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Access to laboratory test results through patient portals is a health equity issue for patients with limited English proficiency (LEP), particularly for Spanish-speaking patients, the largest minority group in the USA. Gaps ranging from linguistic, cultural, and socioeconomic disparities to lack of systematic approaches (e.g., implementation of specific support protocols, policies) are among the identified factors that limit LEP patients’ access to patient portals. This paper summarizes initiatives healthcare providers, laboratory professionals, and portal developers can use to address disparities that affect >26 million LEPs while improving their health equity.

BACKGROUND: 2-Hydroxyethyl mercapturic acid (2HEMA, N-acetyl-S-(2-hydroxyethyl)-L-cysteine) is a urinary metabolite of several volatile organic compounds including acrylonitrile and ethylene oxide, which are found in cigarette smoke. METHODS: We measured 2HEMA concentrations in urine specimens collected during the National Health and Nutrition Examination Survey (2011–2016) from eligible participants aged >12 years (N = 7,416). We developed two multiple linear regression models to characterize the association between cigarette smoking and 2HEMA concentrations wherein the dependent variable was 2HEMA concentrations among participants who exclusively smoked cigarettes at the time of specimen collection and the independent variables included sex, age, race/ethnicity, creatinine, diet, and either cigarettes smoked per day (CPD) or serum cotinine. RESULTS: We detected 2HEMA in 85% of samples tested among exclusive cigarette smokers, and only 40% of specimens from non-smokers. When compared to exclusive cigarette smokers who smoked 1–9 CPD, smoking 10–19 CPD was associated with 36% higher 2HEMA (p < 0.0001) and smoking >19 CPD was associated with 61% higher 2HEMA (p < 0.0001). Additionally, 2HEMA was positively associated with serum cotinine. CONCLUSIONS: This study demonstrates that cigarette smoking intensity is associated with higher urinary 2HEMA concentrations and is likely a major source of acrylonitrile and/or ethylene oxide exposure.

Aldehydes are known carcinogens and irritants that can negatively impact health. They are present in tobacco smoke, the environment, and food. The prevalence of aldehyde exposure and potential health impact warrants a population-wide study of serum aldehydes as exposure biomarkers. We analyzed 12 aldehydes in sera collected from 1843 participants aged 12 years or older in the 2013-2014 National Health and Nutrition Examination Survey. Several aldehydes were detected at high rates, such as isopentanaldehyde (99.2%) and propanaldehyde (88.3%). We used multiple linear regression models to examine the impact of tobacco smoke and dietary variables on serum concentrations of isopentanaldehyde and propanaldehyde. Although 12 serum aldehydes were analyzed and compared to tobacco smoke exposure, only isopentanaldehyde and propanaldehyde showed any significant association with tobacco smoke exposure. Survey participants who smoked 1-10 cigarettes per day (CPD) had 168% higher serum isopentanaldehyde and 28% higher propanaldehyde compared with nonusers. Study participants who smoked 11-20 CPD had higher serum isopentanaldehyde (323%) and propanaldehyde (70%). Similarly, study participants who smoked >20 CPD had 399% higher serum isopentanaldehyde and 110% higher serum propanaldehyde than nonexposed nonusers. The method could not, however, differentiate between nonexposed nonusers and nonusers exposed to secondhand smoke for either of these two aldehydes. No dietary variables were consistently predictive of serum isopentanaldehyde and propanaldehyde concentrations. This report defines baseline concentrations of serum aldehydes in the U.S. population and provides a foundation for future research into the potential health effects of aldehydes. In addition, this study suggests that tobacco smoke is a significant source of exposure to some aldehydes such as isopentanaldehyde and propanaldehyde.

Xylenes are aromatic hydrocarbons used for industrial applications such as the production of petrochemicals and plastics. Acute xylene exposures can negatively impact health through neurotoxicity and irritation of respiratory and dermal tissues. We quantified urinary biomarkers of xylene exposure [2-methylhippuric acid (2MHA) and a mixture of 3- and 4-methylhippuric acids (34MH)] in a representative sample of the U.S. population. Spot urine obtained during the National Health and Nutrition Examination Survey 2005–2006 and 2011–2016 was analyzed using ultra-high-performance liquid chromatography/tandem mass spectrometry. Exclusive smokers were distinguished from non-users using a combination of self-report and serum cotinine data. The median 2MHA and 34MH levels were higher for exclusive smokers (100 μg/g and 748 μg/g creatinine, respectively) than for non-users (27.4 μg/g and 168 μg/g creatinine, respectively). Participants who smoked cigarettes had significantly higher 2MHA and 34MH levels (p20 cigarettes per day (CPD) was significantly associated with 181%, 339% and 393% higher 2MHA levels, respectively. For 34MH, smoking 1–10, 11–20, and >20 CPD was significantly associated with 201%, 398%, and 471% higher 34MH levels, respectively. We confirm that tobacco smoke is a significant source of xylene exposure as measured by urinary 2MHA and 34MH levels.

Terpenes are a class of volatile organic hydrocarbons commonly produced by vegetation and released into the atmosphere. These compounds are responsible for the scents of pine forests, citrus fruits, and some flowers. Human terpene exposure can come from inhalation, diet, smoking, and more recently, using e-cigarettes. Terpenes are present in tobacco smoke and are used as flavor chemicals in e-liquids. The health effects of terpenes are not widely known, though several studies have suggested that they may prove useful in future medical applications. We have developed a novel, high-throughput method of quantifying seven terpenes (α-pinene, β-pinene, β-myrcene, 3-carene, limonene, β-caryophyllene, and α-humulene) in human serum to aid human-exposure investigations. This method employs headspace sampling using solid-phase microextraction (SPME) coupled to gas chromatography-tandem mass spectrometry to detect and quantify five monoterpenes and two sesquiterpenes in the low parts-per-trillion to low parts-per-billion range. The intraday and interday variability (percent error) of the method are ≤2 and ≤11%, respectively. In addition, this method showed excellent recovery in human serum (between 80 and 120% for all analytes). The assay precision ranges between 4.0 and 11%. Limits of detection ranged between 0.032 and 0.162 μg/L. Using serum cotinine values to classify tobacco use showed that smokers have higher serum concentrations of six terpenes compared to nonusers. Terpene concentrations were 14-78% higher in smokers than nonusers. Our method can provide essential biomonitoring data to establish baseline exposure levels for terpenes in humans.

