Gas chromatographic analysis of nicotine and cotinine in hair.

Non-invasive validation of cigarette- or cigar-smoking behaviour is necessary for large population studies. Urine or saliva samples can be used for confirmation of recent nicotine intake by analysis of cotinine, the major metabolite of nicotine. However, this test is not suitable for validation of survey data, since the quantification of cotinine in saliva only reflects nicotine exposure during the preceding week. To validate information on tobacco use, we investigated hair samples for quantifying nicotine and cotinine by gas chromatography-mass spectrometry. Hair (about 50-100 mg) was incubated in 1 M sodium hydroxide at 100 degrees C for 10 min. After cooling, samples were extracted by diethyl ether, using ketamine as an internal standard. Drugs were separated on a 12-m BP-5 capillary column, and detected using selected-ion monitoring (m/z 84, 98 and 180 for nicotine, cotinine and ketamine, respectively). Hair from non-smokers and smokers contained nicotine and cotinine. Although it is difficult to determine an absolute cut-off concentration, more than 2 ng of nicotine per milligram of hair can be used to differentiate smokers from non-smokers. Some applications of this technique are developed to determine the status of passive smokers, the gestational exposure in babies and the pattern of an individual's nicotine use by cutting strands of hair into sections of one-month intervals.     

Determination of thiocyanate in human saliva and urine by ion chromatography.

A simple ion-chromatographic method has been developed for the determination of trace amounts of thiocyanate in human saliva and urine. Thiocyanate separation and detection were carried out on an ODS column coated with cetyldimethylamine and by an ultraviolet detector, respectively. Citrate solution (1 mmol l-1) was used as the mobile phase. Thiocyanate was clearly separated from many organic and inorganic anions found in saliva and urine samples. The analytical results obtained by the proposed method agreed with those of the Fe(3+)-thiocyanate spectrophotometric method. Thiocyanate concentrations in the saliva and urine of smokers were found to be significantly higher than those of non-smokers.     

Detection of tobacco-related biomarkers in urine samples by surface-enhanced Raman spectroscopy coupled with thin-layer chromatography

The nicotine metabolites, cotinine and trans-3′-hydroxycotinine (3HC) are considered as superior biomarkers for identifying tobacco exposure. More importantly, the ratio of 3HC to cotinine is a good indicator to phenotype individuals for cytochrome P450 2A6 activity and to individualize pharmacotherapy for tobacco addiction. In this paper, a simple, robust and novel method based on surface-enhanced Raman spectroscopy coupled with thin-layer chromatography (TLC) was developed to directly quantify the biomarkers in human urine samples. This is the first time surface-enhanced Raman spectroscopy (SERS) was used to detect cotinine and 3HC in urine samples. The linear dynamic range for the detection of cotinine is from 40 nM to 8 μM while that of 3HC is from 1 μM to 15 μM. The detection limits are 10 nM and 0.2 μM for cotinine and 3HC, respectively. The proposed method was further validated by quantifying the concentration of both cotinine and 3HC in smokers’ urine samples. This TLC-SERS method allows the direct detection of cotinine in the urine samples of both active and passive smokers and the detection of 3HC in smokers.

A new molecularly imprinted polymer for selective extraction of cotinine from urine samples by solid-phase extraction

Cotinine, the main metabolite of nicotine in human body, is widely used as a biomarker for assessment of direct or passive exposure to tobacco smoke. A method for molecularly imprinted solid-phase extraction (MISPE) of cotinine from human urine has been investigated. The molecularly imprinted polymer (MIP) with good selectivity and affinity for cotinine was synthesized using cotinine as the template molecule, methacrylic acid as the functional monomer, and ethylene glycol dimethacrylate as the cross-linker. The imprinted polymer was evaluated for use as a SPE sorbent, in tests with aqueous standards, by comparing recovery data obtained using the imprinted form of the polymer and a non-imprinted form (NIP). Extraction from the aqueous solutions resulted in more than 80% recovery. A range of linearity for cotinine between 0.05 and 5 μg mL⁻¹ was obtained by loading 1 mL blank urine samples spiked with cotinine at different concentrations in acetate buffer of pH 9.0, and by using double basic washing and acidic elution. The intra-day coefficient of variation (CV) was below 7% and inter-day CV was below 10%. This investigation has provided a reliable MISPE–HPLC method for determination of cotinine in human urine from both active smokers and passive smokers. Figure     

