A Critical Review of Physicochemical Properties and Analytical Methods Applied to Quantitative Determination of Ebastine.

Allergic diseases are the most common conditions in children and the second most frequent in adults. Currently, there are two well-defined generations of antihistamines, those belonging to first generation, with inherent side effects such as drowsiness and anticholinergic effects. These side effects are often attributed to their high lipophilicity and high affinity for brain H1 receptors. The ebastine is a modern antihistaminic drug belongs to the second generation and has lower lipophilicity, which diminish the undesirable side effects. To ensure the quality, efficacy, safety, and effectiveness of ebastine drug products, efficient and reliable analytical methods are mandatory. Besides official compendial methods, alternative methods are often developed and used in quality control of pharmaceuticals as well as in pharmacokinetic studies. In this work, we present a critical review on characteristics, physicochemical properties, and analytical methods applied in the analysis of ebastine.

Simultaneous Liquid Chromatographic Determination of Ebastine with Two Sympathomimetic Drugs Using a Monolithic Column.

Ebastine (EBS) has been assayed in its laboratory-prepared co-formulated tablets with either pseudoephedrine hydrochloride (PSU) or phenylephrine hydrochloride (PHR) using isocratic reversed-phase chromatography. Separation was conducted using a 50 mm × 4.6 mm i.d., Chromolith® SpeedROD RP-18 end-capped column at ambient temperature. A mobile phase composed of water:acetonitrile in a ratio of 25:75 having a pH of 3.2, has been utilized at 1 mL/min with UV detection at 254 nm for both EBS and PSU and 274 nm for PHR which in turn increased the sensitivity of the proposed method significantly. Symmetric well-separated peaks resulted in a short chromatographic run; <5 min. The proposed method was subjected to detailed validation procedures and proved to be highly sensitive as shown from limit of quantification values which were 4.7, 39.4 and 10.2 µg/mL for EBS, PSU and PHR, respectively. The proposed method was used to analyze EBS in its laboratory-prepared co-formulated tablets; the obtained results were comparable to those resulting from the reference method.

[Determination of Butroxydim in Agricultural Products by LC-MS].

An analytical method for the determination of butroxydim in agricultural products by LC-MS was developed. Butroxydim was extracted with acetonitrile and an aliquot of the crude extract was cleaned up on an octadecyl silanized silica gel (C18) cartridge column (1,000 mg), followed by a salting-out step to remove water. Before purification on a silica gel (SI) cartridge column (690 mg), polar matrices were precipitated by adding ethyl acetate, n-hexane and anhydrous sodium sulfate successively. This process effectively removed caffeine and catechins and improved recovery when analyzing residual butroxydim in tea leaves. Recovery and repeatability were good; the relative standard deviations were less than 5% for all 12 tested agricultural products (brown rice, soybean, potato, spinach, cabbage, apple, orange, grapefruit, lemon, tomato, peas with pods, and tea). Average recoveries for 11 agricultural products, except for lemon, were 74-92%.

Orthogonal projection to latent structures combined with artificial neural networks in non-destructive analysis of ebastine powder.

A new method orthogonal projection to latent structures (O-PLS) combined with artificial neural networks is investigated for non-destructive determination of ebastine powder via near-infrared (NIR) spectroscopy. The modern NIR spectroscopy is efficient, simple and non-destructive technique, which has been used in chemical analysis in diverse fields. Being a preprocessing method, O-PLS provides a way to remove systematic variation from an input data set X not correlated to the response set Y, and does not disturb the correlation between X and Y. In this paper, O-PLS pretreated spectral data was applied to establish the ANN model of ebastine powder, in this model, the concentration of ebastine as the active component was determined. The degree of approximation was employed as the selective criterion of the optimum network parameters. In order to compare the OPLS-ANN model, the calibration models that use first-derivative and second-derivative preprocessing spectra were also designed. Experimental results showed that the OPLS-ANN model was the best.

