Smoking is a major cause of morbidity and mortality worldwide. The adverse health effects of chronic cigarette smoke exposure are widely known. Active smoking increases the risk of developing several pathologies including pulmonary disease, cardiovascular disease and cancer. Importantly, the sequelae of smoking also extend to non-smokers via frequent passive inhalation. Accurate measures of cigarette smoke exposure then are required to draw meaningful conclusions about the healthcare risks to both smokers and non-smokers. Cotinine is the major primary metabolite of nicotine and is the biochemical marker of choice for measuring exposure to cigarette smoke.
by Dr A. Dunlop, Dr B. L. Croal and J. Allison
Background
Chronic exposure to tobacco products is amongst the leading causes of preventable morbidity and mortality worldwide, being responsible for approximately 6 million deaths per annum [1]. Typically this involves inhalation of cigarette smoke which contains in excess of 5000 different chemicals; many of these are known toxins and carcinogens [2]. Upon inhalation of cigarette smoke, nicotine is transported to the lungs within tar droplets, dissolving in the alveolar fluid, and is then absorbed into the bloodstream. Following entry into the pulmonary circulation, nicotine quickly travels to the brain – within a matter of seconds – and exerts its pharmacological effects [3].
Nicotine is the addictive component of tobacco products, stimulating dopamine release in the brain and leading to heightened feelings of pleasure and reward [4]. In active smokers this nicotine dependence sustains chronic exposure to the toxins present in cigarette smoke [5]. Active smokers are therefore at increased risk of developing multiple pathologies including pulmonary disease, cardiovascular disease and cancer [6, 7]. Importantly, non-smokers are also at increased risk via involuntary or passive/second-hand smoke (SHS) exposure [8, 9]. Children are particularly susceptible to involuntary exposure, mainly occurring in enclosed spaces such as the parental home/car, via maternal smoking or passive exposure during pregnancy [10]. The adverse health effects of SHS exposure in children include increased risk of miscarriage, sudden infant death syndrome, lower respiratory tract infections, asthma and invasive meningococcal disease [10].
In addition, an emerging area of interest surrounds involuntary exposure via so-called third-hand smoke (THS). THS is a term used to describe the deposits of tobacco smoke that accumulate on surfaces, objects and in dust particles, persisting long after the dispersal of cigarette smoke. There is some evidence to suggest that atmospheric reactions may lead to re-release of smoke-derived toxins into the environment [11]. However, the health risks of THS are not yet known and remain the subject of ongoing research [12].
Assessing cigarette smoke exposure
The healthcare risks associated with cigarette smoking and SHS exposure ensure that smoking status should always be included in any routine clinical assessment. Monitoring of smoking status may also be indicated in specific circumstances, such as epidemiological studies, smoking cessation programmes, lung transplant patients, employee and health/life insurance screening. The most convenient and cost-effective means of assessing cigarette smoke exposure is by self-report. This may occur either during face-to-face consultation with healthcare professionals or often as part of a generic healthcare questionnaire. However self-report is frequently unreliable in estimating smoking status [13].
Moreover, the risk and extent of SHS exposure to non-smokers cannot be adequately assessed using these methods. For example, self-report cannot reliably quantify exposure in those who co-habit and/or socialise with smokers nor can it inform on fetal exposure in maternal smoking. Consequently, cigarette smoke exposure should be accurately quantified by measuring biomarkers to draw meaningful conclusions between smoking status and health outcomes [14, 15].
Biomarkers of cigarette smoke exposure
Numerous biomarkers have been examined in the analysis of cigarette smoke exposure, e.g. carbon monoxide, carboxyhaemoglobin, thiocyanate and polycyclic aromatic hydrocarbons [4]. However, many are non-specific for tobacco use and contribution from other environmental or dietary sources can cause interference [4]. In contrast, nicotine is a more specific marker of cigarette smoke exposure, being derived solely from tobacco [3]. Biochemical measurements of nicotine and its metabolites then are typically used to provide reliable measures of cigarette smoke exposure. Nicotine largely undergoes hepatic metabolism (with a half-life of approximately 2 h) and the plasma of active smokers typically contains 10–50 ng/mL of nicotine [3]. Cotinine is the major breakdown product of nicotine accounting for around 80% of all metabolites [3]. The half-life of cotinine, at around 16 h, is substantially longer than nicotine and plasma levels in active smokers are approximately 250–300 ng/mL [4]. Consequently, cotinine is the preferred biomarker for measuring cigarette smoke exposure.
