A new enantioselective CE method for determination of oxcarbazepine and licarbazepine after fungal biotransformation.

The present work describes, for the first time, the simultaneous separation of oxcarbazepine (OXC) and its active metabolite 10-hydroxy-10,11-dihydrocarbamazepine (licarbazepine, Lic) by chiral CE. The developed method was employed to monitor the enantioselective biotransformation of OXC into its active metabolite by fungi. The electrophoretic separations were performed using 10 mmol/L of a Tris-phosphate buffer solution (pH 2.5) containing 1% w/v of ß-CD phosphate sodium salt (P-ß-CD) as running electrolyte, -20 kV of applied voltage and a 15°C capillary temperature. The method was linear over the concentration range of 1000-30 000 ng/mL for OXC and 75-900 ng/mL for each Lic enantiomer (r ≥ 0.9952). Within-day precision and accuracy evaluated by RSD and relative errors, respectively, were lower than 15% for all analytes. The validated method was used to evaluate the enantioselective biotransformation of OXC, mediated by fungi, into its active metabolite Lic. This study showed that the fungi Glomerella cingulata (VA1) and Beuveria bassiana were able to enantioselectively metabolize the OXC into Lic after 360 h of incubation. Biotransformation by the fungus Beuveria bassiana showed 79% enantiomeric excess for (S)-(+)-Lic, while VA1 gave an enantiomeric excess of 100% for (S)-(+)-Lic. This study opens a new route to the drug (S)-(+)-licarbazepine.

Dibenzazepin hydrochloride as a new spectrophotometric reagent for determination of hydrogen peroxide in plant extracts.

A rapid, simple, accurate, and sensitive visible spectrophotometric method for the determination of trace amounts of hydrogen peroxide in acidic buffer medium is reported. The proposed method is based on the oxidative coupling of Ampyrone with dibenzazepin hydrochloride by hydrogen peroxide in the buffer medium of pH 4.0 which is catalyzed by ferrous iron. The blue-colored product formed with maximum absorption at 620 nm was found to be stable for 2 h. Beer's law is obeyed for hydrogen peroxide concentration in the range of 0.03-0.42 µg ml(-1). The optimum reaction conditions and other important optical parameters are reported. The molar absorptive and Sandell's sensitivity are found to be 5.89 × 10(4) mol(-1) cm(-1) and 0.57 g/cm(2), respectively. The interference due to diverse ions and complexing agents was studied. The method is successfully applied to the determination of hydrogen peroxide in green plants satisfactorily.

Three-phase hollow fiber microextraction based on two immiscible organic solvents for determination of tricyclic antidepressant drugs: comparison with conventional three-phase hollow fiber microextraction.

The aim of this research was to compare the extraction efficiencies of two modes of three-phase hollow fiber microextraction (HF-LLLME) based on aqueous and organic acceptor phases for analysis of tricyclic antidepressant (TCA) drugs. High-performance liquid chromatography with photodiode array detection (HPLC-DAD) was applied for determination of the drugs. In order to examine the ability of the new concept of HF-LLLME based on organic acceptor solvent in comparison with aqueous acceptor phase to extract the analytes, four TCAs were selected. The effect of different extraction conditions (i.e., type of acceptor phase, hollow fiber length, ionic strength, stirring rate, and extraction time) on the extraction efficiency of the TCAs was investigated and optimized using central composite design (CCD) as a powerful tool. Both methods were characterized by good linearity and high repeatability, but HF-LLLME with organic acceptor provided higher extraction efficiency and thus lower limits of detection (LODs). Calibration curves were linear (r(2)>0.996) in the range of 0.2-200 µgL(-1). LODs for all the TCAs ranged from 0.08 to 0.2 µgL(-1) using HPLC-DAD. Also an improvement in sensitivity of several orders of magnitude was achieved using single-ion monitoring GC-MS analyses (0.04 µgL(-1)) due to compatibility of this technique with GC instrument. The applicability of the proposed HF-LLLME/GC-MS and HPLC-DAD methods was demonstrated by analyzing the drugs in spiked urine and plasma samples. The obtained recoveries of the drugs in the range of 87.9-109.2% indicated the excellent capability of the developed method for extraction of TCAs from complex matrices.

A chiral HPLC-UV method for the quantification of dibenz[b,f]azepine-5-carboxamide derivatives in mouse plasma and brain tissue: eslicarbazepine acetate, carbamazepine and main metabolites.