The in vivo and in vitro metabolism and the detectability in urine of 3',4'-methylenedioxy-alpha-pyrrolidinobutyrophenone (MDPBP), a new pyrrolidinophenone-type designer drug, studied by GC-MS and LC-MS(n.).

3',4'-Methylenedioxy-alpha-pyrrolidinobutyrophenone (MDPBP), a designer drug of the pyrrolidinophenone-type, was first seized in Germany in 2009. It was also identified in 'legal high' samples investigated in the UK. Therefore, the aim of the presented work was to identify its in vivo and in vitro phase I and II metabolites using gas chromatography-mass spectrometry (GC-MS) and liquid chromatography-ion trap mass spectrometry (LC-MS(n) ). Furthermore, detectability of MDPBP in rat and human urine using standard urine screening approaches (SUSA) by GC-MS and LC-MS(n) was studied. The metabolites were isolated either directly or after enzymatic cleavage of conjugates by solid-phase extraction (C18, HCX). The metabolites were then analyzed and structures proposed after GC-MS (phase I) and LC-MS(n) (phase II). Based on these identified metabolites, the following main metabolic steps could be proposed: demethylenation followed by methylation of one hydroxy group, aromatic and side chain hydroxylation, oxidation of the pyrrolidine ring to the corresponding lactam as well as ring opening to the corresponding carboxylic acid. Furthermore, in rat urine after a typical user's dose as well as in human urine, mainly the metabolites could be detected using the authors' SUSA by GC-MS and LC-MS(n) . Thus, it should be possible to monitor an application of MDPBP assuming similar toxicokinetics in humans. Finally, CYP2C19 and CYP2D6 could be identified as the isoenzymes mainly responsible for demethylenation.

Using an innovative Quality-by-Design approach for development of a stability indicating UHPLC method for ebastine in the API and pharmaceutical formulations.

A stability-indicating ultra high performance liquid chromatographic (UHPLC) method has been developed for purity testing of ebastine and its pharmaceutical formulations. Successful chromatographic separation of the API from impurities was achieved on a Waters Acquity UPLC BEH C18, 50 mm × 2.1 mm, 1.7 µm particle size column with gradient elution of 10 mM acetate buffer pH 6.2 and a mixture of acetonitrile/2-propanol (1:1) as the mobile phase. Incorporating Quality by Design (QbD) principles to the method development approach by using the chromatography modeling software DryLab4 allows the visualization of a "Design Space", a region in which changes to method parameters will not significantly affect the results as defined in the ICH guideline Q8 (R2). A verification study demonstrated that the established model for Design Space is accurate with a relative error of prediction of only 0.6%. The method was fully validated for specificity, linearity, accuracy and precision, and robustness in compliance to the ICH guideline Q2 (R1). The method was found to be linear in the concentration range from the quantification limit (LOQ) to 125% of the specification limit for ebastine and each of the impurities with correlation coefficients of not less than 0.999. The recovery rate was between 98.15 and 100.30% for each impurity. The repeatability and intermediate precision (RSD) were less than 3.2% for ebastine and each of the impurities. The robustness of the developed method was studied by varying the six parameters: gradient time, temperature, ternary composition of the eluent, flow rate and start and end concentration of the gradient at 3 levels (+1, 0, -1). The resulting 729 experiments were performed in silico from the previously constructed model for Design Space and showed that the required resolution of 2.0 can be reached in all experiments. To prove the stability-indicating performance of the method, forced degradation (acid and base hydrolysis, oxidation, photolytic and thermal stress conditions) of ebastine was carried out. Baseline separation could be achieved for all peaks of the impurities, the degradation products and the API. Total run time was only 4 min, which is an impressive 40-fold increase in productivity in comparison to the method published in the Ph. Eur. monograph and allowed purity testing of more than 360 samples per day.

Detection of buphedrone in biological and non-biological material--two case reports.

