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Anal. Chem. 1983, 55,2152-2155
Determination of Nonvolatile A/-Nitroso Compounds in Biological Fluids by Liquid Chromatography with Postcolumn Photohydrolysis Detection David E. G. Shuker’ and Steven R. Tannenbaum*
Department of Nutrition and Food Science, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139
A modified photohydrolysls detector (PHD) has been developed for use with reversed-phase HPLC. Wlth the HPLCPHD system nonvolatile N-nitroso compounds (particularly N nltrosamides) were selectlvely determined followlng chromatographlc separatlon. Llght from a hlgh-lntenslty dlscharge lamp photolyzes N-nltroso compounds in aqueous solutlon to glve nitrite ion, whlch was determined colorimetrlcally wlth Grless reagent in a postcoiumn reactor. Several N-nitroso compounds gave a posltive response (detectlon Ilmlt, ngllnJectlon): N-methyl-N’-nltro-N-nltrosoguanldlne (e), N methyl-N-nkrosourea (20), N-methyl-N-nltrosoacetamlde(a), N-nltrosotaurochollc acld ( IOO), N-nitrosoglycocholic acld (60), N-nitrosocimetldine (e), N-nitroso-L-prollne (20). Methods were developed for the analysis of N-nltrosoglycocholic acid In gastrlc julce and N-nltrosoclmetldinein gastric juice and urine. The applicatlon of thls system to the analysis of the products from nltrlte-treated fava beans Is briefly discussed.
Many N-nitroso compounds are carcinogenic in laboratory animals giving rise to concern that they may possess similar activity in man ( I ) . Nitrogenous precursors, e.g., alkylamines, alkylureas, alkylguanidines, and alkylamides, occur widely in nature and potential nitrosating agents, e.g., nitrite (NO2-) and NO, (the gaseous oxides of nitrogen), are similarly widespread. Much interest is therefore directed toward quantifying the various N-nitroso compounds that occur in the environment. The volatile N-nitrosamines, e.g., nitrosodimethylamine and nitrospyrrolidine can be determined at very low levels by gas chromatography in conjunction with a thermal energy analyzer (GC/TEA) (2). There are, however, a large number of compounds, e.g., N-nitrosamides, for which the GC/ or HPLC/ TEA procedure cannot be satisfactorily used because they require pyrolytic generation of nitric oxide from the N-nitroso compound (3). Typically, N-nitrosamides undergo a thermal rearrangement upon heating with the production of molecular nitrogen (4). As part of our work in the possible role of N-nitroso bile acid conjugates in the induction of gastrointestinal cancer, we required an analytical method capable of selectively detecting N-nitrosamides following chromatographic separation by HPLC. Based on work by Daiber and Preussmann ( 5 ) ,Fan and Tannenbaum (6) developed a system for detecting N-nitrosamines that utilized photohydrolysis as the key step. N Nitrosamines upon irradiation in aqueous solution in a quartz capillary tube with UV light generated nitrite, which could be measured colorimetrically by a Griess-type reagent. This was subsequently modified and could be used as an HPLC Current address: MRC Toxicology Unit, Medical Research Laboratories, Woodmansterne Road, Carshalton, Surrey SM5 4EF, England.
