Simultaneous determination of cotinine and trans‐3‐hydroxycotinine in urine by automated solid‐phase extraction using gas chromatography–mass spectrometry
Abstract:A gas chromatography–mass spectrometry method was developed and validated for the simultaneous automated solid-phase extraction and quantification of cotinine and trans-3-hydroxycotinine in human urine. Good linearity was observed over the concentration ranges studied (R2 > 0.99). The limit of quantification was 10 ng/mL for both analytes. The limits of detection were 0.06 ng/mL for cotinine (COT) and 0.02 ng/mL for trans-3-hydroxycotinine (OH-COT). Accuracy for COT ranged from 0.98 to 5.28% and the precision … Show more
“…Each puff is defined as 55 ml over 8 s draw every minute in a British American Tobacco (BAT) exposure chamber (Thorne and Adamson 2013 ) containing warm DMEM circulated by a Fisher Scientific Variable Flow Minipump, for a total of 15 min (Gellatly et al 2020 ). Duration was determined from behavioral studies of patients using an e-cigarette (Garcia-Arcos et al 2016 ) and nicotine serum levels after use (Chiadmi and Schlatter 2014 ; Marsot and Simon 2016 ; Yingst et al 2019 ), as well as modified from International Organization for Standardization (ISO) standards for cigarette smoking experiments. Fig.…”
Section: Methodsmentioning
confidence: 99%
“…Nicotine concentrations in the apical media of e-vapor-exposed SAEC were measured by GC/MS based on previously described methods (Chiadmi and Schlatter 2014 ; Gellatly et al 2020 ). Nicotine in the apical surface of SAEC was detected in a range of 0.86–1.79 µg/ml 24 h after one exposure to e-vape (Fig.…”
Section: Methodsmentioning
confidence: 99%
“…1 b). This concentration is relevant to levels of exposure of humans in vivo, where serum nicotine concentrations were determined to be about 1000 times lower than those in the airway epithelial lining fluid (Chiadmi and Schlatter 2014 ; Marsot and Simon 2016 ; Yingst et al 2019 ).…”
Electronic cigarettes or vaping products have been marketed as a safer alternative to smoking, but very little is known about the health effects in the human lung, particularly in the distal airways, a key site of airway obstruction and destruction in chronic obstructive pulmonary disease that is often exacerbated by viral infections. The aim of this study was to investigate the effects of electronic cigarette vapor (e-vapor) on human distal airway epithelial responses to influenza A virus (IAV) infection. We isolated primary small airway epithelial cells (SAECs) from donor lungs free of lung disease, and cultured them at air–liquid interface (ALI). To measure markers of epithelial injury such as integrity of epithelial barrier structure and function, we selected a regimen of non-toxic, barrier preserving e-vapor exposure of cultured cells to 15 puffs of e-vapor from a commercially available e-cigarette once per day for 3 days, prior to IAV infection. After 72 h of infection, media and cell lysates were collected to measure cytokines involved in inflammatory and antiviral responses. Pre-exposure to e-vapor with IAV infection, compared to IAV infection alone, significantly increased inflammatory and antiviral mediators including IL-8, CXCL10, IFN-beta, and MX1. Our results suggest that e-vapor exposure amplifies human distal airway pro-inflammatory response to IAV infection, independently of the severity of cell injury during viral infection.
“…Each puff is defined as 55 ml over 8 s draw every minute in a British American Tobacco (BAT) exposure chamber (Thorne and Adamson 2013 ) containing warm DMEM circulated by a Fisher Scientific Variable Flow Minipump, for a total of 15 min (Gellatly et al 2020 ). Duration was determined from behavioral studies of patients using an e-cigarette (Garcia-Arcos et al 2016 ) and nicotine serum levels after use (Chiadmi and Schlatter 2014 ; Marsot and Simon 2016 ; Yingst et al 2019 ), as well as modified from International Organization for Standardization (ISO) standards for cigarette smoking experiments. Fig.…”
Section: Methodsmentioning
confidence: 99%
“…Nicotine concentrations in the apical media of e-vapor-exposed SAEC were measured by GC/MS based on previously described methods (Chiadmi and Schlatter 2014 ; Gellatly et al 2020 ). Nicotine in the apical surface of SAEC was detected in a range of 0.86–1.79 µg/ml 24 h after one exposure to e-vape (Fig.…”
Section: Methodsmentioning
confidence: 99%
“…1 b). This concentration is relevant to levels of exposure of humans in vivo, where serum nicotine concentrations were determined to be about 1000 times lower than those in the airway epithelial lining fluid (Chiadmi and Schlatter 2014 ; Marsot and Simon 2016 ; Yingst et al 2019 ).…”
Electronic cigarettes or vaping products have been marketed as a safer alternative to smoking, but very little is known about the health effects in the human lung, particularly in the distal airways, a key site of airway obstruction and destruction in chronic obstructive pulmonary disease that is often exacerbated by viral infections. The aim of this study was to investigate the effects of electronic cigarette vapor (e-vapor) on human distal airway epithelial responses to influenza A virus (IAV) infection. We isolated primary small airway epithelial cells (SAECs) from donor lungs free of lung disease, and cultured them at air–liquid interface (ALI). To measure markers of epithelial injury such as integrity of epithelial barrier structure and function, we selected a regimen of non-toxic, barrier preserving e-vapor exposure of cultured cells to 15 puffs of e-vapor from a commercially available e-cigarette once per day for 3 days, prior to IAV infection. After 72 h of infection, media and cell lysates were collected to measure cytokines involved in inflammatory and antiviral responses. Pre-exposure to e-vapor with IAV infection, compared to IAV infection alone, significantly increased inflammatory and antiviral mediators including IL-8, CXCL10, IFN-beta, and MX1. Our results suggest that e-vapor exposure amplifies human distal airway pro-inflammatory response to IAV infection, independently of the severity of cell injury during viral infection.