Quantifying cotinine in biological matrices
A variety of methods have been developed for quantification of cotinine in several biological matrices including urine, blood, saliva and hair [14, 15]. There is good agreement between cotinine levels in plasma/serum and saliva, whilst levels in urine are typically higher [15].
Immunoassay methods have traditionally been used for the detection of cotinine in urine, offering rapid turnaround with minimal sample preparation. In addition, commercially available immunoassay kits are easily integrated into most core automated analysers available in modern clinical laboratories. However, reagent costs are typically high and it would be fair to say that immunoassays may be susceptible to cross-reactivity with other nicotine and cotinine-derived metabolites and thus may be of questionable accuracy [16, 17].
Gas chromatography–mass spectrometry (GC-MS) methods are also available; although sample preparation is typically labour intensive and time consuming, proving impractical for high sample throughput. Not surprisingly, liquid chromatography-tandem mass spectrometry (LC-MS/MS) methods have emerged as the sine qua non for quantification of cotinine in biological fluids.
LC-MS/MS analyses
Liquid chromatography–tandem mass spectrometry (LC-MS/MS) affords the requisite specificity and sensitivity to detect and quantify cotinine at levels encountered throughout the spectrum of cigarette smoke exposure. The majority of recently published methods now routinely quote lower limits of quantification (LLOQ) in the region of <0.5 ng/mL, in both plasma/serum and in urine [15]. Cut-points to distinguish smokers from non-smokers have been variously proposed from 12 ng/mL down to 3 ng/mL, depending on the population [15]. Nevertheless, regular active smokers can be expected to have serum/plasma cotinine levels in marked excess of 100 ng/mL, although non-smokers are usually comfortably below 10 ng/mL.
The majority of LC-MS/MS methods for cotinine have been developed in-house, an important advantage compared with immunoassay techniques. This not only affords flexibility in the choice of matrix to be analysed but also permits the inclusion of more than one analyte in the assay. Thus nicotine, cotinine and various metabolites thereof may be detected in a multiplexed assay. Published guidelines are also widely available to assist in the development and validation of LC-MS/MS methods [18]. The advent of enhanced chromatographic separation techniques, such as ultra-performance liquid chromatography (UPLC), has significantly shortened run times thereby facilitating higher sample throughput. Development of uncomplicated sample preparation procedures has further simplified analyses.
For example, in our own laboratory we recently developed a rapid and straightforward UPLC-MS/MS protocol for the determination of cotinine in plasma (Fig. 1) [19]. Analytical run time was 4 min per sample with a LLOQ of 0.2 ng/mL and the assay was linear from 0.5 to 1000 ng/mL; comfortably covering the concentration range of active and non-smokers (Fig. 2). A simple 5-step automated SPE process was also developed, permitting minimal sample handling and using only water and methanol, both cheap and readily available. To date we have successfully deployed this method for the analyses of two large patient cohorts (each comprising several hundred samples) associated with independent epidemiological studies.
Although the initial outlay for equipment is high, thereafter LC-MS/MS assays can be run relatively cheaply using readily available inexpensive solvents. Furthermore, sample preparation procedures can usually be streamlined/simplified and therefore easily adapted for high-throughput analyses [19]. Matrix effects, chiefly ion suppression, are a particular disadvantage of LC-MS/MS techniques; however careful consideration and troubleshooting during method development can often overcome this issue [20].
Conclusions and future directions
Despite widespread awareness of the adverse effects of tobacco use and increasing public health initiatives to combat this, cigarette smoking continues to be a major global cause of morbidity and mortality and is likely to remain so for the foreseeable future. Accurate quantification of cigarette smoke exposure via biomarkers is therefore an important measure in stratifying the risk of both active and non-smokers.
The need to quantify ever decreasing amounts of nicotine, cotinine and their metabolites in monitoring exposure to tobacco products ensures that LC-MS/MS techniques and modifications thereof remain at the forefront of detection methods in this field. Similarly, as new biomarkers become available which inform on the detrimental health effects of smoking these methods are ideally placed to keep pace, both in research and in clinical laboratories.