A liquid chromatography-mass spectrometry method for nicotine and cotinine; utility in screening tobacco exposure in patients taking amiodarone

A liquid chromatographic mass spectrometric (LC-MS) assay for the quantification of nicotine and cotinine in human specimens was developed. Human serum and urine (100 μL) were subjected to liquid-liquid extraction. For glucuronidated cotinine, serum was alkalinized and hydrolyzed before extraction. The dried samples were reconstituted and run using gradient flow reverse-phase liquid chromatography with MS detection. The ions utilized for quantification of nicotine, cotinine and milrinone (internal standard) were 162.8, 176.9 and 211.9 m/z, respectively. The mean recoveries were over 80% for cotinine and nicotine with excellent linearity between nominal concentrations and peak area ratios, over a wide concentration range. The percentage coefficient of variation and mean error of the inter- and intra-day validations were <15% for nicotine and cotinine. Analysis of serum from cardiac patients receiving amiodarone suggested that a number of patients were either active smokers or exposed to second-hand smoke. Significant concentrations of nicotine and cotinine were measured in the urine of a known smoking volunteer. The method was highly specific, sensitive and applicable as a tool in detecting and monitoring the passive exposure to tobacco smoke using small specimen volumes (0.1 mL).     

High‐performance liquid chromatography with diode‐array detection cotinine method adapted for the assessment of tobacco smoke exposure

Smoking is considered to be one of the main risk factors for cancer and other diseases and is the second leading cause of death worldwide. As the anti‐tobacco legislation implemented in Europe has reduced secondhand smoke exposure levels, analytical methods must be adapted to these new levels. Recent research has demonstrated that cotinine is the best overall discriminator when biomarkers are used to determine whether a person has ongoing exposure to tobacco smoke. This work proposes a sensitive, simple and low‐cost method based on solid‐phase extraction and liquid chromatography with diode array detection for the assessment of tobacco smoke exposure by cotinine determination in urine. The analytical procedure is simple and fast (20 min) when compared to other similar methods existing in the literature, and it is cheaper than the mass spectrometry techniques usually used to quantify levels in nonsmokers. We obtained a quantification limit of 12.30 μg/L and a recovery of over 90%. The linearity ranges used were 12–250 and 250–4000 μg/L. The method was successfully used to determine cotinine in urine samples collected from different volunteers and is clearly an alternative routine method that allows active and passive smokers to be distinguished.     

Contribution of microextraction in packed sorbent for the analysis of cotinine in human urine by GC–MS

A simple, rapid, sensitive, and non-consuming solvent method for the determination of cotinine in urine was developed, based on sample preparation by the relatively new technique microextraction in packed sorbent (MEPS) and analysis by GC–MS. This optimized method was compared with conventional solid-phase extraction/liquid–liquid extraction method used as reference. The wide linear range (5–5,000 ng/mL) and high sensitivity of the MEPS method (limit of detection 0.8 ng/mL) allow application to analysis of urine from smokers as well as non-smokers susceptible to passive smoking.     

