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.     

Measurement of piperaquine in plasma by liquid chromatography with ultraviolet absorbance detection

Piperaquine (PQ) is an antimalarial drug enjoying a resurgence of use in combination with an artemisinin derivative because of parasite resistance to standard treatments. Its pharmacokinetic properties have not been characterised. An assay for PQ in plasma was developed using solvent extraction and liquid chromatographic separation on a Waters XTerra RP(18) column, with a mobile phase of 7% acetonitrile in water (containing 0.025% trifluoroacetic acid, 0.1% NaCl and 0.008% triethylamine) and UV detection at 340 nm. The assay was linear up to 1000 microg/l. Intra- and inter-day relative standard deviations were <10% (5-500 microg/l) and <21% (5-500 microg/l), respectively. Inter-day limits of quantitation and detection were 5 microg/l and 3 microg/l, respectively. A preliminary pharmacokinetic study in a patient who received 2.56 g of PQ phosphate orally with dihydroartemisinin as four doses over 32 h found an apparent steady-state volume of distribution of 447 l/kg, an apparent oral clearance 0.93 l/h/kg and a terminal half-life of 17.3 days.     

Simple, rapid and sensitive assay method for simultaneous quantification of urinary nicotine and cotinine using gas chromatography-mass spectrometry

Nicotine is a major addictive compound in cigarette. Its smoke is rapidly and extensively metabolized to several metabolites in human. Cotinine as a major metabolite of nicotine is commonly used as a biomarker to determine active and passive smokers. Cotinine has a longer half-life ( approximately 20 h) compared to nicotine ( approximately 2h). A simple, sensitive, rapid and high throughput GC-MS method was developed for simultaneous quantification of urinary nicotine and cotinine in passive and active smokers. In the sample preparation method, the analytes and internal standard were first basified and followed by liquid-liquid extraction. Upon completion, anhydrous sodium sulphate was added to the solvent mixture to trap moistures. The clear extract obtained was directly injected into GC-MS, operating under selective ion monitoring (SIM) mode. Calibration curves in the range of 0.5-5000 ng/mL of the analytes in urine matrix were established with linear correlation coefficients (r(2)) greater than 0.997. The limit of detection for both nicotine and cotinine were 0.20 ng/mL. The mean recoveries for nicotine and cotinine were 93.0 and 100.4%, respectively. The within- and between-assay accuracies were between 2.1 and 7.9% for nicotine and between 0.7 and 11.1% for cotinine. Within- and between-assay precisions of 3.3-9.5% for nicotine and 3.4-9.8% for cotinine were also achieved. The method can be used in routine assessment and monitoring of active smoking and exposure to environmental tobacco smoke. The applicability of the assay was demonstrated in a small-scale comparison study between smokers and non-smokers.     

Determination of nicotine and its metabolites in urine by solid-phase extraction and sample stacking capillary electrophoresis-mass spectrometry

The combination of capillary electrophoresis (CE) and mass spectrometry (MS) with solid-phase extraction (SPE) has been used for the identification of nicotine and eight of its metabolites in urine. The recovery of cotinine from cotinine-spiked urine, by C18 SPE, was found to be 98%. Smokers urine (200 ml) was preconcentrated 200-fold via SPE prior to analysis. The sample stacking mode of CE, when compared to capillary zone electrophoresis, was shown to improve peak efficiency by 132-fold. The combination of hydrodynamic and electrokinetic injection was studied with sample stacking/CE/MS. The on-column limits of detection (LOD) of nicotine and cotinine, by this technique, were found to be 0.11 and 2.25 microg/ml, respectively. Hence, LODs of nicotine and cotinine in urine after 200-fold preconcentration were 0.55 and 11.25 ng/ml, respectively.     

Determination of nicotine and two major metabolites in serum by solid-phase extraction and high-performance liquid chromatography, and high-performance liquid chromatography—particle beam mass spectrometry

A rapid and selective assay of nicotine, cotinine and rans-3'-hydroxycotinine in human serum, based on high-performance liquid chromatography with UV detection has been developed. The compounds were subjected to solid-phase extraction, using Extrelut 1 cartridges. Recoveries were ca. 95% for nicotine, 90% for cotinine and 50–55% for trans-3'-hydroxycotinine. The limit of quantitation observed with this method was 10 ng/ml for nicotine and 5 ng/ml for each of the metabolites. The compounds were also identified using high-performance liquid chromatography with particle beam mass spectrometry, to confirm their presence in human serum.     