Buphedrone (2-(methylamino)-1-phenylbutan-1-one, α-methylamino-butyrophenone, MABP) is a positional isomer of mephedrone. In Poland, it was marketed in the second half of 2010 after the banning of mephedrone. Buphedrone is a stimulant that is snorted, smoked or taken orally. This substance was identified in 15 products seized by law enforcement after August 2010 and analysed in the Institute of Forensic Research (IFR). Buphedrone was the sole psychoactive substance in only 5 products. It was mixed mainly with 4-MEC and MDPV. This paper presents two cases in which both biological and non-biological materials were delivered to the IFR for toxicological analysis. In the first case, a passenger car crashed into a truck. The car driver suffered severe injuries resulting in his death. During external inspection of the deceased the police discovered several packages containing a white powder. In the second case, a man was arrested for possession of illicit drugs. Analysis of powders was carried out using gas chromatography-mass spectrometry (GC-MS) and high-pressure liquid chromatography with diode array detection (HPLC-DAD). The purity of buphedrone found in powder samples was in the range of 58-68%. Analyses of blood were carried out using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Buphedrone was found in the blood of the deceased at a concentration of 127 ng/mL and of the drug user/seller at 3 ng/mL.

[Determination of four n-butyrophenone compounds contained in rhubarb using RP-HPLC].

OBJECTIVE: To establish the method for determining the contets of 4-(4'-hydroxyphenyl)-2-butanone, lindleyin, isolindleyin, and 4-(4'-hydroxyphenyl )-2-butanone-4'-O-beta-D-(2"-O-galloyl-6"-O-cinnamoyl)-glucopyranoside contained in rhubarb. METHOD: Agilent Zorbax SB-C18 analytical column (4.6 mm x 250 mm, 5 microm) was adopted as the chromatographic column, with acetonitrile-0.05% phosphoric acid as mobile phase for gradient elute. The flow rate was 1 mL x min(-1), and detective wavelength was set at 268 nm. RESULT: 4-(4'-hydroxyphenyl)-2-butanone, lindleyin, isolindleyin, and 4-(4'-hydroxyphenyl)-2-butanone-4'-O-beta-D-(2"-O-galloyl-6"-O-cinnamoyl)-glucopyranoside showed good resolutions . The four standard curves displayed good linear relationship within the detection rate (r > 0.9999), with their detection limit of less than 1.76 ng and quantitation limit of less than 4.98 ng. At the high, medium and low levels, their RSDs of inter- and intra-day precision were less than 2.3%, with recovery of more than 91.8%. CONCLUSION: The method is so simple, rapid, accurate and reliable that it can provide reference for comprehensive quality evaluation on rhubarb.

Gradient HPLC-DAD determination of two pharmaceutical mixtures containing the antihistaminic drug ebastine.

This work describes the development, validation and application of a simple and reliable high-performance liquid chromatography-diode array detection (HPLC-DAD) procedure for the analysis of two pharmaceutical mixtures. The first mixture contains the antihistaminic drug ebastine (EBS) and the famous sympathomimetic drug pseudoephedrine hydrochloride (PSD), and the second mixture is composed of EBS and another sympathomimetic agent, phenylephrine hydrochloride (PHR). Effective chromatographic separation of EBS, PSD and PHR was achieved using a Zorbax SB-C8 (4.6 × 250 mm, 5 µm) column with gradient elution of the mobile phase composed of 0.05M phosphoric acid and acetonitrile. The gradient elution started with 20% (by volume) acetonitrile, ramped up linearly to 90% in 5 min, then kept constant until the end of the run. The mobile phase was pumped at a flow rate of 1 mL/min. The multiple wavelength detector was set at 254 (for EBS and PSD) and 274 nm (for PHR) and quantification of the analytes was based on measuring their peak areas. The retention times for PHR, PSD and EBS were approximately 2.5, 2.9 and 7.1 min, respectively. The reliability and analytical performance of the proposed HPLC procedure were statistically validated with respect to linearity, ranges, precision, accuracy, selectivity, robustness and detection and quantification limits. Calibration curves were linear in the ranges 5-100, 100-1,000 and 10-200 µg/mL for EBS, PSD and PHR, respectively, with correlation coefficients > 0.9996. The validated HPLC method was applied to the analysis of the two pharmaceutical mixtures in laboratory-made tablets in which the analytes were successfully quantified with good recovery values and no interfering peaks were encountered from the inactive ingredients. Finally, the proposed method made use of DAD as a tool for peak identity and purity confirmation.

