ISSN 10619348, Journal of Analytical Chemistry, 2015, Vol. 70, No. 9, pp. 1153–1157. © Pleiades Publishing, Ltd., 2015.
ARTICLES
Modified UFLCPDA Method for Determination of Nitrosamines1 Sugandha Sharmaa, Rajesh K. Joshib, *, and Sandeep R. Paib a b
Department of Oral Medicine and Radiology, KLE VK Institute of Dental Sciences Belgaum, Karnataka590010, India Department of Phytochemistry, Regional Medical Research Centre (RMRC), Indian Council of Medical Research (ICMR) Belgaum, Karnataka590010, India *email:
[email protected] Received February 8, 2014; in final form, February 4, 2015
Abstract—Tobaccospecific nitrosamines, viz. N'nitrosonornicotine (NNN) and 4methylnitrosamino1 3pyridyl1butanone (NNK) were determined by using a modified ultra flow liquid chromatographyphoto diode array (UFLCPDA) technique using C18 100A Phenomenex column (Luna, 5 µm, 4.6 × 150 mm) with 10% acetonitrile (ACN) in 1 mM ammonium acetate buffer pH 8 by ammonium hydroxide; ACN and water with 0.75% glacial acetic acid (pH 2.82) solvent system. It was feasible to compute accurate calibration curve for both compounds using the solvent system by determining the peak area as a function of the concentration. Limits of detection of 0.12 µg/mL for NNN and 0.02 µg/mL for NNK were found. This technique allows a reasonably accurate detection of NNN and NNK with the solvent system developed. The study finds an op timum mobile phase for detecting NNN and NNK with effective resolution. Keywords: N'nitrosonornicotine (NNN), 4methylnitrosamino13pyridyl1butanone (NNK), ultra flow liquid chromatographyphoto diode array (UFLCPDA) technique, method development DOI: 10.1134/S1061934815090142 1
Tobacco and its smoke contain more than 2500 and 3800 compounds, respectively [1], which include tu mor initiators such as the polynuclear aromatic hydro carbons [2], tumor promoters, cocarcinogens, and organspecific carcinogens [2, 3]. The large number of tobacco and its smoke make it unlikely that the total carcinogenic activities of tobacco products can be ex plained by individual compound or group of com pounds. Thus, research has focused on major groups of tumorigenic agents, especially those carcinogens that are unique for tobacco and its smoke. Tobacco specific nitrosamines (TSNA) are a group of carcino gens found only in tobacco products. They are formed from nicotine and related alkaloids during the produc tion and processing of tobacco [4]. TSNA are derived from the addiction taking nicotine in high concentra tions and exhibit as powerful carcinogens. However, despite our everincreasing knowledge on the carcino genic effects of TSNA, we can only assume that these agents play an important role for the increasing cancer risk in tobacco chewers and smokers [5]. Nicotine, the precursor for the highly carcinogenic N'nitrosonor nicotine and 4methylnitrosamino13pyridyl1bu tanone (Scheme 1) is considered to be the leading fac tor for the tobacco addiction [6]. The carcinogenic properties of NNN and NNK are partially due to their 1 The article is published in the original.
metabolic conversion to electrophilic intermediates. Among these, methyldiazohydroxide formed from NNK leads to O6methylguanine in DNA. O
N O N
N N O N
N
I
II
Scheme 1. Structures of N'nitrosonornicotine (I) and 4(methylnitrosamino)1(3pyridyl)1butanone (II).
