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The official method for the determination of dirithromycin and related substances in the European Pharmacopoeia (Ph. Eur.) and in the United States Pharmacopeia (USP) is an isocratic liquid chromatographic (LC) method using an ODS column. With this method, the separation of the main component dirithromycin from its epimer is not complete. Moreover, this method suffers sometimes from drift of the baseline and from subsequent quantitation problems. The required resolution is not easy to obtain. Using an adapted method derived from the one prescribed in the pharmacopoeias, the selectivity of a set of more than 40 reversed-phase columns towards dirithromycin components was investigated. The selection of the most suitable column was achieved by the chromatographic response function (CRF) approach. Several changes were introduced to the method in order to improve the separation and to overcome the baseline drift problem. The resulting method uses a Zorbax Extend column maintained at 30 degrees C and a mobile phase containing acetonitrile, methanol, 2-propanol, water and a phosphate buffer at pH 7.5. The method allows a good separation of dirithromycin components, which is much better than that obtained with the existing methods. Several impurities of unknown identity are also separated. The method shows good repeatability, linearity and sensitivity, and it is robust. In addition, it proved to be applicable to a wide number of C18 reversed-phase columns.

A gradient LC method for the determination of indinavir sulfate (IDV) and its impurities has been recently published in a consultation document of the International Pharmacopoeia, WHO Drug Information. The method uses a base-deactivated reversed-phase C18 column (25 cm x 4.6 mm i.d.), 5 microm kept at a temperature of 40 degrees C. The mobile phases consist of acetonitrile, phosphate buffer pH 7.5 and water. The flow rate is 1.0 ml/min. UV detection is performed at 220 nm. A system suitability test (SST) is described to govern the quality of the separation. The separation towards IDV components was investigated on 16 C18 columns and correlation was made with the column classification system developed in our laboratory. The method was evaluated using a Hypersil BDS C18 column (25 cm x 4.6 mm i.d.), 5 microm. A central composite design was applied to examine the robustness of the method. The method shows good precision, linearity, sensitivity and robustness. Six commercial samples were examined using this method.

With a recently developed liquid chromatographic (LC) method, using a phosphate buffer, several unknown impurities present in dirithromycin samples were separated. In this paper, a reversed-phase liquid chromatography-tandem mass spectrometry method is described for the investigation of dirithromycin and related substances. The method employed uses a Zorbax Extend C18 column (250 mm x 4.6 mm I.D.), 5 microm, and a mobile phase consisting of acetonitrile, 2-propanol, water and ammonium acetate solution pH 8.5. Mass spectral data are acquired on an LCQ ion trap mass spectrometer equipped with an electrospray ion (ESI) source operated in the positive ion mode. The LCQ is ideally suited for the identification of related substances because it provides on-line LC/MS(n) capability, which allows efficient identification without time-consuming isolation and purification procedures. Using this method, the fragmentation behavior of dirithromycin and its related substances was studied and the unknown impurities occurring in commercial samples were investigated. In total the structures of nine impurities were elucidated, among which three were different analogues with a modification in the side chain on the oxazine ring. Two impurities showed a different alkyl group in position C13. In two impurities the desosamine sugar was involved with changes in the degrees of methylation of the amino group. One unknown impurity was identified as dirithromycin F and another unknown was characterized as dirithromycin N-oxide.

A selective reversed-phase liquid chromatography/mass spectrometry (LC/MS(n)) method was developed for the characterization of components of the semi-synthetic macrolide clarithromycin. Mass spectral data were acquired online on a LCQ ion trap mass spectrometer equipped with an electrospray ionization source operated in the positive ion mode. One unknown compound was structurally elucidated and two other unknowns were characterized using the MS/MS and MS(n) collision-induced dissociation spectra of reference substances as interpretative templates, combined with knowledge of the nature of functional group fragmentation behaviour. Given the importance attached to the identification of impurities of unknown identity in pharmaceutical substances, this study is useful for companies producing clarithromycin.

In this paper, the performance of a previously developed classification system applied to pharmaceutical chromatographic analyses, is investigated. The separation of seven different drug substances from their respective impurities was studied. The chromatographic procedure for acetylsalicylic acid, clindamycin hydrochloride, buflomedil hydrochloride, chloramphenicol sodium succinate, nimesulide and phenoxymethylpenicillin was performed according to the corresponding European Pharmacopoeia (Ph. Eur.) monograph. The separation of dihydrostreptomycin sulphate was performed according to the literature. It is shown that the column ranking system is a helpful tool in the selection of a suitable column in these analyses.

A simple, isocratic liquid chromatographic (LC) method using volatile mobile phase constituents for the identification of related substances in erythromycin samples is described. For method development, evaporative light scattering detection (ELSD) was used. An XTerra RP18 column was used at 70 degrees C with a mobile phase consisting of acetonitrile-isopropanol-0.2M ammonium acetate pH 7.0-water (165:105:50:680). Mass spectral data were acquired on an ion trap mass spectrometer equipped with an electrospray interface operated in the positive ion mode. First, a library was created using MS/MS and MS(n) spectra of reference substances available in the laboratory. Using these reference spectra as interpretative templates, eight novel related substances in erythromycin samples were identified: N-demethylerythromycin E, erythromycin E N-oxide, anhydroerythromycin C, N-demethylerythromycin B, anhydro-N-demethylerythromycin A, pseudoerythromycin E enol ether, EF lacking the neutral sugar and EA lacking the neutral sugar.