Chemical analysis of two new designer drugs: buphedrone and pentedrone.

The hydrochloride salts of buphedrone and pentedrone, two new designer drugs, have recently been identified in shipments destined for Canada. To confirm their identities, we have synthesized reference materials for these methcathinone analogues and herein provide complete characterization by FTIR, FT-Raman, ¹H NMR, ¹³C NMR, GC/MS and ESI-HRMS.

Stability indicating LC method for the determination of pipamperone.

A high performance liquid chromatographic method for the determination of pipamperone in the presence of one related impurity and its degradation products is described. The method is based on the use of an amide functionalized bonded phase column (LC-ABZ+ Plus) and a mobile phase of acetonitrile-tetrahydrofuran-sodium phosphate monobasic (0.05 M, pH 6.5) (16:11:73, v/v/v). All peaks are eluted in <8 min. The method was demonstrated to be precise, accurate and specific. Degradation study showed that the drug is stable in acidic medium while it degrades under basic and oxidative conditions. The results indicated that the proposed method could be used in a stability assay.

Differential-pulse polarography determination of pipamperone in pharmaceutical formulations.

The electrochemical reduction of pipamperone has been carried out in aqueous solution in KNO(3) (0.1 mol l(-1)) by differential-pulse polarography (DPP). Pipamperone exhibits a well-defined irreversible reduction peak at -1.3 V/ref. The influence of pH on the reduction of pipamperone was studied in Britton-Robinson buffer (pH range 2-10). A method for the analysis of pipamperone in KNO(3) (0.1 mol l(-1)), which allows quantification over the range 1.6x10(-5)-2.0x10(-4) mol l(-1), was proposed and successfully applied to the determination of pipamperone in tablets with mean recovery and relative standard deviation (R.S.D.) of 100.35 and 0.49%, respectively.

Identification of selected psychopharmaceuticals and their metabolites in hair by LC/ESI-CID/MS and LC/MS/MS.

Hair samples of patients of psychiatry and hair samples of suicide cases were analysed by liquid-chromatography/ionspray-mass spectrometry (LC/MS) for antidepressants and neuroleptics. Electrospray ionisation (ESI) with in-source collision induced dissociation (ESI/CID) and tandem-mass spectrometry (MS/MS) were used for drug and metabolite identification. Mass spectra library searching was performed using an ESI/CID mass spectra library and a MS/MS spectra library. Furthermore, extracted ion chromatograms were used for the detection of N-desmethyl-metabolites, which were also identified by their fragment-ion spectra. Three examples using these methods are shown: The tricyclic antidepressant maprotiline, the selective serotonin receptor inhibitor (SSRI) citalopram and their desmethylmetabolites as well as the neuroleptic pipamperone were detected and identified in hair extracts. For extraction powdered hair was treated by ultrasonication in methanol and solid-phase extraction was used for sample clean-up prior to LC/MS or MS/MS analysis. These examples demonstrate the power of LC/MS and LC/MS/MS for the detection and identification of drugs in hair extracts using full-scan mode and ESI/CID with library searching or using highly selective LC/MS/MS-analysis with library searching or in multiple reaction monitoring mode.

Densitometric determination of impurities in pharmaceuticals. Part VI. Determination of 4,4-bis[4-(p-chlorophenyl)- 4-hydroxypiperidino]butyrophenone in haloperidol.

A chromatographic and densitometic method for identification and quantitative determination of 4,4-bis[4-(p-chlorophenyl)-4-hydroxypiperidino]butyrophenone as an impurity in haloperidol pharmaceutical has been developed. The HPTLC plates and chloroform-methanol-ammonium hydroxide 25% (90:9:1) were used for chromatographic separation as stationary and mobile phases respectively. Detection has been carried out in UV at lambda = 350 nm. The determination could be made directly without preliminary component separation by extraction. Based on the statistical analysis of obtained results, it was found that the new method is accurate and repeatable.

