A multidimensional overpressured layer chromatographic method for the characterization of tetrazine libraries

The recent combinatorial approach in synthetic organic chemistry started a new age in drug discovery. The generation of compound libraries in combination with high-throughput screening has become the method of choice for the production of new pharmacological leads for chemical optimization. Characterization and separation of such pool of compounds have been lagging behind the synthetic and screening methodologies. Overpressured layer chromatography (OPLC) is an instrumentalized planar liquid chromatographic technique associated with the use of optimized layers prepared from particles of narrow particle size distribution and small diameter. On one hand, uni-directional OPLC allows the simultaneous separation of large number of samples in minutes. On the other hand, two-dimensional OPLC offers multidimensional separation on a single layer. This paper shows the complete multidimensional separation of a tetrazine library prepared by parallel combinatorial synthesis. In general, this approach may become the method of choice for the characterization of compound libraries.     

Capillary electrophoresis as a verification tool for immunochemical drug screening

BACKGROUnd: The aim of this work was to develop a simple capillary electrophoretic method as the verification and confirmation tool in the screening analysis for amphetamines, opiates, benzodiazepines and cocaine and their metabolites for toxicological applications. METHODS: 50 mM phosphate Tris pH 2.0 with 30% (v/v) of methanol was used as a background electrolyte that enabled fast separation of drugs and their metabolites in saliva and urine. Verification of the data from the electrophoretic method was done by High Performance Thin Layer Chromatography (HPTLC) and the immunochemical screening test QuikScreen. RESULTS: The experimental conditions of the Capillary Electrophoresis (CE) were partially optimized (mainly the influence of concentration and types of additives, e.g. cyclodextrines, organic solvents) and validated; the method was used for analysing samples from drug abusers. CONCLUSIONS: The non-instrumental, immunoassay tests could only confirm qualitative addictions and are mainly employed when the emergency detection of drugs is needed. For quantitative analysis and verification of obtained results the confirmation step is strongly recommended. The simple screening capillary zone electrophoresis method allows recognition of the most abused drugs. The agreement of the results from CE, HPTLC and QuikScreen test was more than 95%.     

High-performance liquid chromatography method for the simultaneous determination of sulfamethoxazole and trimethoprim in bovine milk using an on-line clean-up column

A bidimensional HPLC method for the simultaneous determination of sulfamethoxazole (SMX) and trimethoprim (TMP) in bovine milk has been developed and validated. After centrifugation, aliquots (150 microl) of milk samples were directly injected to a column-switching HPLC system. At the first step a RAM octyl-BSA column was employed to automatically remove proteins that otherwise would interfere with milk analysis. The mobile phase 0.01 M phosphate buffer pH 6.0:acetonitrile (95:5, v/v) was used in the first 5 min for the elution of milk proteins and then 0.01 M phosphate buffer pH 6.0:acetonitrile (83:17, v/v) for transfer SMX and TMP to the analytical column. The separation of SMX and TMP from one another and from other remaining milk components was performed on an octyl column using the mobile phase 0.01 M phosphate buffer pH 5.0:acetonitrile (82:18, v/v), which were detected by UV at 265 nm. The calibration graphs were linear in the concentration ranges of 25-800 ng/ml and 50-400 ng/ml for SMX and TMP, respectively. The intra- and inter-assay coefficients of variation were less than 15% for both drugs. The validated method was applied to the analysis of milk samples of twelve (two groups of six) cows after administration (intramuscular or subcutaneous) of a single recommended therapeutic dose of the SMX-TMP combination.     

Enantioselective analyses of chloroquine and hydroxychloroquine in rat liver microsomes through chiral liquid chromatography–tandem mass spectrometry

