Variation of Benzo[a]pyrene, NNN, and NNK Levels in 16 Commercial Smokeless Tobacco Products.

This study examined the variation of benzo[a]pyrene (B[a]P), N'-nitrosonornicotine (NNN), and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) levels in 16 smokeless tobacco products from several different product subcategories obtained at two different locations and at two different procurement times. B[a]P quantities range from 0.6 to 160 ng/g on a wet-weight basis, whereas NNN and NNK quantities range from 276 to 10473 ng/g and 79 to 28882 ng/g, respectively. The B[a]P, NNN, and NNK quantities vary widely among various smokeless tobacco product categories and among various brands within each product subcategory. Dry snuff products contain the highest B[a]P, NNN, and NNK quantities, whereas loose and portioned snus products contain the lowest B[a]P, NNN, and NNK levels. In general, variation of B[a]P, NNN, and NNK levels across four sets of each product brand purchased six months apart and at two different locations show statistically significant differences (p < 0.05), although with a much narrower product set-to-set variability.

Quantitative measurement of harmful and potentially harmful constituents, pH, and moisture content in 16 commercial smokeless tobacco products.

Smokeless tobacco products expose adult and youth tobacco users to various addictive and carcinogenic constituents that can cause long-term nicotine dependence and oral cancers. In this study, nicotine, benzo[a]pyrene (B[a]P), N'-nitrosonornicotine (NNN), 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), acetaldehyde, crotonaldehyde, formaldehyde, moisture, and pH levels in 16 smokeless tobacco products were measured on a wet-weight basis (wwb). In addition, change in analytical variability with increasing replicate measurements was assessed. Total nicotine in the products varied from 6.2 to 35.5 mg/g. The percentage of total nicotine in the unprotonated form ranged from 0.1 to 62%; whereas, product moisture varied from 7.4 to 57%. The quantities of harmful and potentially harmful constituents (HPHCs) range from 0.46 to 179.9 ng/g for B [a]P, 270-12206 and 81-20716 ng/g for NNN and NNK, respectively, and 0.33-6.85 and 0.13-5.67 µg/g for acetaldehyde and formaldehyde, respectively. This study shows wide variation in smokeless tobacco product HPHC quantities. The results also show that analytical variability stabilizes after seven replicate measurements.

Tobacco-Specific Nitrosamines in the Tobacco and Mainstream Smoke of Commercial Little Cigars.

Cigars are among the broad variety of tobacco products that have not been as extensively studied and characterized as cigarettes. Small cigars wrapped in a tobacco-containing sheet, commonly referred to as little cigars, are a subcategory that are similar to conventional cigarettes with respect to dimensions, filters, and overall appearance. Tobacco-specific nitrosamines (TSNAs) are carcinogens in the tobacco used in both little cigars and cigarettes. This study uses a validated high-performance liquid chromatography-electrospray tandem mass spectrometry (LC-MS/MS) method to measure the TSNAs 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and N'-nitrosonornicotine (NNN) in the tobacco filler and the nonintense International Organization for Standardization smoking regimen, ISO 3308, and the newer ISO 20778 Cigarette Intensive (CI) smoking regimen mainstream smoke of 60 commercial little cigars. Tobacco filler NNK and NNN quantities ranged from 26 to 2950 and 1440 to 12 100 ng/g tobacco, respectively. NNK and NNN by the ISO nonintense smoking regimen ranged from 89 to 879 and 200 to 1540 ng/cigar, respectively; by the CI regimen, NNK and NNN ranged from 138 to 1570 and 445 to 2780 ng/cigar, respectively. The average transfer (%) for NNK and NNN from tobacco filler to mainstream smoke was 24% and 36% by the ISO nonintense and CI smoking regimens, respectively. By the ISO nonintense and CI smoking regimens, mainstream smoke NNK and NNN yields showed a moderate to strong correlation (ISO nonintense, R2 = 0.60-0.68, p < 0.0001; CI, R2 = 0.78-0.81, p < 0.0001) with tobacco filler NNK and NNN quantities. In addition, the mainstream smoke NNK and NNN yields of little cigars were determined to be 3- to 5-fold higher compared to previously tested commercial cigarettes. The mainstream smoke NNK and NNN yields have wide variation among commercial little cigars and suggest that, despite design similarities to cigarettes, machine-smoke yields of carcinogenic TSNAs are higher in little cigars.

Biomarkers of Tobacco Exposure: Summary of an FDA-Sponsored Public Workshop.