Simultaneous estimation of nicotine and cotinine levels in biological fluids using high-resolution capillary-column gas chromatography combined with solid phase extraction work-up

A rapid and sensitive capillary gas-chromatographic method with nitrogen-sensitive detection is reported for the simultaneous analysis of nicotine and cotinine levels occurring in the plasma, saliva, and urine of regular tobacco smokers. The proposed assay has a linear output, has satisfactory accuracy over the range of concentrations of both amines encountered in active smokers, and has also been successful in the analysis of the urine samples of passive smokers. Its lower limit of sensitivity is 0.2 ng of nicotine and 0.5 ng of cotinine per ml of plasma or saliva or per 100 l of urine.The beneficial characteristics of the presented method were achieved by the combination of solid phase extraction of 0.1–1.0 ml of fluid specimens, capillary column gas chromatography with splitless injection and nitrogen sensitive detection, and the use of separate, structurally analogous compounds as internal standards for nicotine. The suitability of the assay is shown by plasma concentration-time curves of nicotine and cotinine in a steady smoker during a 24 hours period.     

Determination of rofecoxib in human plasma and breast milk by high-performance liquid chromatographic assay

A rapid and simple HPLC assay was developed for the determination of rofecoxib in human plasma and breast milk. After solid-phase extraction, rofecoxib was resolved on a C18 column and detected by UV detection at 272 nm. Standard curves were linear over the concentration range 10-2000 microg/L (r2 >0.99). Intra- and inter-day coefficients of variation for both matrices were <10% and the limit of quantification was around 10 microg/L.     

Measurement and stability of FTY720 in human whole blood by high-performance liquid chromatography-atmospheric pressure chemical ionization-tandem mass spectrometry

We report here a validated method for the quantification of a new immunosuppressant drug FTY720, using HPLC-tandem mass spectrometry. Whole blood samples (500 microl) were subjected to liquid-liquid extraction, in the presence of an internal standard (Y-32919). Mass spectrometric detection was by selected reaction monitoring with an atmospheric pressure chemical ionization source in positive ionization mode (FTY720: m/z 308.3-->255.3). The assay was linear from 0.2 to 25 microg/l (r(2)>0.997, n=5). The inter- and intra-day analytical recovery and imprecision for quality control samples (0.5, 7 and 15 microg/l) were 95.8-103.2 and <5.5%, respectively. At the lower limit of quantification (0.2 microg/l) the inter- and intra-day analytical recovery was 99.0-102.8% with imprecision of <7.6% (n=5). The assay had a mean relative recovery of 100.5+/-5.8% (n=15). Extracted samples were stable for 16 h. FTY720 quality control samples were stable at room temperature for 16 h, at 4 degrees C for at least 8 days and when taken through at least three freeze-thaw cycles. In conclusion, the method described displays analytical performance characteristics that are suitable for pharmacokinetic studies in humans.     

Simultaneous determination of nicotine, cotinine, norcotinine, and trans-3'-hydroxycotinine in human oral fluid using solid phase extraction and gas chromatography-mass spectrometry

Nicotine is rapidly and extensively metabolized in humans. We present an analytical method to simultaneously quantify nicotine, cotinine, norcotinine, and trans-3'-hydroxycotinine in human oral fluid. Solid phase extraction (SPE) and GC/MS/EI with selected ion monitoring (SIM) were utilized. Linearity ranged from 5 to 1000 ng/mL of oral fluid; correlation coefficients for calibration curves were >0.99. Recoveries were 90-115% nicotine, 76-117% cotinine, 88-101% norcotinine, and 67-77% trans-3'-hydroxycotinine. Intra-assay precision and accuracy ranged from 1.6 to 5.7% and 1.6 to 17.8%, respectively. Inter-assay precision and accuracy ranged from 4.3 to 10.2% and 0 to 12.8%, respectively. Suitable precision and accuracy were achieved for the simultaneous determination of nicotine and three metabolites in the oral fluid of smokers. This assay is applicable to pharmacokinetic studies of nicotine, cotinine, and trans-3'-hydroxycotinine from tobacco smokers and can be utilized for routine monitoring of tobacco smoke exposure. 3-Hydroxycotinine requires additional investigation to determine its usefulness as a biomarker for tobacco smoke exposure.     

