Analysis of myosmine,cotinine and nicotine in human toenail,plasma and saliva

Myosmine is a minor tobacco alkaloid with widespread occurrence in the human diet. Myosmine is genotoxic in human cells and is readily nitrosated and peroxidated yielding reactive intermediates with carcinogenic potential. For biomonitoring of short-term and long-term exposure, analytical methods were established for determination of myosmine together with nicotine and cotinine in plasma, saliva and toenail by gas chromatography–mass spectrometry (GC/MS). Validation of the method with samples of 14 smokers and 10 non-smokers showed smoking-dependent differences of myosmine in toenails (66?±?56 vs 21?±?15?ng?g^?1, p?<0.01) as well as saliva (2.54?±?2.68 vs 0.73?±?0.65?ng ml^?1, p <0.01). However, these differences were much smaller than those with nicotine (1971?±?818 vs 132?±?82?ng g^?1, p <0.0001) and cotinine (1237?±?818 vs <35?ng?g^?1) in toenail and those of cotinine (97.43?±?84.54 vs 1.85?±?4.50?ng ml^?1, p <0.0001) in saliva. These results were confirmed in plasma samples from 84 patients undergoing gastro-oesophageal endoscopy. Differences between 25 smokers and 59 non-smokers are again much lower for myosmine (0.30?±?0.35 vs 0.16?±?0.18?ng?ml^?1, p <0.05) than for cotinine (54.67?±?29.63 vs 0.61?±?1.82?ng ml^?1, p <0.0001). In conclusion, sources other than tobacco contribute considerably to the human body burden of myosmine.     

The effects of nicotine, cotinine and anabasine on rat adrenal 11 beta-hydroxylase and 21-hydroxylase

The effects of nicotine, cotinine and anabasine on rat adrenal steroidogenesis were examined by spectral and enzymatic techniques. The addition of nicotine, cotinine or anabasine to preparations of rat adrenal mitochondria produced type II cytochrome P-450 difference spectra. The addition of nicotine or anabasine, but not cotinine, to rat adrenal microsomes yielded type II cytochrome P-450 difference spectra. Nicotine and anabasine competitively inhibited rat adrenal mitochondrial 11 beta-hydroxylase and microsomal 21-hydroxylase. Cotinine competitively inhibited mitochondrial 11 beta-hydroxylase, but did not inhibit microsomal 21-hydroxylase. The apparent enzymatic inhibition constants for cotinine, nicotine, anabasine and metyrapone inhibition of the mitochondrial 11 beta-hydroxylase were 32, 96, 120 and 74 microM respectively. These studies suggest that components of cigarette smoke may alter patterns of adrenal steroidogenesis.     

Determination of nicotine and cotinine in tobacco harvesters' urine by solid-phase extraction and liquid chromatography

A solid-phase extraction method using Drug Test-1 column containing chemically modified silica as a solid support for sample clean up and reversed phase ion-paired high-pressure liquid chromatography method have been developed for the simultaneous determination of nicotine and its metabolite cotinine from the urine samples. Mobile phase was consisted of acetate buffer (containing 0.03 M sodium acetate and 0.1 M acetic acid) pH 3.1 and acetonitrile (78:22% (v/v)) containing 0.02 M sodium octanosulfonate as an ion pair agent. pH of the mobile phase was adjusted to 3.6 with triethylamine for better resolution and to prevent peak tailing. The linearity was obtained in the range of 0.5-10 microg/ml concentrations of nicotine and cotinine standards. The correlation coefficients were 0.998 for cotinine and 0.999 for nicotine. The recoveries were obtained in the range of 79-97% with average value of 85% for nicotine and in the range of 82-98% with average value of 88% for cotinine. The limit of detection was 2 ng/ml for cotinine and 5 ng/ml for nicotine with 2 ml urine for extraction, calculated by taking signal to noise ratio 10:3. The intra-day co-efficient of variation (CV) were <4 and 7% and inter-day CV were <9 and 7% for nicotine and cotinine, respectively. The method was applied to the urine samples of tobacco harvesters, who suffer from green tobacco sickness (GTS) to check the absorption of nicotine through dermal route during the various processes of tobacco cultivation due to its good reproducibility and sensitivity.     