The metabolic activation of NNN and NNK occurs in tissues of laboratory animals and humans [4]. Virtually all commercial tobacco products contain NNN and NNK which are formed during processing of tobacco [7]. Since the first reports on NNN and NNK in tobacco [8, 9], many studies have quantified their level in various tobacco products. The level of NNN and NNK are quantified in smoke of research cigarettes made from different tobacco varieties, using the International Standards Organization method [10]. The tobaccospecific NNK is a highly effective lung carcinogen in rats that induces lung tumors in mice and hamsters [11]. It also causes tumor of the pancreas, liver, and nasal mucosa in rats, and tumor of the oral cavity when administered together with Nni trosonornicotine. Two of the nicotine derived nitro
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samines, NNN and NNK, are strong carcinogens to lung and pancreatic cancer in smokers, oral cancer in smokeless tobacco users, and lung cancer in people exposed to environmental tobacco smoke. Very few studies have been reported for quantifica tion of NNN and NNK by using HPLCPDA meth od. Most of the times chiral structures of the com pounds and their affinity towards solvents have pre vented from clear separation in HPLC system. Therefore, use of gradient solvent system, column in series [12] and sophisticated instrumentation like liq uid chromatography (LC) coupled with massspec trometry [13] detectors results in longer retention time (RT) and cost of experiment respectively. Hence, the present study demonstrates and validates an easy method for separation and quantification of NNN and NNK with better resolution. EXPERIMENTAL Materials. The solvents ammonium acetate (NH4OAc), ammonia (NH3), acetronitrle (ACN), water (H2O) and glacial acetic acid (GAA) of HPLC grade (Fischer Scientific, Mumbai, India) were used for the study. HPLC grade NNN [3(1nitroso2pyrrolidinyl) pyridine] and NNK [4(methylnitrosamino)1(3py ridyl)1butanone], (98% pure), were procured from SigmaAldrich, USA and dissolved in ACN at working concentration of 0.125, 1, 10, 50, and 100 µg/mL for NNN and 1, 3, 10, 50, and 100 µg/mL for NNK. Instrumentation. The reversed phase ultra flow liq uid chromatography of Shimadzu chromatographic sys tem (Model no. LC20AD) was used for the determina tion of NNN and NNK. The equipment includes a qua ternary pump, manual injector, degasser (DGU20A5), PDA detector SPDM20A and LCSolution software. Chromatographic separation was achieved on a C18 100A Phenomenex column (Luna, 5 µm, 4.6 × 150 mm) at ambient temperature 25 ± 2°C. The column was selected on its wide pH stability (1.5–10), method flexibility and fast LC results. Chromatographic conditions. Mobile phase con sisting of the following ingredients: A – 10% ACN in 1 mM NH4OAc buffer pH 8 adjusted by NH3; B – ACN; C – H2O with 1% GAA was used for separation in a low pressure gradient mode with injection volume 20 µL. The flow rate was 0.4 and 1.0 mL/min and the detection wavelength of PDA detector beam were set at 229 nm. The analysis time was 13 min for both ana lytes. Limits of detection (LOD) and quantification (LOQ) were determined with the signal/noise ratios of 3.3 and 10, respectively [14]. The calibration curve was obtained for NNN and NNK at 5 concentrations ranging of 0.125–100 µg/mL. Linear calibration was applied for calculation of the calibration function as slope. System suitability. The system suitability test was assessed by three replicate injections of the standard
