1. Introduction.
Tobacco use is a major preventable cause of premature death accounting about 5 million deaths worldwide annually, from cancer, cardiovascular and respiratory diseases to name but few. It poses a significant health risk not only to smokers, but also to those exposed to second-hand smoking (2,3). Serious health effects are reported both in adults and children with increased occurrence of ear and respiratory infections, low birth weight, asthma and sudden infant deaths (3). An accurate assessment of tobacco smoke exposure is critical to the understanding of the processes and prevention of those diseases.
Tobacco smoke contains approximately 4,000 toxic chemicals, including oxidative gases, heavy metals, cyanide and at least 50 carcinogens (4). Nicotine is of one of them and also the most abundant and potent pharmacological alkaloid, with numbers of negative health outcomes (e.g. locomotor activation, increased heart rate and blood pressure, nausea or appetite suppression) (5). Nicotine is also the primary addictive component of tobacco smoke. As it is a liquid soluble, it has a large distribution volume in the body (2-3 litres/kg) and easily penetrates cell membranes (1). It has a half-life of approximately 2-3 hours in the blood and about 5-10% of inhaled nicotine is excreted unchanged in urine, whereas the rest is metabolised in the liver. The major pathway of nicotine metabolism is its oxidation by hepatic cytochrome P450 2A6 to cotinine (80% of nicotine is transformed to the cotinine in that way) (6,5). The nicotine metabolite (cotinine) is the most widely used and considered the best current biomarker with excellent specificity for detecting both active and passive smokers (2,7).
2. Cotinine.
Cotinine is a major proximate metabolic of nicotine and it has become the biomarker of choice for tobacco smoking exposure. Cotinine can both be measured qualitatively (for presence or absence) or quantitatively (for actual concentration). Quantitative testing enables to distinguish between active smokers, those, who have recently quit, non-tobacco users, who have been exposed to significant environmental tobacco smoke and people not exposed at all. The absorbed dose of nicotine is best approximated by cotinine levels in the blood. As the cotinine values in all biological fluids are highly correlated, its levels can be accurately estimated by measuring cotinine in other matrices, such as saliva and urine (6).
Cotinine has longer half-life of 15-20h in different body fluids (plasma, urine and saliva) compared to nicotine (3,6). Because of that, cotinine is eliminated over a longer period of time than nicotine, which results in relatively constant levels of cotinine throughout the day compared to nicotine, which tends to oscillate. Due to this stability of cotinine in the blood over time and its ability to quantify long-term exposure, it became a preferred biomarker of second-hand exposure (6).
It is important to note, that the half-life of cotinine can be 2-3 times higher in infants and children (6,93). Also, there is an individual variation in the relation of cotinine levels in the body fluids and nicotine intake. This is related to the differences in metabolism of nicotine and cotinine and also in the clearance of cotinine itself (6,101) as every individual converts different amounts of nicotine to cotinine (usually between 50 and 90%) and metabolised it at its own rates (8). The cotinine metabolism can also be affected by the pregnancy, liver or kidney diseases, race, age, gender, genetic variation in liver enzyme CYPZAG and other factors (8(7)). Some studies show that plasma cotinine levels vary as much as two-fold due to individual differences in nicotine metabolism (10(3)).
2.1. Materials used in detection of cotinine.
Cotinine concentrations have been determined in a variety of biological matrices, including plasma, serum, urine, saliva, hair and toe nails. Also, use of dried blood spots to screen for metabolic disorders in new-borns have become a popular sampling method for the quantitation of small molecules in blood (3,(7,8)), including cotinine.
2.1. Cotinine in body fluids.
The commonly used body fluids to measure levels of cotinine are blood (serum, plasma), urine and saliva. Cotinine assays using these matrices can accurately distinguish between smokers and non-smokers (6) and also non-smokers exposed to second-hand smoking. The results reflect exposure over a few recent days (Joakkola and Joakkola, 1997).
Cotinine measured in saliva has been preferred method by many researchers as it is non-invasive and still it can discriminate well between active and passive smoking. Salivary cotinine correlates well with plasma cotinine and exceptionally well with serum cotinine, being approximately equal. In urine, because the kidney concentrates cotinine, its levels are several times higher than in saliva (6,47). As urine concentrations are generally much higher, its analyses can provide greater sensitivity for assessing low level exposure, compare to plasma and saliva (2). Urine cotinine also depends on its pH, renal functions and flow rate (6 (47)).