The recent emergence of electronic cigarette devices (e-cigarettes) is currently the subject of much debate. E-cigarettes typically deliver nicotine in a vapour generated via heating a liquid that also contains propylene glycol and other additives e.g. flavouring [21]. Exponents propose e-cigarettes as a safer alternative to smoking associated with tobacco combustion and promote the benefits for smoking cessation. However, some healthcare professionals believe that while e-cigarettes are safer, they may still act as a gateway or as a way of prolonging or even enhancing dependency on nicotine. In addition, the long-term health effects of these products are unknown, as is the need to monitor biomarkers such as nicotine and/or cotinine in so-called ‘e-smokers’.
References
1. WHO report on the global tobacco epidemic 2013. http://www.who.int/tobacco/global_report/2013/en/
2. Talhout R, et al. Hazardous compounds in tobacco smoke. Int J Environ Res Public Health 2011; 8(2): 613–628.
3. Hukkanen J, et al. Metabolism and disposition kinetics of nicotine. Pharmacol Rev. 2005; 57(1): 79–115.
4. Benowitz NL, et al. Nicotine chemistry, metabolism, kinetics and biomarkers. Handb Exp Pharmacol. 2009; 192(192): 29–60.
5. Berrendero F, et al. Neurobiological mechanisms involved in nicotine dependence and reward: participation of the endogenous opioid system. Neurosci Biobehav Rev. 2010; 35(2): 220–231.
6. Doll R, et al. Mortality in relation to smoking: 50 years’ observations on male British doctors. BMJ (Clinical research ed.). 2004; 328(7455): 1519.
7. Jha P. Avoidable global cancer deaths and total deaths from smoking. Nature reviews.Cancer. 2011; 9(9): 655–664.
8. Scientific Committee on Tobacco and Health. Secondhand smoke: Review of evidence since 1998. Update of evidence on health effects of secondhand smoke. Department of Health, UK 2004. http://www.smokefreeengland.co.uk/files/scoth_secondhandsmoke.pdf
9. Vardavas CI, Panagiotakos DB. The causal relationship between passive smoking and inflammation on the development of cardiovascular disease: a review of the evidence. Inflamm Allergy Drug Targets 2009; 8(5): 328–333.
10. Action on Smoking and Health. Research Report. Secondhand smoke: the impact on children. March 2014. http://www.ash.org.uk/files/documents/ASH_596.pdf
11. Sleiman M, et al. Formation of carcinogens indoors by surface-mediated reactions of nicotine with nitrous acid, leading to potential thirdhand smoke hazards. PNAS 2010; 107(15): 6576–6581.
12. Matt GE, et al. Thirdhand tobacco smoke: emerging evidence and arguments for a multidisciplinary research agenda. Environ Health Perspect. 2011; 119(9): 1218–1226.
13. Connor Gorber S, et al. The accuracy of self-reported smoking: a systematic review of the relationship between self-reported and cotinine-assessed smoking status. Nicotine Tob Res. 2009; 11(1): 12–24.
14. Florescu A, et al. Methods for quantification of exposure to cigarette smoking and environmental tobacco smoke: focus on developmental toxicology. Ther Drug Monitg. 2009; 31(1): 14–30.
15. Avila-Tang E, et al. Assesing secondhand smoke using biological markers. Tob Control 2013; 22: 164–171.
16. Schepers G, Walk RA. Cotinine determination by immunoassays may be influenced by other nicotine metabolites. Arch Toxicol. 1988; 62(5): 395–397.
17. Tate J, Ward G. Interferences in immunoassay. Clin Biochem Rev. 2004; 25(2): 105–120.
18. Honour JW. Development and validation of a quantitative assay based on tandem mass spectrometry. Ann Clin Biochem. 2001; 48(2): 97–111.
19. Dunlop AJ, et al. Determination of cotinine by LC-MS-MS with automated solid-phase extraction. J Chromatogr Sci. 2014; 52(4): 351–356.
20. Matuszewski BK, et al. Strategies for the assessment of matrix effect in quantitative bioanalytical methods based on HPLC-MS/MS. Anal Chem. 2003; 75(13): 3019–3030.
