Measuring tobacco smoke exposure: quantifying nicotine/cotinine concentration in biological samples by colorimetry, chromatography and immunoassay methods

doi:10.1016/j.jpba.2004.01.009

Journal of Pharmaceutical and Biomedical Analysis

Volume 35, Issue 1, 1 April 2004, Pages 155-168

https://doi.org/10.1016/j.jpba.2004.01.009 Get rights and content

Abstract

Procedures to assess tobacco smoke exposure are reviewed and biomarkers used for determining the smoking status of an individual are compared. Methods used to extract these biomarkers from saliva, urine, and blood and the advantages and disadvantages of the assays are discussed. Finally, the procedures used to measure the levels of cortisol, a stress hormone speculated to be linked to nicotine metabolism, are discussed.

Introduction

A large body of evidence indicates that tobacco smoking has unfavorable consequences on human health [1], [2], [3], [4], [5], [6], [7]. Chronic smokers run the risk of lung cancer [2], [8], [9], [10], [11], [12], [13], [14], [15], [16], [17], respiratory infections [18], [19], heart disease [2], [20], [21], [22], and pregnancy complications [23], [24] caused by inhalation of nicotine, the principal component of tobacco. The annual worldwide mortality due to tobacco use is estimated to be 3 million [25]. Many hazardous substances in mainstream cigarette smoke are also present in environmental tobacco smoke (ETS). Therefore individuals involuntarily exposed to ETS, called passive smokers, are also adversely affected. For example, there are about 3000 lung cancer deaths per year among nonsmokers [26]. Even infants nursed by smoking mothers are affected by nicotine as it is secreted in the milk [24], [27], [28], [29].

Often, a distinction has to be made between smokers and nonsmokers, and between non smokers exposed to ETS and non smokers not exposed to ETS. For example, life insurance companies are interested in knowing the smoking status of potential insurance customers, since heavy smokers run the risk of decreased life expectancy. In this respect, biochemical measurements with appropriate markers have been found useful [30], [31]. A threshold value of 500 ng ml⁻¹ of cotinine, a major metabolite of nicotine, is used to distinguish smokers from nonsmokers [32].

This article reviews the procedures used to assess tobacco smoke exposure. Specifically, it compares different biomarkers used to determine the smoking status of an individual and the different methods used to extract these biomarkers from saliva, urine, and blood. Advantages and disadvantages of all the assays currently used are discussed. The article also reviews methods for evaluating the cortisol levels in tobacco smokers; cortisol is a stress hormone that appears to be closely linked to nicotine metabolism [33]. Recent review articles regarding tobacco smoke exposure include a mini-review on the use of urinary cotinine as a tobacco-smoke exposure index [34] and a review on the use of cotinine as a biomarker of environmental tobacco smoke exposure [35].

Section snippets

Nicotine metabolites

Nicotine is metabolized to more than 20 different derivatives [36]. In humans, 70% of nicotine is oxidized to cotinine, 4% is oxidized differently, 9% is excreted unchanged in the urine, and the metabolic outcome of the remaining 17% is still unknown [37], [38], [39], [40]. (Scheme 1).

Tobacco smoking also produces metabolites other than those derived from nicotine, such as trans, trans-muconic acid [41], [42] and 1-hydroxy pyrene [43]. These metabolites are produced from benzene [41], [42] and

Biomarkers for assessing smoking status

An ideal marker for assessing the smoking status of individuals should have a reasonable half-life, be specific, be amenable to estimation in body fluids, and be available at concentrations that can be quantified using existing analytical methods. The presence of other compounds should not interfere in the estimation of the marker and the marker should not be influenced by environmental sources other than tobacco smoke. The markers used for assessing the smoking status of individuals, and the

Collection of body fluids

The receptacle for collecting body fluids should be made of either glass or polypropylene, should be disposable, should be kept in a sealed package protected from the environment, and precleaned and siliconized before use [6]. A polypropylene receptacle with a screw cap closure is preferred since it can withstand breakage during transportation to a distant laboratory [65]. Different biological fluids are collected as follows.

