A validated method for the quantification of IOX-2, a potent prolyl hydroxylase inhibitor in equine urine and plasma using liquid chromatography–high-resolution mass spectrometry

IOX-2 is a potent inhibitor of enzyme prolyl hydroxylases-2 (PHD) that plays a critical role in regulating hypoxia inducible factor (HIF) abundance and oxygen homeostasis. Federation for Equestrian Sports has listed HIF activators as prohibited substances to prevent their usage in doping. Consequently, it became essential to develop adequate knowledge and testing methods to detect it in equine sports drug testing samples. The validated method utilizes ultra-high-performance liquid chromatography coupled to high-resolution mass spectrometry in order to detect extremely low concentration of the analyte present in both matrices. Confirmation for the presence of the analyte was achieved by comparing ion ratios, retention time, and accurate mass. Method linear range for plasma was between 0.25 to 100 ng/ml, and limit of detection (LOD) was 0.075 ng/ml. The linear range for urine was 0.125 to 100 ng/ml, and LOD was 0.025 ng/ml. Intraday precision at 0.5, 10, and 50 ng/ml was between 4.0% and 9.7% for plasma and 4.2% and 10.4% for urine. Accuracy at 0.5, 10, and 50 ng/ml was between 91% and 94% for plasma and 99% and 103% for urine. Elimination profile of IOX-2 in equine plasma and urine was carried out using the developed method in which two horses were intravenously administered IOX-2 and samples were collected. Metabolic profile in plasma and urine was investigated. IOX-2 was detected for a minimum of 54 and 151 h of post administration in plasma and urine, respectively, thereby providing a valuable tool for evaluating its misuse in equine racing.     

Pharmacokinetic study of a new angiotensin-AT1 antagonist by HPLC

Recently an innovative novel class angiotensin-AT(1) antagonist has been developed by Rottapharm. In this study, we present a validated method for detecting CR 3834 in biological matrices using high-performance liquid chromatography (HPLC) with diode array detection. After oral administration (30mg/kg) to Wistar rats, the plasma and urine concentrations of CR 3834 and its potential metabolic products were determined. Moreover, the plasmatic time course in rats has been determined after intravenous (IV) administration of CR 3834 (5mg/kg). Biological samples (0.5ml of plasma and 1ml of urine) were purified using solid-phase extraction (SPE) of analytes and the internal standard Idebenone, 2,3-dimethoxy-5-methyl-6-(10-hydroxydecyl)-1-4-benzoquinone. A chromatographic separation was performed on an Adsorboshere C18 at 25 degrees C, with a pre-column of the same matrix; the eluent was made up of acetonitrile/acidified water with CF(3)COOH (pH 2.01) in ratio of 75:25 (v/v); the flow rate was 1.0ml/min and a 100mul loop. The lower limit of detection (LOD) was taken as 25ng/ml in plasma and 50ng/ml in urine samples. The lower limit of quantification (LOQ) was taken as 0.1 and 0.2mug/ml in plasma and urine samples, respectively. The procedures were validated according to international standards with a good reproducibility and linear response (r=0.9916 in plasma; r=0.9997 in urine). The coefficients of variation inter assay ranged between 2.579 and 4.951% in plasma, and between 0.813 and 2.460% in urine. Mean recovery for CR 3834 was 79% in plasma and 97% in urine samples. The experiments performed demonstrated that the method presented was suitable for determining this new angiotensin-AT(1) antagonist in rat plasma and urine.     

Determination of rabeprazole enantiomers and their metabolites by high-performance liquid chromatography with solid-phase extraction

Here, we describe the development of a rapid, simple and sensitive high-performance liquid chromatography (HPLC) method for the simultaneous quantitative determination of rabeprazole enantiomers (1a,b) and their metabolites, rabeprazole-thioether (2) and rabeprazole sulfone (3), in human plasma. Analytes and the internal standard (omeprazole-thioether) were separated using a mobile phase of 0.5 M NaClO4-acetonitrile (6:4, v/v) over a Chiral CD-Ph column. Analysis required only 100 microl of plasma and involved solid-phase extraction with an Oasis HLB cartridge, which gave high recovery (>91.8%) with good selectivity for all analytes. The lower limit of quantification was 5 ng/ml for analytes 1a, 1b and 3 and 10 ng/ml for 2. Linearity of this assay was determined to lie between 5 and 1000 ng/ml for 1a, 1b and 3 and 10 and 1000 ng/ml for 2 (r2>0.982 of the regression line). Inter- and intra-day coefficients of variation were less than 7.8% and accuracies were within 8.4% over the linear range for all analytes. Our results indicate that this method is applicable to the simultaneous monitoring of plasma levels of rabeprazole enantiomers and associated metabolites in human plasma.     