21. Grana R, et al. E-cigarettes: a scientific review. Circulation 2014; 129(19): 1972–1986.
The authors
Allan Dunlop1* PhD, Bernard Croal2 MD and James Allison2 BSc
1Department of Clinical Biochemistry Laboratory, Southern General Hospital, Glasgow G51 4TF, UK
2Department of Clinical Biochemistry, Aberdeen Royal Infirmary, Aberdeen AB25 2ZD, UK
*Corresponding author
E-mail: allandunlop@nhs.net
Tandem mass spectrometer LCMS-8040
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, /in Featured Articles /by 3wmediaViral hepatitis, the silent epidemic
, /in Featured Articles /by 3wmediaGlobally as many people (1.5 million) die each year from viral hepatitis as from HIV/AIDS, but whereas the latter viral disease attracts government and international action and funding, the former is comparatively neglected. It was for this reason that the WHO initiated World Hepatitis Day four years ago, to be observed on the 28th July each year, and the lack of awareness about the repercussions of viral hepatitis was reflected in this year’s theme of ‘Hepatitis: think again’. So far five hepatitis viruses have been identified, though Hepatitis D is only found as a co-infection with B. Whilst the acute infections that food- and water-borne Hepatitis A and E cause are not insignificant in terms of their incidence, morbidity and mortality, it is Hepatitis B and C (HBV and HCV), that are generating a global public health crisis.
These two viral infections have major characteristics in common with HIV/AIDS. The acute infection, acquired by exposure to infectious blood and other body fluids as well as by sexual and vertical transmission, is frequently asymptomatic in the case of HCV. Acute infections can be followed by a period of clinical latency and thus the unwitting transmission of the virus to others. Though such chronic infections with HBV are very uncommon in healthy adults, they occur in over half of young children infected; between 75% – 85% of people infected with HCV develop a chronic infection. After years or even several decades of chronic, asymptomatic infection, cirrhosis of the liver and hepatocellular carcinoma can result. The WHO estimates that there are around 780,000 deaths from acute and chronic HBV infection, and more than 350,000 from chronic HCV infection annually. Even more alarming is that currently 500 million people are chronically infected with either HBV or HCV.
As is the case with HIV/AIDS, avoiding exposure to infectious blood and semen and diagnostic testing of asymptomatic people can help to contain the global viral hepatitis epidemic. However, now the pertinent characteristics of the disease have been elucidated, it should be far more feasible to control viral hepatitis than HIV/AIDS, a disease for which there is no vaccine and no drugs that actually eradicate the virus. There is a highly effective vaccine for HBV, though approved drugs help prevent serious
liver damage but don’t eliminate the virus. Drugs are now available that can eradicate the HCV virus, and clinical trials are currently testing
a vaccine for chronically infected people.
“Hepatitis: think again”. With appropriate education and adequate national and international funding, this looming global health crisis could be averted.
Liquid chromatography-tandem mass spectrometry: an introduction
, /in Featured Articles /by 3wmediaThe use of liquid chromatography-tandem mass spectrometry for clinical analysis is on the increase. This article describes what it is, why it can offer significant improvements over traditional assays and the limitations to be aware of.
by Dr N. Homer
Introduction
Clinical biochemistry laboratories frequently use radioimmunoassays (RIA) and enzyme-linked immunosorbent assays (ELISA) for analysis of blood and urine. However, these techniques are plagued by issues of cross-reactivity and are only suited to look at one analyte at a time [1]. The use of mass spectrometry (MS) techniques has increased since 2007 when the American Endocrine Society recognized the importance of tandem MS and issued a statement recommending the use of liquid chromatography-tandem mass spectrometry (LC-MS/MS) for the determination of endogenous steroid hormones over more traditional technologies such as immunoassays [2]. This has led to the widespread adoption of LC-MS/MS in clinical biochemistry laboratories, in direct response to this recommendation.
What is liquid chromatography-mass spectrometry?
Mass spectrometry is a technique that measures charged molecules or ions in the gaseous state. Samples are introduced into an ion source, ionized and then separated in a mass analyser according to their mass-to-charge ratio (m/z) and then characterized by their relative abundances. Coupled to chromatographic separation techniques such as gas chromatography (GC) or liquid chromatography (LC), MS is considered to be the ‘gold standard’ for validation of quantitative analytical assays. An overview of how a typical chromatograph-mass spectrometer is set up is shown in Figure 1.
Following separation by a chromatography system the sample is introduced into an ion source at the front end of the mass spectrometer. Ionization modes include atmospheric pressure ionization (API), such as electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI), and matrix-assisted laser desorption/ionization (MALDI). ESI is most typically used to ionize the biomolecules encountered in clinical samples.