detector (7). There were some disadvantage to this system as it utilized low-energy fluorescent tubes as a long-wavelength UV light source and required fragile and expensive capillary glass or quartz tubing. In addition relatively long residence times (ca. 30 min) were needed and it was not particularly useful for N-nitrosamides, which have a characteristic absorbance (380-430 nm) higher than that of N-nitrosamines (300-380 nm) . In this paper we describe a photohydrolysis system that is a significant advance over previous ones and is suitable as a detector for high-resolution chromatography. EXPERIMENTAL SECTION Standard Compounds. CAUTION Many N-nitroso compounds are carcinogenic and should be handled with extreme care. N-Nitrosoglycocholic acid (NOGC) (8),N-nitrosotaurocholic acid (NOTC) (8),N-nitrosocimetidine (NC) (9),and N-nitrosoL-proline (NP)(IO)were prepared according to literature methods. N-Methyl-N-nitrosourea (MNU) and N-methyl-N’-nitro-Nnitrosoguanidine (MNNG) were commercially avaiable compounds and used as supplied. N-Methyl-N-nitrosoacetamide(MNA) was kindly supplied by Linda K. Shuker (New England Institute for Life Sciences, Waltham, MA). Chromatography. Chromatographic separations were carried out on a Radial Pak C-18 cartridge (5 mm i.d.), fitted into a Radial Compression Module (RCM-100, Waters Associates), using a mobile phase 30 mM ammonium phosphate at pH 6 and acetonitrile pumped isocratically (proportions determined by the compounds studied) at 0.5 mL/min. Photohydrolysis Detector. The photohydrolysis detector is shown in Figure 1. The outlet (1)of the chromatographic column was connected to in. 0.d. microbore (0.01in. id.) Teflon tubing (3) via a standard Swagelok fitting. .The tubing (50 ft) was wound around a Pyrex water jacket (4) cooled with tap water ( 5 ) designed to prevent the Teflon tube from overheating and to aid in cooling the high intensity discharge (HID) lamp (2). The HID lamp (GTE Sylvania 400-W metal-halide, type MP 400 T16/BD powered by a standard M59 ballasted circuit) was sited concentrically within the water jacket and was cooled by air drawn over it by a fan (6, IMC Magnetics Corp., Rochester, NH, Model WS2107FL). The lamp-water jacket photolysis coil was housed in a box (7), the interior of which was covered with aluminum foil to permit maximum reflectance of emitted light. (CAUTION: The lamp emits very intense light and precautions were taken to keep the box lighttight.) A t a flow rate of 0.5 mL/min the residence time of the column eluant in the coil was approximately 2 min, which was sufficient for complete photolysis of N-nitrosamides. The effluent from the photolysis coil was mixed with Griess reagent (0.170 N-lnaphthylethylenediamine dihydrochloride in waterll70 sulphanilamide in 5% H3P04[1:1, v/v], 9) at 0.5 mL/min (pumped by either a Milton Roy Minipump or a Harvard Instruments high pressure syringe pump) in a low dead volume mixing tee (8 Rainin Instrument Co., Woburn, MA, No. 01-0165). The resulting solution was pumped first into a coil of stainless steel microbore in. 0.d. X 0.01 in. i.d.) immersed in a water tubing (10, 3 f t X bath (65 OC) and then through a coil of similar tubing (11,18 in.) immersed in ice/water and finally into the flow cell of a spectrophotometric detector (12, Schoeffel Instruments Model 770) where the absorbance at 541 nm was recorded. Failure to include this cooling coil resulted in a very unstable spectrophotometric
0003-2700/83/0355-2152$01.50/00 1983 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 55, NO. 13, NOVEMBER 1983
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1
Figure 1, Diagrarnmatic representation of the photohydrolysis detector (PHD). For an explanation of the key numbers see the Experimental Section.
output; presumably the capacity of the heat exchanger in the Schoeffel detec1,or was being exceeded. N-Nitroso compounds were observed ELS peaks on a chart recorder and are quantified by comparison with known concentrations of authentic compound. N-Nitrosoglycocholiic Acid in Gastric Juice. Human gastric juice (2.5 mL) was centriifuged to remove suspended matter and the supernatant adjusted to pH 3. The clear supernatant was eluted through a C-18 Sep-PAK (Waters Associates), which had been wetted with acetonitrile (3 mL) followed by water (5 mL). The eluate was discarded and the Sep-PAK washed with water (2 mL) after which it was dried by forcing a stream of nitrogen (at 10 psi) through it for 10 min. If N-nitrosoglycocholicacid had been prebent in the gastric juice, it was eliuted with acetonitrile (2 mL). The eluant was evaporated to dryness in a stream of nitrogen at room temperature and the residue was dissolved in HPLC mobile p'hase (100 pL). The resulting solution was filtered by use of a centrifugal microfilter (Bioanalytical Services, Inc., West Lafayette, IN) and aliquots (10-25 pL) were analyzed on the HPLC/PHD system. N-Nitrosocimetidine, (a) Gastric Juice. These analyses were carried aut in the same way as that described for N-nitrosoglycocholic acid. ( b ) Urine. Urine (25 imL) was slowly eluted through a wetted C-18 Sep.PAK followed by water (5 mL). The Sep-PAK was dried in a stream of nitrogen as described above. N-Nitrosocimetidine was eluted with acetonitrile (2 mL) which was evaporated to dryness, redissolved in HPLC buffer (250 pL), and filtered with a centrifugal microfilter. Aliquots (10-25 pL) were analyzed on the HPLC/PHD system. Nitrosated Fava Beans. The details of this procedure have been described elsewhere (11).