An efficient, sensitive and selective liquid chromatography–tandem mass spectrometry (LC–MS/MS) chiral analysis method was established for determination of chloroquine and hydroxychloroquine enantiomers in rat liver microsomes. Effects of polysaccharide chiral stationary phases and basic additives on chiral separations of two analytes were discussed in detail. Amylose tris(3, 5-dimethylphenylcarbamate)-coated chiral stationary phase showed the best separation performance for them with acetonitrile-diethylamine-ethanol-diethylamine mixture (90:0.1:10:0.1, v/v/v/v) among four chiral stationary phases. Then, multiple reaction monitoring mode was selected as the data acquisition for determination of two pairs of enantiomers. The proposed LC–MS/MS chiral analysis method was validated in terms of linearity, accuracy, precision, and specificity. Good linearity with correlation coefficient over 0.998 was obtained in the concentration range of 0.05–5 μM. Limits of quantification for chloroquine and hydroxychloroquine enantiomers were 5.0 and 1.0 nM, respectively. The recoveries ranged from 81.14% to 111.09%. The intra-day and inter-day relative standard deviation were less than 6.5%. Moreover, concentrations of chloroquine and hydroxychloroquine enantiomers in rat liver microsomes were determined through the proposed LC–MS/MS analysis method. After incubated with rat liver microsomes for 10 min, the enantiomeric factor of hydroxychloroquine decreased from 0.50 to 0.45 (p < 0.001). In brief, our developed determination method for chloroquine and hydroxychloroquine enantiomers through LC–MS/MS spectrometry showed the characteristics of high-efficiency, fast speed, and very low detection limit, and would be greatly beneficial for screening and quantitation of them in biological matrices.     

Precise gas chromatography with retention time locking in comprehensive toxicological screening for drugs in blood

The long-term precision of three retention parameters, the absolute retention time (RT), the relative retention time related to dibenzepin (RRT), and the internal retention index based on the alkylfluoroaniline series (RI), were studied with 14 basic drugs on HP-5 and DB-17 columns with and without the use of the retention time locking option (RTL). Using the constant flow mode in all experiments, the RTL method was found to produce superior precision with all three retention parameters compared to the non-RTL method on each column. The results showed that RTL offers a significant advantage within a single instrument method, not only between methods, with CV<0.1% by RRT. Consequently, a dual-column gas chromatographic procedure with nitrogen-phosphorus detection was described for comprehensive screening for basic drugs in 1-ml whole blood samples. The method consisted of one-step liquid-liquid extraction with butyl acetate, identification using RRT in the RTL mode, and quantification based on single point calibration. The method allowed reliable screening and quantification of 124 basic drugs at therapeutic and toxic concentration levels in autopsy blood.     

Extraction and isolation of avermectins and milbemycins from liver samples using unmodified supercritical CO2 with in-line trapping on basic alumina

A multi-residue supercritical fluid extraction (SFE) method has been developed for the extraction and isolation of eprinomectin, moxidectin, abamectin, doramectin and ivermectin residues from animal liver. Liver samples are mixed with hydromatrix and packed into a vessel containing 2 g of basic alumina. The samples are extracted at 100°C using unmodified supercritical carbon dioxide (SF-CO₂) at a pressure of 300 bar and flow-rate of 5.0 l/min. The analytes are adsorbed in-line on the basic alumina trap, which is later eluted with 4 ml of methanol–ethyl acetate (70:30, v/v). After evaporating to dryness, sample extracts are derivatised using methylimidazole, trifluoroacetic anhydride and acetic acid at 65°C for 30 min. Derivatised sample extracts are analysed by high-performance liquid chromatography (HPLC) with fluorescence detection. The method was validated using bovine liver fortified at levels of 4 and 20 μg/kg with the drugs. The mean recovery ranged between 76 and 97%. The intra- and inter-assay variations showed RSD values <10 and <16%, respectively. The procedure was also applied to ovine and porcine liver, giving similar results. The limit of quantitation of the method is 2 μg/kg.     

High-performance liquid chromatography assay for simultaneous determination of dextromethorphan and its main metabolites in urine and in microsomal preparations

An HPLC method has been developed and validated for the determination of dextromethorphan, dextrorphan, 3-methoxymorphinan and 3-hydroxymorphinan in urine samples. Deconjugated compounds were extracted on silica cartridges using dichloromethane/hexane (95:05, v/v) as an eluent. Chromatographic separation was accomplished on a Phenyl analytical column serially connected with a Nitrile analytical column. The mobile phase consisted of a mixture of an aqueous solution, containing 1.5% acetic acid and 0.1% triethylamine, and acetonitrile (75:25, v/v). Compounds were monitored using a fluorescence detector. Calibration curves were linear over the range investigated (0.2–8.0 μM) with correlation coefficients >0.999. The method was reproducible and precise. Coefficients of variation and deviations from nominal values were both below 10%. For all the analytes, recoveries exceeded 77% and the limits of detection were 0.01 μM. The validated assay proved to be suitable for the determination of DEM metabolic indexes reported to reflect the enzymatic activity of the cytochrome P450s, CYP2D6 and CYP3A, both in vivo, when applied to urine samples from patients, and in vitro, when applied to samples from the incubation of liver microsomes with dextromethorphan.     

