Determination of tobacco‐specific N‐nitrosamines in rabbit serum by capillary zone electrophoresis and capillary electrophoresis‐electrospray ionization‐mass spectrometry with solid‐phase extraction

Abstract

In this paper, we propose a new strategy for separation and determination of tobacco-specific N-nitrosamines (TSNAs), a group of strong carcinogens found only in tobacco products, by using CZE and CE-MS associated with SPE. Six TSNAs: N'-nitrosonornicotine, N'-nitrosoanatabine, N'-nitrosoanabasine, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol, and 4-(methylnitrosamino)-4-(3-pyridyl)-1-butanol were simultaneously separated by either of two CZE methods, one of which worked with ammonium formate buffer (pH 2.5) and another with citrate buffer (pH 2.4), as well as a CE-MS method. The CZE conditions including pH and concentration of running buffer, capillary length, applied voltage, and capillary temperature were systematically optimized. For CE-MS method, an optimized sheath liquid consisted of methanol-water was used at a flow rate of 10 muL/min. With SPE procedure, our proposed CE-MS method was successfully applied to determine TSNAs after 15 min metabolism in rabbits. A comparison study between CZE and CE-MS methods for quantitative purposes was carried out, showing that both methods provided similar separation efficiency, selectivity, repeatability, linearity, and recovery. However, CE-MS method was better suited for the analysis of TSNAs in complicated biological samples for its sensitivity and extra information on molecular structure. Having good accordance with our previous work by using LC-MS, the new CE-MS method is expected to be an alternative to the LC-MS method and applied to study the metabolism of TSNAs.

Fast Screening for Tobacco-Specific N-nitrosamines by CZE Using Dynamically Coated Capillaries

“…TSNAs have usually been analyzed by liquid extraction followed by high-performance liquid chromatography (HPLC), gas-liquid chromatography (GLC) interfaced with highly specific and sensitive thermal energy analysis (TEA), or liquid chromatography-mass spectrometry (LC-MS) [6][7][8][9]. Compared with these methods, however, capillary electrophoresis (CE) and online coupling of CE with mass spectrometry (CE-MS), which avoids the use of organic solvents as mobile phase with gradient elution and their negative effects on identification of the target analyte, have, in our previous work, been proved to be more effective than convenient techniques [10]. Some new capillary zone electrophoretic (CZE) methods with dynamic coating of the capillary surface have been shown to be even more effective and convenient techniques in a variety of fields [11][12][13][14].…”

Theoretical Explanation of the Peak Splitting of Tobacco-Specific N-Nitrosamines in HPLC

“…Two most widely used methods [2][3][4][5] are gas chromatography coupled with a thermal energy analyzer (GC-TEA) 1 and liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). 6 Recently, methods of gas chromatography coupled to ion-trap tandem mass (GC-ITMS), 7 capillary electrophoresis-electrospray ionization mass spectrometry (CE-MS), 8 and micellar electrokinetic chromatography (MEKC) method combined with cation selective exhaustive injection (CSEI) and sweeping (CSEISweep-MEKC) 3 are also used to quantitate the four TSNAs and even their metabolites.…”

“…Literally, all the peaks of NNN, 8,9 NAT, 9 NNK, 3,8,10 and NNAL 3,10,11 can split into two peaks in certain conditions. In achiral separation condition, the peak-splitting is generally believed to be due to the co-existence of E/Z isomers rather than their enantiomers.…”

“…12 Li and Jansson et al considered that the split-up of the signal was probably due to the basicity of TSNAs and the formation of their protonated isomers. 8 Hecht et al reported that nitrosamines existed as mixture of E and Z isomers. 13 Wu et al found that all TSNAs could exhibit some degree of shoulder effects or split peaks.…”

“…The ratios of the calculated Boltzmann populations of the E and Z isomers of TSNAs coincide well with the reported chromatograms, which E isomers are mainly favored than Z isomers in TSNAs. 3,[8][9][10][11] But it should be noted that all of those chromatogram results are obtained in acidic condition or with acidic mobile phase. Through the procedure of sample preparation for the determination of TSNAs' metabolites in urine, it is shown that TSNAs exist as cations in acidic condition and as free states at pH of 9-10.…”