Determination of Nicotine-Related Alkaloids in Tobacco and Cigarette Smoke by GC-FID

A simple and accurate method, gas chromatography (GC) with flame-ionization detection (FID) has been used for determination of the four main nicotine-related alkaloids in tobacco. Tobacco samples were treated with a small quantity of aqueous ammonia solution, to loosen the tobacco tissue and to convert all alkaloids to free bases, then extracted with 1:3 CH₃OH-CH₂Cl₂. A method for further simultaneous and comprehensive determination of six nicotine-related alkaloids in cigarette smoke was also developed. Because of the complexity of the cigarette smoke matrix and the small amounts of alkaloids, except nicotine, in cigarette smoke, the smoke extract was concentrated after removal of the acidic and neutral fractions. To reduce the adsorption and thermal degradation of alkaloids in the liner, especially for nornicotine, a suitable injector temperature was selected and pulsed injection mode was studied. Different cigarette smokes and tobaccos were analyzed for levels of nicotine-related alkaloids.Revised: 3 January and 21 March 2005     

Protonating and determining myosmine intactly by association with citrate anion

Myosmine can not be separated from nornicotine, nicotine and anabasine intactly by capillary zone electrophoresis (CZE) with a phosphate buffer. Using citrate solution at pH 6.5 as a CZE buffer, myosmine is protonated intactly by H(+), charged positively and then separated from other tobacco alkaloids on the baseline. Its sensitivity is ten times higher than gas chromatography (GC) with a nitrogen-phosphorus detector (NPD). The mechanism for protonating myosmine intactly is discussed and the utility of the new method is testified, too.     

Determination of tobacco alkaloids using solid phase microextraction and GC-NPD

Summary The sampling approaches using solid phase microextraction (SPME) were evaluated for the analysis of tobacco alkaloids. Because of their low volatility and ionic nature, sampling alkaloids from the headspace of dry or wet tobacco samples often required more effort to improve extraction efficiency. Directly dipping the SPME fiber coated with polydimethylsiloxane film into the tobacco extract was proved to be a simple, effective tool for sampling alkaloids from tobacco. When combined with the practice of fast GC and nitrogen-phosphorus detection, nicotine and a group of selected minor alkaloids (i.e., nornicotines, myosmine, anabasine and anatabine) were separated with baseline resolution within 3 min. The detection limits for these alkaloids are below 0.1 μg mL⁻¹. In addition, the carry-over problem frequently occurred in alkaloids analysis was eliminated. The influence of tobacco matrix and fiber aging on the partition of alkaloids, as well as the use of an internal standard to compensate these deviations, were also studied.     

Fast analysis of nicotine related alkaloids in tobacco and cigarette smoke by megabore capillary gas chromatography

A novel fast megabore capillary gas chromatographic (MCGC) method for analysis of 7 nicotine related alkaloids in tobacco and cigarette smoke, including nicotine, nornicotine, myosmine, nicotyrine, anabasine, anatabine and 2,3-dipyridyl, was developed. The use of megabore capillary column GC methodology, equipped with flame ionization detector (FID), provided rapid, unambiguous nicotine related alkaloids analysis. One gram flue-cured tobacco (or Cambridge filter pad), 20 ml ether, and 5 ml 10% sodium hydroxide solution, added with n-heptadecane as the internal standard, were placed in a flask, and the flask was capped and placed in an ultrasonic bath for 15 min. A 1 microl volume was analyzed by capillary GC operating in split-injection mode on a mega bore Simplicity-5 column. This simple procedure was compared with the previously reported packed column GC method and the Griffith still-colorimetric method. The application of the method for analysis of various flue-cured tobaccos and cigarette smoke was discussed.     

The application of thin-layer chromatography and phosphorimetry for the rapid determination of nicotine,nornicotine, and anabasine in tobacco

Tobacco is ground and extracted with chloroform, and thin-layer chromatography is used to separate the 3 major tobacco alkaloids—nicotine, nornicotine and anabasine. Each of the alkaloids is quantitatively removed by scraping the spot into a container and dissolving in ethanol-sulfuric acid. A small volume of the ethanol solution is then measured phosphorimetrically. Excellent recoveries of each of the tobacco alkaloids were obtained; the relative standard deviation of analysis was 6% or less for all analyses. The total time for analysis of the 3 alkaloids was less than 90 min. This method is considerably faster and more accurate than previous methods.     

