ЖУРНАЛ АНАЛИТИЧЕСКОЙ ХИМИИ, 2015, том 70, № 9, с. 998-1002
ОРИГИНАЛЬНЫЕ СТАТЬИ =
УДК 543
MODIFIED UFLC-PDA METHOD FOR DETERMINATION
OF NITROSAMINES
© 2015 Sugandha Sharma*, Rajesh K. Joshi**, 1, Sandeep R. Pai**
*Department of Oral Medicine and Radiology, KLE VK Institute of Dental Sciences Belgaum, Karnataka-590010, India **Department of Phytochemistry, Regional Medical Research Centre (RMRC), Indian Council of Medical Research (ICMR) Belgaum, Karnataka-590010, India 1E-mail: joshirk_natprod@yahoo.com Received 08.02.2014; in final form 04.02.2015
Tobacco-specific nitrosamines, viz. N'-nitrosonornicotine (NNN) and 4-methylnitrosamino-1-3-pyridyl-1-butanone (NNK) were determined by using a modified ultra flow liquid chromatography-photo diode array (UFLC-PDA) technique using C18 100A Phenomenex column (Luna, 5 ^m, 4.6 x 150 mm) with 10% ace-tonitrile (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 p.g/mL for NNN and 0.02 p.g/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 optimum mobile phase for detecting NNN and NNK with effective resolution.
Keywords: N'-nitrosonornicotine (NNN), 4-methylnitrosamino-1-3-pyridyl-1-butanone (NNK), ultra flow liquid chromatography-photo diode array (UFLC-PDA) technique, method development.
DOI: 10.7868/S0044450215090145
Tobacco and its smoke contain more than 2500 and 3800 compounds, respectively [1], which include tumor initiators such as the polynuclear aromatic hydrocarbons [2], tumor promoters, co-carcinogens, and organ-specific carcinogens [2, 3]. The large number of tobacco and its smoke make it unlikely that the total carcinogenic activities of tobacco products can be explained by individual compound or group of compounds. 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 carcinogens found only in tobacco products. They are formed from nicotine and related alkaloids during the production and processing of tobacco [4]. TSNA are derived from the addiction taking nicotine in high concentrations and exhibits as powerful carcinogens. However, despite our ever-increasing knowledge on the carcinogenic 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 4-methylnitrosamino-1-3-pyridyl-1-bu-tanone (Scheme) is considered to be the leading factor for the tobacco addiction [6]. The carcinogenic prop-
erties of NNN and NNK are partially due to their metabolic conversion to electrophilic intermediates. Among these, methyldiazohydroxide formed from NNK leads to O6-methylguanine in DNA.
N
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II
Structures of N'-nitrosonornicotine (I) and 4-(methyl-nitrosamino)-1-(3-pyridyl)-1-butanone (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 tobacco-specific 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 N-ni-trosonornicotine. Two of the nicotine derived nitro-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 quantification of NNN and NNK by using HPLC-PDA method. Most of the times chiral structures of the compounds and their affinity towards solvents have prevented from clear separation in HPLC system. Therefore, use of gradient solvent system, column in series [12] and sophisticated instrumentation like liquid chromatography (LC) coupled with mass-spec -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-(1-nitroso-2-pyrrolidinyl) pyridine] and NNK [4-(methylnitrosamino)-1-(3-py-ridyl)-1-butanone], (98% pure), were procured from Sigma-Aldrich, USA and dissolved in ACN at working concentration of 0.125, 1, 10, 50, and 100 ^g/mL for NNN and 1, 3, 10, 50, and 100 |ig/mL for NNK.
Instrumentation. The reversed phase ultra flow liquid chromatography of Shimadzu chromatographic system (Model no. LC-20AD) was used for the determination of NNN and NNK. The equipment includes a quaternary pump, manual injector, degasser (DGU-20A5), PDA detector SPD-M20A and LC-Solution software. Chromatographic separation was achieved on a C18 100A Phenomenex column (Luna, 5 ^m, 4.6 x x 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 consisting 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 ^L. 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 ^g/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 using UFLC-PDA system can sometimes be difficult, not only because it represents a wide range of applications, 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). 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 HPLC-PDA to detect 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 system 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 ^g/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 ^g/mL) ofNNN and NNK gave no separation and were detected as a single 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 observed. A slight shift in RT appeared from 5.59 to 6.61 min (Table). A ratio of 80 : 20 (A : B) gave no definite 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 appeared at 6.38 and 7.96 min with slight peak broadening at base (Table). Still increase in flow rate up to 1.2 mL/min in the same solvent system, further decreased 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
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UFLC profiles of NNN and NNK at different combinations and concentrations of solvents and at varied retention time. (a) — Profile of NNN at 50% A to 50% B with flow rate 0.4 mL/min; (b) -
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