RIESULTS AND DISCUSSION The principle upon which the photohydrolysis detector works is that irradiation of aqueous solutions of M-nitroso compounds (e.g., N-nitrosamines, N-riitrosoguanidines, N nitrosamides, etc.) with long wavelength light leads to cleavage of the N-NO lbond to give nitric oxide (NO.) (12). NO. is rapidly oxidized to nitrogen dioxide (NOz.) by oxygen (13). NOz., after combination with another mlolecule of NOz*to isive Nz04,or with NO- to give N203,is hydrolyzed to nitrite ion (NO2-). NO2- is then determined via its acid-catalyzed diazotization of sulphanilamide, followed by coupling with N1-naphthylethylenedianninedihydrochloride to give an azo dye, which has its maximum absorbance at 541 nm (e = 5.4 X lo4). I n deaigning the photochemical detector for HPLC, we began by choosing an appropriate light source. Since the compounds of prime interest (N-nitrosamides) have absorbance bands between 380 and 430 nm, a commercially available high- intensity discharge metal halide lamp was chosen which lhas its principle emissioln in the 380-420 nm region of the cipectrum. In addition, the utilization of long wavelength light allowed the use of microbore Teflon tubing (transparent in this region) as the photolysis coil, thus overcoming the problems of fragility which had plagued our earlier efforts.
If -
I d 0 15 min. 0
15
Postcolumn photolysis of N-nitrosobile acid conjugates. Trace A shows a mixture of NOTC and NOGC detected at 245 nm following chromatographic separation and passage through the photolysis coil. Trace B shows the same mixture but with the lamp turned on. Mobile phase NH,HpPO, (30 mM, adjusted to pH 6):CH3CN, 2:l (v/v) at 0.5 mL/min. Flgure 2.
Using the system shown in Figure 1 and described in the Experimental Section, we found that the N-nitrosamides studied were completely destroyed during the residence time (ca. 2 min at 0.5 mL/min flow rate) of the HPLC column effluent in the photolysis coil. This was demonstrated by monitoring the effluent from the photolysis coil at 245 nm with the lamp on or off. Peaks due to N-nitrosamides were observed with the lamp switched off and disappeared when it was turned on (Figure 2). Conversely, when the entire photohydrolysis system was assembled, peaks could only be seen when the lamp was turned on (Figure 3). These observations clearly demonstrate that the peaks which were observed arose from the photohydrolytic generation of nitrite from the N-nitrosamide compounds studied. Despite the use of microbore tubing throughout the postcolumn photolysis coil and mixing coils, we found that a noticeable loss of chromatographic resolution occurred when normal spectrophotometric detection was replaced by photohydrolysis (Figure 4). The maximum sensitivity of the detector for N-nitrosamides was similar to that when using spectrophotometric detection despite the fact that the absorbance of the azo dye (Arna 541, e = 5.4 X lo4) is 5-10 times greater than the corresponding N-nitrosamides,,X,( 240-250 nm, t = (5-10) X lo3). It is apparent therefore that stoichiometric conversion of the N-nitrosamides to nitrite ion and thence to the azo dye is not being achieved. Our attempts to increase this response have, so far, met with failure. However, the fact that the PHD only responds to photolabile NO compounds confers a significant advantage on this system over the less selective spectrophotometric detection.