Analyses of tobacco alkaloids by cation-selective exhaustive injection sweeping microemulsion electrokinetic chromatography

In this study, an on-line concentration method which coupled cation-selective exhaustive injection (CSEI) sweeping technology with microemulsion electrokinetic chromatography (MEEKC) was used to detect and analyze several tobacco alkaloids (nornicotine, anabasine, anatabine, nicotine, myosmine and cotinine) that are commonly found in various tobacco products. First, the effects of microemulsion compositions (oil, cosurfactant and solution pH) were examined in order to optimize the alkaloid separations in conventional MEEKC. The pH value and the injection length of basic plug were found to be the predominant influences on the alkaloid stacking. This optimal CSEI sweeping MEEKC method provided approximately 180- to 540-fold increase in detection sensitivity in terms of peak height without any loss in separation efficiency when compared to normal MEEKC separation. Furthermore, this proposed CSEI sweeping MEEKC method was applied successfully for the detection of the minor alkaloids nornicotine, anabasine and anatabine in tobacco products.     

全扫描-选择离子数据采集技术应用于烟草中生物碱的快速测定

建立了应用气相色谱-质谱(GC-MS)全扫描-选择离子监测(Scan-SIM)数据采集方式同时测定烟草中8种含量相差较大的生物碱的方法,采用Scan模式分析烟碱、降烟碱、新烟碱、去氢新烟碱,同时采用SIM模式分析麦斯明、二烯烟碱、2,3′-联吡啶、可铁宁。结果表明,烟草中各生物碱的回收率为94.8%～98.8%,5次测定的相对标准偏差均小于6%。该方法具有简单、快速、准确的特点,应用于烟草样品测定,结果令人满意。     

Simultaneous Determination of Tobacco Alkaloids,Tobacco-Specific Nitrosamines,and Solanesol in Consumer Products Using UPLC–ESI-MS/MS

The US Alcohol and Tobacco Tax and Trade Bureau (TTB) is responsible for collecting Federal excise taxes on tobacco products. Tobacco products in the USA may fall into several taxable categories including cigars, cigarettes, snuff, chewing tobacco, pipe tobacco, and roll-your-own. The existence of these taxable categories means that the TTB is also responsible for the determination of proper tax classification. Not only does proper classification determine the amount of tax owed, but comprehensive classification procedures must also determine if a consumer product is subject to the tobacco excise tax. Since a product must contain tobacco to be subject to the excise tax, laboratory methods that test for the presence of tobacco can provide useful information to ascertain the taxable status of a product. To test for the presence of chemical markers associated with tobacco, an analytical method was developed that permits the simultaneous determination of nicotine and related alkaloids, tobacco-specific nitrosamines (TSNA), and solanesol in methanolic extracts of tobacco. The method utilizes ultra performance liquid chromatography with electrospray ionization–tandem mass spectrometric detection (UPLC–ESI-MS/MS) and was optimized for the analysis of nicotine, cotinine, nornicotine, anatabine, myosmine, anabasine, isonicoteine, nornicotyrine, nicotyrine, N-nitrosoanatabine (NAT), N-nitrosoanabasine (NAB), 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), N′-nitrosonornicotine (NNN) and solanesol. The analytical method was designed to attenuate the instrument response of nicotine, which is overwhelming, to permit simultaneous analysis of all analytes.     

Aqueous aldol catalysis by a nicotine metabolite

Nornicotine, an endogenous tobacco alkaloid and minor nicotine metabolite, can catalyze aldol reactions at physiological pH. Catalysis appears to be due to a covalent enamine mechanism, an unprecedented reaction with small organic molecule catalysts in aqueous buffer. Kinetic parameters for nornicotine as well as other related alkaloids were measured and demonstrate that both the pyrrolidine and pyridine rings are critical for optimal catalysis. Substrate compatibility of this catalyst and its implications in vivo are discussed.     