Fluorimetric determination of phytic acid based on the activation of the oxidation of 2,2'-dipyridyl ketone hydrazone catalysed by Cu(II)

Phytic acid exerts an activation effect on the oxidation of 2,2'-dipyridyl ketone hydrazone catalysed by Cu(II) ion and the oxidation product is highly fluorescent. A fixed time method for the fluorimetric determination of phytic acid based on this effect is described. The calibration graph is linear over the range 0.05-0.6 mg l-1 phytic acid, resulting in a limit of detection of 0.03 mg l-1 phytic acid. The relative standard deviation is in the range 1.4-1.8%, depending on the sample analysed. The method was successfully applied to the determination of phytic acid in human urine (20 samples) and food samples (nine different products). The results obtained for urine samples ranged from 0.31 to 3.6 mg l-1 phytic acid and for food samples from 3.8 to 22 mg g-1 phytic acid. This is the first procedure to be reported for the determination of phytic acid based on fluorimetric measurements.     

Application of response surface methodology to vortex‐assisted dispersive liquid–liquid extraction for the determination of nicotine and cotinine in urine by gas chromatography–tandem mass spectrometry

A method of vortex‐assisted dispersive liquid–liquid extraction coupled with gas chromatography and tandem mass spectrometry for the determination of nicotine and cotinine in urine was developed. Response surface methodology was applied to obtain the optimum extraction conditions. In this method, Plackett–Burman design was utilized to evaluate the impact of five selected factors on pretreatment procedure. Then, three main factors were optimized using a Box–Behnken design. The optimized method showed good linearities at 1–2000 μg/L with correlation coefficients of 0.9998 for nicotine and 0.9986 for cotinine. Recovery was 91.4–106 and 91.7–108% for nicotine and cotinine, respectively. The intraday relative standard derivations of determination were 1.47–4.06% for nicotine and 0.41–3.16% for cotinine, and interday relative standard derivations were 3.03–6.70% for nicotine and 1.64–6.38% for cotinine. The method detection limits for nicotine and cotinine were 0.33 and 0.34 μg/L, respectively. A total of 87 urine samples from smokers and nonsmokers were tested with the proposed method. Urinary nicotine and cotinine were 23.0–6.67 × 10³ and 18.4–4.17 × 10³ μg/(g·cr) for smokers and 1.31–286 and 1.39–131 μg/(g·cr) for nonsmokers, respectively. The method is sensitive, suitable and reliable for the determination of nicotine and cotinine in urine and meets the requirements for evaluating short‐term tobacco exposure.     

A New Molecularly Imprinted Polymer for Solid-phase Extraction of Cotinine from Human Urine

A molecularly imprinted polymer （MIP）, prepared around a cotinine template, has been synthesized. The feasibility of using the polymer for solid-phase extraction （SPE） of cotinine from biological samples has been investigated. The results show that cotinine can be quantitatively retained and eluted from the polymer. Experiments with human urine samples indicate that clean target analyte is obtained for HPLC with UV detection using the protocol.     

Rapid and sensitive quantification of urinary N7-methylguanine by isotope-dilution liquid chromatography/electrospray ionization tandem mass spectrometry with on-line solid-phase extraction

A rapid, highly specific and sensitive isotope-dilution liquid chromatography/tandem mass spectrometry (LC/MS/MS) method coupled with an on-line solid-phase extraction (SPE) system was developed to measure N7-methylguanine (N7-MeG) in urine. 15N5-Labeled N7-MeG was synthesized to serve as an internal standard, and an on-line SPE cartridge was used for on-line sample cleanup and enrichment. The urine sample can be directly analyzed within 15 min without prior sample purification. The detection limit for this method was estimated as 8.0 pg/mL (4.8 pmol) on-column. This method was further applied to study exposure to methylating agents arising from cigarette smoke. Sixty-seven volunteers were recruited, including 32 regular smokers and 35 nonsmokers. Urinary cotinine, a major metabolite of nicotine, was also determined using an isotope-dilution LC/MS/MS method. The results showed that urinary levels of N7-MeG observed in smokers (4215 +/- 1739 ng/mg creatinine) were significantly (P < 0.01) higher than those in nonsmokers (3035 +/- 720 ng/mg creatinine). It was further noted that the urinary level of N7-MeG was found to be correlated with that of cotinine for smokers, implying that cigarette smoking resulted in increased DNA methylation, followed by depurination and excretion of N7-MeG in urine. As a result of the on-line extraction system, this method is capable of routine high-throughput analysis and accurate quantitation of N7-MeG, and could be a useful tool for health surveillance of methylating agent exposure.     