Detection of cotinine in body fluids is relatively easy, but there are some limitations in measurements at low levels of cotinine relevant to second hand smoking. As they have a short half-life in body fluids, they are able to reflect only recent exposure to smoke – up to 3 days preceding sample collection (6).
2.1.2. Cotinine in the hair.
Since 1983, when presence of nicotine in human hair was reported, a number of studies have investigated the use of hair for measuring exposure to nicotine and its metabolities (1). Hair analysis proved to be a good method as (????) is non-invasive, the samples are stable, can be collected easily and provide a wide window of detection (6). But the major advantage is its ability to quantify long-term exposure to tobacco (for up to several months) (6,7). As the average hair growth rate has been calculated as 1.1cm /month, bearing in mind variability related to gender, race and age (scalp grows faster in women than in men), each centimetre of scalp hair reflects approximately one month of past exposure. The recommended site for sample collection is the back of the scalp, where hairs have most uniform growth pattern. Scalp grows more quickly than pubic or axillary hair and 85-90% of it is continuously found in the growing stage. That continuous growth enables and provides updated information on exposure and the limited percentage of non-growing hair reduces variability of results (1). The poor correlation between hair cotinine and serum cotinine levels can be expected, because the serum levels depend on the time the blood sample was taken – whether it was during the peak serum level or after cotinine levels had dropped down – while cotinine levels in the hair are not related to such irregularities, because of the slow hair growth rate (1).
The mechanism of actual drug incorporation into hair is unsure. Some studies say that external substances like nicotine and its metabolities reach the hair through systematic circulation through the hair bulb blood supply. It is believed, that nicotine moves by passive diffusion from the bloodstream into the growing hair cells at the base of the follicle and then becomes tightly bound in the interior of the hair shaft during subsequent keratogenesis (1). Nicotine incorporation into hair is dependent on the average concentration in blood over time, which depends on the dose inhaled or ingested on the other hand (1). Other studies suggest, that drugs secreted from sweat, sebum or mucous glands, are incorporated into hair (6,136). In addition, drugs present in a vapour phase may be absorbed from the environment or may deposit on the external surface of the hair shaft. That is why in the laboratory all hair samples are washed away by cleansing solutions before analysis, to avoid contamination from nicotine externally bound to the hair shaft and false results (6).
Regardless of whether the uptake is systemic or external, nicotine and its metabolities are consistently incorporated in the hair shaft and are available for analysis and has been found to be well correlated with reports of exposure (1). However, cotinine levels in hair are much lower than nicotine levels and this may slightly reduce the detection of second-hand exposure to non-smokers (1).
As with other biomarkers (matrices??), the cotinine measured in the hair have some limitations, as hair cotinine may be affected by factors such as irregular hair growth, hair type and colour. Hair colour may influence the cotinine uptake. It was reported, that as nicotine have a higher affinity to melanin, it is found at higher concentrations in dark hair compared to white or fair hair (1,6). Also, studies of Pichini et al (6,146) found that in dyed and treated hair, the concentrations of cotinine are reduced. Chemical and physical treatments, such as strong hair detergents and permanent waves may influence the integrity of the hair shaft’s outer cuticle layer and cause leakage of cotinine from hair (1).
3. Methods for detection of cotinine.
A number of methods have been developed to measure cotinine in biological fluids and tissues. The four main techniques are colorimetry, chromatography, radioimmunoassay and enzyme- linked immunosorbent assay ( ELISA). Also, there are some various methods for sample preparation before analysis such as solid-phase extraction (SPE) or liquid-liquid extraction (LLE) techniques.
3.1. Chromatography.
Techniques of chromatography were modified and developed to enhance detection of the cotinine and reduce the time of run. To improve sensitivity two types of chromatography – gas and liquid, were coupled with mass spectrometry due to the sensitive and highly specific character of this technique.