Rapid high-performance liquid chromatographic method for the separation of hydroxylated testosterone metabolites

A rapid high-performance liquid chromatography (HPLC) method is described for the quantitation of hydroxytestosterone metabolites. The method combines a Hypersil BDS C₁₈ analytical column (10 cm×0.46 cm) and a linear mobile phase (1.25 ml/min) gradient of tetrahydrofuran–acetonitrile–water (10:10:80, v/v) changing to tetrahydrofuran–acetonitrile–water (14:14:72, v/v) over 10 min then remaining isocratic for 3 min. The total run time for the chromatographic separation of eight metabolites of testosterone is 15 min. Detection by UV is linear between 300 ng/ml and 10 μg/ml with a limit of detection on column of 300 ng/ml. A method for the direct HPLC analysis of liver microsomal incubates of [¹⁴C]testosterone is also briefly described and when combined with the HPLC method, offers a distinct advantage over previously reported methods for the rapid screening of testosterone hydroxylase activity in rat and human liver microsomes.     

Development of a RP-HPLC method for screening potentially counterfeit anti-diabetic drugs

Pharmaceutical counterfeiting is becoming a serious problem in the world, especially in developing countries including China. Herein an isocratic reversed-phase high performance liquid chromatography (RP-HPLC) method was developed for screening counterfeit medicines and adulterated dietary supplement products. The developed method could be employed to separate and determine simultaneously six anti-diabetic drugs (glipizide, gliclazide, glibenclamide, glimepiride, gliquidone, repaglinide) on an isocratic solvent system using an Alltima C18 column (5 microm, 150 mmx4.6 mm) with an isocratic mobile phase of methanol-phosphate buffer (pH 3.0; 0.01 mol/L) (70:30, v/v), at a flow rate of 1.0 mL/min and at a wavelength of 230 nm. The proposed method was successfully applied to the analysis of medicinal and dietary supplement samples purchased from the local market in China.     

Simultaneous determination of amiodarone and its major metabolite desethylamiodarone in plasma, urine and tissues by high-performance liquid chromatography

A simple and sensitive high-performance liquid chromatographic method for the simultaneous assay of amiodarone and desethylarniodarone in plasma, urine and tissues has been developed. The method for plasma samples and tissue samples after homogenizing with 50% ethanol, involves deproteinization with acetonitrile containing the internal standard followed by centrifugation and direct injection of the supernatant into the liquid chromatograph. The method for urine specimens includes extraction with a diisopropyl ether—acetonitrile (95:5, v/v) mixture at pH 7.0 using disposable Clin-Elut 1003 columns, followed by evaporation of the eluate, reconstitution of the residue in methanol—acetonitrile (1:2, v/v) mixture and injection into the chromatograph. Separation was obtained using a Radial-Pak C₁₈ column operating in combination with a radial compression separation unit and a methanol–25% ammonia (99.3:0.7, v/v) mobile phase. A wavelength of 242 nm was used to monitor amiodarone, desethylamiodarone and the internal standard. The influence of the ammonia concentration in the mobile phase on the capacity factors of amiodarone, desethylamiodarone and two other potential metabolites, monoiodoamiodarone (L6355) and desiodoamiodarone (L3937) were investigated. Endogenous substances or a variety of drugs concomitantly used in amiodarone therapy did not interfere with the assay.The limit of sensitivity of the assay was 0.025 μg/ml with a precision of ± 17%. The inter- and intra-day coefficient of variation for replicate analyses of spiked plasma samples was less than 6%. This method has been demonstrated to be suitable for pharmacokinetic and metabolism studies of amiodarone in man.     

Quantitation of itopride in human serum by high-performance liquid chromatography with fluorescence detection and its application to a bioequivalence study

A new method was developed for determination of itopride in human serum by reversed phase high-performance liquid chromatography (HPLC) with fluorescence detection (excitation at 291 nm and emission at 342 nm). The method employed one-step extraction of itopride from serum matrix with a mixture of tert-butyl methyl ether and dichloromethane (70:30, v/v) using etoricoxib as an internal standard. Chromatographic separation was obtained within 12.0 min using a reverse phase YMC-Pack AM ODS column (250 mm x 4.6 mm, 5 microm) and an isocratic mobile phase constituting of a mixture of 0.05% tri-fluoro acetic acid in water and acetonitrile (75:25, v/v) flowing at a flow rate of 1.0 ml/min. The method was linear in the range of 14.0 ng/ml to 1000.0 ng/ml. The lower limit of quantitation (LLOQ) was 14.0 ng/ml. Average recovery of itopride and the internal standard from the biological matrix was more than 66.04 and 64.57%, respectively. The inter-day accuracy of the drug containing serum samples was more than 97.81% with a precision of 2.31-3.68%. The intra-day accuracy was 96.91% or more with a precision of 5.17-9.50%. Serum samples containing itopride were stable for 180.0 days at -70+/-5 degrees C and for 24.0 h at ambient temperature (25+/-5 degrees C). The method was successfully applied to the bioequivalence study of itopride in healthy, male human subjects.     