Hair analysis of nicotine and cotinine for evaluating tobacco smoke exposure by liquid chromatography-mass spectrometry

A simple liquid chromatography-electrospray ionization mass spectrometry (LC-ESI-MS) method for the determination of nicotine and cotinine in human hair was established. In the procedure, a hair sample (10 mg) was washed with dichloromethane and digested in 2.5 M sodium hydroxide. The digest was extracted with dichloromethane and then 25 mM hydrochloric acid in methanol was added to the extract, to prevent loss of analytes. The solution was evaporated and redissolved in the mobile phase, methanol/10 mM ammonium acetate (30/70, v/v). A 20 microL aliquot of redissolved solution was subjected to analysis. Nicotine and cotinine in human hair were quantified by using deuterated analytes as internal standards. The quantification limits were 8 microg/L for nicotine and 0.9 microg/L for cotinine. The proposed method was applied to measure the concentrations of nicotine and cotinine in hair of smokers and non-smokers to evaluate their self-reported smoking and exposure to environmental tobacco smoke. In both cases, the method provided good selectivity, accuracy and precision.     

Determination of Cotinine in Urine and Wastewaters by High Performance Liquid Chromatography Coupled with Tandem Mass-Spectrometric Detection

A technique of extracting cotinine in urine and wastewaters, followed by its quantitative determination, using high performance liquid chromatography combined with tandem mass-spectrometric detection is presented. The method is characterized by low detection limits and high levels of efficiency and sensitivity. The optimal conditions for the solid-phase extraction of cotinine from urine and wastewaters are found. This technique makes it possible to reliably estimate the content of cotinine in the urine of active and passive smokers and in wastewaters.     

Determination of the nicotine metabolite trans-3'-hydroxycotinine in urine of smokers using gas chromatography with nitrogen-selective detection or selected ion monitoring.

A gas chromatographic method for the determination of the nicotine metabolite trans-3'-hydroxycotinine is described. The method involves conversion of the metabolite to the tert.-butyldimethylsilyl derivative, chromatography on a fused-silica capillary column, and determination using nitrogen-phosphorus detection or electron ionization mass spectrometry with selected ion monitoring. A structural analogue, trans-3-hydroxy-1-methyl-5-(2-pyridyl)pyrrolidin-2-one (trans-3'-hydroxy-ortho-cotinine), was used as an internal standard. Using selected ion monitoring, good precision and accuracy were obtained for determination of trans-3'-hydroxycotinine in urine over the concentration range 10-10,000 ng/ml. There was a good correlation between concentrations determined by selected ion monitoring and by nitrogen-phosphorus detection in urine of smokers, although low concentrations determined using nitrogen-phosphorus detection tended to be somewhat higher, suggesting some interference from urinary constituents. Concentrations and 24-h excretion of trans-3'-hydroxycotinine in the urine of 22 cigarette smokers are reported and compared to concentrations and excretion of nicotine, cotinine, nicotine 1'-N-oxide, nornicotine, and cotinine N-oxide.     

