Gary D. Byrd

Quantitation of isoprostane isomers in human urine from smokers and nonsmokers by LC-MS/MS

A simple, rapid liquid chromatography-tandem mass spectrometry method was developed to identify and quantitate in human urine the isoprostanes iPF2α-III, 15-epi-iPF2α-III, iPF2α-VI, and 8,12-iso-iPF2α-VI along with the prostaglandin PGF2α and 2,3-dinor-iPF2α-III, a metabolite of iPF2α-III. Assay specificity, linearity, precision, and accuracy met the required criteria for most analytes. The urine sample storage stability and standard solution stability were also tested. The methodology was applied to analyze 24 h urine samples collected from smokers and nonsmokers on controlled diets. The results for iPF2α-III obtained by our method were significantly correlated with results by an ELISA, although an ∼2-fold high bias was observed for the ELISA data. For iPF2α-III and its metabolite 2,3-dinor-iPF2α-III, smokers had significantly higher concentrations than nonsmokers (513 ± 275 vs. 294 ± 104 pg/mg creatinine; 3,030 ± 1,546 vs. 2,046 ± 836 pg/mg creatinine, respectively). The concentration of iPF2α-VI tended to be higher in smokers than in nonsmokers; however, the increase was not statistically significant in this sample set. Concentrations of the other three isoprostane isomers showed no trends toward differences between smokers and nonsmokers. Among smokers, the daily output of two type VI isoprostanes showed a weak correlation with the amount of tobacco smoke exposure, as determined by urinary excretion of total nicotine equivalents.

A further study of FTC yield and nicotine absorption in smokers

Abstract The relationship between nicotine yield as determined by the FTC method and nicotine absorption was examined in 72 smokers in a more rigorous repetition of a previous study of 33 smokers. For this study, 113 smokers evenly distributed across four FTC “tar” yield ranges were recruited; only 72 demonstrated reasonable compliance with the study criteria with regard to sample collections and cigarette brand style consistency. Subjects recorded the number of cigarettes smoked daily and collected a 24-h urine sample and a saliva sample on 3 consecutive days. Nicotine absorption was determined by monitoring urinary excretion of nicotine and its metabolites. In addition, saliva samples were monitored for cotinine using radioimmunoassay (RIA). The correlation of the relationship for nicotine absorbed per cigarette was positive and significant (r = 0.31, P = 0.008) but weaker than in the previous study. Only smokers in the highest yield range showed any statistical difference from smokers in the lower ranges. Our results suggest that FTC nicotine yield is weakly related to nicotine absorption and that smoker-controlled factors exert a great influence on the amount of nicotine absorbed by smokers. Compensation is substantial but incomplete for the minority (by market share) of smokers at the low end of the yield scale. It is uncertain how well any alternative set of machine parameters would predict nicotine absorption for the majority of smokers, even if it were more predictive for the small number of smokers at the lower yield part of the range.

Comparison of measured and FTC.predicted nicotine uptake in smokers

Cigarette smokers have a wide variety of “tar” and nicotine yields to choose from in the current market, ranging from 0.5 mg “tar” and less than 0.05 mg nicotine to 27 mg “tar” and 1.8 mg nicotine by the Federal Trade Commission (FTC) method. To understand better the relationship between FTC nicotine yields and actual nicotine uptake in smokers, we have studied nicotine uptake in 33 smokers of self-selected products representing four “tar” groupings: 1 mg “tar” (1 MG), ultra-low “tar” (ULT), full-flavor low “tar” (FFLT), and full flavor (FF) cigarettes. These cigarette categories had mean FTC nicotine yields of 0.14, 0.49, 0.67, and 1.13 mg/cigarette, respectively. The subjects smoked their usual brand of cigarette ad libitum and provided a 24-h urine sample for total nicotine uptake analysis over a period during which the number of cigarettes smoked was recorded. Nicotine uptake was determined by monitoring urinary nicotine and its metabolites, including the glucuronide conjugates. Daily nicotine uptake was 9.1±7.3 mg (range 1–21 mg) for 1 MG, 19.2±10.0 mg (range 4–42 mg) for ULT, 21.8±9.4 mg (range 13–38 mg) for FFLT, and 37.1±14.4 mg (range 21–60 mg) for FF smokers. On a per cigarette basis, yields were 0.23±0.11, 0.56±0.23, 0.60±0.18, and 1.19±0.43 mg nicotine, respectively. Although individual variability was fairly large (CVs of 0.39–0.80), means for the different groups showed that lower FTC yield smokers not only absorb less nicotine per 24-h period, but also per cigarette smoked. These data suggest that nicotine uptake is a function of individual smoking behavior within product design limits. We conclude from these data that, while FTC yield cannot precisely predict nicotine uptake for an individual smoker, it is useful in predicting and comparing actual nicotine uptake by smokers who select cigarettes with a particular FTC yield.