Simple and simultaneous determination for 12 phenothiazines in human serum by reversed-phase high-performance liquid chromatography

A high-performance liquid chromatographic method has been developed for the simultaneous analysis of the 12 phenothiazines (chlorpromazine, fluphenazine, levomepromazine, perazine, perphenazine, prochlorperazine, profenamine, promethazine, propericiazine, thioproperazine, thioridazine and trifluoperazine) in human serum using HPLC/UV. The separation was achieved using a C(18) reversed-phase column (250 mm x 4.6 mm I.D., particle size 5 microm, Inersil ODS-SP). The mobile phase, consisting of acetonitrile-methanol-30 mM NaH(2)PO(4) (pH 5.6) (300:200:500, v/v/v), was delivered at a flow rate of 0.9 mL/min and UV detection was carried out at 250 nm. The recoveries of the 12 phenothiazines spiked into serum samples were 87.6-99.8%. Regression equations for the 12 phenothiazines showed excellent linearity, with detection limits of 3.2-5.5 ng/mL for serum. The inter-day and intra-day coefficients of variation for serum samples were commonly below 8.8%. The selectivity, accuracy and precision of this method are satisfactory for clinical and forensic purposes. This sensitive and selective method offers the opportunity for simultaneous screening and quantification of almost all phenothiazines available in Japan for the purposes of clinical and forensic applications.     

Sensitivity improvement of circular dichroism detection in HPLC by using a low-pass electronic noise filter: Application to the enantiomeric determination purity of a basic drug

The quality control of chiral drugs requires the determination of their enantiomeric purity. Nowadays, circular dichroism (CD) spectroscopy is gaining increasing importance in pharmaceutical analysis because of the commercially available CD detector in liquid chromatography. The separation of the two enantiomers of a basic drug (efaroxan) was achieved by high performance liquid chromatography using an amylose-derivated column with both UV and CD detections. A baseline-resolved separation (resolution: 5) was obtained after optimization of the mobile phase composition with hexane-ethanol-diethylamine (90:10:0.05; v/v/v). The use of a commercial low-pass electronic noise filter of the CD signal has improved the signal-to-noise ratio by a factor twelve and allowed the quantitation of each enantiomer in the 1.25-300 microg ml(-1) concentration range. The CD linear calibration curve, expressed in terms of stereoisomer height ratio versus concentration ratio, was plotted over the 0.4-6% range. A correlation coefficient greater than 0.999 was obtained by least-squares regression and the limit of detection for the distomer/eutomer ratio was estimated at 0.14%. Although the method validation showed good repeatability on the retention times (RSD < 0.9%), on the peak height ratios (RSD < 8.7%) of each enantiomer only up to 99.2% enantiomeric purity was achieved.     

Development of an LC-MS/MS method for the quantitation of 55 compounds prescribed in combined cardiovascular therapy

This paper reports an LC–MS/MS method with positive electrospray ionization for the screening of commonly prescribed cardiovascular drugs in human plasma, including compounds with antihypertensive (57), antidiabetic (12), hypolipemiant (5), anticoagulant (2) and platelet anti-aggregation (2) effects. Sample treatment consisted of a simple protein precipitation with MeOH/0.1 M ZnSO₄ (4:1, v/v) solution after the addition of internal standard, followed by evaporation and reconstitution. Analytes separation was performed on a Polar-RP column (150 mm × 2 mm, 4 μm) using a gradient elution of 15 min. The MS system was operated in MRM mode, monitoring one quantitation and one confirmation transition for each analyte. The recovery of the protein precipitation step ranged from 50 to 70% for most of the compounds, while some were considerably affected by matrix effects. Since several analytes fulfilled the linearity, accuracy and precision values required by the ICH guidelines, the method proved to be suitable for their quantitative analysis. The limits of quantitation varied from 0.38 to 9.1 μg/L and the limits of detection from 0.12 to 5.34 μg/L. The method showed to be suitable for the detection of plasma samples of patients under cardiovascular treatment with the studied drugs, and for 55 compounds reliable quantitative results could be obtained.     