Urinary analysis of 8-oxoguanine, 8-oxoguanosine, fapy-guanine and 8-oxo-2'-deoxyguanosine by high-performance liquid chromatography-electrospray tandem mass spectrometry as a measure of oxidative stress

A sensitive and specific assay aimed at measuring the oxidized nucleic acids, 8-oxoguanine (8-oxoGua), fapy-guanine (Fapy-Gua), 8-oxoguanosine (8-oxoGuo), 8-oxo-2'-deoxyguanosine (8-oxodG) has been developed by coupling reversed phase liquid chromatography (HPLC) with electrospray tandem mass spectrometry detection (MS/MS) and isotope dilution. The HPLC-MS/MS approach with multiple reaction monitoring (MRM) allowed for the sensitive determination of 8-oxoGua, Fapy-Gua, 8-oxoGuo, and 8-oxodG in human urine samples. There is no sample preparation needed except for the addition of buffer and (13)C- and (15)N-labeled internal standards to the urine prior to sample injection into the HPLC-MS/MS system. This method was tested in urine samples from non-smokers, smokers, non-smokers with chronic kidney disease (CKD) and smokers with CKD, to assess the level of oxidative damage to nucleic acids. Markers of both RNA and DNA damage were significantly increased in the smokers with and without CKD compared to their respective control subjects. These findings suggest that a highly specific and sensitive analytical method such as isotope dilution HPLC-MS/MS may represent a valuable tool for the measurement of oxidative stress in human subjects.     

Simultaneous analysis of 28 urinary VOC metabolites using ultra high performance liquid chromatography coupled with electrospray ionization tandem mass spectrometry (UPLC-ESI/MSMS)

Volatile organic compounds (VOCs) are ubiquitous in the environment, originating from many different natural and anthropogenic sources, including tobacco smoke. Long-term exposure to certain VOCs may increase the risk for cancer, birth defects, and neurocognitive impairment. Therefore, VOC exposure is an area of significant public health concern. Urinary VOC metabolites are useful biomarkers for assessing VOC exposure because of non-invasiveness of sampling and longer physiological half-lives of urinary metabolites compared with VOCs in blood and breath. We developed a method using reversed-phase ultra high performance liquid chromatography (UPLC) coupled with electrospray ionization tandem mass spectrometry (ESI/MSMS) to simultaneously quantify 28 urinary VOC metabolites as biomarkers of exposure. We describe a method that monitors metabolites of acrolein, acrylamide, acrylonitrile, benzene, 1-bromopropane, 1,3-butadiene, carbon-disulfide, crotonaldehyde, cyanide, N,N-dimethylformamide, ethylbenzene, ethylene oxide, propylene oxide, styrene, tetrachloroethylene, toluene, trichloroethylene, vinyl chloride and xylene. The method is accurate (mean accuracy for spiked matrix ranged from 84 to104%), sensitive (limit of detection ranged from 0.5 to 20 ng mL⁻¹) and precise (the relative standard deviations ranged from 2.5 to 11%). We applied this method to urine samples collected from 1203 non-smokers and 347 smokers and demonstrated that smokers have significantly elevated levels of tobacco-related biomarkers compared to non-smokers. We found significant (p < 0.0001) correlations between serum cotinine and most of the tobacco-related biomarkers measured. These findings confirm that this method can effectively quantify urinary VOC metabolites in a population exposed to volatile organics.     

Determination of trichloroacetate in human serum and urine by ion chromatography

A simple and rapid ion chromatographic method has been developed for the determination of trichloroacetate in human serum and urine. Two short (50 x 5 mm i.d.) low-capacity anion-exchange resin columns in series were used as the analytical column and a mixture of 35 mM sodium hydroxide, 20 mM p-hydroxybenzonitrile and 2% V/V acetonitrile (pH 12.3) as the eluent. The calibration graph was rectilinear from 0.3 to 33 mg l-1 of trichloroacetate at 10 microS with a detection limit (3 sigma) of 0.08 mg l-1. The recovery of trichloroacetate, obtained by analysing spiked serum and urine samples, was in the range 98.2-101% and the coefficient of variation was always less than 3.0%.     