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ANALYTICAL CHEMISTRY, VOL. 55, NO. 13,NOVEMBER 1983
-
0
15
rnin
0
15
Figure 3. Postcolumn photohydrolytic determinations of MNNG: (Trace
A) the chromatogram obtained with MNNG using the fully assembled photohydrolytic detector with the lamp on; (Trace B) the results of turning the lamp off. Note that a small peak is observed presumably due to some acid catalyzed nitrite generation in the reagent. Mobile phase NH,H,PO, (30 mM, pH 6):CH,CN, 2:0.75 (v/v) at 1 mL/min; colorimetrlc reagent introduced at 0.5 mL/min.
Table I. N-Nitroso Compounds That Give a Positive Response on the Postcolumn Photohydrolysis Detector
N-nitroso compound N-methyl-" -nitro-Nnitrosoguanidine (MNNG) N-methyl-N-nitrosourea(MNU) N-methyl-N-nitrosoacetamide(MNA) N-nitrosotaurocholic acid (NOTC) N-nitrosoglycocholic acid (NOGC) N-nitrosocimetidine (NC) N-nitroso-L-proline (NP)
approx detection unit nmol ng 8
20 8 100 60 6 20
0.05 0.2 0.8
0.18 0.12 0.02
0.14
A number of N-nitroso compounds were examined in the RP-HPLC/PHD system and all were found to give a response (Table I). The detection limit was defined as the minimum amount required to give a peak height equivalent to three times the noise level. The variation in the detection limit was mainly due to chromatographic differences (e.g., peak shape and retention time) between various compounds. Since being constructed the HPLC/PHD system has found a number of application in our laboratory in which the specific detection of N-nitroso compounds was required. N-Nitrosoglycocholic Acid. Bile acid conjugates (e.g., glycocholic acid) are naturally occurring amides normally present in the upper intestine as part of the body's mechanisms for the absorption of dietary fats. In certain cases bile reflux occurs and the bile acid conjugates are to be found in the stomach where they can be exposed to nitrosating agents generated from nitrite ion under acidic conditions. We have reasoned (8)that the formation of N-nitroso bile acid conjugates, such as N-nitrosoglycocholic acid (NOGC) is possible under such conditions. Added N-nitrosoglycocholic acid can be extracted from human gastric juice with excellent recoveries (>go%) by using a C-18 Sep-PAK. Normal human gastric juice shows no interfering peaks on the HPLC/PHD. N-Nitrosoglycocholic acid (2 pM, 0.1% yield) was found in human gastric juice (at pH 1.26), which had been treated with glycocholic acid (2 mM) and nitrite (2 mM) for 3 h at 37 "C. The addition of glycocholic acid or nitrite alone under the same conditions did not give a detectable peak for N-nitrosoglycocholic acid. N-Nitrosocimetidine (NC). N-Nitrosocimetidine (NC) is the product of nitrosation of the widely used H2-antagonist
OT c
A
B
I
L1 5--I
1
0
I
1
15
0
15
0
15 rnin.
Figure 4. Effect of detector volume on chromatographic resolution:
(Trace A) a mixture of N-nitrosobile acid conjugates detected in the usual manner at 245 nm; (Trace B) the effect of introducing the photolysis coil between the column and the detector; (Trace C) the effect of adding the photolysis coll and the mixing tee and reaction coils. Conditions: Mobile phase NH4H,P0, (30mM, pH 6):CH,CN, 2:l (v/v) at 0.5 mL/min; water (in place of the colorimetric reagent) at 0.5 mL/min. Peak variance data ( 1 4 ) for the various configurations above, with respect to the NOTC peak, are as follows: (Trace A) u2 = 22 500 pL2; (Trace B) n* = 47 400 pL2; (Trace C) u2 = 140 500 pL2. For the entire photohydrolysis detector A(r2 = 118 000 pL2.