Sensitive quantification of ranolazine in human plasma by liquid chromatography--tandem mass spectrometry with positive electrospray ionization

A rapid, selective and sensitive liquid chromatography-tandem mass spectrometry (LC-MS-MS) method with positive electrospray ionization (ESI) was developed for the quantification of ranolazine in human plasma. After liquid-liquid extraction of ranolazine and internal standard (ISTD) phenoprolamine from a 100 microl specimen of plasma, HPLC separation was achieved on a Nova-Pak C(18) column, using acetonitrile-water-formic acid-10% n-butylamine (70:30:0.5:0.08, v/v/v/v) as the mobile phase. The mass spectrometer was operated in multiple reaction monitoring (MRM) mode using the transition m/z 428.5-->m/z 279.1 for ranolazine and m/z 344.3-->m/z 165.1 for the internal standard, respectively. Linear calibration curves were obtained in the concentration range of 5-4000 ng/ml, with a lower limit of quantitation (LLOQ) of 5 ng/ml. The intra- and inter-day precision values were below 3.7% and accuracy was within +/-3.2% at all three quality control (QC) levels. This method was found suitable for the analysis of plasma samples collected during the phase I pharmacokinetic studies of ranolazine performed in 28 healthy volunteers after single oral doses from 200 mg to 800 mg.     

Enantiomeric separation of norgestrel by reversed phase high-performance liquid chromatography using eluents containing hydroxypropyl-beta-cyclodextrin in stereoselective skin permeation study

The chiral separation of norgestrel enantiomers using reversed-phase high-performance liquid chromatography (RP-HPLC) was studied with hydroxypropyl-beta-cyclodextrin (HP-beta-CD) as chiral mobile phase additive. The effect of mobile phase composition, concentration of HP-beta-CD and column temperature on enantioselective separation were investigated. The quantification properties of the developed RP-HPLC method were examined. A baseline separation of norgestrel enantiomers was achieved on a Agilent ZORBAX Eclipse XDB-C8 column (150 mm x 4.6 mm i.d., 5 microm). The mobile phase was a mixture of acetonitrile and phosphate buffer (pH 5.0, 20 mM) containing 25 mM HP-beta-CD (30:70, v/v) with a flow rate of 1.0 ml/min. The UV detector was set at 240 nm. Calibration curves were linear (n=8) in the range of 0.2-25 microg/ml, the limit of detection and quantitation were 0.10 and 0.20 microg/ml, respectively, for racemic norgestrel. The values of RSD of repeatability and intermediate precision for spiked sample were less than 4.8%. The method was successfully applied to the enantioselective determination of this drug in stereoselective skin permeation study.     

Rapid determination of tamsulosin in human plasma by high-performance liquid chromatography using extraction with butyl acetate

A high-performance liquid chromatographic method with fluorescence detection for the determination of tamsulosin in human plasma is reported. The sample preparation involved liquid-liquid extraction of tamsulosin from alkalised plasma with butyl acetate and back-extraction of the drug to the phosphate buffer (pH 2). Butyl acetate is preferable to more commonly used ethyl acetate because of its much lower solubility in water. Liquid chromatography was performed on an octadecylsilica column (55 mm x 4 mm, 3 microm particles), the mobile phase consisted of acetonitrile-30 mM dihydrogenpotassium phosphate (25:75 v/v). The run time was 3.5 min. The fluorimetric detector was operated at 228/326 nm (excitation/emission wavelength). An analogue of tamsulosin, (R)-5-[2-[(3-(2-ethoxyphenoxy)propyl)amino]-2-methylethyl]-2-methoxybenzensulfonamide was used as the internal standard. The limit of quantitation was 0.4 ng/ml using 1 ml of plasma. Within-day and between-day precision expressed by relative standard deviation was less than 10% and inaccuracy did not exceed 5%. The assay was applied to the analysis of samples from several pharmacokinetic studies.     

