Tobacco cigarette smoking is the leading cause of preventable diseases and death in the US. Exposure to secondhand smoke (SHS) can also cause heart disease, lung cancer, and respiratory illness. Cotinine (COT) and trans-3'-hydroxycotinine (HCT) are the primary metabolites of nicotine, the main addictive alkaloid in tobacco products. For many years, we have measured serum levels of COT and HCT in National Health and Nutritional Examination Survey (NHANES) participants to monitor exposure of the U.S. population to active smoking and SHS. As exposure to SHS is decreasing, a more sensitive analytical method is needed to detect the lower levels of these biomarkers for SHS assessment. We developed and validated a new automated method for the detection of COT and HCT in human serum. We implemented a new liquid handling automation system to aliquot and prepare samples using supported liquid extraction. Samples were analyzed by liquid chromatography-tandem mass spectrometry. The new automated sample preparation method increases sample throughput by reducing sample cleanup time to 2 hours for preparing a 96-well plate. The method has excellent sensitivity, specificity, precision (<10%), and accuracy (±15%). We were able to lower the estimated limit of detection (LOD) for COT by 33% and HCT by 73% from our previous LOD. The new LODs for COT and HCT are 0.010 ng/mL and 0.004 ng/mL, respectively. These lower LODs would enable better detection of SHS in future NHANES surveys.

BACKGROUND: Cigarette smoking increases the risk of cancer, cardiovascular diseases, and premature death. Aromatic amines (AA) are found in cigarette smoke and are well-established human bladder carcinogens. METHODS: We measured and compared total urinary levels of 1-aminonaphthalene (1AMN), 2-aminonaphthalene (2AMN), and 4-aminobiphenyl (4ABP) in adults who smoked cigarettes exclusively and in adult nonusers of tobacco products from a nationally representative sample of non-institutionalized U.S. population in the 2013-2014 National Health and Nutrition Examination Survey. RESULTS: Sample-weighted geometric mean concentrations of AAs in adults who smoked cigarettes exclusively compared with adult nonusers were 30 times higher for 1AMN and 4 to 6 times higher for 2AMN and 4ABP. We evaluated the association of tobacco-smoke exposure with urinary AAs using sample-weighted multiple linear regression models to control for age, sex, race/ethnicity, diet, and urinary creatinine. Secondhand smoke exposure status was categorized using serum cotinine (SCOT) among adult nonusers (SCOT ≤ 10 ng/mL). The exposure for adults who smoked cigarettes exclusively (SCOT > 10 ng/mL) was categorized on the basis of the average number of self-reported cigarettes smoked per day (CPD) in the five days prior to urine collection. The regression models show AAs concentration increased with increasing CPD (P < 0.001). Dietary-intake variables derived from the 24-hours recall questionnaire were not consistently significant predictors of urinary AAs. CONCLUSIONS: This is the first characterized total urinary AA concentrations of the U.S. adult non-institutionalized population. Our analyses show that smoking status is a major contributor to AA exposures. IMPACT: These data provide a crucial baseline for exposure to three AAs in U.S. non-institutionalized adults.

Several aromatic amines (AAs) are established by the International Agency for Research on Cancer as carcinogenic (group 1) or probable/possible carcinogens to humans (group 2A/2B). AAs can be found in mainstream and sidestream smoke from combustible tobacco products, as well as in certain environmental pollution and occupational exposure from several chemical industry sectors. Exposure to AAs can be estimated by measuring their concentrations in urine; however, information about the short-term and long-term stabilities of AAs in urine need to be characterized before conducting large-scale population studies on AA exposure and the potentially harmful effects of AA exposure. In this report, the storage stability of o-toluidine, 2,6-dimethylaniline, o-anisidine, 1-aminonaphthalene, 2-aminonaphthalene, and 4-aminobiphenyl fortified in pooled, filtered, non-smokers' urine is analyzed by isotope dilution gas chromatography-triple quadrupole mass spectrometry (ID GC-MS/MS). The six AAs were measured in urine samples stored at ~20 °C (collection temperature), 4 °C and 10 °C (short-term transit temperatures), and -20 °C and -70 °C (long-term storage temperatures) over a 10-day period. All six analytes were stable for 10 days at transit and long-term storage temperatures but showed reduced recovery at 20 °C. The instability of the target AAs at 20 °C suggests that immediate storage of freshly voided urine at low temperatures is needed to attenuate degradation. A subset of the urine samples was analyzed following a longer storage duration at -70 °C: all AAs were stable for up to 14 months at this temperature. The stability of the six AAs in urine samples can be maintained at the various temperature levels and storage times expected in a typical study set.