Since 2009, the FDA Center for Tobacco Products (CTP) has had the authority to regulate the manufacturing, distribution, and marketing of tobacco products in order to reduce the death and disease caused by tobacco use. Biomarkers of exposure pertain to actual human exposure to chemicals arising from tobacco use and could play an important role across a number of FDA regulatory activities, including assessing new and modified-risk tobacco products and identifying and evaluating potential product standards. On August 3-4, 2015, FDA/CTP hosted a public workshop focused on biomarkers of exposure with participants from government, industry, academia, and other organizations. The workshop was divided into four sessions focused on: (i) approaches to evaluating and selecting biomarkers; (ii) biomarkers of exposure and relationship to disease risk; (iii) currently used biomarkers of exposure and biomarkers in development; and (iv) biomarkers of exposure and the assessment of smokeless tobacco and electronic nicotine delivery systems. This article synthesizes the main findings from the workshop and highlights research areas that could further strengthen the science around biomarkers of exposure and help determine their application in tobacco product regulation. Cancer Epidemiol Biomarkers Prev; 26(3); 291-302. ©2016 AACR.

Tobacco-Specific Nitrosamines in the Tobacco and Mainstream Smoke of U.S. Commercial Cigarettes.

Tobacco-specific nitrosamines (TSNAs) are N-nitroso-derivatives of pyridine-alkaloids (e.g., nicotine) present in tobacco and cigarette smoke. Two TSNAs, N'-nitrosonornicotine (NNN) and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), are included on the Food and Drug Administration's list of harmful and potentially harmful constituents (HPHCs) in tobacco products and tobacco. The amounts of four TSNAs (NNK, NNN, N-nitrosoanabasine (NAB), and N'-nitrosoanatabine (NAT)) in the tobacco and mainstream smoke from 50 U.S. commercial cigarette brands were measured from November 15, 2011 to January 4, 2012 using a validated HPLC/MS/MS method. Smoke samples were generated using the International Organization of Standardization (ISO) and Canadian Intense (CI) machine-smoking regimens. NNN and NAT were the most abundant TSNAs in tobacco filler and smoke across all cigarette brands, whereas NNK and NAB were present in lesser amounts. The average ratios for each TSNA in mainstream smoke to filler content is 29% by the CI smoking regimen and 13% for the ISO machine-smoking regimen. The reliability of individual TSNAs to predict total TSNA amounts in the filler and smoke was examined. NNN, NAT, and NAB have a moderate to high correlation (R2 = 0.61-0.98, p < 0.0001), and all three TSNAs individually predict total TSNAs with minimal difference between measured and predicted total TSNA amounts (error < 7.4%). NNK has weaker correlation (R2 = 0.56-0.82; p < 0.0001) and is a less reliable predictor of total TSNA quantities. Tobacco weight and levels of TSNAs in filler influence TSNA levels in smoke from the CI machine-smoking regimen. In contrast, filter ventilation is a major determinant of levels of TSNAs in smoke by the ISO machine-smoking regimen. Comparative analysis demonstrates substantial variability in TSNA amounts in tobacco filler and mainstream smoke yields under ISO and CI machine-smoking regimens among U.S. commercial cigarette brands.

Measurements for 8 common analytes in native sera identify inadequate standardization among 6 routine laboratory assays.

BACKGROUND: External quality assessment (EQA) with commutable samples is essential for assessing the quality of assays performed by laboratories, particularly when the emphasis is on their standardization status and interchangeability of results. METHODS: We used a panel of 20 fresh-frozen single-donation serum samples to assess assays for the measurement of creatinine, glucose, phosphate, uric acid, total cholesterol, HDL cholesterol, LDL cholesterol, and triglycerides. The commercial random access platforms included: Abbott Architect, Beckman Coulter AU, Ortho Vitros, Roche Cobas, Siemens Advia, and Thermo Scientific Konelab. The assessment was done at the peer group level and by comparison against the all-method trimmed mean or reference method values, where available. The considered quality indicators were intraassay imprecision, combined imprecision (including sample-matrix interference), bias, and total error. Fail/pass decisions were based on limits reflecting state-of-the-art performance, but also limits related to biological variation. RESULTS: Most assays showed excellent peer performance attributes, except for HDL- and LDL cholesterol. Cases in which individual assays had biases exceeding the used limits were the Siemens Advia creatinine (-4.2%), Ortho Vitros phosphate (8.9%), Beckman Coulter AU triglycerides (5.4%), and Thermo Scientific Konelab uric acid (6.4%), which lead to considerable interassay discrepancies. Additionally, large laboratory effects were observed that caused interlaboratory differences of >30%. CONCLUSIONS: The design of the EQA study was well suited for monitoring different quality attributes of assays performed in daily laboratory practice. There is a need for improvement, even for simple clinical chemistry analytes. In particular, the interchangeability of results remains jeopardized both by assay standardization issues and individual laboratory effects.

Reference measurement procedure for total glycerides by isotope dilution GC-MS.