Quantitative determination of paclitaxel in human plasma using semi-automated liquid-liquid extraction in conjunction with liquid chromatography/tandem mass spectrometry

This paper describes a high-throughput sample preparation procedure combined with LC-MS/MS analysis to measure paclitaxel in human plasma. Paclitaxel and an internal standard were extracted from plasma by a semi-automated robotic method using liquid-liquid extraction. Thereafter compounds were separated on a RP C18 column. Detection was by a PE Sciex API 3000 mass spectrometer equipped with a TurboIonSpray interface. The compounds were detected in positive ion mode using the mass transition m/z 854.6-->286.2 and m/z 831.6-->263.2 for paclitaxel and the internal standard, respectively. The limit of quantitation for paclitaxel was 1 ng/ml with an imprecision of 5.2% following extraction of 0.1 ml of plasma. Linearity was confirmed over the whole calibration range (1-1000 ng/ml) with correlation coefficients higher than 0.99 indicating good fits of the regression models. The inter and intra-day precision was better than 9.5% and the accuracy ranged from 90.3 to 104.4%. The assay was simple, fast, specific and exhibited excellent ruggedness.     

Liquid chromatographic-tandem mass spectrometric urine assay for a highly metabolized cyclic ureidobenzenesulfonamide: issues concerning assay specificity and quality control preparation

An LC–MS–MS method was validated for the quantitation of a β₃ agonist (A) in human urine to support Phase I studies. A was designed to accelerate metabolism for weight reduction. During assay development a significant loss of A was apparent from frozen urine quality control samples. The addition of 0.75% bovine serum albumin (BSA) in urine (v/v) was required to maximize the recovery of A from urine. Urine samples were basified and extracted into methyl t-butyl ether–isopropyl alcohol (90:10, v/v). The organic layer was washed, evaporated, reconstituted, and injected onto a 5 cm, C8 HPLC column prior to MS–MS analysis. The standard curve was linear from 5 to 500 ng/ml. Intraday precision for peak area ratios from BSA urine samples at seven separate concentrations over a range of 5–500 ng/ml (n=5) was <4.0% and calculated concentrations were within 91–115% of nominal concentrations. Interday precision for BSA urine quality control (QC) samples at four separate concentrations (n=10 of each) was <5.0% and individual calculated concentrations were within 90–111% of nominal concentrations. This work emphasizes that potential metabolites and quality control standards should be prepared and assayed as early as possible in method development, especially before the sample collection section of the clinical protocol is prepared. The methods described here have wide utility to other compounds containing basic benzene sulfonamides and to β₃ agonist candidates.     

Determination of 3'-C-ethynylcytidine in human plasma and urine by liquid chromatographic-electrospray ionization tandem mass spectrometry

A liquid chromatographic-electrospray ionization tandem mass spectrometric (LC-ESI/MS/MS) method was developed for the quantitative analysis of a novel anticancer drug, 3'-C-ethynylcytidine (I) in human plasma and urine. I and its stable isotope-labeled internal standard (II) were extracted from human plasma and urine samples using a polymer-based cation-exchange cartridge, and LC-ESI/MS/MS analysis was performed by monitoring the positive fragment ions of I and II. The linear ranges are 1-500 ng/ml in plasma and 10-5000 ng/ml in urine. The limits of quantitation for I were 1 ng/ml in plasma and 10 ng/ml in urine. The relative errors (RE) for I ranged from -8.4 to 3.0% in plasma and from 0.8 to 4.4% in urine. The relative standard deviations (RSD) for I ranged from 1.2 to 8.9% in plasma and from 0.7 to 2.8% in urine. This validated analytical method is demonstrated to be useful for the analysis of I in human plasma and urine in clinical studies.     