Once ionized the mass analyser separates the ions according to their m/z. Mass analysers include magnetic or electric sectors, time-of-flight (ToF) tubes, quadrupoles and two-dimensional and three-dimensional ion traps. Softer ionization techniques, which generally leave the molecule intact, such as ESI, have led to the use of quadrupole mass analysers. These consist of four parallel rods or poles, generally of hyperbolic cross-section, through which ions are passed and separated.
Tandem mass spectrometry (often termed MS/MS) technology increases the specificity of MS significantly. There are a number of modes that a tandem mass spectrometer can be operated under, depending on the requirement of the experiment (Fig. 2). Tandem MS requires two or more mass analysers to be placed in sequence and the ions are fragmented in a collision cell to give structural information. Trace analysis of complex biological matrices is ideally suited to tandem MS instruments, operated in selected reaction monitoring (SRM) mode. In addition, linear ion traps as the third mass analyser are also increasing in popularity as they offer additional structural identification and specificity.
Sample preparation and liquid chromatography method development
Clinical samples are complex biological matrices and contain interferences that can lead to so-called matrix effects within the mass spectrometer. For validated assays, samples are prepared by addition of an internal standard followed by extraction to remove as much of the interferences as possible. The internal standard is either a closely related analogue of the compound of interest, or a stable isotope labelled version of the compound, enriched with at least two atoms of 13C, 2H or 15N.
Sample preparation methods commonly applied to clinical samples include protein precipitation with an organic solvent, liquid–liquid extraction (LLE) or solid-phase extraction (SPE). If sample clean-up is not sufficient it can lead to matrix effects, including ion suppression of the analyte, usually observed as a loss of response. This affects the detection limits, accuracy and precision of the assay. Various ion suppression tests have been developed and these are an important part of the method validation set-up required for clinical MS assays. The two most effective ways of avoiding ion suppression are improved sample extraction and optimized chromatographic selectivity.
On-line multidimensional chromatography technology allows an unextracted sample to be introduced into the chromatography apparatus and can lead to faster analysis. These systems generally consist of multi-channel switching valves, on-line SPE cartridges and analytical columns ahead of ESI-LC-MS/MS. Steroids and isoprostanes are often analysed in this manner [3, 4].
Liquid chromatography
Once prepared, a sample is introduced into the LC system which consists of a pump and an analytical column. The purpose of the chromatography system is to separate the components of the sample as much as possible, before introduction into the mass spectrometer. Analytical LC columns are stainless steel tubes that are packed with tiny silica beads. The type of LC used in clinical analysis is usually reversed-phase chromatography as the silica beads are generally chemically modified. Typically, samples are introduced onto the column in a highly aqueous phase, the analytes associate with the chemically-modified packed silica beads and are washed off the column with a high organic solvent such as methanol or acetonitrile.
Once the ionization and mass spectrometer parameters have been optimized, much of the method development falls to the chromatography and the importance of this stage should not be underestimated. It is imperative that co-eluting compounds do not interfere with the analytical peaks of interest. In recent years there has been a trend for fast analysis in LC-MS/MS; however, this does not always give a robust assay. In addition, it is important to be aware of isobaric compounds (same mass) and [M+2] isotopomers. An example of isotopomers is that of the stress hormone cortisol (m/z 363) and its inactive form cortisone (m/z 361). In an LC-MS/MS assay for cortisol it is essential to have two separate peaks for cortisol and cortisone, otherwise the risk of isotopomers of cortisone contributing to cortisol would lead to an over-estimation of cortisol in the sample (Fig. 3).
There are a number of parameters that can be altered in LC and these in turn alter the selectivity of the column, that is the order and rate at which the components elute. Parameters that can be influenced include column temperature, mobile phase pH, composition and flow rate, column dimensions, column particle size and the nature of course the chemical modification of the particles too.
Analytical LC column technology is continuously improving. The better the resolution, which is simply how well separated each peak is, the better the assay. Sub-2 µm particles have been introduced in the past decade, which generate sharp peaks and excellent resolution with improved capacity over the more traditional 3–5 µm particles. However, the smaller particle size leads to high backpressure and requires specific LC pumps that can withstand these ultra-high pressures (UHPLC, ultra-high performance LC). To reduce the need for new instrumentation, LC columns packed with fused-core particles ~2.5 µm have been developed to allow separation comparable to sub-2 µm particles. The backpressure generated by these fused-core particles is significantly less than the sub-2 µm particle columns and exclude the requirement of high-pressure capable LC pumps and fittings.