Background Electronic cigarette (e-cigarette) conventions regularly bring together thousands of users around the world. In these environments, secondhand exposures to high concentrations of e-cigarette emissions are prevalent. Some biomarkers for tobacco smoke exposure may be used to characterize secondhand e-cigarette exposures in such an environment. Methods Participants who did not use any tobacco product attended four separate e-cigarette events for approximately six hours. Urine and saliva samples were collected from participants prior to the event, immediately after the event, 4-h after the event, and the next morning (first void). Urine samples from 34 participants were analyzed for cotinine, trans-3′-hydroxycotinine, S-(3-hydroxypropyl)-N-acetylcysteine (3-HPMA), S-carboxyethyl-N-acetylcysteine (CEMA), select tobacco-specific nitrosamines (TSNAs), and 8-isoprostane. Saliva samples were analyzed for cotinine and trans-3′-hydroxycotinine. Results Data from 28 of 34 participants were used in the data analysis. Creatinine-adjusted urinary cotinine concentrations increased up to 13-fold and peaked 4-h after completed exposure (range of adjusted geometric means [AGMs] = 0.352–2.31 μg/g creatinine). Salivary cotinine concentrations were also the highest 4-h after completed exposure (range of AGMs = 0.0373–0.167 ng/mL). Salivary cotinine and creatinine-corrected concentrations of urinary cotinine, trans-3′-hydroxycotinine, CEMA, and 3-HPMA varied significantly across sampling times. Urinary and salivary cotinine, urinary trans-3′-hydroxycotinine, and urinary 3-HPMA concentrations also varied significantly across events. Conclusion Secondhand e-cigarette exposures lasting six hours resulted in significant changes in exposure biomarker concentrations of both nicotine and acrolein but did not change exposure to tobacco-specific nitrosamines. Additional research is needed to understand the relationship between biomarker concentrations and environmental concentrations of toxicants in e-cigarette emissions.

Biomarkers of tobacco exposure are known to be associated with disease risk but previous studies are limited in number and restricted to certain regions. We conducted a nested case–control study examining baseline levels and subsequent lung cancer incidence among current male exclusive cigarette smokers in the Golestan Cohort Study in Iran. We calculated geometric mean biomarker concentrations for 28 matched cases and 52 controls for the correlation of biomarker levels among controls and for adjusted odds’ ratios (ORs) for lung cancer incidence by biomarker concentration, accounting for demographic characteristics, smoking quantity and duration, and opium use. Lung cancer cases had higher average levels of most biomarkers including total nicotine equivalents (TNE-2), 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL), and 3-hydroxyfluorene (3-FLU). Many biomarkers correlated highly with one another including TNE-2 with NNAL and N-Acetyl-S-(2-cyanoethyl)-L-cysteine (2CYEMA), and N-Acetyl-S-(4-hydroxy-2-buten-1-yl)-L-cysteine (t4HBEMA) with N-Acetyl-S-(3-hydroxypropyl-1-methyl)-L-cysteine (3HMPMA) and N-Acetyl-S-(4-hydroxy-2-methyl-2-buten-1-yl)-L-cysteine (4HMBEMA). Lung cancer risk increased with concentration for several biomarkers, including TNE-2 (OR = 2.22, 95% CI = 1.03, 4.78) and NNN (OR = 2.44, 95% CI = 1.13, 5.27), and estimates were significant after further adjustment for demographic and smoking characteristics for 2CYEMA (OR = 2.17, 95% CI = 1.03, 4.55), N-Acetyl-S-(2-carbamoylethyl)-L-cysteine (2CAEMA) (OR = 2.14, 95% CI = 1.01, 4.55), and N-Acetyl-S-(2-hydroxypropyl)-L-cysteine (2HPMA) (OR = 2.85, 95% CI = 1.04, 7.81). Estimates were not significant with adjustment for opium use. Concentrations of many biomarkers were higher at the baseline for participants who subsequently developed lung cancer than among the matched controls. Odds of lung cancer were higher for several biomarkers including with adjustment for smoking exposure for some but not with adjustment for opium use.

Introduction Cotinine is a widely used biomarker for classifying cigarette smoking status. However, cotinine does not differentiate between the use of combustible and noncombustible tobacco products. The increasing use of noncombustible tobacco drives the need for a complementary biomarker for distinguishing cigarette smokers from users of noncombustible tobacco products. Aims and Methods We evaluated the urinary acrylonitrile metabolite, 2CyEMA, as a biomarker of exposure to cigarette smoke in the US population-representative data from the National Health and Nutritional Examination Survey (NHANES). Smoking status was categorized based on the recent tobacco use questionnaire. The receiver operating characteristic (ROC) curve analysis was performed to identify optimal cutoff concentrations by maximizing Youden’s J index. The area under the curve (AUC) was used to compare 2CyEMA effectiveness with respect to serum cotinine. Results The overall cutoff concentration for the classification of cigarette smokers from nonsmokers was 7.32 ng/ml with high sensitivity and specificity (≥0.925). When stratified by demographic variables, the cutoff concentrations varied among subgroups based on age, sex, and race/Hispanic origin. Non-Hispanic Blacks had the highest cutoff concentration (15.3 ng/ml), and Hispanics had the lowest (4.63 ng/ml). Females had higher cutoff concentrations (8.80 ng/ml) compared to males (6.10 ng/ml). Among different age groups, the cutoff concentrations varied between 4.63 ng/ml (21–39 years old) and 10.6 ng/ml (for ≥60 years old). We also explored the creatinine adjusted cutoff values. Conclusions 2CyEMA is an effective biomarker for distinguishing cigarette smokers from nonsmokers (users of noncombustible tobacco products or nonusers). Implications Distinguishes smokers from noncombustible tobacco product users.

1,3-Butadiene is a volatile organic compound with a gasoline-like odour that is primarily used as a monomer in the production of synthetic rubber. The International Agency for Research on Cancer has classified 1,3-butadiene as a human carcinogen. We assessed 1,3-butadiene exposure in the U.S. population by measuring its urinary metabolites N-acetyl-S-(3,4-dihydroxybutyl)-L-cysteine (34HBMA), N-acetyl-S-(1-hydroxymethyl-2-propenyl)-L-cysteine (1HMPeMA), N-acetyl-S-(2-hydroxy-3-butenyl)-L-cysteine (2HBeMA), and N-acetyl-S-(4-hydroxy-2-buten-1-yl)-L-cysteine (4HBeMA). Urine samples from the 2011 to 2016 National Health and Nutrition Examination Survey were analysed for 1,3-butadiene metabolites using ultrahigh-performance liquid chromatography/tandem mass spectrometry. 34HBMA and 4HBeMA were detected in >96% of the samples; 1HMPeMA and 2HBeMA were detected in 0.66% and 9.84% of the samples, respectively. We used sample-weighted linear regression models to examine the influence of smoking status (using a combination of self-reporting and serum-cotinine data), demographic variables, and diet on biomarker levels. The median 4HBeMA among exclusive smokers (31.5 μg/g creatinine) was higher than in non-users (4.11 μg/g creatinine). Similarly, the median 34HBMA among exclusive smokers (391 μg/g creatinine) was higher than in non-users (296 μg/g creatinine). Furthermore, smoking 1–10, 11–20, and >20 cigarettes per day (CPD) was associated with 475%, 849%, and 1143% higher 4HBeMA (p < 0.0001), respectively. Additionally, smoking 1–10, 11–20, and >20 CPD was associated with 33%, 44%, and 102% higher 34HBMA (p < 0.0001). These results provide significant baseline data for 1,3-butadiene exposure in the U.S. population, and demonstrate that tobacco smoke is a major exposure source.