UrinarypH and urine flow independent renal clearance of methotrexate in dogs

The effects of urine flow and pH on methotrexate renal clearance were studied in seven conditioned male Beagle-Mongrel dogs. Steady-state plasma methotrexate and inulin concentrations were achieved by i.v. infusions preceded by i.v. bolus doses. Plasma and urine concentrations of methotrexate were quantitated by a sensitive high-performance liquid Chromatographic assay, while those of inulin were measured by a colorimetric method. Since plasma protein binding of methotrexate was pH and concentration independent, methotrexate/inulin renal clearance without correcting for plasma binding was used for most of the data analyses. The results showed that the renal clearance ratios at the plasma methotrexate levels (approximately 0.1, 1.0, 20.0, and 100 g/ml) studied remained relatively constant when urine pH (differences of up to about 2.5 units) and flow rate (differences of up to approximately 30 times) were changed. This indicated that renal reabsorption of methotrexate in these dogs was negligible. However, concentration-dependent renal clearance was observed. The mean renal clearances were 3.84, 3.94, 2.73, and 2.72 ml/min/kg at plasma concentrations of about 0.1, 1.0, 20.0, and 100.0 g/ml, respectively, when urine was alkalized by sodium bicarbonate. The corresponding clearances were 4.02, 4.28, 2.62, and 2.65 ml/min/kg when urine was acidified by ammonium chloride. These showed the existence of saturable tubular secretion of methotrexate. No 7-hydroxy-methotrexate, a metabolite found in other species, was detected in the urine and plasma of the dogs.This research was supported in part by a grant from the National Cancer Institute, CA-29754. Abstracted from a dissertation submitted in 1984 by Chung Y. Lui to the Graduate College, University of Illinois at Chicago in partial fulfillment of Doctor of Philosophy Degree requirements.     

Sensitive determination of nefopam and its metabolite desmethyl-nefopam in human biological fluids by HPLC

Nefopam (NEF) and desmethyl-nefopam (DMN) were assayed simultaneously in plasma, globule and urine samples using imipramine as internal standard. A liquid-liquid extraction procedure was coupled with a reverse phase high-performance liquid chromatography system. This system requires a mobile phase containing buffer (15 mM KH(2)PO(4) with 5 mM octane sulfonic acid: pH 3.7) and acetonitrile (77:33, v/v) through (flow rate=1.5 ml/min) a C(18) Symmetry column (150x4.6 I.D., 5 micrometer particle size: Waters) and a UV detector set at 210 nm. Internal standard was added to 1 ml of plasma or globule sample or 0.5 ml of urine sample, prior to the extraction under alkaline ambiance with n-hexane. The limits of quantification were 1 and 2 ng/ml for both molecules in plasma and globule, respectively; 5 and 10 ng/ml for NEF and DMN in urine, respectively. The method proved to be accurate and precise: the relative error at three concentrations ranged from -13.0 to +12.3% of the nominal concentration for all molecule and biological fluid; the within-day and between-day precision (relative standard deviation %) ranged from 1.0 to 10.1% for all the molecules and biological fluids. The method was linear between 1 and 60 ng/ml for both molecules in the plasma; 2 and 25 ng/ml for both molecules in the globule; 25 and 250 ng/ml for NEF and 50 and 500 ng/ml for DMN in the urine: correlation coefficients of calibration curves (determined by least-squares regression) of each molecule were higher than 0.992 whatever the biological fluid and during the pre-study and in-study validations. This method was successfully applied to a bio-availability study of NEF in healthy subjects.     

GLC determination of phendimetrazine in human plasma, serum, or urine

A sensitive, specific, and quantitative GLC method is described for the determination of phendimetrazine in plasma, serum, or urine. An internal standard was used, which was extracted along with the drug. This mixture then was acetylated to improve the chromatographic separation. A concentration as low as 2 ng/ml of phendimetrazine could be measured from 2 ml of sample using a nitrogen-phosphorous detector. Linearity extended from 2 to 500 ng/ml, and the coefficient of variation was 7%. The method was shown to be applicable to a single-dose bioavailability study.     