solutions at a particular concentration. The peak areas were used to evaluate repeatability of the proposed method, and their peaks were analyzed for resolution. Parameters such as linearity, precision and resolution were also studied for the proposed method. RESULTS AND DISCUSSION Determination of NNN and NNK together by us ing UFLCPDA system can sometimes be difficult, not only because it represents a wide range of applica tions, but sometimes analytes tend to retain in column and may also have great affinity towards polar solvents. Chemically, the basic structure of the studied analytes consists of a single pyridine ring (Scheme 1). It is well documented that C18 columns are compatible even with 100% aqueous mobile phase and have wide pH stability up to 1.5 for method flexibility. There are very few eluent systems that are using HPLCPDA to de tect NNN and NNK by such system with success. One of them was A: 10% ACN in 1 mM NH4OAc buffer, pH 8 adjusted by NH3 and B: acetonitrile where, A : B were in ratio of 90 : 10 [13] and other given by Carmel la and Hecht [12, 15] with 20 min linear gradient sys tem using 24 mM aqueous NaOH, pH 6 with acetic acid for NNN as solvent A and 50% CH3OH in H2O as solvent B. In present study, individual injections of 50 µg/mL of NNN and NNK yielded peak heights of 1195 and 444 mAU, with RT of 4.96 ± 0.05 and 5.07 ± 0.05 min, respectively, at a flow rate of 0.4 mL/min on 50 : 50 (A : B) solvent system (Figs. a and b). Interestingly in this solvent system the difference of the RT for both the compounds was only 0.117 min. Equal amounts (50 µg/mL) of NNN and NNK gave no separation and were detected as a sin gle peak at RT 4.97 min (Table, Fig. c). At the ratios of 60 : 40 and 70 : 30 of solvent A and B with flow rate of 0.4 mL/min, no separation of NNN and NNK was ob served. A slight shift in RT appeared from 5.59 to 6.61 min (Table). A ratio of 80 : 20 (A : B) gave no def inite peak separation of NNN from NNK at RT 9.05 and 10.13 min respectively (Table). Moreover, a ratio of 90 : 10 (A : B) at flow rate of 0.4 mL/min showed better separation (Table, Fig. d) than previous, but was evident with increase in RT and peak tailing (NNN 15.46 and NNK 19.28 min). Similarly, increase in flow rate from 0.4 to 1.0 mL/min decreased RT which ap peared at 6.38 and 7.96 min with slight peak broaden ing at base (Table). Still increase in flow rate up to 1.2 mL/min in the same solvent system, further de creased RT by about 1 min compared to previous for both compounds (NNN 5.33 and NNK 6.65 min) with peak broadening (Table). Flow rate of 1.0 mL/min of 100% solvent A gave better separation than all previous solvent systems in gradient, but elut ed late with detection at 19.63 and 32.47 min for NNN and NNK, respectively (Table). Thus, to reduce the RT we added water (solvent C) to make the system concentration 85 : 10 : 5 (A : B : C). This reduced RT
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400 300
500
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100
0
0 0
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0
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750 500 250
2.5
5.0 7.5 Time, min (d)
5
10 15 Time, min
mAU
NNN + NNK 4.975
mAU
5.0 7.5 Time, min (c)
10.0
NNN 15.459
750
NNK 5.026
NNN 4.959
1000
(b)
mAU
100 75 50 25
NNK 19.276
(a)
mAU
1155
0
0 2.5
5.0 Time, min
7.5
0
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20
(e) NNN 3.092
mAU 300 200 100
NNK 4.133
0
0 0
1
2
3 5 4 Time, min
6
7
8
UFLC profiles of NNN and NNK at different combinations and concentrations of solvents and at varied retention time. (a) Pro file of NNN at 50% A to 50% B with flow rate 0.4 mL/min; (b) profile of NNK at 50% A to 50% B with flow rate 0.4 mL/min; (c) profile of NNN and NNK mix at 50% A to 50% B with flow rate 0.4 mL/min; (d) profile of NNN and NNK mix at 90% A to 10% B with flow rate 0.4 mL/min; (e) profile of NNN and NNK mix at 70% A : 20% B : 10% C with flow rate 1.0 mL/min.