Cigarette smoking significantly increases the risk of cancer and cardiovascular diseases as well as premature death. Aromatic amines (AAs) such as o-toluidine, 2-aminonaphthalene and 4-aminobiphenyl are found in cigarette smoke and are well-established human bladder carcinogens presumably acting via the formation of DNA adducts. These amines may be metabolized in the liver to acetylated or glucuronidated forms or oxidized to a hydroxylamine which may react with protein and DNA to form adducts. Free, acetylated and glucuronidated AAs are excreted in urine and can be measured as exposure biomarkers. Using isotope dilution GC-MS/MS, our laboratory quantifies six urinary AAs that are known or suspected carcinogens-o-toluidine, 2,6-dimethylaniline, o-anisidine, 1-aminonaphthalene, 2-aminonaphthalene and 4-aminobiphenyl-for large population studies such as the National Health and Nutrition Examination Survey (NHANES). We also monitor two additional corresponding structural isomers-2-aminobiphenyl and 3-aminobiphenyl-to verify isomer separation. A new and improved automated sample preparation method was developed to quantify these AAs, in which, sample cleanup was done via Supported Liquid Extraction (SLE+ ISOLUTE(R)) on a Hamilton STAR workstation. This automated method increased sample throughput by reducing sample cleanup time from 8 to 4 h while maintaining precision (intra and inter-run coefficient of variation <7%) and accuracy (+/-17%). Recent improvements in our GC/MS method have enhanced our assay sensitivity and specificity, resulting in longer analytical column life and maintaining or reducing the limit of detection for all six analytes. Indigo ASCENTTM software (3.7.1, Indigo BioAutomation, Inc.) is used for peak integration, calibration and quantification. A streamlined sample data flow was created in parallel with the automated method, in which samples can be tracked from receiving to final laboratory information management system output with minimal human intervention, minimizing potential human error. This newly validated, automated method and sample data flow are currently applied in biomonitoring of AAs in the US noninstitutionalized population NHANES 2013-2014 cycle.

BACKGROUND: Polio eradication efforts have been hampered by low responses to trivalent oral poliovirus vaccine (tOPV) in some developing countries. Since stomach acidity may neutralize vaccine viruses, we assessed whether administration of a buffer solution could improve the immunogenicity of tOPV. METHODS: Healthy infants 4-6 weeks old in Sylhet, Bangladesh, were randomized to receive tOPV with or without a sodium bicarbonate and sodium citrate buffer at age 6, 10, and 14 weeks. Levels of serum neutralizing antibodies for poliovirus types 1, 2, and 3 were measured before and after vaccination, at 6 and 18 weeks of age, respectively. FINDINGS: Serologic response rates following 3 doses of tOPV for buffer recipients and control infants were 95% and 88% (P = .065), respectively, for type 1 poliovirus; 95% and 97% (P = .543), respectively, for type 2 poliovirus; and 90% and 89% (P = .79), respectively, for type 3 poliovirus. CONCLUSIONS: Administration of a buffer solution prior to vaccination was not associated with statistically significant increases in the immune response to tOPV; however, a marginal 7% increase (P = .065) in serologic response to poliovirus type 1 was observed.