Methods for the extraction of biomarkers

Several organic solvents have been employed to extract organic constituents in plasma, saliva, and urine. From the organic solvent the basic constituents, namely, nicotine and cotinine are recovered by salt formation with a mineral acid (HCl/H₂SO₄/H₃PO₄) [73]. Cotinine and/or nicotine can be re-extracted from the corresponding salts by basification by NaOH (or Na₂CO₃–NaHCO₃ [79], [80], [81], [82]/K₂CO₃–NH₄OH [74]). These are then assayed either by GLC [79], [83], [84], HPLC [39], [65], GC–MS

Colorimetry

For monitoring the smoking status of humans, colorimetry is a desirable method of analysis. It is simple, inexpensive, and gives (after taking appropriate precautions) [87], [88] an estimate of total metabolites produced from nicotine inhaled during smoking [77], [89], [90]. It, however, lacks specificity and the estimated urinary concentration is reported to be higher than that obtained by gas or liquid chromatography. Moreover, drugs containing the pyridine nucleus (e.g., isoniazid,

Cortisol

Cortisol is one of the most frequently used steroid hormones for assessing adrenal disorders. Serum and urinary cortisol concentrations have been used to monitor adrenocortical function as well as in the diagnosis of chronic fatigue and depression [152], [153]. Cortisol is also used as a biomarker of stress [154], [155], [156], [157]. A study performed on rats concluded that stress lowers circulating nicotine levels [33]. In humans, the relationship between stress and smoking is well documented

Conclusion

Of the different body fluids, saliva is the matrix of choice for assessing the presence of nicotine and its metabolites in humans exposed to ETS. Of the different biomarkers, cotinine appears to be the analyte of choice, as it fulfills the prerequisites of specificity and retention time and is found in detectable concentrations in all three matrices. GC–MS is preferred for analyzing nicotine and its metabolites in smokers whereas GLC is preferred for studies related to passive smokers.

Acknowledgements

The author thanks Christina Quadraccio and Steven Gilday for assisting in the literature search.

References (189)

M.L. Brown
J. Pediatr. Health Care
(2001)
K.M. Emmons et al.
Prev. Med.
(1994)
S.S. Hecht
Lancet Oncol.
(2002)
P. Correa et al.
Lancet
(1983)
A.S. Mayer et al.
Respir. Physiol.
(2001)
M.V. Djordjevic et al.
Prev. Med.
(1997)
P. Hill et al.
J. Chronic Dis.
(1983)
D. Schwartz-Bickenbach et al.
Toxicol. Lett.
(1987)
W. Luck et al.
J. Pediatr.
(1985)
P.P. Rop et al.
J. Chromatogr. Biomed. Appl.
(1993)

M. Meger et al.

J. Chromatogr. B Anal. Echnol. Biomed. Life Sci.

(2002)

J.R. Idle

J. Clin. Epidemiol.

(1990)

S. Zevin et al.

Drug Alcohol Depend.

(2000)

W. Schramm et al.

Prev. Med.

(1992)

J.K. Yao et al.

Clin. Biochem.

(1998)

S.A. McAdams et al.

J. Chromatogr. Biomed. Appl.

(1993)

H.W. Kuo et al.

J. Chromatogr. B Anal. Technol. Biomed. Life Sci.

(2002)

M. Nakajima et al.

J. Chromatogr. B

(2000)

F. Ceppa et al.

J. Chromatogr. B Biomed. Sci. Appl.

(2000)

P. Jacob et al.

J. Pharm. Biomed. Anal.

(2000)

H.S. Shin et al.

J. Chromatogr. B. Anal. Technol. Biomed. Life Sci.

(2002)

P. Jacob et al.

J. Chromatogr.

(1993)

P. Jacob et al.

J. Chromatogr.

(1981)

M. Curvall et al.

J. Chromatogr.

(1982)

H.D. Harvey et al.

Int. J. Environ. Health Res.

(2002)

M. Bartal

Monaldi Arch. Chest Dis.

(2001)

H.D. Harvey et al.

J.R. Soc. Health

(2001)

B.H. Marcus et al.

J. Public Health Policy

(1992)

T.M. Ezzati et al.

Vital Health Stat.

(1992)

J.P. Wisnivesky et al.