For the first time, a selective and sensitive chiral HPLC-UV method was developed and fully validated for the simultaneous quantification of eslicarbazepine acetate (ESL), carbamazepine (CBZ), S-licarbazepine (S-Lic), R-licarbazepine (R-Lic), oxcarbazepine (OXC) and carbamazepine-10,11-epoxide (CBZ-E), in mouse plasma and brain homogenate supernatant. After the addition of chloramphenicol as the internal standard, samples were processed using an SPE procedure. The chiral chromatographic analysis was carried out on a LiChroCART 250-4 ChiraDex column, employing a mobile phase of water and methanol (88:12, v/v) pumped at 0.9 mL/min and the UV detector set at 235 nm. The assay was linear (r(2) ≥0.995) for ESL, CBZ, OXC, S-Lic, R-Lic and CBZ-E in the range of, respectively, 0.2-4, 0.4-30, 0.1-60, 0.2-60, 0.2-60 and 0.2-30 µg/mL, in plasma, and of 0.06-1.5 µg/mL for ESL, 0.12-15 µg/mL for CBZ and CBZ-E and 0.06-15 µg/mL for OXC and both licarbazepine (Lic) enantiomers in brain homogenate supernatant. The overall precision was within 8.71% and accuracy ranged from -7.55 to 8.97%. The recoveries of all the compounds were over 92.1%. Afterwards, the application of the method was demonstrated using real plasma and brain samples obtained from mice administered simultaneously with ESL and CBZ.

Evaluation of a high resolving power time-of-flight mass spectrometer for drug analysis in terms of resolving power and acquisition rate.

Liquid chromatography time-of-flight mass spectrometry (LC-TOFMS) is applied increasingly to various fields of small molecule analysis. The moderate resolving power (RP) of standard TOFMS instruments poses a risk of false negative results when complex biological matrices are to be analyzed. In this study, the performance of a high resolving power TOFMS instrument (maXis by Bruker Daltonik, Bremen, Germany) was evaluated for drug analysis. By flow injection analysis of critical drug mixtures, including a total of 17 compounds with nominal masses of 212-415 Da and with mass differences of 8.8-23.5 mDa, RP varied from 34,400 to 51,900 (FWHM). The effect of acquisition rate on RP, mass accuracy, and isotopic pattern fit was studied by applying 1, 2, 5, 10, and 20 Hz acquisition rates in a 16 min gradient elution LC separation. All three variables were independent of the acquisition rate, with an average mass accuracy and isotopic pattern fit factor (mSigma) of 0.33 ppm and 5.9, respectively. The average relative standard deviation of RP was 1.8%, showing high repeatability. The performance was tested further with authentic urine extracts containing a co-eluting compound pair with a nominal mass of 296 Da and an 11.2 mDa mass difference. The authentic sample components were readily resolved and correctly identified by the automated data analysis. The average RP, mass accuracy, and isotopic pattern fit were 36,600, 0.9 ppm, and 7.3 mSigma, respectively.

Determination of carbamazepine and its impurities iminostilbene and iminodibenzyl in solid dosage form by column high-performance liquid chromatography.

An accurate and precise RP-HPLC method was developed and validated for the determination of carbamazepine and its impurities iminostilbene and iminodibenzyl in a tablet formulation with fluphenazine as an internal standard. Buffer-methanol (50 + 50, v/v) was used as the mobile phase. During validation, specificity, linearity, precision, accuracy, LOD, LOQ, and robustness of the method were tested. The method was proven to be specific against placebo interference. Linearity was evaluated over the concentration range of 100-500, 0.05-0.25, and 0.1-0.5 microg/mL, and the r values were 0.9994, 0.9997, and 0.9979 for carbamazepine, iminostilbene, and iminodibenzyl, respectively. Intraday precision of the method was good, and RSD was below 2% for all analytes. The accuracy of the method ranged from 100.69 to 102.10, 99.76 to 102.66, and 99.26 to 100.08% for carbamazepine, iminostilbene, and iminodibenzyl, respectively. LOD was 0.0125, 0.025, and 0.05 microg/mL and LOQ was 0.05, 0.05, and 0.1 microg/mL for carbamazepine, iminostilbene, and iminodibenzyl, respectiviely. Robustness of the method was proven by using a chemometric approach. The method was successfully applied to the analysis of commercially available carbamazepine tablets and showed good repeatability, with RSD below 2%.

Development and validation of liquid chromatographic and ultraviolet derivative spectrophotometric methods for determination of epinastine hydrochloride in coated tablets.

High-performance liquid chromatographic (LC) and ultraviolet derivative spectrophotometric (UVD) methods were developed and validated for the quantitative determination of epinastine hydrochloride in coated tablets. LC was performed on a reversed-phase RP-18 column with a mobile phase composed of 0.3% triethylamine (pH adjusted to 4.0 with 10% orthophosphoric acid)-methanol (60 + 40, v/v). The first-order derivative method was performed at 243.8 nm using HCI and methanol as the solvent. The methods were validated according to U.S. Pharmacopoeia and International Conference on Harmonization guidelines. The statistical analysis by Student's t-test showed no significant difference between the results obtained by the 2 methods. The proposed methods were found to be simple, rapid, precise, accurate, robust, and sensitive, allowing perfect interchange. The LC and UVD methods can be used in the routine quantitative determination of the epinastine hydrochloride in coated tablets.