A method to achieve accurate measurement of unmetabolized volatile organic compounds (VOCs) in urine was developed and characterized. The method incorporates a novel preanalytical approach of adding isotopically labeled internal standard (ISTD) analogues directly to the collection container at the point of collection to compensate for analyte loss to the headspace and the collection container surfaces. Using this approach, 45 toxic VOCs ranging in water solubility and boiling point were evaluated and analyzed by headspace solid-phase microextraction/gas chromatography-mass spectrometry. Results show that urine VOCs could be equally lost to the container headspace as to the container surface suggesting similarity of these two regions as partition phases. Surface adsorption loss was found to trend with compound water solubility. In particular, with no headspace, more nonpolar VOCs experienced substantial losses (e.g., 48% for hexane) in a standard 120 mL urine cup at concentrations in the low- and sub-ppb range. The most polar VOCs evaluated (e.g., tetrahydrofuran) showed no significant loss. Other commonly practiced methods for urine sample collection and analysis such as aliquoting, specimen freezing, and use of surrogate ISTD were found to significantly bias results. With this method, we achieved errors ranging from -8.0 to 4.8% of spiked urine specimens. Paired urine and blood specimens from cigarette smokers were compared to assess this method.

Background: Marijuana smoke contains some of the same toxicants present in tobacco smoke. Marijuana smoking is prevalent among HIV+ individuals, but few studies have characterized smoke-related toxicants or associated health outcomes in exclusive marijuana users. Methods: This longitudinal study included 245 participants over age 40 (76% HIV+). 33 plasma and 28 urine metabolites of nicotine, ∆-9-trans-tetrahydrocannabinol, polycyclic aromatic hydrocarbons, and volatile organic compounds were assayed by liquid or gas chromatography/mass spectrometry. Exposures and health outcomes were assessed from surveys and medical records. Findings: At baseline, 18% of participants were marijuana-only smokers, 20% tobacco-only smokers, and 24% dual marijuana-tobacco smokers (median (IQR) age 53 (47–60) years, 78% male, 54% white race). Marijuana smoking was independently associated with elevated plasma naphthalenes, 2-hydroxyfluorene sulfate, 4-vinylphenol sulfate, and o-cresol sulfate (p

Increased interest in volatile organic compound (VOC) exposure has led to an increased need for consistent, systematic, and informative naming of VOC metabolites. As analytical methods have expanded to include many metabolites in a single assay, the number of acronyms in use for a single metabolite has expanded in an unplanned and inconsistent manner due to a lack of guidance or group consensus. Even though the measurement of VOC metabolites is a well-established means to investigate exposure to VOCs, a formal attempt to harmonize acronyms amongst investigators has not been published. The aim of this work is to establish a system of acronym naming that provides consistency in current acronym usage and a foundation for creating acronyms for future VOC metabolites.

Cannabis use through smoking, vaping, or ingestion is increasing, but only limited studies have investigated the resulting exposure to harmful chemicals. N-acetyl-S-(2-cyanoethyl)-L-cysteine (2CYEMA), a urinary metabolite of acrylonitrile, a possible carcinogen, is elevated in the urine of past-30-day cannabis users compared to non-cannabis users. Five frequent and five occasional cannabis users smoked and vaped cannabis on separate days, one also consumed cannabis orally. Urine samples were collected before and up to 72 h post dose and urinary 2CYEMA was quantified. We compared 2CYEMA pre-exposure levels, maximum concentration, time at maximum concentration for occasional versus frequent users following different exposure routes, and measured half-life of elimination. Smoking cannabis joints rapidly (within 10 min) increased 2CYEMA in the urine of occasional cannabis users, but not in frequent users. Urine 2CYEMA did not consistently increase following vaping or ingestion in either study group. Cigarette smokers had high pre-exposure concentrations of 2CYEMA. Following cannabis smoking, the half-lives of 2CYEMA ranged from 2.5 to 9.0 h. 2CYEMA is an effective biomarker of cannabis smoke exposure, including smoke from a single cannabis joint, however, not from vaping or when consumed orally. When using 2CYEMA to evaluate exposure in cannabis users, investigators should collect the details about tobacco smoking, route of consumption, and time since last use as possible covariates.

Hookah smoking has become common in the USA, especially among young adults. This study measured biomarkers of exposure to known tobacco product toxicants in a population-based sample of exclusive, established hookah users. Urinary biomarker data from 1753 adults in Wave 1 of the Population Assessment of Tobacco and Health (PATH) Study were used to compare geometric mean concentrations of biomarkers of exposure in exclusive, established past 30-day hookah users to never users of tobacco. Geometric mean ratios were calculated comparing hookah user groups with never users adjusting for age, sex, race/ethnicity, education, past 30-day marijuana use, secondhand smoke exposure and creatinine. Past 30-day hookah users (n = 98) had 10.6 times the urinary cotinine level of never tobacco users. Compared to never tobacco users, past 30-day hookah users had 2.3 times the level of the carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL), a metabolite of the tobacco-specific nitrosamine (TSNA) 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), 1.3 times higher polycyclic aromatic hydrocarbons (PAHs) 3-hydroxyfluorene and 1-hydroxypyrene, 1.8 times higher levels of acrylonitrile, 1.3 times higher levels of acrylamide, and 1.2 times higher levels of acrolein exposure. These data indicate that hookah use is a significant source of exposure to nicotine, carcinogens, and respiratory toxicants.

Methylcarbamoyl mercapturic acid (MCAMA, N-acetyl-S-(N-methylcarbamoyl)-L-cysteine) is a urinary metabolite of N,N-dimethylformamide and methyl isocyanate, which are volatile organic compounds that are harmful to humans. N,N-dimethylformamide exposure causes liver damage, and methyl isocyanate inhalation damages the lining of the respiratory tract, which can increase risk of chronic obstructive pulmonary disease and asthma. This study characterizes urinary MCAMA levels in the US population and explores associations of MCAMA concentrations with select demographic and environmental factors. We used liquid chromatography tandem mass spectrometry to measure MCAMA in urine collected from study participants ≥ 12 years old (N = 8272) as part of the National Health and Nutrition Examination Survey 2005–2006 and 2011–2016. We produced multiple regression models with MCAMA concentrations as the dependent variable and sex, age, fasting time, race/ethnicity, diet, and cigarette smoking as independent variables. Cigarette smokers and nonsmokers had median urinary MCAMA concentrations of 517 μg/g creatinine and 127 μg/g creatinine, respectively. Sample-weighted multiple regression analysis showed that MCAMA was positively associated with serum cotinine (p < 0.0001). Compared to non-exposed participants (serum cotinine ≤ 0.015 ng/mL), presumptive exposure to second-hand tobacco smoke (serum cotinine > 0.015–≤ 10 ng/mL and 0 cigarettes smoked per day) was associated with 20% higher MCAMA (p < 0.0001). Additionally, smoking 1–10 cigarettes per day was associated with 261% higher MCAMA (p < 0.0001), smoking 11–20 cigarettes per day was associated with 357% higher MCAMA (p < 0.0001), and smoking > 20 cigarettes per day was associated with 416% higher MCAMA (p < 0.0001). These findings underscore the strong association of tobacco smoke exposure with urinary MCAMA biomarker levels.