Chromatography separating a mixture of chemicals, being in gas or liquid form, help to identify them one by one. Solutions prepared from samples are injected into a column that comprises a narrow stainless steel tube packed with a highly absorbent solid, such as silica gel or crystals. The compounds are separated on the basis of their relative interaction with the chemical coating (stationary phase) and the solvent eluting through the column (mobile phase). Then, the components eluted from the chromatographic column are insert to the mass spectrometer by a specialised interface. The most common used in mass spectrometry are the electrospray ionisation (ESI) and atmospheric pressure chemical ionisation interfaces (APCI). Mass spectrometer alone operate by converting the analyte molecules to a charged (ionised) state and analyse the ions and fragment ions, that are produced during the ionisation process, measuring their mass-to-charge ratio. Several different technologies are available for ionisation and ion analysis, resulting in various types of mass spectrometers and different combinations of these two processes. In typical Mass Spectrometry procedure, a sample loaded onto MS instrument, goes into vaporization, components are ionized to ions, which are separated by electromagnetic fields and detected usually by a quantitative method (James, 2009).
3.1.1. Liquid chromatography/mass spectrometry.
Liquid chromatography can be performed using a conventional HPLC (High Performance Liquid Chromatography) system or UPLC (Ultra Performance Liquid Chromatography). UPLC being able to separate particles smaller than 2 um in diameter archive superior sensitivity and resolution in short periods of time compared to HPLC and it’s a method of choice of many researchers.
The method is successfully used for a few decades now and is still modified in order to increase its sensitivity. De Cremer et al, (2013) was the first to published report in which he proposed the coupling of on-line solid phase extraction (SPE) with ultra-performance chromatography tandem-mass spectrometry (UPLC-MS/MS) for detection of cotinine and its metabolites in human urine. The advantage of this method is efficient and economical biomonitoring of active and passive smokers and those who are trying to stop smoking. The modified procedure is less time consuming and labour intensive. It eliminates the human errors during processing the sample and reduces loss of analyte during drying or dissolving steps as it is developed with the least possible manual sample preparation. (8) UPLC, as a separation technique, provides an enhanced resolution with narrower peaks resulting in greater peak heights leading to a better sensitivity at a reduced analysis time. The main disadvantage of the on-line SPE is the building up of contaminants from solvents and tubing on the SPE column during low aqueous conditions. The authors of this novel technique argue that their method is always faster compere to other methods as avoid time consuming sample preparations, use less urine sample volume ( 100ml) and injection volume ( 10 ul vs 30-1000 ul) and the 1:10 dilution method, has comparable or even better sensitivity at ug/L level depending on the analyte (8).
3.1.2. Gas chromatography.
The second most common chromatography technique used in detection of cotinine is gas chromatography. In this (the standard procedure) method a sample is vaporised before injected into head of the chromatography column. The method was adapted and modified by Iwan et al in measuring the cotinine levels in urine using samples prepared by micro-extraction by packed sorbent (MEPS) and subjected to gas chromatography- mass spectrometry (GC/MS) analysis. The method provides a good reproducibility, with a detection limit of cotinine of 20 µg/ml.
Micro- extraction by packed sorbent (MEPS) technique was developed by miniaturization of conventional SPE (Solid-phase extraction) devices. It was proved to be a simple method used for detection of drugs in variety of biological fluids such as plasma or urine. Its modification allowed to reduce the sorbent bed volume which makes it suitable for a large sample volume range (even as low as 10-1000 ml) and also simplify the procedures compere to standard SPE techniques.
A typical MEPS assay is designed in a syringe format, in which a few mg of the sorbent is packed inside a syringe (100-250ml) as a plug, or between the barrel and the needle as a cartridge. Any kind of sorbent material can be used, such as the silica-based materials, molecular imprinted polymers, or strong cation exchange (as packing bed or coating) (Iwan et al, 2013). In the final elution, the reduction of the concentrations of phospholipids resulted in the improvement of the matrix effect by the sample preparation technique (MEPS), which made this method even more selective.
In the research the urine was chosen as a specimen as it has a higher concentration in urine matrix compared to other matrices and also results in accurate detection in samples. The MEPS was performed according to procedures and the elute was subjected to gas chromatography-mass spectrometry (GC-MS) for analysis. Because of use of MEPS assay method, no evaporation of solvent or salting was necessary, as the very small volume of solvent utilised and also use of the correct composition of the solvent mixture for GC-MS.
The big advantage of the MEPS method is that the extract is directly subjected to GC-MS without need for evaporation. The technique is simple and convenient and can be performed by inexperienced personel.