Sensitive and simple method for the determination of nicotine and cotinine in human urine, plasma and saliva by gas chromatography-mass spectrometry

A method is proposed for the determination of nicotine and cotinine in human urine, plasma and saliva. Nicotine and cotinine were extracted from alkalinized sample with ethyl ether and concentrated to minimum volume with nitrogen stream. The volatility of nicotine was prevented by the addition of acetic acid to the organic solvent during evaporation. Peak shapes and quantitation of nicotine and cotinine are excellent, with linear calibration curves over a wide range of 1-10,000 ng/ml. The detection limits of nicotine and cotinine are 0.2 ng/ml in urine and 1.0 ng/ml in plasma and saliva. The intra-day precision of nicotine and cotinine in all samples was <5% relative standard deviation (RSD). Urine, plasma and saliva samples of 303 non-smoking and 41 smoking volunteers from a girl's high school in Korea were quantified by the described procedure. As a result, the concentrations of nicotine and cotinine in plasma ranged from 6 to 498 ng/ml and 4 to 96 ng/ml. Otherwise, those of nicotine and cotinine in saliva ranged from 0 to 207 ng/ml and 0 to 42 ng/ml, and those of nicotine and cotinine in urine ranged from 0 to 1,590 ng/ml and 0 to 2,986 ng/ml, respectively. We found that the concentration of cotinine in plasma was successfully predicted from the salivary cotinine concentration by the equation y=2.31x+4.76 (x=the concentration of cotinine in saliva, y=the concentration of cotinine in plasma). The results show that through the accurate determination of cotinine in saliva, the risk of ETS-exposed human can be predicted.     

Determination of dichloromethane, trichloroethylene and perchloroethylene in urine samples by headspace solid phase microextraction gas chromatography-mass spectrometry

A method for the determination of volatile chlorinated hydrocarbons, namely dichloromethane (DCM), trichloroethylene (TCE), and perchloroethylene (PCE), in urine samples was developed using headspace solid phase microextraction (HS-SPME) gas chromatography-mass spectrometry (GC-MS). HS-SPME was performed using a 75 microm Carboxen-polydimethylsiloxane fiber. Factors, which affect the HS-SPME process, such as adsorption and desorption times, stirring, salting-out effect, and temperature of sampling have been evaluated and optimized. The highest extraction efficiency was obtained when sampling was performed at room temperature (22 degrees C), from samples saturated with salt and under agitation. Linearity of the HS-SPME-GC-MS method was established over four orders of magnitude and the limit of detection was 0.005 microg/l for all the compounds. Precision, calculated as %R.S.D. at three different concentration levels, was within 1-8% for all intra- and inter-day determinations. The method was applied to the quantitative determination of TCE and PCE in human urine samples from exposed (TCE, n=5; median, 9.32 microg/l and PCE, n=39; median, 0.58 microg/l) and non-exposed individuals (n=120; median concentrations, 0.64, 0.22 and 0.11 microg/l for DCM, TCE and PCE, respectively. In addition, two cases of acute accidental exposure to DCM are reported, and the elimination kinetics in blood and urine was followed up. The calculated half-lives of urinary and blood DCM were, respectively, 7.5 and 8.1 h for one subject and 3.8 and 4.3 h for the other.     

Ultrasonic-assisted derivatization reaction of amino acids prior to their determination in urine by using single-drop microextraction in conjunction with gas chromatography

A derivatization-extraction method that avoids tedious preconcentration steps is established in order to determine amino acids accurately at nanogram levels. The method involves conversion of the analytes of concern to N(O,S)-ethoxycarbonyl amino acid ethyl esters and subsequent extraction by single-drop microextraction (SDME) followed by GC analysis. The reaction proceeds smoothly and rapidly under ultrasonication which removes the bubbles from the bulk solution. Precision is acceptable and 12 non-hydrolyzed amino acids can be determined in urine in this manner. As long as the extraction conditions are consistently applied, quantitative analysis can be performed accurately. The limits of detection were satisfactory in the range 0.010-0.025 microg/ml for GC-FID and 0.26-68 ng/ml for GC-MS(SIM) with 1 ml sample volume.     

Determination of tramadol in human plasma and urine samples using liquid phase microextraction with back extraction combined with high performance liquid chromatography

Liquid phase microextraction by back extraction (LPME-BE) combined with high performance liquid chromatography (HPLC)-fluorescence detection was developed for the determination of tramadol in human plasma. Tramadol was extracted from 2 mL of basic sample solution (donor phase) with pH 11.5 through a micro liter-size organic solvent phase (100 microL n-octane) for 25 min and finally into a 3.5 microL acidic aqueous acceptor microdrop with pH 2.5 suspended in the organic phase from the tip of a HPLC microsyringe needle for 15 min with the stirring rate of 1250 rpm. After extraction for a period of time, the microdrop was taken back into the syringe and injected into HPLC. Effected the experimental parameters such as the nature of the extracting solvent and its volume, sample temperature, stirring rate, volume of the acceptor phase, pH and extraction time on LPME-BE efficiency was investigated. At the optimized condition, enrichment factor of 366 and detection limit (LOD) of 0.12 microg L(-1) were obtained. The calibration curve was linear (r=0.999) in the concentration range of 0.3-130 microg L(-1). Within-day relative standard deviation RSD (S/N=3) and between-day RSD were 3.16% and 6.29%, respectively. The method was successfully applied to determine the concentration of tramadol in the plasma and urine samples and satisfactory results were obtained.     