Rationale Over 2800 e-cigarette, or vaping, product use-associated lung injury (EVALI) cases were reported to the Centers for Disease Control and Prevention (CDC) during August 2019 to February 2020. Bronchoalveolar lavage (BAL) fluid samples from 51 EVALI and 99 non-EVALI cases were analyzed for toxicants including terpenes. We describe a novel method to measure selected terpenes in BAL fluid by gas chromatography/tandem mass spectrometry (GC/MS/MS). Methods α-Pinene, β-pinene, β-myrcene, 3-carene, and limonene were measured in BAL fluid specimens by headspace solid-phase microextraction/gas chromatography/tandem mass spectrometry. We created and characterized BAL fluid pools from non-EVALI individuals to determine assay accuracy, precision, linearity, limits of detection, and analytical specificity. All measurements were conducted in accordance with the CDC's Division of Laboratory Sciences rigorous method validation procedures. Results Matrix validation experiments showed that calibration curves in BAL fluid and saline had similar slopes, with differences of less than 7%. The assay precision ranged from 2.52% to 5.30%. In addition, the limits of detection for the five analytes ranged from 1.80 to 16.8 ng/L, and the linearity was confirmed with R2 values >0.99. Conclusions We developed and validated a method to quantify selected terpenes in BAL fluid specimens using GC/MS/MS. The assay provided accurate and precise analyses of EVALI and non-EVALI BAL fluid specimens in support of CDC's EVALI response. This method is applicable to the determination of a broad range of terpenes in BAL fluid specimens.

Background: Acrylonitrile is a possible human carcinogen that is used in polymers and formed in tobacco smoke. We assessed acrylonitrile exposure in the US population by measuring its urinary metabolites N-acetyl-S-(4-hydroxy-2-methyl-2-buten-1-yl)-L-cysteine (2CYEMA) and N-acetyl-S-(1-cyano-2-hydroxyethyl)-L-cysteine (1CYHEMA) in participants from the 2011–2016 National Health and Nutrition Examination Survey. Objective: To assessed acrylonitrile exposure using population-based biomonitoring data of the US civilian, non-institutionalized population. Methods: Laboratory data for 8,057 participants were reported for 2CYEMA and 1CYHEMA using ultrahigh-performance liquid chromatography / tandem mass spectrometry. Exclusive tobacco smokers were distinguished from non-users using a combination of self-reporting and serum cotinine data. We used multiple linear regression models to fit 2CYEMA concentrations with sex, age, race/Hispanic origin, and tobacco user group as predictor variables. Results: The median 2CYEMA level was higher for exclusive cigarette smokers (145 μg/g creatinine) than for non-users (1.38 μg/g creatinine). Compared to unexposed individuals (serum cotinine ≤ 0.015 ng/ml) and controlling for confounders, presumptive second-hand tobacco smoke exposure (serum cotinine > 0.015 – ≤ 10 ng/ml and 0 cigarettes per day, CPD) was significantly associated with 36% higher 2CYEMA levels (p

Volatile organic compounds (VOCs) are ubiquitous in the environment. In the United States (U.S.), tobacco smoke is the major non-occupational source of exposure to many harmful VOCs. Exposure to VOCs can be assessed by measuring their urinary metabolites (VOCMs). The Population Assessment of Tobacco and Health (PATH) Study is a U.S. national longitudinal study of tobacco use in the adult and youth civilian non-institutionalized population. We measured 20 VOCMs in urine specimens from a subsample of adults in Wave 1 (W1) (2013&ndash, 2014) to characterize VOC exposures among tobacco product users and non-users. We calculated weighted geometric means (GMs) and percentiles of each VOCM for exclusive combustible product users (smokers), exclusive electronic cigarette (e-cigarette) users, exclusive smokeless product users, and tobacco product never users. We produced linear regression models for six VOCMs with sex, age, race, and tobacco user group as predictor variables. Creatinine-ratioed levels of VOCMs from exposure to acrolein, crotonaldehyde, isoprene, acrylonitrile, and 1,3-butadiene were significantly higher in smokers than in never users. Small differences of VOCM levels among exclusive e-cigarette users and smokeless users were observed when compared to never users. Smokers showed higher VOCM concentrations than e-cigarette, smokeless, and never users. Urinary VOC metabolites are useful biomarkers of exposure to harmful VOCs.

Adenosine deaminase severe combined immunodeficiency (ADA-SCID) is an autosomal recessive disorder in which a lack of ADA enzyme prevents the maturation of T- and B-cells; early intervention is crucial for restoring immune function in affected neonates. ADA is responsible for purine metabolism and-in its absence-adenosine, deoxyadenosine, and S-adenosylhomocysteine build up and can be detected in the blood. Preparing dried blood spot (DBS) quality control (QC) materials for these analytes is challenging because enrichments are quickly metabolized by the endogenous ADA in normal donor blood. Adding an inhibitor, erythro-9-(2-hydroxy-3-nonyl) adenine (EHNA), has been previously reported to minimize enzyme activity, although this adds additional cost and complexity. We describe an alternative method using unnatural L-enantiomer nucleosides (L-adenosine and 2'-deoxy-L-adenosine) which eliminates the need for enzyme inhibition. We also present a novel method for characterization of the materials using liquid chromatography mass spectrometry to quantify the analytes of interest.

Benzene is a known genotoxic carcinogen linked to many hematological abnormalities. S-phenylmercapturic acid (PHMA, N-acetyl-S-(phenyl)-L-cysteine, CAS# 4775-80-8) is a urinary metabolite of benzene and is used as a biomarker to assess benzene exposure. Pre-S-phenylmercapturic acid (pre-PHMA) is a PHMA precursor that dehydrates to PHMA at acidic pH. Published analytical methods that measure urinary PHMA adjust urine samples to a wide range of pH values using several types of acid, potentially leading to highly variable results depending on the concentration of pre-PHMA in a sample. Information is lacking on the variation in sample preparation among laboratories regularly measuring PHMA and the effect of those differences on PHMA quantitation in human urine samples. To investigate the differences in PHMA quantitation, we conducted an inter-laboratory comparison that included the analysis of 50 anonymous human urine samples (25 self-identified smokers and 25 self-identified non-smokers), quality control samples and commercially available reference samples in five laboratories using different analytical methods. Observed urinary PHMA concentrations were proportionally higher at lower pH, and results for anonymous urine samples varied widely among the methods. The method with the neutral preparation pH yielded results about 60% lower than the method using the most acidic conditions. Samples spiked with PHMA showed little variation, suggesting that the variability in results in human urine samples across methods is driven by the acid-mediated conversion of pre-PHMA to PHMA.