The determination of nadolol in biological samples using high-performance liquid chromatography

A method has been developed for the determination of nadolol in biological samples by reversed-phase high-performance liquid chromatography with fluorimetric detection. The method has been applied to plasma, serum and urine samples, which are prepared by extraction with diethyl ether-dichloromethane (5:2,v/v), evaporation of the organic solvent, and dissolution of the resultant residue in the chromatographic eluent. The sample is then subjected to chromatography on a C(18)-silica column, with an eluent of water-acetonitrile-triethylamine (800:200:1,v/v) adjusted to pH 3.0 with orthophosphoric acid. A single point external standard is used for quantitation. The working ranges were 1-400 ng/ml for plasma/serum, and 0.1-40 mug/ml for urine, although a detection limit of 0.1 ng/ml appears to be readily attainable. The sample size was 0.5 ml, and for both types of sample the method showed good correlation with a previously published fluorimetric method (for plasma, r = 0.9544, n = 70; for urine, r = 0.9919, n = 35).     

Determination of clarithromycin in human plasma by liquid chromatography-electrospray ionization tandem mass spectrometry

A rapid and sensitive method has been developed for the determination of clarithromycin in human plasma with liquid chromatography-tandem mass spectrometry. Clarithromycin and the internal standard, telmisartan were precipitated from the matrix (50 microl) with 200 microl acetonitrile and separated by HPLC using formic acid:10 mM ammonium acetate:methanol (1:99:400, v/v/v) as the mobile phase. The assay based on detection by electrospray positive ionization mass spectrometry in the multiple-reaction monitoring mode was finished within 2.4 min. Linearity was over the concentration range 10-5000 ng/ml with a limit of detection of 0.50 ng/ml. Intra- and inter-day precision measured as relative standard deviation were <3.73% and <9.93%, respectively. The method was applied in a bioequivalence study of two tablet formulations of clarithromycin.     

Determination of a new phosphodiesterase V inhibitor,DA-8159, in plasma and urine by high-performance liquid chromatography

A high-performance liquid chromatographic (HPLC) method using liquid-liquid extraction for sample preparation was developed for the determination of a new phosphodiesterase V inhibitor, DA-8159, in rat plasma and urine using sildenafil citrate as an internal standard. A 100 microl aliquot of 0.1 M Na(2)CO(3) (containing sildenafil citrate, 3 microg/ml as free sildenafil) and a 1 ml aliquot of ether were added to a 100 microl aliquot of biological samples (urine samples were diluted 20 times with distilled water). After vortex centrifugation at 9000 x g for 3 min, the ether layer was collected and dried under nitrogen gas. The residue was reconstituted with a 150 microl aliquot of the mobile phase, centrifuged, and a 100 microl aliquot of the supernatant was injected onto a reversed-phase column. The mobile phases, 20 mM KH(2)PO(4) (pH 4.7):acetonitrile (70:30, v/v for plasma and tissue samples, and 75:25, v/v for urine samples), were run at a flow rate of 1.0 ml/min. The column effluent was monitored by an ultraviolet detector set at 292 nm. The retention times for DA-8159 and the internal standard were approximately 10.7 and 9.1 min, respectively, in plasma and tissue samples and the corresponding values in urine samples were 47 and 33 min. The detection limits for DA-8159 in rat plasma and urine were 20 and 100 ng/ml, respectively. The coefficients of variation of the assay were generally low: below 10% for plasma and 9.9% for urine. No interferences from endogenous substances were found.     

High-Performance Liquid Chromatographic Determination of 2′, 3′ -Didehydro-3′ -deoxythymidine (d4T) in Human and Rabbit Plasma and Urine and Its Application to Pharmacokinetic Studies in the Rabbit

A rapid and sensitive liquid chromatographic assay for 2,3-didehydro-3-deoxythymidine (d4T) in plasma and urine is described. This assay uses thymidine oxetane (TO), a synthetic precursor of d4T, as internal standard. Sample preparation involves a simple extraction of plasma or urine with 5% isopropyl alcohol in methylene chloride. The method is specific and sensitive, allowing a linear response over a 2000-fold range of concentrations in human plasma (5 ng/ml to 10 µg/ml) and urine (50 ng/ml to 100 µg/ml). This assay, developed for human plasma and urine, is also applicable to rabbit samples with minor modification. Intravenous bolus doses of 10 mg/kg d4T to rabbits showed that the plasma concentration–time profile followed a biexponential decay. Estimates of the distribution and elimination half-lives were 6.7 ± 0.9 and 51 ± 6 min, respectively. The total-body and renal clearances were 23.4 ± 3.6 and 8.82 ± 3.9 ml/min · kg, respectively. That the renal clearance exceeds the glomerular filtration rate in the rabbit suggests that d4T is actively secreted in the renal tubule. The fraction excreted unchanged in the urine was 36 ± 8%. Similar results were obtained in the same rabbits at steady state during constant-rate intravenous infusion. Noncompartmental analysis estimates of the MRT and V _dss were 46 ± 5 min and 1.08 ± 0.13 L/kg, respectively.     