Determination of nicotine and cotinine in human serum by means of LC/MS

As part of a joint clinical research project to study the effects of nicotine on the brain, a HPLC electrospray ionisation mass spectrometry method with a solid-phase extraction sample preparation was developed for the quantitative determination of nicotine and cotinine in human serum in volunteers. The measured concentrations of nicotine and cotinine were used as control for smoking behaviour. A X-Bridge-column from Waters, and a SSQ 7000 single quadropole mass spectrometer with a TSP liquid chromatographic system were used. The method includes a simple and robust sample preparation and this assay has been shown to be of a sufficient sensitivity for this application. The limits of quantification were 5 and 2 ng/ml for cotinine and nicotine, respectively. A simultaneous study was conducted to measure nicotine receptor availability and the vigilance in the same group of volunteers.     

Determination of colistin in human plasma, urine and other biological samples using LC-MS/MS

A liquid chromatography-tandem mass spectrometric (LC-MS/MS) method was developed to quantify colistin in human plasma and urine, and perfusate and urine from the isolated perfused rat kidney (IPK). Solid phase extraction (SPE) preceded chromatography on a Synergi Fusion-RP column with a mobile phase of acetonitrile, water and acetic acid (80/19/1) at 0.2mL/min. Ions were generated using electrospray ionization and detected in the positive-ion mode. Multiple reaction monitoring was performed using precursor-product ion combinations. Calibration curves were linear from 0.028microg/mL (human plasma, IPK perfusate and urine)/0.056microg/mL (human urine) to 1.78microg/mL (all four media) for colistin A sulfate; corresponding values for colistin B sulfate were 0.016/0.032 to 1.01microg/mL. Accuracy and precision were within 10%. The LLOQ for colistin A sulfate was 0.028microg/mL in human plasma, IPK perfusate and urine and 0.056microg/mL in human urine; corresponding values for colistin B sulfate were 0.016 and 0.032microg/mL. The low sample volume, short analysis time and low LLOQ are ideal for pre-clinical and human pharmacokinetic studies of colistin.     

Extraction and determination of opium alkaloids in urine samples using dispersive liquid-liquid microextraction followed by high-performance liquid chromatography

A simple, rapid and sensitive method based on dispersive liquid-liquid microextraction (DLLME) combined with high-performance liquid chromatography-ultraviolet detection (HPLC-UV) was used to determine opium alkaloids in urine samples. Some effective parameters on extraction were studied and optimized. Under the optimum conditions, enrichment factors and recoveries for different opiates are in the range of 63.0-104.5 and 31.5-52.2%, respectively. The calibration graphs are linear in the range of 0.50-500 μg L(-1) and limit of detections (LODs) are in the range of 0.2-10 μg L(-1). The relative standard deviations (RSDs) for 200 μg L(-1) of morphine, codeine and thebaine, 5.0 μg L(-1) of papaverine and 10.0 μg L(-1) of noscapine in diluted urine sample are in the range of 2.8-6.1% (n=7). The relative recoveries of urine samples spiked with alkaloids are 84.3-106.0%. The obtained results show that DLLME combined with HPLC-UV is a fast and simple method for the determination of opium alkaloids in urine samples.     

Analysis of biological samples using solid-phase microextraction

Solid-phase microextraction (SPME) has gained widespread acceptance for analyte-matrix separation and preconcentration. SPME is a simple, effective adsorption/desorption technique that eliminates the need for solvents or complicated apparatus for concentrating volatile or non-volatile compounds in liquid samples or headspace. SPME is compatible with analyte separation/detection by gas chromatography and high performance liquid chromatography and provides linear results for a wide range of concentrations of analytes. By controlling the polarity and thickness of the coating on the fiber, maintaining consistent sampling time, and adjusting several other extraction parameters, an analyst can ensure highly reliable results for low concentrations of analytes. This review provides updated information on SPME with chromatographic separation for the extraction and measurement of different analytes in biological fluids and materials. Firstly the background to the technique is given in terms of apparatus, fibers used, extraction conditions and derivatisation procedures. Then the different matrices, urine, blood, breast milk, hair and saliva are considered separately. Finally, the future potential of SPME for the analysis of biological samples in terms of the development of new devices and fiber chemistries as well as applications for in vivo studies are discussed.     

