Search

An atomic emission detector (AED) for a gas chromatograph (GC) can be used to selectively detect compounds labeled with stable isotopes, such as D, 13C, and 15N. This capability can be used to determine precursor-fate relationships within complex matrixes, using stable isotopes rather than radiolabeled isotopes. Employing stable isotopes removes the safety consideration associated with radiolabeling studies. Two previous reports have employed GC-AED in fate studies for 13C-labeled precursors. The goal of this study was to evaluate the utility of GC-AED for precursor-fate determinations in tobacco science. In this work, GC-AED was used to determine the fate of nicotine-[d.sub.4] in a burning cigarette. GC-MSD was then employed to identify the compounds that the AED indicated contained D. Spectral confirmation of the presence of D was performed on each chromatographic peak of interest with both AED and MSD to ensure that the identification of the products was correct. Nicotine and nicotine-[d.sub.4] standards were used to evaluate the effect of coelution of unlabeled compounds with the labeled compounds on the AED response for D. It was shown that the AED response for D at [Lambda] = 308 nm decreases with increased concentration of unlabeled compound. Detection at [Lambda] = 656 nm, however, is unaffected by the presence of the unlabeled compound. Compound-independent calibration is also possible at this wavelength. GC-AED studies with nicotine-[d.sub.4] core injected into cigarettes demonstrated that most of the nicotine (79%) is distilled, unchanged, into the mainstream (MS) and sidestream (SS) smoke. The degradation products that do occur include 3-substituted pyridines and nicotine-oxidation products. These are found only in the SS smoke.

Gas chromatography with atomic emission detection is a useful tool for the detection of stable isotope labels in complex samples. While papers involving the analysis of D and 13C are numerous, little work has been done in the area of 15N detection. For 15N isotope detection, three reagent gases are used: [H.sub.2], [O.sub.2], and C[H.sub.4]. In this work, the reagent gas flows were varied to optimize the sensitivity of 15N detection without sacrificing isotope selectivity. The optimal gas flows determined in this work produce the following ratios of the spectral peak areas: O 725 area/He 728 area = 0.039 with only [O.sub.2] flowing; H 486 area/He 492 area = 12 with only [H.sub.2] flowing; C 496 area/He 502 area = 0.41 with [O.sub.2], [H.sub.2], and C[H.sub.4] flowing for C and no gases flowing for He. When using these gas settings, the 15N sensitivity is increased by nearly 2 orders of magnitude relative to the manufacturer-recommended settings. It was also demonstrated that the presence of a compound in both the labeled and unlabeled forms in the same sample does not affect the response. The ratios of 15N to 14N in standards, calculated from calibration plots (which are linear for both isotopes), agree well with the actual values. A tobacco smoke sample containing various 15N-labeled compounds was used to show the utility of the GC-AED for indicating which compounds in a complex sample contain the label. This sample also demonstrates the necessity for optimal sensitivity when dealing with samples containing small amounts of compounds with low incorporation levels.

Compound-independent calibration (CIC) is a theoretical concept in which a single compound is used to quantitate other compounds within a sample. Gas chromatography with atomic emission detection (AED) is one technique in which CIC can theoretically be applied. Reports are present in the literature, however, that both support CIC and indicate dependence of the AED response on compound structure. In this investigation, the effects of two gas chromatographic parameters (injection mode and column flow rate) on the atomic emission detector elemental response factor (ERF, peak area per nanogram of element injected) were studied. Seven compounds containing various nitrogen functional groups and three detection wavelengths (C at 179 nm, H at 486 nm, and N at 174 nm) were used in this work to explore CIC with GC-AED. When on-column injection was used, the calibration plots for the seven compounds fell on the same line (R = 0.9995), indicating that CIC was possible. However, when splitless injection was employed, an apparent compound dependence of the AED response was observed due to discrimination at the injector. The flow rate dependence of AED response was demonstrated through studies performed with on-column injection and constant flow rates of 1.0, 2.8, and 5.0 mL/min. All three conditions produce compound-independent response, yet the average ERFs varied with the different flow rates. When a constant head pressure of 30 psi was used, the ERFs for the seven compounds became more variable due to the change in flow rate during a temperature-programmed chromatographic experiment. Thus, the experimental parameters of injection mode and column flow rate must be considered in order to prevent apparent compound dependence of AED response.

A new direct method using liquid chromatography-tandem mass spectrometry has been developed and validated for quantitation of 3-hydroxypropylmercapturic acid (3-HPMA) in urine. The method is fast, simple, and does not require extraction from urine. Analyte was separated on a hydrophilic interaction liquid chromatography column. Severe ion suppression was circumvented by a fast gradient after separation. Assay specificity, linearity, precision, and accuracy met the required FDA/CDER bioanalytical method criteria. Matrix effect and carryover of the assay were assessed. Urine sample storage stability and standard solution stability were also tested. The limit of quantitation was 22.0 ng/mL. The results for 3-HPMA obtained by our method were significantly correlated with results obtained by a contract lab.

A tiered testing strategy has been developed to evaluate the potential for new ingredients, tobacco processes, and technological developments to increase or reduce the biological activity that results from burning tobacco. In the manufacture of cigarettes, honey is used as a casing ingredient to impart both aroma and taste. The primary objective of this document is to summarize and interpret chemical and toxicological studies that have been conducted to evaluate the potential impact of honey on the biological activity of either mainstream cigarette smoke or cigarette smoke condensate. As part of ongoing stewardship efforts, cigarettes produced with honey (5% wet weight) as an alternative to invert sugar in tobacco casing material were subjected to extensive evaluation. Principal components of this evaluation were a determination of selected mainstream smoke constituent yields, Ames assay, sister chromatid exchange assay in Chinese hamster ovary cells, a 30-wk dermal tumor promotion evaluation of cigarette smoke condensate in SENCAR mice, and a 13-wk inhalation study of cigarette smoke in Sprague-Dawley rats. Comparative analytical evaluations demonstrated that the substitution of honey for invert sugar as a casing material in cigarettes had no significant impact on mainstream smoke chemistry. In addition, in vitro and in vivo studies demonstrated that cigarettes containing tobacco cased with honey had comparable biological activity to cigarettes containing invert sugar. Collectively, these data demonstrate that the use of honey as an alternative casing material in the manufacture of cigarettes does not alter the potential toxicity of cigarette smoke condensate (CSC) or cigarette smoke; therefore the use of honey as an ingredient added to cigarette tobacco is acceptable from a toxicological perspective.