LC-MS-MS determination of nemorubicin (methoxymorpholinyldoxorubicin,PNU-152243A) and its 13-OH metabolite (PNU-155051A) in human plasma

A selective and sensitive liquid chromatography-tandem mass spectrometry (LC-MS-MS) method for quantitative determination of nemorubicin, (PNU-152243A, 3'-deamino-3'[2(S)-methoxy-4-morpholinyl]doxorubicin) hydrocloride and its reduced metabolite PNU-155051 in human plasma has been developed and validated. The method involved solid phase extraction (SPE) in 96-well plates. Plasma samples (0.5 ml plasma, spiked with doxorubicin as internal standard and diluted with 0.5 ml of 0.01 M borate buffer, pH 8.4) were extracted using Oasis HLB SPE material. The elution of PNU-152243, PNU-155051 and of IS was performed with 1 ml of methanol:0.1 M formic acid mixture (90:10, v/v). The organic phase was reduced to dryness under a stream of nitrogen at 20 degrees C and the residue was reconstituted with 0.25 ml of 10 mM ammonium formate buffer pH 4.15:acetonitrile mixture (90:10, v/v). Aliquots of 60 microl of the resulting solution were injected onto the LC-MS-MS system. A Zorbax SB C18 column (2.1 x 150 mm, 3.5 microm) was used to perform the chromatographic analysis. The mobile phase consisted of ammonium formate buffer 10 mM pH 4.15:acetonitrile (73:27, v/v) with a flow-rate of 0.2 ml/min. Detection was achieved by a PE-SCIEX API 3000 with Turbo IonSpray interface, and multiple reaction monitoring (645 --> 321 for PNU-152243, 647 --> 363 for PNU-155051 and 545 --> 345 m/z for doxorubicin) operated in positive ion mode. A weighted linear regression was used to calculate PNU-152243 and PNU-155051 concentrations in QC and unknown samples. Linearity, precision, accuracy and recovery of the method were evaluated over the concentration range of 0.1-5 ng/ml for both compounds. No interference from blank human plasma was observed. The suitability of the method for in vivo samples was assessed by the analysis of samples obtained from patients who had received a single intrahepatic artery dose of PNU-152243A.     

HPLC method for the determination of fluvoxamine in human plasma and urine for application to pharmacokinetic studies

A simple, specific and sensitive high-performance liquid chromatographic (HPLC) method has been developed for the assay of fluvoxamine in human plasma and urine. The method was based on reaction of fluvoxamine with 1,2-naphthoquinone-4-sulphonic acid sodium salt (NQS) forming orange colored product. The fluvoxamine-NQ derivative was separated by isocratic reversed-phase HPLC and detected at 450 nm. The chromatographic conditions were as follows: Phenomenex C(18) (250 mm x 4.6 mm i.d., 5 microm) column, mobile phase consisting of acetonitrile/water (80:20 v/v) at a flow rate of 1 ml/min. Tryptamine was selected as an internal standard. The assay was linear over the concentration range of 5-145 and 2-100 ng/ml for plasma and urine, respectively. The limits of detection (LOD) were 1.4 and 1 ng/ml for plasma and urine estimation at a signal-to-noise (S/N) ratio of 3. The limits of quantification (LOQ) were 5 and 2 ng/ml for plasma and urine, respectively. The extraction recoveries were found to be 96.66+/-0.69 and 96.73+/-2.17% for plasma and urine, respectively. The intra-day and inter-day standard deviations (S.D.) were less than 1. The method indicated good performance in terms of specificity, linearity, detection and quantification limits, precision and accuracy. This assay was demonstrated to be applicable for clinical pharmacokinetic studies.     