Supported liquid membranes in hollow fiber liquid-phase microextraction (LPME)--practical considerations in the three-phase mode.

In this work, three-phase liquid-phase microextraction (LPME) based on a supported liquid membrane (SLM) sustained in the wall of a hollow fiber was investigated with special focus on optimization of the experimental procedures in terms of recovery and repeatability. Recovery data for doxepin, amitriptyline, clomipramine, and mianserin were in the range of 67.8-79.8%. Within-day repeatability data for the four basic drugs were in the range of 4.1-7.7%. No single factor was found to be responsible for these variations, and the variability was caused by several factors related to the LPME extractions as well as to the final HPLC determination. Although the volume of the SLM varied within 0.4-3.1% RSD depending on the preparation procedure, and the volume of the acceptor solution varied within 4.8% RSD, both recoveries and repeatability were found to be relative insensitive to these variations. Thus, the handling of microliters of liquid in LPME was not a very critical factor, and the preparation of the SLM was accomplished in several different ways with comparable performance. Reuse of hollow fibers was found to suffer from matrix effects due to built-up of analytes in the SLM, whereas washing of the hollow fibers in acetone was beneficial in terms of recovery, especially for the extraction of the most hydrophobic substances. Several of the organic solvents used in the literature as SLM suffered from poor long-term stability, but silicone oil AR 20 (polyphenylmethylsiloxane), 2-nitrophenyl octyl ether (NPOE), and dodecyl acetate (DDA) all extracted with unaltered performance even after 60 days of storage at room temperature.

Simultaneous and enantioselective liquid chromatographic determination of eslicarbazepine acetate, S-licarbazepine, R-licarbazepine and oxcarbazepine in mouse tissue samples using ultraviolet detection.

Herein is reported, for the first time, a simple and reliable chiral reversed-phase liquid chromatographic method coupled to ultraviolet (UV) detection for simultaneous determination of eslicarbazepine acetate (ESL) and its metabolites, S-licarbazepine (S-LC), R-licarbazepine (R-LC) and oxcarbazepine (OXC), in mouse plasma and brain, liver and kidney tissue homogenates. All analytes and the internal standard were extracted from plasma and tissue homogenates by a solid-phase extraction procedure using Waters Oasis hydrophilic-lipophilic balance cartridges. The chromatographic separation was performed by isocratic elution with water/methanol (88:12, v/v), pumped at a flow rate of 0.7 mL min(-1), on a LichroCART 250-4 ChiraDex (beta-cyclodextrin, 5 microm) column at 30 degrees C. The UV detector was set at 225 nm. Calibration curves were linear (r2 > or = 0.996) in the ranges 0.4-8 microg mL(-1), 0.1-1.5 microg mL(-1) and 0.1-2 microg mL(-1) for ESL and OXC and in the ranges 0.4-80 microg mL(-1), 0.1-15 microg mL(-1) and 0.1-20 microg mL(-1) for R-LC and S-LC in plasma, brain and liver/kidney homogenates, respectively. The overall precision not exceeded 11.6% (%CV) and the accuracy ranged from -3.79 to 3.84% (%bias), considering all analytes in all matrices. Hence, this method will be a useful tool to characterize the pharmacokinetic disposition of ESL in mice.

Simultaneous multiresponse optimization applied to epinastine determination in human serum by using capillary electrophoresis.

Experimental design and optimization techniques were implemented for the development of a rapid and simple capillary zone electrophoresis method (CZE) for the determination of epinastine hydrochloride in human serum. The effects of five factors were studied on the resolution between the peaks for the target analyte (epinastine hydrochloride) and lidocaine hydrochloride, used as internal standard, as well as on the analysis time. The factors were the concentration and pH of the buffer, the injection time, the injection voltage and the separation voltage. The separation was carried out by using an uncoated silica capillary with 50 microm i.d. and total length 64.5 cm (150 microm of path length) and UV detection (200 nm). Multiple response simultaneous optimization by using the desirability function was used to find experimental conditions where the system generates desirable results. The optimum conditions were: sodium phosphate buffer solution, 16.0 mmol L(-1); pH 8.50; injection voltage, 20.0 kV; injection time, 30 s; separation voltage, 26.7 kV. The method was confirmed to be linear in the range of 2.0-12 ng mL(-1). The injection repeatability of the method was evaluated by six injections at three concentration levels, while intra-assay precision was assessed by analysing a single concentration level, yielding a CV's of ca. 1% for standard and 2% for serum samples. Accuracy was evaluated by recovery assays and by comparing with an HPLC method, the results being acceptable according to regulatory agencies. The rudgeness was evaluated by means of an experimental Plackett-Burman design, in which the accuracy was assessed when small changes were set in the studied parameters. Clean-up of human serum samples was carried out by means of a liquid-liquid extraction procedure, which gave a high extraction yield for epinastine hydrochloride (93.00%).