Forensic analysis of eleven cyclic antidepressants in human biological samples using a new reversed-phase chromatographic column of 2 μm porous microspherical silica gel

A high-performance liquid chromatographic method has been developed for the forensic analysis of eleven frequently used cyclic antidepressant drugs (ADSs) (amitriptyline, amoxapine, clomipramine, desipramine, dosulepine, doxepin, imipramine, maprotiline, melitracen, mianserine and nortriptyline) using a recently developed reversed-phase column with 2 μm particles for the analysis of biological samples. The separation was carried out using two different C₈ reversed-phase columns (column 1: 100 mm × 4.6 mm I.D., particle size 2 μm, TSK gel Super-Octyl; column 2: 100 mm × 4.6 mm I.D., particle size 5 μm, Hypersil MOS-C₈) for comparison. The mobile phase was composed of methanol-20 mM KH₂PO₄ (pH 7) (60:40, v/v) and the flow-rate was 0.6 ml/min for both columns. The absorbance of the eluent was monitored at 254 nm. When the eleven drugs were determined, the sensitivity with the 2 μm particles was about five times greater than with the 5 μm particles. Retention times on column 1 were shorter than those on column 2. These results show that the new ODS column packing with a particle size of 2 μm gives higher sensitivity and a shorter analysis time than the conventional ODS column packing when applied to the analysis of biological samples.     

Determination of midazolam and its major metabolite 1'-hydroxymidazolam by high-performance liquid chromatography-electrospray mass spectrometry in plasma from children

We have developed a sensitive, selective and reproducible reversed-phase high-performance liquid chromatography method coupled with electrospray ionization mass spectrometry (HPLC-ESI-MS) for the simultaneous quantification of midazolam (MDZ) and its major metabolite, 1'-hydroxymidazolam (1'-OHM) in a small volume (200 microl) of human plasma. Midazolam, 1'-OHM and 1'-chlordiazepoxide (internal standard) were extracted from alkalinised (pH 9.5) spiked and clinical plasma samples using a single step liquid-liquid extraction with 1-chlorobutane. The chromatographic separation was performed on a reversed-phase HyPURITY Elite C18 (5 microm particle size; 100 mm x 2.1mm i.d.) analytical column using an acidic (pH 2.8) mobile phase (water-acetonitrile; 75:25% (v/v) containing formic acid (0.1%, v/v)) delivered at a flow-rate of 200 microl/min. The mass spectrometer was operated in the positive ion mode at the protonated-molecular ions [M+l]+ of parent drug and metabolite. Calibration curves in spiked plasma were linear (r2 > or = 0.99) from 15 to 600 ng/ml (MDZ) and 5-200 ng/ml (1'-OHM). The limits of detection and quantification were 2 and 5 ng/ml, respectively, for both MDZ and 1'-OHM. The mean relative recoveries at 40 and 600 ng/ml (MDZ) were 79.4+/-3.1% (n = 6) and 84.2+/-4.7% (n = 8), respectively; for 1'-OHM at 30 and 200 ng/ml the values were 89.9+/-7.2% (n = 6) and 86.9+/-5.6% (n = 8), respectively. The intra-assay and inter-assay coefficients of variation (CVs) for MDZ were less than 8%, and for 1'-OHM were less than 13%. There was no interference from other commonly used antimalarials, antipyretic drugs and antibiotics. The method was successfully applied to a pharmacokinetic study of MDZ and 1'-OHM in children with severe malaria and convulsions following administration of MDZ either intravenously (i.v.) or intramuscularly (i.m.).     

Extraction and determination of oxybutynin in human bladder samples by reversed-phase high-performance liquid chromatography

A reversed-phase high-performance liquid chromatography method is described for the determination of oxybutynin (OXB) in human bladder samples. Following homogenization, tissue samples underwent double extraction with hexane and eventually were concentrated by freeze–drying before analysis. Chromatographic separation was performed with a mobile phase of acetonitrile–water–1 M ammonium acetate, pH 7.0 (85:13:2, v/v/v) at a flow-rate of 0.5 ml/min and double (electrochemical and UV) detection was applied. The retention time of oxybutynin eluting peak was around 18 min. Using a standard curve range of 10 to 500 ng/ml the quantification limit with electrochemical detection was 5 ng/ml with an injection volume of 100 μl. Within-day and day-to-day relative standard deviation values were 4.9 and 9.81%, respectively, while a 94% accuracy and a 72% recovery was attained. We applied this method to compare the OXB levels into bladder wall tissue samples after passive diffusion and after electromotive drug administration (EMDA), using a two-chambered poly(vinyl chloride) diffusion cell designed and developed in our laboratory. The results obtained show that EMDA enhanced OXB penetration into bladder wall and that this novel way of local drug administration can be potentially used in patients with neurogenic bladder dysfunction or urinary incontinence.