Biliary excretion and enterohepatic recycling of proscillaridin a after oral administration to man

Summary A single oral dose of proscillaridin A (1.0–1.5 mg) was given to six patients with T-tube drainage of the common bile duct, and simultaneous samples of bile and plasma were collected at various times during the following 24 hours. Glycoside activity was assayed by the⁸⁶Rb-uptake inhibition technique. Peak activities in plasma (mean 0.80 ng/ml) were attained after 0.5–2 h, and in bile (mean 6.9 ng/ml) after 1–4 h. Subsequently, proscillaridin activity in bile was less than 5 ng/ml for the remainder of the sampling period, and 10–100 times higher than that in plasma. Bile samples treated with -glucuronidase and sulphatase showed 100–200 fold increase in glycoside activity. Deconjugation was also produced by treatment with enteric contents. The results suggest that conjugation of unchanged proscillaridin is a major metabolic route. After excretion in the bile, the conjugates may be split in the intestine and reabsorbed as active glycoside.     

HPLC-紫外法测定人血浆中芬太尼浓度

目的 :建立高效液相色谱法 -紫外检测器测定人血浆中芬太尼浓度的方法。方法 :本实验采用外标法 ,以Shim -PackCLC -ODS(6 0mm×150mm ,5μm )为固定相 ,含0 015mol/LNaH2PO4 的乙腈 -水溶液 (30∶70 ,v/v)为流动相 ,流速1 5ml/min ,紫外检测波长195nm。结果 :标准曲线在2 0～100ng/ml范围内线性关系良好 (r=0 999) ,最低检测浓度为1ng/ml,方法回收率为(91 70±4 70) % ,提取回收率为 (97 38±3 69) % ,日内变异RSD (6 50±2 79) % ,日间变异RSD (6 70±3 04) %。结论 :本方法简便 ,准确 ,检测浓度低 ,能够满足血浆中低浓度芬太尼的测定及临床药代动力学研究的要求。     

Urinary pH and urine flow independent renal clearance of methotrexate in dogs

The effects of urine flow and pH on methotrexate renal clearance were studied in seven conditioned male Beagle-Mongrel dogs. Steady-state plasma methotrexate and inulin concentrations were achieved by i.v. infusions preceded by i.v. bolus doses. Plasma and urine concentrations of methotrexate were quantitated by a sensitive high-performance liquid chromatographic assay, while those of inulin were measured by a colorimetric method. Since plasma protein binding of methotrexate was pH and concentration independent, methotrexate/inulin renal clearance without correcting for plasma binding was used for most of the data analyses. The results showed that the renal clearance ratios at the plasma methotrexate levels (approximately 0.1, 1.0, 20.0 and 100 micrograms/ml) studied remained relatively constant when urine pH (differences of up to about 2.5 units) and flow rate (differences of up to approximately 30 times) were changed. This indicated that renal reabsorption of methotrexate in these dogs was negligible. However, concentration-dependent renal clearance was observed. The mean renal clearances were 3.84, 3.94, 2.73, and 2.72 ml/min/kg at plasma concentrations of about 0.1, 1.0, 20.0, and 100.0 micrograms/ml, respectively, when urine was alkalized by sodium bicarbonate. The corresponding clearances were 4.02, 4.28, 2.62,and 2.65 ml/min/kg when urine was acidified by ammonium chloride. These showed the existence of saturable tubular secretion of methotrexate. No 7-hydroxy-methotrexate, a metabolite found in other species, was detected in the urine and plasma of the dogs.     

High performance liquid chromatographic determination of nicotine and cotinine in plasma and nicotine and cotinine, simultaneously, in urine

Three analytical procedures were developed to determine nicotine in plasma, cotinine in plasma and, simultaneously, nicotine and cotinine in urine. After liquid or solid-phase extraction, the purified aqueous phase is injected into a high performance liquid chromatograph equipped with an ultra-violet detector using a CN Spheri-5 micron cartridge-column with an inner diameter of 4.6 mm and a length of 10 or 22 cm. The limit of quantitation for nicotine in plasma was around 8 to 15 ng/ml, that of cotinine in plasma around 50 ng/ml and that of nicotine and cotinine in urine around 170 ng/ml and 70 ng/ml, respectively. The limit of detection of nicotine in plasma was around 1 ng/ml and that of nicotine and cotinine in urine around 20 ng/ml and 10 ng/ml, respectively. The passive exposure to cigarette smoke by non-smokers and the "resting levels" of nicotine in plasma and urine of smokers were studied. The analytical methods were set up to study the pharmacokinetics and bioavailability of nicotine in healthy volunteers following single and repeated administrations of different doses of transdermal nicotine systems.