Determination of trace cadmium in saliva samples using spray assisted droplet formation-liquid phase microextraction prior to the measurement by slotted quartz tube-flame atomic absorption spectrophotometry

BackgroundAn effective, green and rapid analytical strategy namely the simultaneous spray assisted droplet formation-liquid phase microextraction (S-SADF-LPME) method was developed for the determination of trace quantity of cadmium in saliva samples by using the slotted quartz tube-flame atomic absorption spectrophotometry (SQT-FAAS). By the developed method, external dispersive solvent usage for droplet formation was reduced to obtain a more environmental-friendly method.MethodsMethod consists of a simultaneous complexing and extraction step, which was based on spraying an extraction solvent containing a solid ligand into the aqueous sample solution, forming fine droplets without the use of dispersive solvent. The procedure was implemented using a customized, cost effective and portable spray apparatus to minimize the consumption of reagent, analysis time and operation steps. Thus, this methodology ensures better repeatability and accuracy while minimizing the relative errors caused by the experimental steps. Parameters including the buffer amount, extractant/ligand concentration, extraction solvent type, extraction/ligand solution volume, spraying number and vortex period were systemically optimized to lower the detection limit.ResultsUnder the optimal extraction conditions, 96.9-folds enhancement in the detection power of the traditional FAAS was achieved. The limit of detection and limit of quantification values of presented method were calculated to be 0.65 and 2.17 ng mL⁻¹, respectively. Accuracy and applicability of the optimized method was investigated by collecting saliva samples from smokers. Satisfactory percent recovery values wereachieved for cadmium with a low standard deviation in the acceptable range of 84.9–109.6 %.ConclusionThe developed dispersive solvent-free S-SADF-LPME technique presents a fast, simple, cost-effective and eco-friendly microextraction method based on the use of an easily accessible and functional spray apparatus.     

Determination of aconitine,hypaconitine and mesaconitine in urine using hollow fiber liquid-phase microextraction combined with high-performance liquid chromatography

Hollow fiber liquid-phase microextraction (HF-LPME) coupled with high-performance liquid chromatography was used to simultaneously determine three Aconitum alkaloids, including aconitine (AC), hypaconitine (HA) and mesaconitine (MA) in human urine sample. Analytes were extracted from 5 mL urine sample containing 1.0 mmol/L NaOH into 1-octanol membrane phase impregnated in the pores of hollow fiber wall, and then back extracted into acidified aqueous solution in the lumen of the hollow fiber. After extraction, 10 μL of the acceptor phase was analyzed directly by HPLC. In this method, some important extraction parameters, such as organic solvent, extraction time, stirring rate, pH of donor phase and acceptor phase, temperature, and the volume of acceptor phase were optimized. This method provided 98- to 288-fold enrichment factors within 60 min of extraction and good repeatability with RSDs of 0.99–7.22%. The calibration curves were linear over the ranges of 16.0–128.0 μg/L for AC, 11.0–88.0 μg/L for HA and 8.1–64.8 μg/L for MA in human urine sample, with correlation coefficients of 0.9949, 0.9969 and 0.9904, respectively. Limits of detection were from 0.7 to 1.5 μg/L, and recoveries from spiked urine sample varied from 84.4% to 106.2% for AC, 77.3% to 85.6% for HA and 90.1% to 100.8% for MA.     

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°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.     

Verification of smoking history in patients after infarction using urinary nicotine and cotinine measurements.

Urinary concentrations of nicotine and its major metabolite cotinine were measured in volunteers whose smoking habits were known to test the reliability of the measurements as indicators of current smoking. In the non-smokers detectable concentrations were always below the confidence limits set for the method, while in smokers the concentrations were always above these limits. After subjects stopped smoking cotinine appeared in the urine for longer than nicotine and was still detectable at least 36 hours after the last cigarette had been smoked. When this method was used to verify the smoking histories given by patients attending an infarction clinic it was estimated that 46-53% of previous smokers had actually stopped smoking compared with the 63% who said that they had done so. It is suggested that simultaneous assays of urinary nicotine and cotinine may be a useful means of verifying patients'' current smoking habits.     

Determination of some benzodiazepines and metabolites in serum,urine and saliva by high-performance liquid chromatography

The performance of a number of liquid—solid systems, consisting of mixtures of buffers (0.05 M) and methanol as mobile phase and methyl-silica as stationary phase, were investigated with respect to their use in the separation of 1,4-benzodiazepines by reversed-phase high-performance liquid chromatography with UV detection at 254 nm. Phase system selectivities and column efficiencies were determined. A nomogram is presented from which the chromatographic parameters can be calculated.A complete separation of nine benzodiazepines within 12 min has been achieved, using methyl-silica as the stationary phase and 50% methanol as the eluent.The results were applied to the development of a method for the determination of therapeutic levels of diazepam and its metabolites in human serum, urine and saliva. The first step in the analysis, the extraction of diazepam and its metabolites from serum and urine, was also investigated and good recoveries were achieved. A low detection limit (0.2 ng) and high precision were obtained. The concentrations of diazepam and its metabolites in human serum, urine and saliva were determined after both single and multiple oral doses of diazepam (and oxazepam).