3.2. Enzyme -linked immunosorbent assay.
Gas and liquid chromatography are the most validated and reliable assays for detection of cotinine. However, these methods are not always suitable as they require expensive equipment, large amount of samples, skilled operators and they are both time consuming (13, 4-7). The practical alternative for (gas and liquid chromatography in indication of ) indication of tobacco smoke exposure is the enzyme- linked immunosorbent assay (ELISA). It can be used in large numbers of samples especially when they need to be analysed rapidly and when other tests are not available.
ELISA is an immunoassay technique widely used as a diagnostic and analytical tool in medicine and biomedical research, for detection and quantification of specific antigens and antibodies in fluid samples. It allows detection of all types of biological molecules such as proteins, hormones or peptides at very low quantities and concentration. The mechanism for this assay is that unknown antigens are immobilised in the plastic multitier plate (usually 96-well). The antigen is allowed to bind to a specific antibody, which is added in conjunction with the enzyme. After the incubation period, any unbound antibody is washed away. Then, secondary antibody conjugated to the enzyme is added, which recognises and binds to the first antibody. As the second antibody is covalently attached to the enzyme such as peroxidase or alkaline phosphatase, it can catalyse a chromogenic reaction. Visible colour change or fluorescence indicating the presence of an antigen can be measured quantitatively or qualitatively by spectrometer specially designed to monitor ELISA plates. The intensity of the colour or fluorescence will depend on the amount of enzyme, which depends on the amount of protein factor, which was originally attached to the bottom of the plate (15).
Matsumoto at al in their research validated a commercially available ELISA test as a practical alternative to chromatographic methods. As an specimen they used a urine samples from children. Comparing the ELISA with GC-MS and HPLC, they found close correlations between immunoreactive (IR) cotinine (both free and total cotinine) with those measured in that internationally validated procedures. However, they noticed that the levels of cotinine were much higher when determined by ELISA than by GC-MS. Also, the ELISA assay showed closer correlation between levels of cotinine and number of tobacco cigarettes smoked by the parents than the results from GC-MS measures.
The important advantage of the ELISA is that the detection limit for IR- cotinine is 1,3 ng/ml, which enable monitoring of passive smoking. However, the limitation of the ELISA is difficulty to compare IR cotinine with cotinine analysed by chromatographic methods and these issues should be take into consideration when interpreting results.
4. Discussion.
Several different analytical techniques have been formulated for determination of cotinine in biological fluids and tissues. Between all of them, tests using chromatographic procedures, such as gas chromatography or liquid chromatography are the most trusted and reliable methods. Some researches claim, that the ultimate standard of reference in analysis of cotinine from different body fluids is gas chromatography – mass spectrometry for smokers and gas-liquid chromatography for people exposed to passive smoking (6).
Most of these assays require a long chromatographic analysis time to achieve better separation of the analytes from matrix and that’s why loads of them were modified to improve procedures, making them simpler and less time consuming. To enhance detection, the chromatography assay, whether gas or liquid, was coupled with mass spectrometry, which has high specificity and sensitivity. Also, standard methods for sample preparation before analysis, such as solid phase extraction (SPE) or liquid-liquid extraction (LLE) techniques, were modified (like SPE method, improved by adopting on-line SPE procedures), or replaced (by micro-extraction by packed sorbent – MEPS). Transformation of those procedures reduced the number of steps typically involved in traditional techniques and so the potential human errors and sample material losses by reducing the manual sample preparation.
Despite all the advantages of methods using chromatography/mass spectrometry techniques, they are not always possible to run, as they require trained personnel and expensive equipment. In that situation, a commercially available enzyme-linked immunosorbent assay (ELISA) can be an alternative to the chromatographic methods. It was proved, that levels of cotinine detected using ELISA closely correlates with those of cotinine, measured by gas chromatography – mass spectrometry (GC-MS) and even reflects the smoking behaviour of person’s parents more precisely than cotinine levels measured by GC-MS. Multiple and portable ELISA as a ready-to-use, relatively cheap lab kit, is ideal for large population screening, especially in low-budget settings.
All described methods were carried out of urine specimen. As it is easy to obtain, it can be collected non-invasively, without the need of medically trained personnel. Also, there is strong correlation between urine cotinine concentrations and the rest of fluids, like plasma or saliva.