“…Nicotine and cotinine concentrations in the apical media of e-vaporexposed SAEC were detected and measured by GC/MS based on a previously described method. 26 The instruments used were a Focus GC coupled to a DSQ II mass spectrometer and an AS 3000 autosampler. A 15 m RTX-5MS column (0.25 mm i.d., 0.25 µm film thickness; Restek, Bellefonte, PA) was used with ultrahigh purity helium as the carrier gas at a constant flow rate of 1.0mL/min.…”
Section: Nicotine and Cotinine Measurements In The Media Of E-vapor-exposed Saecmentioning
Purpose: Electronic cigarettes (e-cigs) are relatively new devices that allow the user to inhale a heated and aerosolized solution. At present, little is known about their health effects in the human lung, particularly in the small airways (<2 mm in diameter), a key site of airway obstruction and destruction in chronic obstructive pulmonary disease and other acute and chronic lung conditions. The aim of this study was to investigate the effect of e-cigarettes on human distal airway inflammation and remodeling. Methods: We isolated primary small airway epithelial cells from donor lungs without known lung disease. Small airway epithelial cells were cultured at air-liquid interface and exposed to 15 puffs vapor obtained by heating a commercially available e-cigarette solution (e-vapor) with or without nicotine. After 24 hrs of e-vapor exposure, basolateral and apical media as well as cell lysates were collected to measure the pleiotropic cytokine interleukin 6 (IL6) and MUC5AC, one of the major components in mucus. Results: Unlike the nicotine-containing e-vapor, nicotine-free e-vapor significantly increased the amount of IL6, which was coupled with increased levels of intracellular MUC5AC protein. Importantly, a neutralizing IL6 antibody (vs an IgG isotype control) significantly inhibited the production of MUC5AC induced by nicotine-free e-vapor.
Conclusion:Our results suggest that human small airway epithelial cells exposed to nicotine-free e-vapor increase the inflammatory response and mucin production, which may contribute to distal lung airflow limitation and airway obstruction.
“…In contrast to urine, human plasma or serum is easier to obtain from intoxicated persons and the concentration of the drug in blood is more closely correlated to clinical situation of the patient. During recent years, SPE has become a widely used sample pretreatment technique, for example, for the enrichment of compounds of interest in human body fluids, because of its ability to produce cleaner extracts, ideal for bioanalytical analysis (Kwok et al ., ; Mueller et al ., , Mueller and Rentsch, ; Navarrete et al ., ; Chiadmi and Schlatter, ).…”
An automated multi-analyte screening method for the identification and quantification of 92 drugs and metabolites based on on-line solid-phase extraction-high-performance liquid chromatography-diode array detection technique was developed and successfully validated. In addition, a database with 870 entries including UV-spectra, relative/retention times and response factors of toxicologically relevant compounds was created. Plasma samples (0.2 mL) were treated with methanol, diluted with buffer and on-line extracted (Strata X, 20 ×2 mm, 25 µm) at pH 9. Analytical separation was carried out on a Gemini NX column (150 ×4.6 mm, 3 µm) using gradient elution with acetonitrile-water (90:10,v/v) and 0.05 m potassium dihydrogen phosphate buffer (pH 2.3). Linear calibration curves with correlation coefficients ≥0.9950 were obtained for 78 analytes. As an additional benefit, the newly developed method allows the quantification of 42 analytes (e.g. antidepressants, neuroleptics and anticonvulsants) in a concentration range suitable for therapeutic drug monitoring. Limits of quantitation ranged from 0.02 mg/L (chlordiazepoxide) to 3.4 mg/L (mexiletine). Inter- and intra-day precisions of quality control samples (low/high) were better than 15% (zolpidem) and accuracy (bias) ranged from -11% (opipramol, venlafaxine) to 11% (venlafaxine, trazodone). Tests for carry-over and sample stability under different storage conditions were also performed and stability was adequate. Four cases of poisoning analysis are presented.
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