen as an illustrative application for the analysis of NDMA in beer. Because there have been numerous investigations of NDMA in beer, this application can be used as a direct comparison with other methods. In the late 1970s, the mean NDMA values in beer were reported to be approximately 2.7 ppb.38 The NDMA-acid complex from a spiked (2 ppb) beer sample is easily detected at a retention time similar to that for the standard (29) Lurie, I. S.; Cooper, D. A.; Krull, I. S. J. Chromatogr. 1993, 629, 143. (30) Reference 11, Chapter 1. (31) Sander, J. Z. Physiol. Chem. 1967, 348, 852. (32) Challis, B. C.; Kyrtopoulos, S. A. J. Chem. Soc., Perkin Trans. 1979, 1, 299. (33) Shuker, D. E. G.; Tannebaum, S. R. Anal. Chem. 1983, 55, 2152. (34) Axenrod, T.; Milne, G. W. A. Tetrahedron 1968, 24, 5777. (35) Reference 22; p D-77. (36) Lias, S. G.; Liebman, J. F.; Levin, R. D. J. Phys. Chem. Ref. Data 1984, 13, 695. (37) Increased sensitivities for formation of nitrate and nitrite from N-nitrosodimethylamine are known to occur in the pH range of 5-8; for discussion, see: Fan, T.-Y.; Tannenbaum, S. R. J. Agric. Food Chem. 1971, 19, 1267. (38) Spielgelhalder, B.; Eisenbrand, G.; Preussmann, R. Food Cosmet. Toxicol. 1979, 17, 29.

552 Analytical Chemistry, Vol. 68, No. 3, February 1, 1996

Figure 6. Extracted ion current profile, in the SIM mode, for NDMAacid complex (m/z 75) and that of its dissociative photolysis products, [(CH3)2NH2]+ (m/z 46) and [(CH3)(CH2)NH]+ (m/z 44), obtained from a 2 ppb (∼25 ng detected) spiked beer sample. See Experimental Section for LC-hν-MS conditions.

(see Figure 6). Alas, there are several matrix interferences in the low-mass region that prevent unambiguous confirmation of NDMA in beer. These problems, however, can be directly related to the sample extraction/preconcentration procedure. Analysis of an unspiked sample gives similar interferences (figure not shown), whereas that of a control sample (i.e., solvent blank) shows no such interferences (figure not shown). Better procedures for extraction/preconcentration will significantly decrease the interferences, thereby increasing the sensitivity in the lowmass region. The LC-hν-MS method should also be adequate for detection and confirmation of other N-nitrosodialkylamines, especially the nonvolatiles, in varying types of food or nonfood substrates, provided more rigorous extraction/preconcentration procedures than the one reported here are utilized. This will be the focus of future research. ACKNOWLEDGMENT D.L.V. and D.A.V. are supported by an appointment to the ORAU research program at the NCTR, which is administered by the Oak Ridge Associated Universities through an interagency agreement between the U.S. Department of Energy and the U.S. Food and Drug Administration.

Received for review August 22, 1995. Accepted October 25, 1995.X AC9508614 X

Abstract published in Advance ACS Abstracts, December 15, 1995.