Optimisation of programmable temperature vaporizer-based large volume injection for determination of pesticide residues in fruits and vegetables using gas chromatography-mass spectrometry

The applicability of programmable temperature vaporizer (PTV) solvent vent injection to the gas chromatographic (GC) determination of pesticide residues in fruits and vegetables was evaluated with the aim of miniaturizing the current multiresidue method. For that purpose 24 pesticides representing different chemical classes were initially chosen for optimisation of the large volume injection (LVI) parameters. Various parameters related to the optimum injector performance were tested for several types of packed and empty liners using both fast (at-once) and speed-controlled PTV solvent vent injection of standard solutions in ethyl acetate. In the next step, several packed and empty liners were evaluated for their suitability for pesticide multiresidue analysis. Parameters identified as optimal were then applied for PTV solvent vent injection of sample extracts prepared using the miniaturized multiresidue method to assess the long-term stability of the system. The combined use of large volume injection of 10 microl ethyl acetate extract into an empty multi-baffled or a CarboFrit packed liner using PTV injectors and GC-MS analysis enabled the detection and quantification of 124 pesticides in fruit and vegetable samples at the 0.01 mg/kg level using miniaturized reversed-phase solid-phase extraction (RP-SPE) of diluted acetone extract and clean-up on a small anion-exchange SPE column.     

Simultaneous determination of six alkaloids in tobacco and tobacco products by direct analysis of real‐time triple quadrupole mass spectrometry with a modified pretreatment method

In order to determine six alkaloids (mass fraction) of nicotine, nornicotine, myosmine, anatabine, anabasine, and nicotyrine in tobacco and tobacco products quickly, accurately, and simultaneously, a novel method based on direct analysis of real‐time model in situ ionization technique combined tandem mass spectrometry with a modified sample pretreatment was established, in which experimental parameters such as the type and amount of extraction solvent and injection rate were optimized, respectively. The samples of five commercial cigarettes and five kinds of tobacco leaves were analyzed by the established method, and the determined values were compared with those obtained using a gas chromatography with mass spectrometry method: (1) Under optimized conditions (30 mL ultrapure water as extraction solvent and with extraction rate of 0.6 mm/s), analysis could be completed within 10 min. (2) The linear range of the method was 0.002–2000 μg/g with, the recovery ranged from 86.8 to 105.6%, and the limit of detection and the limit of quantification were 0.004–0.835 μg/g and 0.013–2.787 μg/g, respectively. (3) The relative standard deviation between direct analysis of real‐time method and the gas chromatography with mass spectrometry method was 0.34–8.83%. The established method is rapid, reliable, and suitable for the ultrafast determination of six alkaloids in tobacco and tobacco products.     

Optimization study for the reversed-phase ion-pair liquid chromatographic determination of nicotine in commercial tobacco products.

The availability of published methods for the determination of nicotine in commercial tobacco products based on state-of-the-art chromatographic methods is limited. Nicotine is a diprotic base with pKa's of 3.12 (pyridine ring) and 8.02 (pyrrolidine ring). Other monoprotic and diprotic bases are also present in commercial tobacco including anatabine, nornicotine, anabasine, and cotinine. In this paper, the chromatography of nicotine and the minor tobacco alkaloids under reversed-phase ion-pairing conditions is thoroughly studied. The results of this study are used to understand the retention mechanisms of the tobacco alkaloids, to examine their observed elution order with respect to fundamental analyte properties (size, functionality, and acid-base strength), and to select optimum chromatographic conditions for the determination of nicotine in commercial tobacco products.     

Comparison of methods for extraction of tobacco alkaloids

Ultrasound and microwave techniques were used to extract tobacco alkaloids, and response surface methodology was used to optimize extraction conditions. Ultrasonic technique factors were temperature, 30-85 degrees C; time, 3-45 min; solvent volume, 8-80 mL. Microwave extraction factors were pressure, 15-75 psi; time, 3-40 min; power, 30-90% of the maximum magnetron power of 650 W. Soxhlet and solvent AOAC-modified extraction methods were also applied after some improvements. Nicotine, nornicotine, anabasine, and anatabine were quantified by gas chromatography. A steam distillation International Standards Organization method for total alkaloid evaluation was used as reference. The results obtained by the different methods were compared using a least squares deviation test. The ultrasonic and the proposed modified-AOAC extraction method were the more convenient with regard to practicability and precision. The relative deviations (n = 5) were as follows: For the ultrasonic method in low-level alkaloid tobaccos, 0.7% nicotine and 1.4-14% minor alkaloids; in high-level alkaloid tobaccos, 2.4% nicotine and 4.5-5.1% minor alkaloids. For the modified AOAC method in low-level alkaloid tobaccos, 0.9% nicotine and 2.4-11.6% minor alkaloids; and in high-level alkaloid tobaccos, 1.7% nicotine and 2.0-2.4% minor alkaloids.     