Microbial volatile organic compounds (MVOCs) are primary and secondary metabolites of fungal and bacterial growth. Changes in environmental conditions (e.g., humidity, light, oxygen, and carbon dioxide) influence microbial growth in indoor environments. Prolonged human exposure to MVOCs has been directly associated with sick building syndrome (SBS), respiratory irritation, and asthma-like symptoms. However, no method exists for assessing MVOC exposure by quantifying them in human serum. We developed a novel, high-throughput automated method for quantifying seven MVOCs (3-methylfuran, 2-hexanone, 2-heptanone, 3-octanone, 1-octen-3-ol, 2-ethyl-1-hexanol, and geosmin) in human serum. The method quantifies the target analytes using solid-phase microextraction gas chromatography-tandem mass spectrometry at low parts-per-billion levels. Limits of detection ranged from 0.076 to 2.77 μg/L. This method provides excellent linearity over the concentration range for the analytes, with coefficients of determination >0.992. Recovery in human serum was between 84.5% and 113%, and analyte precision ranged from 0.38% to 8.78%. The intra-day and inter-day reproducibility showed coefficients of variation ≤11% and ≤8%, respectively. Accurate and precise quantification of MVOCs is necessary for detecting and quantifying harmful human exposures in environments with active microbial growth. The method is well suited for high-throughput analysis to aid investigations of unhealthy exposures to microbial emissions.

Background: How carcinogen exposure varies across users of different, particularly noncigarette, tobacco products remains poorly understood. Methods: We randomly selected 165 participants of the Golestan Cohort Study from northeastern Iran: 60 never users of any tobacco, 35 exclusive cigarette, 40 exclusive (78% daily) waterpipe, and 30 exclusive smokeless tobacco (nass) users. We measured concentrations of 39 biomarkers of exposure in 4 chemical classes in baseline urine samples: tobacco alkaloids, tobacco-specific nitrosamines (TSNA), polycyclic aromatic hydrocarbons (PAH), and volatile organic compounds (VOC). We also quantified the same biomarkers in a second urine sample, obtained 5 years later, among continuing cigarette smokers and never tobacco users. Results: Nass users had the highest concentrations of tobacco alkaloids. All tobacco users had elevated TSNA concentrations, which correlated with nicotine dose. In both cigarette and waterpipe smokers, PAH and VOC biomarkers were higher than never tobacco users and nass users, and highly correlated with nicotine dose. PAH biomarkers of phenanthrene and pyrene and two VOC metabolites (phenylmercapturic acid and phenylglyoxylic acid) were higher in waterpipe smokers than in all other groups. PAH biomarkers among Golestan never tobacco users were comparable to those in U.S. cigarette smokers. All biomarkers had moderate to good correlations over 5 years, particularly in continuing cigarette smokers. Conclusions: We observed two patterns of exposure biomarkers that differentiated the use of the combustible products (cigarettes and waterpipe) from the smokeless product. Environmental exposure from nontobacco sources appeared to contribute to the presence of high levels of PAH metabolites in the Golestan Cohort. Impact: Most of these biomarkers would be useful for exposure assessment in a longitudinal study.

Nitromethane is a known toxicant and suspected human carcinogen. Exposure to nitromethane in a representative sample of the civilian, noninstitutionalized population in the United States ≥12 years old was assessed using 2007-2012 National Health and Nutritional Examination Survey (NHANES) data. Nitromethane was detected in all 8000 human blood samples collected, of which 6730 were used for analyses reported here. Sample-weighted median blood nitromethane was higher among exclusive combusted tobacco users (exclusive smokers; 774 ng/L) than nonusers of tobacco products (625 ng/L). In stratified sample-weighted regression analysis, smoking 0.5 pack of cigarettes per day was associated with a statistically significant increase in blood nitromethane by 150 ng/L, and secondhand smoke exposure (serum cotinine0.05 ng/mL and10 ng/mL) was statistically significant with a 31.1 ng/L increase in blood nitromethane. Certain dietary sources were associated with small but statistically significant increases in blood nitromethane. At median consumption levels, blood nitromethane was associated with an increase of 7.55 ng/L (meat/poultry), 9.32 ng/L (grain products), and 14.5 ng/L (vegetables). This is the first assessment of the magnitude and relative source apportionment of nitromethane exposure in the U.S. population.

Sources of human aldehyde exposure include food additives, combustion of organic matter (tobacco smoke), water disinfection byproducts via ozonation, and endogenous processes. Aldehydes are potentially carcinogenic and mutagenic, and chronic human aldehyde exposure has raised concerns about potential deleterious health effects. To aid investigations of human aldehyde exposure, we developed a novel method to measure 19 aldehydes released from Schiff base protein adducts in serum using controlled acid hydrolysis, solid-phase microextraction (SPME), gas chromatography (GC), and high-resolution mass spectrometry (HRMS). Aldehydes are released from Schiff base protein adducts through acid hydrolysis, and are quantified in trace amounts (μg/L) using stable isotope dilution. Detection limits range from 0.1 to 50 μg/L, with calibration curves spanning 3 orders of magnitude. The analysis of fortified quality control material over a three-month period showed excellent precision and long-term stability (3-22% CV) for samples stored at -70 °C. The intraday precision is also excellent (CV, 1-10%). The method accuracy ranges from 89 to 108% for all measured aldehydes, except acrolein and crotonaldehyde, two aldehydes present in tobacco smoke; their analysis by this method is not considered robust due in part to their reactivity in vivo. However, results strongly suggest that propanal, butanal, isobutanal, and isopentanal levels in smokers are higher than levels in nonsmokers, and thus may be useful as biomarkers of tobacco smoke exposure. This method will facilitate large epidemiological studies involving aldehyde biomonitoring to examine nonoccupational environmental exposures.