of NNN and NNK (6.51 and 8.02 min) with peak broadening (Table). To reduce peak broadening, in crease absorbance and decrease retention time, 0.50% of GAA was added in solvent C, which gave pH 2.92. At a flow rate of 1.0 mL/min and ratio of 70 : 20 : 10 (0.50% GAA) (A : B : C), NNN and NNK were de tected at 3.27 and 4.21 min, respectively, with good separation and resolution (Table). Again, further in crease of GAA (0.75%) in solvent C gave better sepa ration and resolution of NNN and NNK at RT of 3.33 JOURNAL OF ANALYTICAL CHEMISTRY
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and 4.21 min (Table, Fig. e), respectively, while other parameters were same as previous. Despite this still further increase in GAA (1.0%) decreases RT of NNN and NNK (3.16 and 4.15 min), but reduces resolution and absorbance of the analytes (Table). Therefore, peaks achieved with the solvent system containing 0.75% GAA were confirmed by measuring width at 50% height of peak. Both sides at 50% height were more or less equal indicating a pure peak. No peak tailing was observed (Fig. e). There was no peak shoul No. 9
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Optimization of solvent system and pH to allow separation of NNN and NNK A 50 50 50 60 70 80 90 90 90 100 85 70 70 70
B 50 50 50 40 30 20 10 10 10 0 10 20 20 20
C 0 0 0 0 0 0 0 0 0 0 5 10 10 10
GAA in C, %
pH of C
– – – – – – – – – – – 0.50 0.75 1.00
– – – – – – – – – – – 2.92 2.82 2.76
RT (min) ± SD
Peak height (mAU) ± SD
Flow rate, mL/min
NNN
NNK
NNN
NNK
0.4 0.4 0.4 0.4 0.4 0.4 0.4 1.0 1.2 1.0 1.0 1.0 1.0 1.0
– 4.96 ± 0.05 4.97 ± 0.05* 5.59 ± 0.05* 6.61 ± 0.06* 9.05 ± 0.09 15.46 ± 0.15 6.38 ± 0.06 5.33 ± 0.05 19.63 ± 0.19 6.51 ± 0.06 3.27 ± 0.03 3.33 ± 0.03 3.16 ± 0.03
5.07 ± 0.05 – 4.97 ± 0.05* 5.59 ± 0.05* 6.61 ± 0.06* 10.13 ± 0.10 19.28 ± 0.19 7.96 ± 0.08 6.65 ± 0.06 32.47 ± 0.32 8.02 ± 0.08 4.21 ± 0.04 4.21 ± 0.04 4.15 ± 0.04
– 1195 ± 24 796 ± 15 587 ± 11 386 ± 7 218 ± 4 116 ± 2 130 ± 2 121 ± 2 59 ± 1 132 ± 2 293 ± 5 300 ± 6 279 ± 5
444 ± 8 – 796 ± 15 587 ± 11 386 ± 7 80 ± 1 40 ± 1 44 ± 1 42 ± 1 13 ± 0.2 47 ± 1 81 ± 2 84 ± 2 77 ± 1
Figure (a) (b) (c) – – – (d) – – – – – (e) –
* Retention times for both were same, as peaks were overlapping.
dering as evident from the profile obtained using mod ified solvent system considering wavelength, time and peak intensity (Fig. e). Though the compounds are present in low concentrations and/or sample size is small (e.g. blood, saliva etc.), selection of solvents should be optimized taking parameter of quality sepa ration and better resolution for compound of interest. Mere using of buffer and solvents sometimes does not perform good resolution if small sample size ob tained and compound are present in very low concen tration. Results of Table revealed that selection of buff er and solvent however gave separation of compounds but increased the LOD of NNN and NNK. For sol vent system 80 : 20 (A : B) at flow rate 0.4 mL/min peak height of NNN and NNK was 218 and 80 mAU, respectively, while 90 : 10 (A : B) peak height was de creased for NNN and NNK to 116 and 40 mAU. In creasing of flow rate (1.0 mL/min) in this ratio slightly increased peak height (NNN 130 and NNK 44 mAU) but in significantly. Whereas, at 100% of solvent A, re duced peak height with poor resolution was observed (Table). Changing of gradient system of solvent A, B and C at a ratio of 85 : 10 : 5 has improved the peak height of NNN and NNK then using 100% of buffer. Addition of GAA improves separation and resolution of NNN and NNK. Hence, in this modified method solvent system A : B : C at a ratio of 85 : 10 : 5 with 0.75% GAA and 1.0 mL/min flow rate achieves better separation, resolution and reproducibility. Moreover, improvement of resolution of the compound can be supportive substantiation for trace level detection of carcinogenic constituents.