Programmed temperature vaporizer based method for the sensitive determination of trihalomethanes and benzene,toluene, ethylbenzene and xylenes in soils

A methodology based on the coupling of a headspace autosampler with a GC and a MS detector operating in SIM mode has been developed for the determination of volatile organic compounds (THMs and BTEX) in soils. The GC device used is equipped with a programmable temperature vaporizer (PTV) packed with Tenax-TA^® to introduce the samples (the injection mode used was solvent vent), and a modular accelerated column heater (MACH™) to control column temperature. The proposed measurement procedure reduces the sample pretreatment step to a minimum. Combined use of solvent vent injection mode and mass spectrometry detection allows a highly sensitive method to be proposed, with limits of detection of the order of ng/kg for all the target compounds. Furthermore, the capillary column used allows rapid separations of compounds in less than 4.60 min, affording a very short total analysis cycle time of 9 min.     

Headspace-programmed temperature vaporizer-fast gas chromatography-mass spectrometry coupling for the determination of trihalomethanes in water

A new method based on the use of a headspace autosampler in combination with a GC equipped with a programmable temperature vaporizer (PTV) and an MS detector has been developed for the screening and quantitative determination of trihalomethanes (THMs) in different aqueous matrices. The use of headspace generation to introduce the sample has the advantage that no prior sample treatment is required, thus minimizing the creation of analytical artifacts and the errors associated with this step of the analytical process. The PTV inlet used was packed with Tenax-TA. The injection mode was solvent vent, in which the analytes are retained in the hydrophobic insert packing by cold trapping, while the water vapour is eliminated through the split line. This allows rapid injection of the sample in splitless mode, very low detection limits being achieved without the critical problem of initial sample bandwidth. The capillary column used allowed rapid separations with half-height widths ranging from 1.68 s (chloroform) to 0.66 s (bromoform). The GC run time was 7.3 min. The use of mass spectrometry allows the identification and quantification of the analytes at the low ppt level. The S/N ratio was at least 10-fold higher when the SIM mode was used in data acquisition as compared to the scan mode. The proposed method is extremely sensitive, with detection limits ranging from 0.4 to 2.6 ppt.     

Nitrogen-phosphorus detection and nitrogen chemiluminescence detection of volatile nitrosamines in water matrices: optimization and performance comparison

Two nitrogen-specific detection methods, nitrogen-phosphorus detection (NPD) and nitrogen chemiluminescence detection (NCD), were investigated as low cost alternatives to mass spectrometry (MS) with chemical ionization (CI) for analysis of nitrosamines in aqueous samples. NCD showed greater sensitivity to N-nitrosodimethylamine (NDMA) and seven other volatile nitrosamines than did NPD. Instrument detection levels for NDMA were established at 2.6 microg/L and 4.0 microg/L in solvent with 3 microL splitless gas chromatograph (GC) injection for NCD and NPD, respectively. Using a dual-column confirmation method, both NCD and NPD compared favorably with CI-MS results for NDMA analysis in a variety of water sample types. For seven other nitrosamines, both detectors showed excellent accuracy in analyzing high concentrations (greater than 300 ng/L) in complex wastewater matrices, while the accuracy of spike recoveries of very low levels (less than 15 ng/L) in clean matrices varied for each nitrosamine and detection method.     