INTRODUCTION: Crotonaldehyde is an α,β-unsaturated carbonyl compound that is a potent eye, respiratory, and skin irritant. Crotonaldehyde is a major constituent of tobacco smoke and its exposure can be quantified using its urinary metabolite N-acetyl-S-(3-hydroxypropyl-1-methyl)-L-cysteine (HPMM). A large-scale biomonitoring study is needed to determine HPMM levels, as a measure of crotonaldehyde exposure, in the general U.S. population. MATERIALS AND METHODS: Urine samples were obtained as part of the National Health and Nutrition Examination Survey 2005–2006 and 2011–2012 from participants who were at least six-years-old (N = 4,692). Samples were analyzed for HPMM using ultra performance liquid chromatography - tandem mass spectrometry. Exclusive tobacco smokers were distinguished from non-tobacco users through a combination of self-reporting and serum cotinine data. RESULTS: Detection rate of HPMM among eligible samples was 99.9%. Sample-weighted, median urinary HPMM levels for smokers and non-users were 1.61 and 0.313 mg/g creatinine, respectively. Multivariable regression analysis among smokers showed that HPMM was positively associated with serum cotinine, after controlling for survey year, urinary creatinine, age, sex, race, poverty level, body mass index, pre-exam fasting time, and food intake. Other significant predictors of urinary HPMM include sex (female > male), age (children > non-user adults), race (non-Hispanic Blacks < non-Hispanic Whites). CONCLUSIONS: This study characterizes U.S. population exposure to crotonaldehyde and confirms that tobacco smoke is a major exposure source. Urinary HPMM levels were significantly higher among exclusive combusted tobacco users compared to non-users, and serum cotinine and cigarettes per day were significant predictors of increased urinary HPMM. This study also found that sex, age, ethnicity, pre-exam fasting time, and fruit consumption are related to urinary HPMM levels.

This work describes a quantitative high-throughput analytical method for the simultaneous measurement of small aliphatic nitrogenous biomarkers, i.e., 1,6-hexamethylenediamine (HDA), isophoronediamine (IPDA), ß-methylamino-L-alanine (BMAA), and trimethylamine N-oxide (TMAO), in human urine. Urinary aliphatic diamines, HDA and IPDA, are potential biomarkers of environmental exposure to their corresponding diisocyanates. Urinary BMAA forms as a result of human exposure to blue-green algae contaminated food. And, TMAO is excreted in urine due to the consumption of carnitine- and choline-rich diets. These urinary biomarkers represent classes of small aliphatic nitrogen-containing compounds (N-compounds) that have a high aqueous solubility, low logP, and/or high basic pK(a). Because of the highly polar characteristics, analysis of these compounds in complex sample matrices is often challenging. We report on the development of ion-pairing chemistry based ultra-performance liquid chromatography–electrospray ionization–tandem mass spectrometry (UPLC–ESI–MS/MS) method for the simultaneous measurement of these biomarkers in human urine. Chromatographic separation was optimized using heptafluorobutyric acid- (HFBA-) based mobile phase and a reversed-phase C18 column. All four analytes were baseline separated within 2.6 minutes with an overall run time of five minutes per sample injection. Sample preparation involved four hours of acid hydrolysis followed by automated solid phase extraction (SPE) performed using strong cation exchange sorbent bed with 7 N ammonia solution in methanol as eluent. Limits of detection ranged from 0.05 ng/mL to 1.60 ng/mL. The inter-day and intra-day accuracy were within 10%, and reproducibility within 15%. The method is accurate, fast, and well-suited for biomonitoring studies within targeted groups, as well as larger population-based studies such as the U. S. National Health and Nutrition Examination Survey (NHANES).

The importance of hemoglobin A2 (HbA2) as an indicator of the presence of β-thalassemia was established many years ago. However, clinical application of recommended HbA2 cut off values is often hampered due to poor equivalence of HbA2 results among methods and laboratories. Thus, the IFCC standardization program for HbA2 was initiated in 2004 with the goal of achieving a complete reference system for this measurand. HbA2 standardization efforts are still in progress, including the development of a higher-order HbA2 reference measurement procedure and the preparation of a certified reference material in collaboration with the IRMM. Here, we review the past, present and future of HbA2 standardization and describe the current status of HbA2 testing.

Isoprene is the 2-methyl analog of 1,3-butadiene and is a possible human carcinogen (IARC Group 2B). We assessed isoprene exposure in the general US population by measuring its urinary metabolite, N-acetyl-S-(4-hydroxy-2-methyl-2-buten-1-yl)-L-cysteine (IPM3) in participants (≥3 years old) from the 2015-2016 National Health and Nutrition Examination Survey. Spot urine samples were analyzed for IPM3 using ultra-high performance liquid chromatography coupled with electrospray ionization tandem mass spectrometry. Exclusive tobacco smokers were distinguished from non-users using a combination of self-reporting and serum cotinine data. IPM3 was detected in 80.2% of samples. The median IPM3 level was higher for exclusive cigarette smokers (39.8 μg/g creatinine) than for non-users (3.05 μg/g creatinine). Sample weighted regression analysis—controlling for creatinine, sex, age, race, body mass index, and diet—showed that IPM3 was positively and significantly associated with serum cotinine. Smoking 1-10 cigarettes per day (CPD, 0.5 pack) was significantly associated with an IPM3 increase of 596% (p < .0001), and smoking >20 CPD (>1 pack) was significantly associated with an IPM3 increase of 1,640% (p < .0001), controlling for confounding variables. Drinking beer/ale at median and 90(th) percentile levels (compared to zero consumption) was associated (p < 0.05) with 0% and 2.9%, respectively, increase in IPM3 in non-users. We conclude that tobacco smoke is a major source of isoprene exposure in the US population. This study provides important public health biomonitoring data on isoprene exposure in the general US population.

Disinfection is critical for maintaining a safe water supply, but the use of chlorine or chloramine leads to exposure to disinfection byproducts (DBPs), including trihalomethanes (THMs), which have been associated with adverse reproductive outcomes and bladder cancer. The U.S. Environmental Protection Agency revised the DBP regulations starting in 1998 to further limit levels of THMs in household water. We analyzed data from the National Health and Nutrition Examination Survey (NHANES) collected between 2001 and 2012 (with 2 years per cycle) using models with and without water-related predictors to examine the utility of including these measures. Median blood chloroform levels (25th-75th percentiles) were 16.2 (9.13-31.2) ng/L in 2001-2002 and 5.97 (2.92-12.3) ng/L in 2011-2012. Median blood bromodichloromethane (BDCM) levels (25th-75th percentiles) were 2.22 (1.06-4.61) ng/L in 2001-2002 and 1.18 (