Simultaneous determination of inulin and p-aminohippuric acid (PAH) in human plasma and urine by high-performance liquid chromatography

Inulin and p-aminohippuric acid (PAH) clearances are used for the estimation of glomerular filtration rate (GFR) and effective renal plasma flow (ERPF). A simple and rapid high-performance liquid chromatography (HPLC) method with UV detection is described for the simultaneous determination of inulin and PAH in the same chromatogram in the plasma and urine of humans. Plasma and urine samples were hydrolyzed with perchloric acid (0.7%) in boiling water. The mobile phase consisted of 0.01 M potassium dihydrogenphosphate with 0.02 M tetramethylammonium chloride and o-phosphoric acid (pH 3)-acetonitrile (94:6, v/v), pumped at a rate of 1.2 ml min-1 on a C8 reversed-phase column. Tannic acid was used as the internal standard and UV detection at 285 nm was employed. The calibration curves were linear over the concentration range of 12.5-100 mg l-1 for inulin and 6.25-50 mg l-1 for PAH with determination coefficients greater than 0.997. The method is accurate (bias < 13%) and reproducible (intra- and inter-day relative standard deviation less than 11%), with a limit of quantitation of 12.5 mg l-1 and 6.25 mg l-1 for inulin and PAH, respectively. Analytical recoveries from urine and plasma were ranged from 81 to 108% for both compounds. This fully validated method, which allows the simultaneous determination of inulin and PAH clearances, is simple, rapid (total run time < 10 min) and requires only a 200 microliters plasma or urine sample.     

Development of a highly efficient in-tube solid-phase microextraction system coupled with UHPLC-MS/MS for analyzing trace hydroxyl polycyclic aromatic hydrocarbons in biological samples

Hydroxyl polycyclic aromatic hydrocarbons are considered active mutagenic and carcinogenic substances and are found in extremely low levels (ng/g) in biological samples. As a result, their determination in urine and blood samples is challenging, and a sensitive and effective method for the analysis of trace hydroxyl polycyclic aromatic hydrocarbons in complex biological matrices is required. In this work, a novel macroporous in-tube solid-phase microextraction monolith was prepared via a thiol-yne click reaction, and a highly efficient analytical method based on in-tube solid-phase microextraction coupled with UHPLC-MS/MS was developed to determine hydroxyl polycyclic aromatic hydrocarbons with low detection limits of 0.137–11.0 ng/L in complex biological samples. Four hydroxyl polycyclic aromatic hydrocarbons, namely, 2-hydroxyanthraquinone, 1-hydroxypyrene, 1,8-dihydroxyanthraquinone, and 6-hydroxychrysene, were determined in the urine samples of smokers, non-smokers, and whole blood samples of mice. Satisfactory recoveries were achieved in the range of 83.1–113% with relative standard deviations of 3.2–9.7%. It was found that implementation of the macroporous monolith gave a highly efficient approach for enriching trace hydroxyl polycyclic aromatic hydrocarbons in biological samples.     

On-line molecularly imprinted solid-phase extraction for the selective spectrophotometric determination of nicotine in the urine of smokers

This work describes an on-line molecularly imprinted solid-phase extraction (MISPE) method for spectrophotometric determination of nicotine in urine samples of smokers. This method is based on manganese (VII) to manganese (VI) reduction in an alkaline medium, promoted by nicotine. Two wash solutions (1:4 (v/v) acetonitrile:sodium hydroxide - pH 11.4, and nitric acid - pH 2.5) were employed to circumvent interferences. Aqueous solutions containing nicotine plus different possible concomitants (cotinine, anabasine, norcotinine and caffeine) were tested individually. The analytical calibration curve was prepared in urine samples collected from non-smokers and spiked with nicotine standard from 1.1 to 60 μmol L⁻¹ (r² > 0.998). The limit of quantification and the analytical frequency were 1.1 μmol L⁻¹ and 11 h⁻¹, respectively. The precision, evaluated using 3, 10 and 30 μmol L⁻¹ nicotine in urine, was 10, 10 and 4% (intra-day precision) and 12, 13 and 5% (inter-day precision), respectively. Accuracy was checked through high performance liquid chromatography and the results did not present significant differences at the 95% confidence level according to the Student's t-test.