Considerations when establishing an LC-MS/MS clinical biochemistry method
As with all techniques, there are drawbacks to LC-MS/MS. The instrumentation and software can be complex and requires regular maintenance, although manufacturers are addressing this perception by introducing simpler software interfaces with dedicated instrument support for method development and even fool-proof methods, guaranteed by the provider. Also, some compounds are not amenable to ionization due to their chemical nature, but chemical modification before analysis can improve the chance of ionization efficiency, so all is not lost.
Summary
The benefits that MS offers over other traditional assay techniques have seen an increase in the number of assays using this methodology. The analysis of steroid hormones by MS is a well-documented area. Other commonly encountered uses include newborn screening for congenital metabolic diseases such as aminoacidopathies and fatty acid oxidation disorders, multi-analyte therapeutic drug monitoring, oncology drugs, anti-virals, toxicants and drugs of abuse screening and analysis of endogenous peptides [3, 4, 5].
One area that is continuing to gain interest in clinical research is high-resolution MS (HRMS) [5]. This allows for accurate mass determination over a defined mass range, which differs from the targeted analysis approach used by triple quadrupole MS. With technological improvement in the linear range of HRMS instruments to match that of triple quadrupoles, it seems likely that the benefits of HRMS will also be exploited by the clinical biochemistry field, in addition to LC-MS/MS analysis.
The range of clinical applications of MS outlined is broad and constantly expanding. Much research is being conducted in the pioneering fields of proteomics and metabolomics. In recent years the emergence of imaging mass spectrometry also offers exciting possibilities for the future and there is no doubt that MS will continue to feature heavily in the clinical biochemistry laboratory and function as an important clinical research tool.
References
1. Penning TM, et al. Liquid chromatography-mass spectrometry (LC-MS) of steroid hormone metabolites and its applications. J Ster Biochem Mol Biol. 2010; 121: 546–555.
2. Rosner W, et al. Position statement: utility, limitations, and pitfalls in measuring testosterone: an Endocrine Society position statement. J Clin Endocrinol Metab. 2007; 92: 405–413.
3. Shushan B. A review of clinical diagnostic applications of liquid chromatography-tandem mass spectrometry. Mass Spectrom Rev. 2010; 29: 930–944.
4. Chace DH, et al. Use of tandem mass spectrometry for multianalyte screening of dried blood specimens from newborns. Clin Chem. 2003; 49: 1797–1817.
5. Jiwan J-LH, et al. HPLC-high resolution mass spectrometry in clinical laboratory? Clin Biochem. 2011; 44: 136–147.
The author
Natalie Homer PhD
CRF Mass Spectrometry Core Laboratory, Queen’s Medical Research Institute, University of Edinburgh
E-mail: n.z.m.homer@ed.ac.uk
Measuring cigarette smoke exposure: quantification of cotinine by LC-MS/MS
, /in Featured Articles /by 3wmediaSmoking is a major cause of morbidity and mortality worldwide. The adverse health effects of chronic cigarette smoke exposure are widely known. Active smoking increases the risk of developing several pathologies including pulmonary disease, cardiovascular disease and cancer. Importantly, the sequelae of smoking also extend to non-smokers via frequent passive inhalation. Accurate measures of cigarette smoke exposure then are required to draw meaningful conclusions about the healthcare risks to both smokers and non-smokers. Cotinine is the major primary metabolite of nicotine and is the biochemical marker of choice for measuring exposure to cigarette smoke.
by Dr A. Dunlop, Dr B. L. Croal and J. Allison
Background
Chronic exposure to tobacco products is amongst the leading causes of preventable morbidity and mortality worldwide, being responsible for approximately 6 million deaths per annum [1]. Typically this involves inhalation of cigarette smoke which contains in excess of 5000 different chemicals; many of these are known toxins and carcinogens [2]. Upon inhalation of cigarette smoke, nicotine is transported to the lungs within tar droplets, dissolving in the alveolar fluid, and is then absorbed into the bloodstream. Following entry into the pulmonary circulation, nicotine quickly travels to the brain – within a matter of seconds – and exerts its pharmacological effects [3].