Identification of rat faecal metabolites of ebastine by B/E linked scanning liquid secondary ion mass spectrometry.

The identification of rat faecal metabolites of a new antihistaminic agent, ebastine, 4'-tert-butyl-4-[4-(diphenylmethoxy)piperidino]butyrophenone, is presented. After oral administration of (14C)ebastine (20 mg kg-1) to rats, 84% of the radioactive dose was excreted in the 24 h faeces. Unchanged drug and five metabolites were isolated from the faeces by thin-layer chromatography and solid-phase extraction, and their structures were identified by liquid secondary ion mass spectrometry using the B/E linked scanning technique. The main metabolic pathways were oxidation of a terminal methyl group to give the hydroxymethyl and carboxyl derivatives, and hydroxylation of a phenyl ring in the diphenylmethoxy moiety. In addition to the oxidative mechanism, metabolism of ebastine involved sulphate conjugation. It is noteworthy that M-4, having both phenolic and alcoholic hydroxyl groups, was sulphated selectively in the latter position.

Spectrophotometric determination of pharmaceutical dosages by partial least-squares calibration.

Partial least-squares calibration was used for the spectrophotometric determination of the active compound and preservative in a syrup also containing several light absorbing excipients. The calibration matrix was constructed from laboratory-made mixtures of the analytes and excipients and was used to quantify three samples from different batches. The most suitable conditions for quantitation were determined and the results obtained are compared with those provided by HPLC.

Determination of some butyrophenones in body fluids by gas chromatography with surface ionization detection.

Haloperidol, moperone, pipamperone and bromperidol were found measurable with high sensitivity by gas chromatography (GC) with surface ionization detection (SID). The calibration curves using moperone as internal standard were linear in the range of 0.4-4 pmol on column for haloperidol, pipamperone, and bromperidol. The detection limit of the drugs was about 0.1 pmol on column. For their actual determination with forensic samples, a detailed procedure for their extraction from human whole blood and urine was established with Sep-Pak C18 cartridges. The recovery of the 4 drugs, which had been added to whole blood and urine, was more than 90%. Haloperidol in whole blood and urine of a schizophrenic patient, who had been administered with this drug (3 mg/day p.o.), could be quantitated by the present GC-SID, and the levels were 7.18 and 43.2 pmol/ml, respectively.

In vivo 19F nuclear magnetic resonance of a monofluorinated neuroleptic in the rat.

In vivo 19F NMR measurements of the fluorinated neuroleptic melperone [4'-fluoro-4-(4-methylpiperidino)-butyrophenone hydrochloride] in the rat brain were performed using a geometrically optimized surface coil at 4.7 T. It was possible for the first time to detect a signal of a monofluorinated neuroleptic drug with a time resolution of 30 min after i.p. application. The kinetic time course of the investigated neuroleptic melperone was recorded over 6 h and showed that the half-life in the rat brain is 4.3 h. The total amount of drug and its metabolites in the brain was estimated to be 50 microM. the chemical shift of the 19F NMR signal shows the same upfield shift relative to that in aqueous solution as has been reported for trifluorinated neuroleptic drugs.

Localization of the active principles of the male fern (Dryopteris filix-mas (L.) Schott.) by fluorescence microscopy.

The active principles of the male fern, Dryopteris filix-mas (L.) Schott., consisting of a mixture of phloroglucinol derivatives called "crude filicin", are localized by means of fluorescence microscopy under U.V. 365 nm and monochromatic blue 435-490 nm lights. Crude filicin is autofluorescent, and therefore may be easily localized in the internal glandular hairs of the intercellular spaces of rhizome and leaf bases parenchyma, without pre-treatment of sections. Localization is confirmed both morphologically and chemically, and is possible on a fresh material, as well as on a desiccated one. The test allows also a complete anatomical examination of sections, and may be useful, for its easiness and quickness, in further taxonomic or pharmacognostic studies.