cimetidine (Tagamet). It is mutagenic (15) and has been found to be formed under simulated gastric conditions from nitrite and cimetidine (16). As yet, there is no published method for the selective analysis of N-nitrosocimetidine in biological fluids. We have found the N-nitrosocimetidine can be extracted from aqueous buffers, gastric juice, and urine by use of a C-18 Sep-PAK and subsequently analyzed by HPLC/PHD. Recoveries of standards from aqueous buffers and gastric juice were >75% and no significant interfering peaks were observed (Figure 5 ) . Urinary analysis proved to be more difficult due to interfering peaks but recoveries were still good ( > 7 5 % ) . Clearly this method has great potential for use in the determination of N-nitrosocimetidine in biological fluids.
A Nonvolatile, Mutagenic N-Nitroso Compound from Nitrite-Treated Fava Beans (Vicia faba). Recently we have produced evidence (11)that the mutagenic principle in an extract of nitrite-treated fava beans (Vicia f a b a ) is an activated N-nitroso compound. After a number of cleanup steps the mutagenic activity occurs in a fraction which gives a single peak on the HPLC/PHD system. Further fractionation shows that the mutagenic activity cochromatographs with the peak. This observation had not been possible with existing available methods for detecting N-nitroso compounds following HPLC, namely, HPLC/UV and HPLC/TEA. Further work is currently under way to examine the occurrence of nonvolatile N-nitroso compounds in physiological
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Anal. Chem. 1983, 55, 2155-2159
4
1
I II 15 min. 0 15
0
Figure 5. Determinatlon of N-nitrosocimetidine (NC) in gastric juice: (Trace A) normal human gastric juice; (Trace El) the same gastric Juice spiked with authentic N-nitrosocimetidine. Conditions: mablie phase, NH,H,PO, (30 mM, pH 6):CH,CN, 2O:O.g (vlv) at 0.5 mL/min, colarimetric reagent at 0.5 mL/imin, detector at 541 nm.
fluids, particularly gastric juice, using the procedure described in this paper.
ACKNOWLEDGMENT We gratefully acknowledge the advice and material help provided by P a d Ulcickas of GTE Sylvania,Manchester, NH, in connection with the high intensity discharge lamp. We thank a reviewer for helpful suggesti,ons concerning the evaluation of chromatographic resolution. This investiglition was supported by the National Institute of Environmental Health Science Grant No. 2-Pol-ES00597-13 and by PHS Grant No. 1-Pol-CA26733.-04, awarded by the National Cancer Institute, DHHS. A preliminary account of
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some of this work was given at the 7th International Meeting on the Analysis and Formation of N-Nitroso Compounds, Tokyo, Japan, Sept 18-0ct 1, 1981. Registry No. MNNG, 70-25-7; MNU, 684-93-5; MNA, 7417-67-6;NOTC, 82660-96-6;NOGC, 76757-85-2;NC, 73785-40-7; NP, 86941-92-6.