Background: Methyl tertiary-butyl ether (MTBE) was used as a gasoline additive in the United States during 1995–2006. Because of concerns about potential exposure and health effects, some U.S. states began banning MTBE use in 2002, leading to a nationwide phaseout in 2006. Objectives: We investigated the change in blood MTBE that occurred during the years in which MTBE was being phased out of gasoline. Methods: We used data from the National Health and Nutrition Examination Survey (NHANES) from 2001–2012 to assess the change in blood MTBE over this period. We fit sample-weighted multivariate linear regression models to 12,597 human blood MTBE concentrations from the NHANES 2001–2002 to 2011–2012 survey cycles. Results: The unweighted proportion of the individuals with MTBE blood levels above the limit of detection (LOD) of 1.4 ng/L was 93.9% for 2001–2002. This portion dropped to 25.4% for the period 2011–2012. Weighted blood MTBE median levels (ng/L) (25th and 75th percentiles) decreased from 25.8 (6.08, 68.1) ng/L for the period from 2001–2002 to 4.57 (1.44, 19.1) ng/L for the period from 2005–2006. For the entire postban period (2007–2012), MTBE median levels were below the detection limit of 1.4 ng/L. Discussion: These decreases in blood MTBE coincided with multiple statewide bans that began in 2002 and a nationwide ban in 2006. The multivariate log-linear regression model for the NHANES 2003–2004 data showed significantly higher blood MTBE concentrations in the group who pumped gasoline less than 7 h before questionnaire administration compared to those who pumped gasoline more than 12 h before questionnaire administration (p=0.032). This study is the first large-scale, national-level confirmation of substantial decrease in blood MTBE levels in the general population following the phaseout of the use of MTBE as a fuel additive. https://doi.org/10.1289/EHP5572

BACKGROUND: Over 19.5 million people worldwide abuse natural opiates, such as opium-derived products common in Central Asia and Middle East, and many of them also smoke cigarettes. However, there is little information on human exposure to carcinogens and other toxicants related to opiate use, alone or in combination with tobacco use. METHODS: Based on self-reported information, we randomly selected four groups of participants of the Golestan Cohort Study in Northeast Iran: 60 never users of either opiates or tobacco, 35 exclusive current cigarette smokers, 30 exclusive current opiate users, and 30 current opiate users who also smoked cigarettes (dual users; 21 smoked opiates and 9 took them by mouth). We quantified urinary concentrations of 39 exposure biomarkers in four chemical classes: tobacco alkaloids, tobacco specific nitrosamines (TSNAs), polycyclic aromatic hydrocarbons (PAHs), and volatile organic compounds (VOCs). Total nicotine equivalent (TNE) was used as a measure of nicotine dose. We used Oaxaca-Blinder decomposition to parse out the share of the biomarker concentrations explained by opiate use and nicotine dose. RESULTS: Exclusive opiate users and exclusive cigarette smokers had substantially higher concentrations of PAH and VOC biomarkers than never users of either product, but dual users had the highest concentrations. Decomposition analysis showed that opiate use contributed a larger part of the PAH concentrations than nicotine dose, and the sum of 2- and 3-hydroxyphenanthrene (∑(2,3)-phe) resulted almost completely (92%) from opiate use. Concentrations of most VOC biomarkers were explained by both nicotine dose and opiate use, but nicotine dose contributed more. Two acrylamide metabolites (AAMA: 90%, GAMA: 91%), the 1,3-butadiene metabolite (DHBM: 73%), and the dimethylformamide metabolite (AMCA: 72%) were more strongly explained by opiate use. Acrylamide metabolites and ∑(2,3)-phe were significantly higher in opiate smokers than opiate eaters; other biomarkers did not vary by the route of opiate intake. CONCLUSION: Both opiate users and cigarette smokers are exposed to several toxicants and carcinogens. Most biomarkers in opiate users were independent of exposure route, but a few were higher among opiate smokers than eaters. As opiates are widely used worldwide, exposure to some of these toxicants, including PAHs and VOCs, may have substantial global public health impact.

Background: The FDA is considering a mandated reduction in the nicotine content of cigarettes. Clinical trials have been limited by non-study cigarette use (noncompliance), which could mask compensation. The goal of this study was to assess whether compensation occurs when smokers provided with very low nicotine cigarettes cannot access normal nicotine cigarettes. Methods: In a within-subjects, crossover design, current smokers (n = 16) were confined to a hotel for two 4-night hotel stays during which they were only able to access the research cigarettes provided. The hotel stays offered normal nicotine cigarettes or very low nicotine content (VLNC) cigarettes, in an unblinded design, available for “purchase” via a study bank. Results: In the context of complete compliance with the study cigarettes (n = 16), there was not a significant increase during the VLNC condition for cigarettes smoked per day, expired carbon monoxide, or N-acetyl-S-(cyanoethyl)-l-cysteine (cyanoethyl-MA, metabolite of acrylonitrile). There was a significant nicotine × time interaction on urine N-acetyl-S-(3-hydroxypropyl)-l-cysteine (hydroxypropyl-MA, metabolite of acrolein), driven by an increase in the VLNC condition during the first 24 hours. By the end of the VLNC condition, there was no evidence of compensation across any measure of smoking or smoke exposure. Conclusions: Among current smokers who exclusively used VLNC cigarettes for 4 days, there was no significant compensatory smoking behavior. Impact: These data, combined with the larger body of work, suggest that a mandated reduction in nicotine content is unlikely to result in an increase in smoking behavior to obtain more nicotine.

We report on the development of an ultra-performance liquid chromatography–tandem mass spectrometry (UPLC-MS/MS) method for simultaneously measuring eight biomarkers of volatile organic compound (VOC) exposure, with potential application to e-cigarette aerosol biomonitoring. Phenylmercapturic acid (PMA) and trans, trans-muconic acid (tt-MA) are metabolites of benzene; 2-aminothiazoline-4-carboxylic acid (ATCA) is a metabolite of cyanide; N-2-furoylglycine (N2FG) is a metabolite of furfural and furfuryl alcohol; 5-hydroxymethylfuroic acid (HMFA), 5-hydroxymethyl-2-furoylglycine (HMFG), and 2,5-furandicarboxylic acid (FDCA) are metabolites of 5-hydroxymethylfurfural; and 5-hydroxy-N-methylpyrrolidone (5HMP) is a metabolite of N-methyl-2-pyrrolidone. A pentafluorophenyl-modified silica column was used for chromatographic separation. The overall run time for the method is about 6 min per sample injection. The method has low to sub-nanograms per milliliter sensitivity, linearity over 3 orders of magnitude, and precision and accuracy within 15%. The method was used to measure human urine samples. Results showed that people with known benzene exposure (daily cigarette smokers) had higher levels of tt-MA and PMA compared with non-smokers. The method is advantageous for high-throughput analysis of selected VOC metabolites in large-scale, population-based studies such as the National Health and Nutrition Examination Survey (NHANES). Quantifying these urinary biomarkers is important to public health efforts to understand human exposure to VOCs from various sources, including tobacco products and electronic nicotine delivery systems.