Geriatrics

(2002)

N. Yanagawa et al.

Jpn. J. Cancer Res.

(2002)

W.C. Vial

Am. J. Med. Sci.

(1986)

T. Hirayama

Br. Med. J.

(1981)

D. Trichopoulos et al.

Int. J. Cancer

(1981)

L. Garfunkel

J. Natl. Cancer Inst.

(1981)

S. Akiba et al.

Cancer Res.

(1986)

N.A. Dalager et al.

Cancer Res.

(1986)

M.D. Winniford

J. Hypertens. Suppl.

(1990)

S.G. West et al.

Ann. Behav. Med.

(2001)

K.O. Haustein

Int. J. Clin. Pharmacol. Ther.

(1999)

Killer Environment. Environmental Health Perspectives 107 (1999)...

C.A. Pope et al.

JAMA

(2002)

W. Luck et al.

Br. J. Clin. Pharmacol.

(1984)

B. Schultze-Hobein et al.

Acta Pediatr.

(1992)

K.M. Cummings et al.

Arch. Environ. Health

(1990)

K.M. Emmons et al.

Am. J. Public Health

(1992)

G. Apseloff et al.

Clin. Pharmacol. Ther.

(1994)

S.E. Winders et al.

Psycopharmacology

(1998)

V. Haufroid et al.

Int. Arch. Occup. Environ. Health

(1998)

N.L. Benowitz

Epidemiol. Rev.

(1996)

Cited by (115)