Nicotine is the addictive component of tobacco products, stimulating dopamine release in the brain and leading to heightened feelings of pleasure and reward [4]. In active smokers this nicotine dependence sustains chronic exposure to the toxins present in cigarette smoke [5]. Active smokers are therefore at increased risk of developing multiple pathologies including pulmonary disease, cardiovascular disease and cancer [6, 7]. Importantly, non-smokers are also at increased risk via involuntary or passive/second-hand smoke (SHS) exposure [8, 9]. Children are particularly susceptible to involuntary exposure, mainly occurring in enclosed spaces such as the parental home/car, via maternal smoking or passive exposure during pregnancy [10]. The adverse health effects of SHS exposure in children include increased risk of miscarriage, sudden infant death syndrome, lower respiratory tract infections, asthma and invasive meningococcal disease [10].
In addition, an emerging area of interest surrounds involuntary exposure via so-called third-hand smoke (THS). THS is a term used to describe the deposits of tobacco smoke that accumulate on surfaces, objects and in dust particles, persisting long after the dispersal of cigarette smoke. There is some evidence to suggest that atmospheric reactions may lead to re-release of smoke-derived toxins into the environment [11]. However, the health risks of THS are not yet known and remain the subject of ongoing research [12].
Assessing cigarette smoke exposure
The healthcare risks associated with cigarette smoking and SHS exposure ensure that smoking status should always be included in any routine clinical assessment. Monitoring of smoking status may also be indicated in specific circumstances, such as epidemiological studies, smoking cessation programmes, lung transplant patients, employee and health/life insurance screening. The most convenient and cost-effective means of assessing cigarette smoke exposure is by self-report. This may occur either during face-to-face consultation with healthcare professionals or often as part of a generic healthcare questionnaire. However self-report is frequently unreliable in estimating smoking status [13].
Moreover, the risk and extent of SHS exposure to non-smokers cannot be adequately assessed using these methods. For example, self-report cannot reliably quantify exposure in those who co-habit and/or socialise with smokers nor can it inform on fetal exposure in maternal smoking. Consequently, cigarette smoke exposure should be accurately quantified by measuring biomarkers to draw meaningful conclusions between smoking status and health outcomes [14, 15].
Biomarkers of cigarette smoke exposure
Numerous biomarkers have been examined in the analysis of cigarette smoke exposure, e.g. carbon monoxide, carboxyhaemoglobin, thiocyanate and polycyclic aromatic hydrocarbons [4]. However, many are non-specific for tobacco use and contribution from other environmental or dietary sources can cause interference [4]. In contrast, nicotine is a more specific marker of cigarette smoke exposure, being derived solely from tobacco [3]. Biochemical measurements of nicotine and its metabolites then are typically used to provide reliable measures of cigarette smoke exposure. Nicotine largely undergoes hepatic metabolism (with a half-life of approximately 2 h) and the plasma of active smokers typically contains 10–50 ng/mL of nicotine [3]. Cotinine is the major breakdown product of nicotine accounting for around 80% of all metabolites [3]. The half-life of cotinine, at around 16 h, is substantially longer than nicotine and plasma levels in active smokers are approximately 250–300 ng/mL [4]. Consequently, cotinine is the preferred biomarker for measuring cigarette smoke exposure.
Quantifying cotinine in biological matrices
A variety of methods have been developed for quantification of cotinine in several biological matrices including urine, blood, saliva and hair [14, 15]. There is good agreement between cotinine levels in plasma/serum and saliva, whilst levels in urine are typically higher [15].
Immunoassay methods have traditionally been used for the detection of cotinine in urine, offering rapid turnaround with minimal sample preparation. In addition, commercially available immunoassay kits are easily integrated into most core automated analysers available in modern clinical laboratories. However, reagent costs are typically high and it would be fair to say that immunoassays may be susceptible to cross-reactivity with other nicotine and cotinine-derived metabolites and thus may be of questionable accuracy [16, 17].
Gas chromatography–mass spectrometry (GC-MS) methods are also available; although sample preparation is typically labour intensive and time consuming, proving impractical for high sample throughput. Not surprisingly, liquid chromatography-tandem mass spectrometry (LC-MS/MS) methods have emerged as the sine qua non for quantification of cotinine in biological fluids.