Quantitative trace analysis of estriol in human plasma by negative ion chemical ionization gas chromatography-mass spectrometry using a deuterated internal standard

A stable isotope dilution gas chromatography-mass spectrometry (GC-MS) assay for the trace level determination of estriol in human plasma is described. Negative ion chemical ionization (NICI) MS is used for highly specific detection. The method involves derivatization of the phenolic hydroxyl to the pentafluorobenzyl ether derivative and subsequent reaction of the remaining hydroxyls with heptafluorobutyric anhydride. This derivative allows detection of the strikingly abundant phenolate ion under NICI conditions. [2,4,17beta]-2H(3)-labeled estriol was used as an internal standard. For high-level measurements (>313 ng/l) plasma was directly derivatized by extractive alkylation followed by heptafluorobutylation prior to analysis. A rapid and simple sample work up procedure was elaborated for trace level determinations (>5 ng/l plasma) using solid-phase extraction on C(18) with an absolute recovery of 92.9%. For low-level measurements, the calibration curve was linear in the range of 5 to 625 ng/l (r=0.99993). Inter-assay analytical precisions (RSDs) were 1.29, 2.30 and 2.89% at 39, 156 and 650 ng/l plasma, respectively. For high-level measurements, calibration curve linearity was observed in the range of 0.313 to 20 microg/l (r=0.99998). Inter-assay analytical precisions (RSDs) were 5.17, 1.92, 2.57 and 2.74% at 0.313, 0.625, 2.5 and 10 microg/l plasma, respectively. Postmenopausal plasma was used for spiked plasma samples. Sensitivity and specificity of the presented method allows adequate determination of estriol in human plasma samples.     

Quantification and stability of everolimus (SDZ RAD) in human blood by high-performance liquid chromatography-electrospray tandem mass spectrometry

We report here a validated method for the quantification of a new immunosuppressant drug, everolimus (SDZ RAD), using HPLC-tandem mass spectrometry. Whole blood samples (500 microl) were prepared by protein precipitation, followed by C(18) solid-phase extraction. Mass spectrometric detection was by selected reaction monitoring with an electrospray interface operating in positive ionization mode. The assay was linear from 0.5 to 100 microg/l (r(2) > 0.996, n = 9). The analytical recovery and inter-day imprecision, determined using whole blood quality control samples (n = 5) at 0.5, 1.2, 20.0, and 75.0 microg/l, was 100.3 - 105.4% and < or = 7.6%, respectively. The assay had a mean relative recovery of 94.8 +/- 3.8%. Extracted samples were stable for up to 24 h. Fortified everolimus blood samples were stable at -80 degrees C for at least 8 months and everolimus was found to be stable in blood when taken through at least three freeze-thaw cycles. The reported method provides accurate, precise and specific measurement of everolimus in blood over a wide analytical range and is currently supporting phase II and III clinical trials.     

Rapid and simple high-performance liquid chromatographic assay for the determination of metformin in human plasma and breast milk

A rapid and simple high-performance liquid chromatographic (HPLC) assay for the determination of metformin in human plasma and breast milk is described. After proteins were precipitated with acetonitrile, metformin and the internal standard buformin were resolved on a cation-exchange column and detected by UV detection at 236 nm. Standard curves were linear over the concentration range 20.0-4000 microg/l. Intra- and inter-day coefficients of variation were <9.0% and the limit of quantification was around 20 microg/l.     

Rapid method for the simultaneous measurement of nicotine and cotinine in urine and serum by gas chromatography–mass spectrometry

A simple, sensitive, and rapid gas chromatographic–mass spectrometric method is described for the simultaneous detection and quantitation of nicotine and its metabolite, cotinine, in urine and serum. The analytes and their respective deuterated internal standards were extracted by liquid–liquid extraction coupled to centrifugation and evaporation. The detection limit of the assay was 0.16 ng/ml for both nicotine and cotinine. The limit of quantitation for each analyte was 1.25 ng/ml.     