2,2'-Bipyridine as a new and sensitive spectrophotometric reagent for the determination of nanoamounts of certain dibenzazepine class of tricyclic antidepressant drugs.

2,2'-bipyridine is proposed as new and sensitive spectrophotometric reagent for the determination of certain dibenzazepine class of tricyclic antidepressants. The spectrophotometric method is based on the reaction of imipramine hydrochloride (IPH), desipramine hydrochloride (DPH), clomipramine hydrochloride (CPH), trimipramine maleate (TPM) and opipramol (OPP) with iron (III) and subsequent reaction with 2,2'-bipyridine in an acetic acid medium to yield a pink color with maximum absorption at 530 nm. The color developed was stable over 3-4 h at room temperature (approximately 27 degrees C). The commonly encountered excipients and additives did not interfere with the determination. Results from the analysis of pure drugs and commercial tablets agreed well with those of the official method (United States Pharmacopoeia, 24, USP Convention, Rockville 2000, pp. 505-506, 865-867.).

Synthesis, characterization, and analytical studies of adosupine, a potential new drug for urinary incontinence.

The synthesis and the spectroscopic characterization of a new potential drug for urinary incontinence, adosupine, is described. Adosupine and its potential synthesis impurities were analyzed by a new HPLC method that was developed with a C18 reversed-phase column. The analysis was made under isocratic conditions, with a mobile phase of acetonitrile:water (15:85, v/v). Resolution of all synthesis impurities was allowed. The method was also applied to stability studies of adosupine in solid state and in solution under different conditions. With the conditions used, only one degradation product was shown by HPLC analysis; it was isolated, characterized, and identified as the hydrolysis product of the lactam ring present in the adosupine structure.

[UV-spectrophotometry in drug control. 47. Drug substances with chromophores and auxochromes in cyclic and tricyclic systems (benzodiazepine, dibenzoazepine and dibenzodiazepine)]. / UV-Spektrophotometrie in der Arzneimittelkontrolle. 47. Mitteilung: Arzneistoffe mit Chromophoren und Auxochromen in bicyclischen und tricyclischen Systemen (Benzodiazepin, Dibenzoazepin und Dibenzodiazepin.

The results of a systematic examination of the UV/VIS spectra of 10 drug substances with chromophores and auxochromes in bicyclic and tricyclic systems (benzodiazepine, dibenzoazepine and dibenzodiazepine) are described. Influences of substituents and solvents on shifts of the E, K, B and R bands are discussed.

[A comparative study of the pharmacokinetics and pharmacodynamics of bonnecor]. / Sravnitel'noe izuchenie farmakokinetiki i farmakodinamiki bonnekora.

The pharmacokinetics and pharmacodynamics of bonnecor were studied simultaneously in animals with experimental arrhythmia. It was shown that irrespective of the animal species and individual features of the drug elimination kinetics the level of bonnecor concentration correlated with the antiarrhythmic effect. The data on the excretion of bonnecor and its metabolites in the urine in the dog and man were obtained. The decrease of bioavailability at oral administration of bonnecor was demonstrated to be related to its intensive conversion in metabolite M-I.

[Drugs from the classes of tricyclic antidepressives and antiepileptics, nitrosatable under simulated human gastric conditions]. / Unter simulierten Bedingungen des menschlichen Magens nitrosierbare Arzneimittel aus den Gruppen der trizyklischen Antidepressiva und Antiepileptika.

The nitrosatability of Pryleugan (imipramine), Herphonal (trimipramine), and Finlepsin (carbamazepine) was investigated under simulated human gastric conditions using a colorimetric measuring method. All of them proved to be nitrosatable even at very low nitrite concentrations. In the presence of ascorbic acid, the formation of N-nitroso compounds under model conditions was inhibited markedly. N-nitroso-dihydrodibenzazepine and N-nitroso-dibenzazepine could be identified by thin layer chromatography as main products. The biological effects of these N-nitroso compounds are not known up to now.

Experience with routine applications of liquid chromatography-mass spectrometry in the pharmaceutical industry.

The combination of liquid chromatography and mass spectrometry (LC-MS) has been established to complement gas chromatography (GC)-MS in the analysis of non-volatile and labile drugs in complex materials. The possibilities of LC-MS in the pharmaceutical industry for the analysis of drug substances and dosage forms, metabolism studies and the elucidation of the structures of materials of biological origin are discussed. Instrumental requirements, limitations and applications of LC-MS are considered and experiences with LC-MS in routine applications are reported. Preliminary results obtained with thermospray LC-MS are compared with those using a direct liquid inlet interface.