LC-MS/MS analyses
Liquid chromatography–tandem mass spectrometry (LC-MS/MS) affords the requisite specificity and sensitivity to detect and quantify cotinine at levels encountered throughout the spectrum of cigarette smoke exposure. The majority of recently published methods now routinely quote lower limits of quantification (LLOQ) in the region of <0.5 ng/mL, in both plasma/serum and in urine [15]. Cut-points to distinguish smokers from non-smokers have been variously proposed from 12 ng/mL down to 3 ng/mL, depending on the population [15]. Nevertheless, regular active smokers can be expected to have serum/plasma cotinine levels in marked excess of 100 ng/mL, although non-smokers are usually comfortably below 10 ng/mL. The majority of LC-MS/MS methods for cotinine have been developed in-house, an important advantage compared with immunoassay techniques. This not only affords flexibility in the choice of matrix to be analysed but also permits the inclusion of more than one analyte in the assay. Thus nicotine, cotinine and various metabolites thereof may be detected in a multiplexed assay. Published guidelines are also widely available to assist in the development and validation of LC-MS/MS methods [18]. The advent of enhanced chromatographic separation techniques, such as ultra-performance liquid chromatography (UPLC), has significantly shortened run times thereby facilitating higher sample throughput. Development of uncomplicated sample preparation procedures has further simplified analyses. For example, in our own laboratory we recently developed a rapid and straightforward UPLC-MS/MS protocol for the determination of cotinine in plasma (Fig. 1) [19]. Analytical run time was 4 min per sample with a LLOQ of 0.2 ng/mL and the assay was linear from 0.5 to 1000 ng/mL; comfortably covering the concentration range of active and non-smokers (Fig. 2). A simple 5-step automated SPE process was also developed, permitting minimal sample handling and using only water and methanol, both cheap and readily available. To date we have successfully deployed this method for the analyses of two large patient cohorts (each comprising several hundred samples) associated with independent epidemiological studies. Although the initial outlay for equipment is high, thereafter LC-MS/MS assays can be run relatively cheaply using readily available inexpensive solvents. Furthermore, sample preparation procedures can usually be streamlined/simplified and therefore easily adapted for high-throughput analyses [19]. Matrix effects, chiefly ion suppression, are a particular disadvantage of LC-MS/MS techniques; however careful consideration and troubleshooting during method development can often overcome this issue [20]. Conclusions and future directions
Despite widespread awareness of the adverse effects of tobacco use and increasing public health initiatives to combat this, cigarette smoking continues to be a major global cause of morbidity and mortality and is likely to remain so for the foreseeable future. Accurate quantification of cigarette smoke exposure via biomarkers is therefore an important measure in stratifying the risk of both active and non-smokers.
The need to quantify ever decreasing amounts of nicotine, cotinine and their metabolites in monitoring exposure to tobacco products ensures that LC-MS/MS techniques and modifications thereof remain at the forefront of detection methods in this field. Similarly, as new biomarkers become available which inform on the detrimental health effects of smoking these methods are ideally placed to keep pace, both in research and in clinical laboratories.
The recent emergence of electronic cigarette devices (e-cigarettes) is currently the subject of much debate. E-cigarettes typically deliver nicotine in a vapour generated via heating a liquid that also contains propylene glycol and other additives e.g. flavouring [21]. Exponents propose e-cigarettes as a safer alternative to smoking associated with tobacco combustion and promote the benefits for smoking cessation. However, some healthcare professionals believe that while e-cigarettes are safer, they may still act as a gateway or as a way of prolonging or even enhancing dependency on nicotine. In addition, the long-term health effects of these products are unknown, as is the need to monitor biomarkers such as nicotine and/or cotinine in so-called ‘e-smokers’.
References
1. WHO report on the global tobacco epidemic 2013. http://www.who.int/tobacco/global_report/2013/en/
2. Talhout R, et al. Hazardous compounds in tobacco smoke. Int J Environ Res Public Health 2011; 8(2): 613–628.
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The authors
Allan Dunlop1* PhD, Bernard Croal2 MD and James Allison2 BSc
1Department of Clinical Biochemistry Laboratory, Southern General Hospital, Glasgow G51 4TF, UK
2Department of Clinical Biochemistry, Aberdeen Royal Infirmary, Aberdeen AB25 2ZD, UK
*Corresponding author
E-mail: allandunlop@nhs.net