Feasibility of mailed biomarker data collection among U.S. young adults: Saliva-based cotinine and self-reported nicotine use
2023, Drug and Alcohol Dependence
Nationally representative self-report studies are the standard for data on the prevalence of substance use. Newly emerging biomarker assessments can add objective measurements of exposure. However, biomarker assessment has typically depended on in-person sample collection. The current study examined whether young adults in a national sample would be willing and able to provide a saliva sample via mail, and the correspondence of cotinine in the saliva sample with self-reported vaping and smoking.
Data collection for the Monitoring the Future (MTF) Vaping Supplement was from September to November 2020. Eligible participants (N = 4358) were selected from a nationally-representative sample of US 12th-grade students in MTF in spring 2019. The MTF Vaping Supplement surveyed individuals nationally about one year after the 12th grade MTF survey (in 2020, mean age = 19.6 years; N = 1244). Survey weights accounted for design and attrition.
Of those surveyed, 66.2% consented to provide a saliva sample and, of those, 73.8% mailed a sample. There were no significant differences in providing a saliva sample across any demographic characteristic, but those who reported nicotine use were less likely to provide a sample. Cotinine cut-off measures of > 3 ng/mL and > 10 ng/mL had good correspondence with self-reported measures.
Results support the feasibility of collecting saliva via the mail in a national sample and the validity of data collected in this way. These findings support future research innovations to expand existing survey research protocols to include biomarker data collection in representative samples of young adults.
Development of an Ultra-High Performance Liquid Chromatography method for the simultaneous mass detection of tobacco biomarkers in urine
2022, Journal of Chromatography B: Analytical Technologies in the Biomedical and Life Sciences
The quantification of tobacco exposure biomarkers is relevant to follow the patients’ tobacco use. They allow to discriminate between tobacco users, non-users, passive smokers, and nicotine products users, such as in nicotine replacement therapy. The aim of this study was to develop and validate a quantification method of tobacco biomarkers of choice – nicotine, cotinine, trans-3′-hydroxycotinine, anatabine and anabasine – in urine. The challenge was to develop an easy and rapid liquid chromatography method requiring only one extraction step and allowing simultaneous detections. Some methods are described in the literature but need specific investment in terms of instrumentation and users training. Here, the developed method had to be carried out with instrumentation easily accessible for medical laboratories. The extraction of the analytes was performed by Supported Liquid Extraction (SLE), which consists in liquid–liquid extraction but supported by a sorbent. It allows to insure efficient neutrals extraction with less organic solvent and without any emulsion formation. 200 µl of basified urine – analytes of interest are neutral in this condition – were loaded on Novum SLE 96-Well Plates (Phenomenex) and analytes were eluted with 1 % formic acid in dichloromethane/propan-2-ol (95/5). After solvent evaporation, samples were reconstituted with 100 µl of water for injection. A mass detector (QDa, Waters) was used to detect analytes, this pre-optimised quadrupole mass analyser being less expensive and requiring less adjustments than traditional mass spectrometers while benefiting of the reliability of mass spectral data. This detector was integrated after an Ultra-high performance liquid chromatography (UHPLC) separation on a BEH C18 column (Waters) at a flow rate of 0.5 ml/min. A gradient elution of H₂O (pH 10 with NH₄OH) and CH₃CN was used. Finally, the developed method was validated. This new method is conclusive to assess the patients’ tobacco exposure and is easy to implement in medical laboratories.
Unlocking the potential of forensic traces: Analytical approaches to generate investigative leads
2022, Science and Justice
Forensic investigation involves gathering the information necessary to understand the criminal events as well as linking objects or individuals to an item, location or other individual(s) for investigative purposes. For years techniques such as presumptive chemical tests, DNA profiling or fingermark analysis have been of great value to this process. However, these techniques have their limitations, whether it is a lack of confidence in the results obtained due to cross-reactivity, subjectivity and low sensitivity; or because they are dependent on holding reference samples in a pre-existing database. There is currently a need to devise new ways to gather as much information as possible from a single trace, particularly from biological traces commonly encountered in forensic casework. This review outlines the most recent advancements in the forensic analysis of biological fluids, fingermarks and hair. Special emphasis is placed on analytical methods that can expand the information obtained from the trace beyond what is achieved in the usual practices. Special attention is paid to those methods that accurately determine the nature of the sample, as well as how long it has been at the crime scene, along with individualising information regarding the donor source of the trace.
Recent advancements in sampling, power management strategies and development in applications for non-invasive wearable electrochemical sensors
2022, Journal of Electroanalytical Chemistry
Wearable electrochemical sensors that can detect chemical analytes non-invasively are a fast developing next-generation digital-health technology. Because of their excellent performance, intrinsic compactness, and low cost, electrochemical-based sensors are capable of detecting biomarkers through a non-invasive approach for the continuous monitoring of real-time health status, which holds a lot of potential as wearable sensors for a tremendous promise for a plethora of applications. These wearable electrochemical sensors have been incorporated into various materials systems and even directly on the epidermis for different monitoring purposes because of their unique potential to process chemical analytes in a minimal/non-invasive and non-obtrusive manner. With ongoing innovation and a focus on critical challenges, such minimal/non-invasive electrochemical biosensors are anticipated to open up a new exciting path in the field of wearable wireless sensing devices and body-sensor networks, and thus find abundant use in a wide range of personal healthcare monitoring as well as in sport and military applications. Hence, this review article examines current advancements and discoveries in the field of wearable biosensors, with a focus on a subset of these devices that can conduct very sensitive electrochemical analysis. Recent insights into novel sampling strategies, various electrochemical sensing mechanisms and power management techniques have been discussed in detail in this review article. Finally, present unmet challenges and opportunities in wearable electrochemical biosensors are explored to motivate future technological breakthroughs.
Electrochemical determination of nicotine in smokers’ sweat
2020, Microchemical Journal
In this work, we report the demonstration of the capability of electrochemical sensors to provide measurements directly on the sweat of volunteers to successfully discriminate heavy smokers by light ones. Since its importance as pharmaceutical treatment-agent for nicotine replacement therapy for purposes of tobacco cessation, and since that is the key compound for the development of reduced-risk cigarettes by tobacco industry, nicotine is one of those markers that are of interest for monitoring in human sweat. Especially in therapies for purposes of smoking cessation, easy methods for an easy measure of nicotine on the patients is highly required for a fine adjustment of the nicotine dosage as release, e.g., by through therapeutic patches. Therefore, we have checked the use of a simple, inexpensive and very sensitive electrochemical sensor for quantification of nicotine in smokers’ sweat. Proposed sensor is capable for fast, label-free and pretreatment-free detection of nicotine in human sweat as collected directly from the skin of heavy and light smokers. Our finding demonstrates as we can detect nicotine from human skin with sweat-collecting patches thanks to its lower detection limit (0.59 µM) as compared to the physiological limit of nicotine in human blood (12.33 µM), while keeping under control the selectivity with respect various interfering compounds find in sweat too.
Biocompatible SPME fibers for direct monitoring of nicotine and its metabolites at ultra trace concentration in rabbit plasma following the application of smoking cessation formulations
2020, Journal of Chromatography A
The ultra-trace determination of nicotine and its 4 major metabolites (cotinine, nornicotine, norcotinine and anabasine) from rabbit plasma was achieved by a newly developed solid phase microextraction–liquid chromatography–tandem mass spectrometry method. Extraction of the target analytes was performed with hydrophilic/lipophilic balance-polyacrylonitrile SPME fibers. Dual fiber extraction was necessary to guarantee improved recovery at parts-per-trillion levels. Liquid chromatographic analysis was achieved in a 6-min run using a C18 (1.9 µm C18, 50 mm x 2.1 mm) column with a mobile phase flow rate of 0.4 mL/min. Tandem mass spectrometry was used for detection and quantification in positive electrospray ionization (ESI+) mode for all the targeted analytes. Two stable isotope-labeled internal standards were used for signal correction and accurate quantification. The mass spectrometer with laminar flow ion flux transport, guaranteed improved signal stability, minimal contamination of the ion guide and reproducibility into the first quadrupole analyzer. The method was validated in line with the Food and Drug Administration (FDA) guidelines for bioanalytical method validation. The results met the acceptance criteria as proposed by the FDA: accuracy was tested at 0.35, 10 and 75 µg L ⁻ ¹ and ranged between 98.3–112.2% for nicotine, 94.1–101.9% for cotinine, 94.7–107.0% for nornicotine, 81.1–107.2% for norcotinine and 94.3–115.2% for anabasine, with precision up to 14.2%. Stability tests indicated that all the targeted analytes were stable in the desorption solution for at least 1 week. LOQs ranged from 0.05 to 1 µg L⁻¹. The method was successfully applied to analyze plasma samples obtained from rabbits following transdermal application of a smoking cessation formulation loaded with solid lipid nanoparticles containing a nicotine-stearic acid conjugate.