Ethylbenzene and styrene are air toxicants with widespread nonoccupational exposure sources, including tobacco smoke and diet. Ethylbenzene and styrene (EB/S) exposure was quantified from their common metabolites measured in spot urine samples obtained from participants (≥6 years old) in the 2005–2006 and 2011–2012 cycles of the National Health and Nutrition Examination Survey (NHANES; N = 4690). EB/S metabolites mandelic acid (MA) and phenylglyoxylic acid (PGA) were measured using ultra-high performance liquid chromatography coupled with electrospray ionization tandem mass spectrometry (UPLC-ESI-MS/MS). MA and PGA were detected in 98.9% and 90.6% of tested urine specimens, respectively. Exclusive smokers had 2-fold and 1.6-fold higher median urinary MA and PGA, respectively, compared with non-users. Sampleweighted regression analysis among exclusive smokers showed that smoking 0.5 pack cigarettes per day significantly increased MA (+97.9 μg/L) and PGA (+69.3 μg/L), controlling for potential confounders. In comparison, exposure from the median daily dietary intake of grain products increased MA by 1.95 μg/L and was not associated with statistically significant changes in urinary PGA levels. Conversely, consuming vegetables and fruit was associated with decreased MA and PGA. These results confirm tobacco smoke as a major source of ethylbenzene and styrene exposure for the general U.S. population.

For over three decades our laboratory has been developing and improving methods for quantifying toxic volatile organic compounds (VOCs) in blood in support of numerous national and regional studies...

The goal of this study was to include the quantitation of hexacosanoyl lysophosphatidylcholine, a biomarker for X-linked adrenoleukodystrophy and other peroxisomal disorders, in the routine extraction and analysis procedure used to quantitate amino acids, acylcarnitines, and succinylacetone during newborn screening. Criteria for the method included use of a single punch from a dried blood spot, one simple extraction of the punch, no high-performance liquid chromatography, and utilizing tandem mass spectrometry to quantitate the analytes.Dried blood spot punches were extracted with a methanolic solution of stable-isotope labeled internal standards, formic acid, and hydrazine, followed by flow injection analysis-electrospray ionization-tandem mass spectrometry.Quantitation of amino acids, acylcarnitines, and hexacosanoyl lysophosphatidylcholine using this combined method was similar to results obtained using two separate methods.A single dried blood spot punch extracted by a rapid (45min), simple procedure can be analyzed with high throughput (2min per sample) to quantitate amino acids, acylcarnitines, succinylacetone, and hexacosanoyl lysophosphatidylcholine.

Objective Prior to initial distribution of Glucose-6-phosphate dehydrogenase (G6PD) proficiency testing (PT) materials, we evaluated G6PD enzyme stability in dried blood spots (DBS) under various temperature and humidity environments to develop storage and usage guidelines for our new materials. Design & methods We prepared fresh G6PD-normal DBS materials and conducted stability evaluations of daily use and short and long-term storage under various temperature and humidity environments. Results G6PD DBS PT materials retained 92% of initial activity after 30 days of use at 4 °C. Materials stored at − 20 °C and 4 °C with desiccant for 30 days retained 95% and 90% of initial activity, respectively. When stored for one year at − 20 °C or six months at 4 °C specimens retained > 90% of initial activity. Specimens stored at 37 °C with desiccant lost 10% activity in three days. At the end of 30 days, specimens stored under ‘Extreme’—humidity > 50% without desiccant— conditions at 37 °C assayed below the NSQAP cut off for G6PD. Humidity exacerbated loss of enzyme activity with increasing temperature and time duration. Conclusion Data suggest that G6PD PT materials can be stored at 4 °C and used for up to one month and can be stored at − 20 °C for one year and yield > 90% enzyme activity. Exposure to warm temperatures, especially with elevated humidity, should be avoided. Desiccant should always be used to mitigate humidity effects.

To best understand the use of dried blood spots (DBS) in clinical chemistry, we must first differentiate laboratory screening from diagnostics. Newborn screening (NBS) utilizes DBS to detect and quantify biomarkers indicative of more than 50 congenital diseases primarily of metabolic origin [1]. Many different methods are used, including modern analytical technologies such as tandem mass spectrometry (MS/MS) [2, 3] and molecular analyses [4, 5]. Each method is designed specifically to analyze DBS specimens with laboratory protocols and systems that begin with a paper hole puncher rather than a pipette. Unfortunately, NBS is often differentiated from clinical chemistry because it is “screening” tool rather than a “diagnostic” application. This is a common misconception because no method is diagnostic, but rather leads to a physician diagnosis that is based, in part, on laboratory results. Like any clinical test, NBS results take into consideration additional data such as age of newborn, birth weight, gestational age, and nutritional status [6–8]. Additionally, NBS is regulated by a comprehensive quality assurance/quality control (QA/QC) network that is shared worldwide [9]. There are a small number of traditional clinical laboratories that use DBS in a diagnostic setting, primarily in specialized metabolic applications, further supporting that DBS utilization is not restricted to “screening” laboratories, nor does it involve relaxed laboratory standards. The question is: why are DBS not used to a greater extent in traditional clinical laboratories? It is likely due to the analytical challenges and common misperceptions of the DBS. Specifically, the challenges of DBS implementation such as cross-validation of a DBS assay from a liquid sample analysis, logistical systems limitations for handling DBS versus liquid samples, and clinical laboratory bias towards single metabolite analysis versus metabolic profiles.

Orotic aciduria in the presence of hyperammonemia is a key indicator for a defect in the urea cycle, specifically ornithine transcarbamylase (OTC) deficiency. Current newborn screening (NBS) protocols can detect several defects of the urea cycle, but screening for OTC deficiency remains a challenge due to the lack of a suitable assay. The purpose of this study was to develop a high-throughput assay to measure orotic acid in dried blood spot (DBS) specimens as an indicator for urea cycle dysfunction, which can be readily incorporated into routine NBS.Orotic acid was extracted from DBS punches and analyzed using flow-injection analysis tandem mass spectrometry (FIA-MS/MS) with negative-mode ionization, requiring2 min/sample run time. This method was then multiplexed into a conventional newborn screening assay for analysis of amino acids, acylcarnitines, and orotic acid.We describe 2 assays which can quantify orotic acid in DBS: a stand-alone method and a combined method for analysis of orotic acid, amino acids, and acylcarnitines. Both methods demonstrated orotic acid recovery of 75-85% at multiple levels of enrichment. Precision was also comparable to traditional FIA-MS/MS methods. Analysis of residual presumptively normal NBS specimens demonstrated a 5:1 signal to noise ratio and the average concentration of orotic acid was approximately 1.2 μmol/l. The concentration of amino acids and acylcarnitines as measured by the combined method showed no significant differences when compared to the conventional newborn screening assay. In addition, retrospective analysis of confirmed patients and presumptively normal newborn screening specimens suggests potential for the methods to identify patients with OTC deficiency, as well as other urea cycle defects.The assays described here quantify orotic acid in DBS using a simple extraction and FIA-MS/MS analysis procedures that can be implemented into current NBS protocols.