BACKGROUND: The CDC's Lipid Standardization Program established the chromotropic acid (CA) reference measurement procedure (RMP) as the accuracy base for standardization and metrological traceability for triglyceride testing. The CA RMP has several disadvantages, including lack of ruggedness. It uses obsolete instrumentation and hazardous reagents. To overcome these problems the CDC developed an isotope dilution GC-MS (ID-GC-MS) RMP for total glycerides in serum. METHODS: We diluted serum samples with Tris-HCl buffer solution and spiked 200-µL aliquots with [(13)C(3)]-glycerol. These samples were incubated and hydrolyzed under basic conditions. The samples were dried, derivatized with acetic anhydride and pyridine, extracted with ethyl acetate, and analyzed by ID-GC-MS. Linearity, imprecision, and accuracy were evaluated by analyzing calibrator solutions, 10 serum pools, and a standard reference material (SRM 1951b). RESULTS: The calibration response was linear for the range of calibrator concentrations examined (0-1.24 mmol/L) with a slope and intercept of 0.717 (95% CI, 0.7123-0.7225) and 0.3122 (95% CI, 0.3096-0.3140), respectively. The limit of detection was 14.8 µmol/L. The mean %CV for the sample set (serum pools and SRM) was 1.2%. The mean %bias from NIST isotope dilution MS values for SRM 1951b was 0.7%. CONCLUSIONS: This ID-GC-MS RMP has the specificity and ruggedness to accurately quantify total glycerides in the serum pools used in the CDC's Lipid Standardization Program and demonstrates sufficiently acceptable agreement with the NIST primary RMP for total glyceride measurement.

Proposed serum cholesterol reference measurement procedure by gas chromatography-isotope dilution mass spectrometry.

BACKGROUND: Our purpose was to establish a mass spectrometry reference measurement procedure (RMP) for cholesterol to use in the CDC's standardization programs. We explored a gas chromatography-isotope dilution mass spectrometry (GC-IDMS) procedure using a multilevel standard calibration curve to quantify samples with varying cholesterol concentrations. METHODS: We calibrated the mass spectrometry instrument by isotope dilution with a pure primary standard reference material and an isotopically enriched cholesterol analog as the internal standard (IS). We diluted the serum samples with Tris-HCl buffer (pH 7.4, 0.05 mol/L, 0.25% Triton X-100) before analysis. We used 17 serum pools, 10 native samples, and 2 standard reference materials (SRMs). We compared the GC-IDMS measurements with the CDC's modified Abell-Levy-Brodie-Kendall (AK) RMP measurements and assessed method accuracy by analyzing 2 SRMs. We evaluated the procedure for lack of interference by analyzing serum spiked with a mixture of 7 sterols. RESULTS: The mean percent bias between the AK and the GC-IDMS RMP was 1.6% for all samples examined. The mean percent bias from NIST's RMP was 0.5% for the SRMs. The total %CVs for SRM 1951b levels I and II were 0.61 and 0.73%, respectively. We found that none of the sterols investigated interfered with the cholesterol measurement. CONCLUSIONS: The low imprecision, linear response, lack of interferences, and acceptable bias vs the NIST primary RMP qualifies this procedure as an RMP for determining serum cholesterol. The CDC will adopt and implement this GC-IDMS procedure for cholesterol standardization.

Chiral separation of polychlorinated biphenyls using a combination of hydroxypropyl-gamma-cyclodextrin and a polymeric chiral surfactant.

Chiral separation of moderately to highly hydrophobic polychlorinated biphenyls (PCBs) using a conventional chiral micelle or a polymeric chiral surfactant, as the single chiral selector is very difficult since the hydrophobic interactions between the chiral PCB and the monomeric or polymeric surfactant is very strong. Combined use of a polymeric chiral surfactant, polysodium N-undecanoyl-D-valinate (poly-D-SUV) with hydroxypropyl-gamma-cyclodextrin (HP-gamma-CD) was successful in cyclodextrin modified electrokinetic chromatography (CD-EKC) enantioseparation of PCB congeners. Addition of HP-gamma-CD to the background electrolyte containing poly-D-SUV functioned to improved chiral resolution for the PCBs and reduce the analysis time for these congeners. In addition, concentration of methanol, concentration of 2-(N-cyclohexylamino) ethanesulfonic acid (CHES) buffer and separation voltage was also varied to optimize multicomponent separation of five chiral PCBs. Simultaneous separation and enantioseparation of all five PCBs was possible in less than 50 min under optimized conditions that requires a 5 mM CHES solution buffered at about pH 10 with 1.5% w/v (ca. 60 mM) poly-D-SUV and 16 mM HP-gamma-CD. In addition, 1 M urea and 20% v/v methanol should be added as organic modifier and the capillary temperature maintained at 45 degrees C. As expected the polymeric surfactant showed improved chiral resolution of PCBs over conventional micelles of SUV. Under optimized conditions, when CD-EKC of chiral PCBs using poly-D-SUV was compared to sodium dodecyl sulfate (SDS), better resolution, higher efficiency and shorter analysis time was achieved with poly-D-SUV.