LITERATURE CITED (1) Magee, P. N.; Montesano, R.; Preussmann, R . "Chemical Carcinogens"; Searle, C. W., Ed.; American Chemical Society: Washington, DC, 1976; American Chemical Society Monograph No. 173; Chapter 11. (2) Fine, D. H.; Roundbehler, D. P. J . Chromatogr. 1975, 109, 271. (3) Hansen, T. J.; Archer, M. C.; Tannenbaum, S.R. Anal. Chem. 1979, 51, 1526. (4) White, E. H.; Woodcock, D. J. "Chemistry of the Amino Group"; Patai, S., Ed.; Wiley: New York, 1966; Chapter 6. (5) Daiber, D.; Preussmann, R. 2.Anal. Chem. 1984, 206, 344. (6) Fan, T.-Y.; Tannenbaum, S. R. J . Agrlc. FoodChem. 1971, 19, 1267. (7) Iwaoka, W.; Tannenbaum, S. R. "Environmental N-Nitroso Compounds. Analysis and Formation"; Walker, E. A., Bogovski, P., GriciUte, L., Eds.; International Agency for Research on Cancer; Lyon, 1976 Sci. Publ. No. 14, p 51. (8) Shuker, D. E. G.; Tannenbaum, S.R.; Wishnok, J. S.J . Org. Chem. 1981, 4 6 , 2092. (9) Foster, A. 8.; Jarman, M.; Manson, D. Cancer Lett. 1980, 9 ,47. ( I O ) Hansen, T. J.; Iwaoka, W. T.; Archer, M. C. J . LabelledCompd. 1974, IO. 669. (11) Piacek-Lianes, B. G.; Tannenbaum, S. R. Carcinogenesis 1982, 3 . 1379. (12) Chow, Y. L. "N-Nitrosamines"; Anseime, J. P., Ed.; American Chumical Society: Washington, DC, 1979; ACS Symposium Series No. I O I , p 13. (13) Challis, B. C.; Kyrtopoulos, S. A. J . Chem. Soc., Perkin Trans. 1 1979, 299. (14) Snyder, L. R.; Kirkland, J. J. "Introduction to Modern Liquid Chromatography", 2nd ed.; Wiiey: New York, 1979, pp 31-33. (15) Ichinotsubo, D.; McKlnnon, E. A,; Liu, C.; Rice, S.;Mower, H. F. Carcinogenesis 1981, 2, 261. (,6) DeFiora, s,; picclotto, A, Carcinogenesis Isso, , 925,
,
RECEIVED for review May 25,1983. Accepted August 12,1983.
Identification of Moriomethylated Polycyclic Aromatic Hydrocarbons in Crude Oils by Liquid Chromatography and High-Resolution Shpol'skii Effect Fluorescence Spectrometry Philippe Garrigues* and Marc Ewatld Groupe d'oce'anographie Physico-Chimique d u LA 348 (CNRS), Laboratoire de Chimie Physique A, UniversitB de Bordeaux I , 33405 Taleiltce Cedex, France High-resolution spectrometry (HRS) of polycyclic aromatic compounds (PAC) In n-alkanes frozen at 15 K (Shpol'skli effect) is applied to the Identification of monomethyiated Isomers In pyrene, phenanthrene, and chrysene. Best results for identification and estimation of the relatlve dlstrikution of Isomers are obtained after fractlonatlon of the crude oil by a now classlcal way, the high-performance llquid chromatography (HPLC) procedure which includes two steps: first, to Isolate compounds according to the degree of aromatlclty, and second, to separate parent compounds accordlng to the degree of alkylation. The chromatographic fractions suspected of contalnlng the studled PAH are then analyzed by HRS at dHferenX levels of fractlonatlon. The results presented here illustrate the capability of this technique for the complete ldentlflcation off methylated Isomers In natural extracts, ailowlng further quantification.
An increasing number of studies on polycyclic aromatic
compounds (PAC) by high-resolution spectrometry (HRS) in frozen n-alkane matrices (Shpol'skii effect) have been reported during the last years (1-3). The high sensitivity and selectivity of such a technique offers an alternative approach to other analytical methodologies (i.e., gas capillary chromatography coupled or not with mass spectrometry (GC/MS) or highperformance liquid chromatography (HPLC)). However, preliminary chromatographic separations of natural samples are often required for specific molecular identification of PAC by low-temperature spectrometry (2, 4). Recent developments in HPLC (normal and reverse phase) allow rapid and powerful separations of the studied series of PAC (5, 6) even if the raw material is very complex, as for crude oils. Complete identification and relative quantification of each isomer are important, because the toxic activity of PAC is often related, for example, to a specific position of the methyl group on the parent molecule. For instance, 5-methylchrysene is one of the stxongest carcinogenic products while the other isomers are only moderate carcinogens (7). In the same way,
0003-2700/63/0355-2155$01.50/00 1963 American Chemical Society