Quantification of nicotine, cotinine, trans-3'-hydroxycotinine and varenicline in human plasma by a sensitive and specific UPLC-tandem mass-spectrometry procedure for a clinical study on smoking cessation

A sensitive and specific ultra performance liquid chromatography-tandem mass spectrometry method for the simultaneous quantification of nicotine, its metabolites cotinine and trans-3'-hydroxycotinine and varenicline in human plasma was developed and validated. Sample preparation was realized by solid phase extraction of the target compounds and of the internal standards (nicotine-d4, cotinine-d3, trans-3'-hydroxycotinine-d3 and CP-533,633, a structural analog of varenicline) from 0.5 mL of plasma, using a mixed-mode cation exchange support. Chromatographic separations were performed on a hydrophilic interaction liquid chromatography column (HILIC BEH 2.1×100 mm, 1.7 μm). A gradient program was used, with a 10 mM ammonium formate buffer pH 3/acetonitrile mobile phase at a flow of 0.4 mL/min. The compounds were detected on a triple quadrupole mass spectrometer, operated with an electrospray interface in positive ionization mode and quantification was performed using multiple reaction monitoring. Matrix effects were quantitatively evaluated with success, with coefficients of variation inferior to 8%. The procedure was fully validated according to Food and Drug Administration guidelines and to Société Fran?aise des Sciences et Techniques Pharmaceutiques. The concentration range was 2-500 ng/mL for nicotine, 1-1000 ng/mL for cotinine, 2-1000 ng/mL for trans-3'-hydroxycotinine and 1-500 ng/mL for varenicline, according to levels usually measured in plasma. Trueness (86.2-113.6%), repeatability (1.9-12.3%) and intermediate precision (4.4-15.9%) were found to be satisfactory, as well as stability in plasma. The procedure was successfully used to quantify nicotine, its metabolites and varenicline in more than 400 plasma samples from participants in a clinical study on smoking cessation.     

Determination of celecoxib in human plasma and breast milk by high-performance liquid chromatographic assay

A rapid and simple HPLC assay was developed for the determination of celecoxib in human plasma and breast milk. After proteins were precipitated with acetonitrile, celecoxib was resolved on a C18 column and detected by UV detection at 254 nm. Standard curves were linear over the concentration range 10-2000 microg/L (r(2)>0.99). Bias was 相似文献   

High-performance liquid chromatographic determination of cotinine in urine in isocratic mode

A simple procedure for the determination of cotinine, major metabolite of nicotine in urine, is described. The assay involved a liquid–liquid extraction with dichloromethane in alkaline environment. The extract was dried at ambient temperature under a gentle stream of nitrogen. The residue was dissolved in 300 μl of mobile phase and 30 μl aliquot was injected via an automatic sampler into the liquid chromatograph and eluted with the mobile phase (10–9%, v/v methanol and acetonitrile, respectively in potassium dihydrogenphosphate buffer adjusted to pH 3.4) at a flow rate of 1 ml/min on a C₈ Symmetry cartridge column (5 μm, 150 mm×3.9 mm, Waters) at 25°C. The eluate was detected at 260 nm. Internal standard was 2-phenylimidazole. Sensitive and specific, this technique was performed to test urine of diabetic patients (smokers and non-smokers) admitted in an endocrinology service. Urinary cotinine seems to be a better marker of smoking status than thiocyanates.     

Quantification of free mycophenolic acid and its glucuronide metabolite in human plasma by liquid-chromatography using mass spectrometric and ultraviolet absorbance detection

The immunosuppressant drug mycophenolic acid (MPA) and its major metabolite, mycophenolic acid glucuronide (MPAG), are highly bound to albumin. An HPLC-tandem-MS (HPLC/MS/MS) and an HPLC-UV assay were developed to measure free (unbound) concentrations of MPA and MPAG, respectively. Ultrafiltrate was prepared from plasma (500 microl) by ultrafiltration at 3000 x g for 20 min (20 degrees C). Both MPA and MPAG were isolated from ultrafiltrate (100 microl) by acidification and C18 solid-phase extraction. Free MPA was measured by electrospray tandem mass spectrometry using selected reactant monitoring (MPA: m/z 338.2--> 206.9) in positive ionisation mode. Chromatography was performed on a PFPP column (50 mm x 2 mm, 5 microm). Total analysis time was 7 min. The assay was linear over the range 1-200 microg/l with a limit of quantification of 1 microg/l. The inter-day accuracy and imprecision of quality controls (7.5, 40, 150 microg/l) were 94-99% and < 7%, respectively. Free MPAG was chromatographed on a C18 Nova-Pak column (150 mm x 3.9 mm, 5 microm) using a binary gradient over 20 min. The eluent was monitored at 254 nm. The assay was linear over the range 1-50 mg/l with the limit of quantification at 2.5 mg/l. The inter-day accuracy and imprecision of quality controls (5, 20, 45 mg/l) was 101-107% and < 8% (n = 4), respectively. For both methods no interfering substances were found in ultrafiltrate from patients not receiving MPA. The methods described have a suitable dynamic linear range to facilitate the investigation of free MPA and MPAG pharmacokinetics in transplant patients. Further, this is the first reported HPLC-UV method to determine free MPAG concentrations.     