View all citing articles on Scopus

View full text

Measuring tobacco smoke exposure: quantifying nicotine/cotinine concentration in biological samples by colorimetry, chromatography and immunoassay methods

Abstract

Introduction

Section snippets

Nicotine metabolites

Biomarkers for assessing smoking status

Collection of body fluids

Methods for the extraction of biomarkers

Colorimetry

Cortisol

Conclusion

Acknowledgements

J. Pediatr. Health Care

Prev. Med.

Lancet Oncol.

Lancet

Respir. Physiol.

Prev. Med.

J. Chronic Dis.

Toxicol. Lett.

J. Pediatr.

J. Chromatogr. Biomed. Appl.

J. Chromatogr. B Anal. Echnol. Biomed. Life Sci.

J. Clin. Epidemiol.

Drug Alcohol Depend.

Prev. Med.

Clin. Biochem.

J. Chromatogr. Biomed. Appl.

J. Chromatogr. B Anal. Technol. Biomed. Life Sci.

J. Chromatogr. B

J. Chromatogr. B Biomed. Sci. Appl.

J. Pharm. Biomed. Anal.

J. Chromatogr. B. Anal. Technol. Biomed. Life Sci.

J. Chromatogr.

J. Chromatogr.

J. Chromatogr.

Int. J. Environ. Health Res.

Monaldi Arch. Chest Dis.

J.R. Soc. Health

J. Public Health Policy

Vital Health Stat.

Geriatrics

Jpn. J. Cancer Res.

Am. J. Med. Sci.

Br. Med. J.

Int. J. Cancer

J. Natl. Cancer Inst.

Cancer Res.

Cancer Res.

J. Hypertens. Suppl.

Ann. Behav. Med.

Int. J. Clin. Pharmacol. Ther.

JAMA

Br. J. Clin. Pharmacol.

Acta Pediatr.

Arch. Environ. Health

Am. J. Public Health

Clin. Pharmacol. Ther.

Psycopharmacology

Int. Arch. Occup. Environ. Health

Epidemiol. Rev.