Chapter 6 – Use of gas chromatographic and mass spectrometric techniques for the determination of nicotine and its metabolites

This chapter examines the application of gas chromatographic methods with emphasis on mass spectrometric detection for the determination of nicotine and its metabolites. The coupling of a high resolution separation technique, such as gas chromatography (GC) with mass spectrometry (MS), produces one of the most effective analytical tools used in chemistry today. Fundamental descriptions of GC and MS methodologies are presented in the chapter. Qualitative and quantitative strategies for GC–MS are discussed, and applications are presented to illustrate these points. GC is a highly suitable method for determination of nicotine, and has been employed for many years to measure nicotine concentrations in a variety of matrices. MS has been widely applied to the study of nicotine and its metabolites in biological samples. The coupling of mass spectrometry to GC seemed possible because both techniques were restricted to volatile samples.

Chapter 7 – Use of high-performance liquid chromatographic–mass spectrometric (LC–MS) techniques for the determination of nicotine and its metabolites

This chapter discusses the application of high-performance liquid chromatographic (HPLC) methods for the determination of nicotine and its metabolites with emphasis on mass spectrometric detection. Other important detectors, such as radiometric and diode arrays detectors, are covered in brief. The role of HPLC in studying nicotine metabolism and the operation of several liquid chromatography–mass spectrometry (LC–MS) interfaces are described in the chapter. Qualitative strategies for LC–MS are discussed and applications that determine nicotine metabolites are presented to illustrate these points. Finally, practical considerations of LC–MS operation and cost are given. Improvements in commercial instruments have made LC–MS as easy as gas chromatography (GC)–MS in operation. There are two important considerations to get the most out of an LC–MS system. First of all, familiarity with HPLC is important, particularly, for quantitative applications. Second, most of the interfaces produce molecular adducts that provide superb molecular weight information but little structural information. Thus, tandem mass spectrometry, as a triple stage system or an ion trap, may be worth the extra expenditure.

Synthesis of (E)-N-[Methyl-d3]- 4-(3-pyridinyl)-3-buten-1-amine, a deuterated analogue of the nicotinic agonist RJR-2403

The synthesis of (E)-N-[methyl-d3]-4-(3-pyridinyl)-3-buten-1-amine ([methyl-d3]RJR 2403; [methyl-d3]metanicotine) is reported. The incorporation of deuterium was performed during the first step of the synthesis via N-methylation of the pyrrolidine nitrogen of racemic nornicotine with [methyl-d3]iodomethane, in the presence of n-BuLi at −70°C to afford racemic [methyl-d3]nicotine in high yield (91%). The pyrrolidine ring was then cleaved with ethyl chloroformate to give (E)-N-[methyl-d3]-N-ethyloxycarbonyl-4-(3-pyridinyl)-3-buten-1-amine; in this reaction, elimination of HCl occurred during heating of the intermediate N-[methyl-d3]-N-ethyloxycarbonyl-4-chloro-4-(3-pyridinyl)butan-1-amine under vacuum (0.5 mm Hg). The last step of the synthesis, i.e. the removal of the N-carbamoyl group, was achieved via acidic hydrolysis with concentrated aqueous hydrochloric acid, to afford [methyl-d3]metanicotine in 82% overall yield. The isotopic purity of the sample was determined by mass spectrometry and calculated to be 97.6 atom % deuterium. Copyright