Determination of nicotine,anabasine, and cotinine in urine and saliva samples using single-drop microextraction

A simple, sensitive, and inexpensive singe-drop microextraction (SDME) followed by gas chromatography and flame-ionization detection (GC-FID) was developed for determination of nicotine, anabasine, and cotinine in human urine and saliva samples. The target compounds were extracted from alkaline aqueous sample solution into an organic acceptor drop suspended on the tip of a 25-μL GC microsyringe in the aqueous sample solution. This microsyringe was also used for direct injection after extraction. Under optimized experimental conditions, calibration plots were found to be linear in the range of 0.5–25.0, 0.5–65.0, and 0.5–45.0 mg L^?1 for nicotine, anabasines and cotinine, respectively. The method detection limit values were in the range of 0.33–0.45 mg L^?1. Intra-day and inter-day precisions for peak area ratios were in the range of 1.3–9.2% and 2.0–7.0%, respectively. The proposed procedure was successfully applied to the determination of analytes in spiked urine and saliva samples with satisfactory results. The mean relative recoveries of spiked water samples ranged over 71.2–111.0%, with relative standard deviations varying from 2.3% to 10.0%.     

Measurement of total mirtazapine and normirtazapine in plasma/serum by liquid chromatography with fluorescence detection

A simple high performance liquid chromatography (HPLC) method for the measurement of the new antidepressant mirtazapine and its N-demethyl metabolite, normirtazapine, in human plasma or serum during low dose mirtazapine therapy has been developed. A Waters Spherisorb S5 SCX column was used with ammonium perchlorate (50 mmol/l) in methanol/water (95 + 5 (v/v)), apparent pH 6.7, as eluent, and fluorescence detection. Only small volumes of sample (0.2 ml) and extraction solvent are used. An interference study found no significant co-elution with drug or metabolite, although paroxetine co-elutes with the internal standard. The recovery of mirtazapine and normirtazapine (mean +/- S.D.) was 79 +/- 2, and 64 +/- 3%, respectively. The LOD was estimated as 0.5 microg/l, LLOQ was 1 microg/l, with a linear response over the concentration range 4-1000 microg/l (both analytes). The analytes were stable in serum for at least 10 months when stored at -20 degrees C. Intra- and inter-day accuracy were in the range 91-107 and 93-103%, respectively. In clinical samples (n = 14, median mirtazapine dose 45 mg per day, range 15-45 mg per day) the median (range) mirtazapine and normirtazapine concentrations were 26 (8-40) and 21 (8-32) microg/l, respectively.     

Nicotine and cotinine inhibit steroidogenesis in mouse Leydig cells

Cigarette smoking alters plasma testosterone concentrations in men. The objectives of this study were to determine if nicotine and cotinine, two alkaloid products of cigarettes, affect luteinizing hormone(LH)-stimulated steroidogenesis in isolated adult mouse Leydig cells. Leydig cells from adult Swiss-Webster mice were isolated by linear density gradient and incubated (95% O2, 5% CO2) in minimum essential medium at 37 C for 3 hours with LH (10 ng) and with or without nicotine or cotinine (10(-5)-10(-7) M). Both nicotine and cotinine produced dose response inhibition (P less than 0.05) of LH-stimulated testosterone production (50-70%). The addition of 8-bromo-3',5'-cyclic monophosphate (cAMP, 500 uM) stimulated steroidogenesis comparable to LH in the absence of the alkaloids, but both nicotine and cotinine significantly (P less than 0.05) reduced testosterone production in response to cAMP, suggesting that the alkaloids inhibit testosterone production in response to LH distal to the formation of cAMP. In MEM without calcium, LH-stimulated testosterone synthesis was decreased, and neither nicotine nor cotinine significantly affected steroidogenesis. The addition of a calcium ionophore in MEM with normal calcium content enhanced (P less than 0.05) the inhibitory effects of nicotine and cotinine on LH-responsive steroidogenesis. A calcium channel blocking agent, verapamil, at 10uM significantly (P less than 0.05) reversed the inhibition of LH-stimulated testosterone production produced by both alkaloids when incubated in the medium with a normal calcium concentration. These results suggest that nicotine and cotinine either affect intracellular calcium content or block the effects of calcium on steroidogenesis in mouse Leydig cells.