Determination of substituted benzenes in water samples by fiber-in-tube liquid phase microextraction coupled with gas chromatography

A method for determination of toluene, ethylbenzene, p-xylene, o-xylene, 1,3,5-trimethylbenzene and 1,2,4-trimethylbenzene in water samples was developed by a fiber-in-tube liquid phase microextraction technique (fiber-in-tube LPME) coupled with GC-flame ionization detector (FID). The method used a tube packed with polytetrafluoroethylene (PTFE) fibers as an extraction medium, improving the stableness of the solvent and the performance of extraction. Certain amounts of curled PTFE fibers were packed into a section of PTFE tube. Because the fibers were curled, they formed network structure in the tube. The fiber packed tube was firstly immersed into organic solvent to be filled with organic solvent and then was exposing to an aqueous solution to extract the target compounds. The extract was then retracted by a conventional GC microsyringe and analyzed by GC-FID. Extraction of the analytes in 8 ml aqueous solution for 15 min yielded enrichment factors of 224-361. The precision (R.S.D., n = 5) was 3.6-8.1% for peak area. The limit of detection (LOD, S/N = 3) for the six substituted benzenes were in the range of 0.3-5.0 μg l⁻¹.     

Determination of acrylamide formed in asparagine/D-glucose maillard model systems by using gas chromatography with headspace solid-phase microextraction

A gas chromatographic method, along with a headspace solid-phase microextraction (HS-SPME), was developed for the determination of acrylamide formed in Maillard reaction model systems. The developed method was validated by liquid chromatography/mass spectrometry. A headspace sample was collected from an aqueous acrylamide solution (100 microg/mL) by SPME and directly injected into a gas chromatograph equipped with a nitrogen-phosphorus detector. The recovery of acrylamide from an aqueous solution was satisfactory, i.e, >93% under the conditions used. Acrylamide formed in an asparagine/D-glucose (molar ratio, 1/2) Maillard reaction model system heated at 150 and 170 degrees C for 20 min was collected and analyzed by the newly developed method using gas chromatography with nitrogen-phosphorus detection and HS-SPME. The amounts of acrylamide were 318 +/- 33 microg/g asparagine from a sample heated at 150 degrees C and 3329 +/- 176 microg/g asparagine from a sample heated at 170 degrees C. Addition of cysteamine or glutathione to the above model system reduced acrylamide formation. Acrylamide formation was not observed when cysteamine or glutathione was added to asparagine in the above model systems to obtain equimolar concentrations of both compounds. This newly developed method is simple and sensitive, and requires no solvent extraction.     

Rapid sensitive speciation analysis of butyl- and phenyltin compounds in water by capillary gas chromatography atomic emission spectrometry (GC-AES) after in-situ ethylation and in-liner preconcentration

Summary A rapid, simple and sensitive method has been developed for the simultaneous determination of butyl- and phenyltin species in environmental waters. The ionic organotin compounds are ethylated in the aqueous phase using sodium tetraethylborate (NaBEt₄) and extracted with hexane. A 25 l aliquot of the extract is injected at a low temperature into a Tenax filled liner. After solvent venting the analytes are transferred onto the capillary column using programmed temperature vaporization (PTV) injection. Detection is done by means of a microwave induced plasma atomic emission detector (MIP AED). The method allows the determination of butyl- and phenyltin compounds in water samples down to the level of 0.1 ng/l (as Sn) while 50 ml of sample is sufficient for analysis. The accuracy of the method was confirmed by GC-AAS after chelation and Grignard derivatization.     

Large volume splitless injection with concurrent solvent recondensation: keeping the sample in place in the hot vaporizing chamber

An injector liner packed with a plug of glass wool is compared with a laminar and a mini laminar liner for large volume (20-50 microL) splitless injection with concurrent solvent recondensation (CSR-LV splitless injection). Videos from experiments with perylene solutions injected into imitation injectors show that glass wool perfectly arrested the sample liquid and kept it in place until the solvent had evaporated. The sample must be transferred from the needle to the glass wool as a band, avoiding 'thermospraying' by partial solvent evaporation inside the needle. The liquid contacted the liner wall when the band was directed towards it, but from there it was largely diverted to the glass wool. In the laminar liners, part of the liquid remained and evaporated at the entrance of the obstacle, while the other proceeded to the center cavity. Vapors formed in the center cavity drove liquid from the entrance of the obstacle upwards, but the importance of such problems could not be verified in the real injector. Some liquid split into small droplets broke through the obstacle and entered the column. Breakthrough through the laminar liners was confirmed by a chromatographic experiment. An improved design of a laminar liner for large volume injection is discussed as a promising alternative if glass wool causes problems originating from insufficient inertness.