The linearity and sensitivity of the method was an alyzed using above set chromatographic conditions. Three independent calibration curves for each analyte were plotted correlating the detector signals with con centrations of the compound of interest. Regression equation was obtained using leastsquare method, and the standard deviation did not exceed 2%. The results revealed good linear calibration fit in the range of 1– 100 µg/mL for NNN and 0.125–100 µg/mL for NNK. The calibration plots were described by the equations y = 6.92 × 104 c(µg/mL) + 1.15 × 104 (n = 5) and y = 1.99 × 104 c(µg/mL) + 7.62 × 104 (n = 5) and the corresponding coefficients of determination (R 2) were 0.995 and 0.999 for NNN and NNK, respective ly. Here the higher R 2 values indicated good linearity and the values of standard deviations of the intercept and the slopes were indicative for the significant valid ity of the calibration points used for the study. LOD for NNN and NNK were found to be 0.12 and 0.02 µg/mL, respectively, while LOQ of NNN and NNK were 0.40 and 0.05 μg/mL, respectively. Five injections, at three different concentrations of NNN (1, 10, and 50 µg/mL) and NNK (3, 10, and 50 µg/mL), showed ex cellent interday precision with RSD of retention time less than 0.11%. Similarly, the intraday precision was de termined using triplicate readings at same concentra tion ranges of analytes. Lower RSD (0.16%) of reten tion time here indicated acceptable reproducibility of the method [16]. The resolution for the principle peaks was found to be 1.51, indicating good separation of the analytes. The theoretical plates number (N) was found to be 1372 (NNN) and 845 (NNK) for the col umn used in the study (150 mm × 4.6 mm i.d., particle
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size 5 µm), thus demonstrating acceptable column ef ficiency. All these results assure the competence of the proposed UFLC method for routine analysis of NNN and NNK. This study emphasized that low cost and good res olution of the analytes responsible for many diseases like cancer. The developed method will be supportive to the researchers working in determination of toxic compounds in various biological samples. ACKNOWLEDGEMENT The authors are indebted to Indian Council of Medical Research (ICMR), New Delhi, India for pro viding necessary facilities. REFERENCES 1. Dube, M.F., Rec. Adv. Tob. Sci.,1982, vol. 8, p. 42. 2. Wynder, E.L. and Hoffmann, D., N. Engl. J. Med., 1979, vol. 300, p. 894. 3. United States Department of Health and Human Servic es. The health consequences of smoking. Cancer. USPHS publication No. 8250179. Washington, DC, Govern ment Printing Office, 1982, p. 322. 4. Hecht, S.S. and Hoffmann, D., Carcinogenesis (Lond.), 1988, vol. 9, p. 875. 5. Hoffmann, D. and Hecht, S.S., Cancer Res., 1985, vol. 45, p. 935.
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6. Krasnagor, N.A., Natl. Inst. Drug Abuse Res. Monogr. Ser., 1979, vol. 23, p. 194. 7. International Agency for Research on Cancer. Tobacco Smoke and Involuntary Smoking. IARC Monographs on the Evaluation of Carcinogenic Risks to Humans, Ly on, France: IARC, 2003, vol. 83. 8. Hoffmann, D., Hecht, S.S., Ornaf, R.M., and Wynder, E.L., Science, 1974, vol. 186, p. 265. 9. Hecht, S.S., Chen, C.B., Hirota, N., Omef, R.M., Tso, T.C., and Hoffmann, D., J. Natl. Cancer Inst., 1978, vol. 60, p. 819. 10. Ding, Y.S., Zhang, L., Jain, R.B., Jain, N.R., Wang, Y., Ashley, D.L., and Watson, C.H., Cancer Epidemiol. Biomarkers Prevent. 2008, vol. 17, p. 3366. 11. Hecht, S.S., Chem. Res. Toxicol., 1988, vol. 11, p. 559. 12. Carmella, S.G. and Hecht, S.S., Anal. Biochem., 1985, vol. 145, p. 239. 13. Wang, L., Henday, S., and Schnute, B., Robust and fast analysis with an RSLC PA2 column of four tobacco specif ic nitrosamines in cigarettes by LC–MS/MS, Dionex Application Note 242, 2009. 14. Urban, M., Scherer, G., Kavvadias, D., Hagedorn, H., Feng, S., Serafin, R., Kapur, S., Muhammad, R., Jin, Y., Mendes, P., and Roethig, H., J. Anal. Toxicol., 2009, vol. 33, p. 260. 15. Carmella, S.G. and Hecht, S.S., Cancer Res., 1987, vol. 47, p. 2626. 16. ICH. Guideline, Q2 (R1): Validation of Analytical Proce dure: Text and Methodology. London: International Conference on Harmonisation (ICH), 2005.L
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