An improved LC method is described for the separation of doxycycline and its impurities. The separation of 2-acetyl-2-decarboxamidodoxycycline from the main peak doxycycline is much better than that obtained with official pharmacopoeia methods. The method is robust and shows good selectivity, repeatability, linearity and sensitivity. Eight commercial samples were examined using the method developed. The method uses an XTerra RP-18, 5 μm (25 cm × 4.6 mm I. D.) column kept at a temperature of 35 °C. UV detection is performed at 280 nm. The mobile phase consists of acetonitrile – 0.2 M tetrabutylammonium hydrogen sulphate pH 7.0 – 0.3 M ethylenediaminetetraacetate pH 7.0 – water (130:350:350:170, v/v/v/v).

Gels containing a combination of erythromycin and benzoylperoxide are frequently used in the treatment of acne vulgaris. A method was developed to determine the content of both erythromycin and benzoylperoxide in these gels. Erythromycin was extracted from the gel in conditions where the oxidative power of benzoylperoxide was neutralised by addition of ascorbic acid and this extract was analysed on an Xterra RP(18) column, with a mobile phase containing acetonitrile-0.2 M K2HPO4-water (35:5:60, v/v/v). The detection wavelength was 215 nm. A second extraction procedure was developed for the analysis of benzoylperoxide. The extraction solution was analysed on a Hypersil C(18) BDS column and a mobile phase containing acetonitrile-water (58:42, v/v). Detection was performed at 254 nm. The flow rate was 1.0 ml/min in both methods. The selectivity, repeatability, linearity and recovery of both methods were examined. Special attention was given to determination of the recovery and the uncertainty on the recovery. This allowed evaluation of the bias of the extraction method. The method developed was used to examine the stability of a gel for topical use.

The application of liquid chromatography–ion trap mass spectrometry for the characterization of linear and cyclic polypeptide antibiotics was investigated. The aim was on-line identification of impurities in those antibiotic complexes without recourse to time-consuming isolation and purification procedures. Hyphenated techniques, such as liquid chromatography coupled to mass spectrometry, are ideally suited for this purpose. Characterization was performed with an ion trap mass spectrometer offering MS n capability; this enables more structural information to be obtained. Liquid chromatography in combination with ion trap mass spectrometry was successfully applied for the characterization of impurities in gramicidin, polymyxin B, polymyxin E, and bacitracin and the study of the degradation products of polymyxins B and E.

A selective reversed-phase liquid chromatography/mass spectrometry (LC/MSn) method is described for the characterization of related compounds in commercial bacitracin samples. Mass spectral data for these polypeptide antibiotics were acquired on a LCQ ion trap mass spectrometer equipped with an electrospray ionization probe operated in the positive and negative ion mode. The LCQ ion trap is ideally suited for the sequencing of those linear side-chain cyclized peptides because it provides on-line LC/MSn capability. Using this method bacitracin A, 1-epibacitracin A, bacitracins B1, B2, B3 and bacitracin F were sequenced and previous sequencing was confirmed. Bacitracins C1, C2, C3, D, H2 and H3 were resolved chromatographically and their ring portion was sequenced for the first time. Four components not described in the literature (1-epibacitracin B1, 1-epibacitracin B2, 1-epibacitracin C1 and H4) were sequenced completely for the first time. The main advantage of this hyphenated LC/MSn technique is the characterization of the related substances without time-consuming isolation and purification procedures. Copyright © 2003 John Wiley & Sons, Ltd.

Analysis of nitrite, the indicator of nitric oxide (NO) generation in vivo, provides a useful tool to study NO synthesis in vivo. A fast and sensitive fluorometric CE method was developed for determination of nitrite in human plasma through its derivatization with 2,3-diaminonaphthalene (DAN). Nitrite in human plasma was easily reacted with DAN under acid conditions to yield the highly fluorescent 2,3-naphthotriazole (NAT). Fluorescence detection was optimized to achieve subnanomolar detection which allows a direct analysis of plasma samples unlike most CE–UV methods using sample stacking. Acetonitrile was used to remove the protein. Short-end injection and a high voltage (−30 kV) were used to shorten the analysis time. The good separation was achieved with 20 mM borate buffer at pH 9.23. The separation of NAT was obtained within 1.4 min. The deproteinized plasma sample was injected hydrodynamically for 5 s at −50 mbar into a 60 cm×75 μm internal diameter uncoated fused-silica capillary. Excitation wavelength was selected with a broad-band filter (240–400 nm), and the emitted light was measured at 418 nm by the use of a cutoff filter. A good linearity (R2=0.9975) was obtained in the range from 2 to 500 nM. The detection limit of nitrite was 0.6 nM in original plasma samples, which is 750 times lower than our previous CE–UV method. The developed fluorometric CE method offers the advantages of more simple system and lower cost compared with the current fluorometric HPLC methods without losing sensitivity. The detected mean nitrite concentration in human plasma by this method was consistent with the most frequently reported values.

The high shear wet granulation upscaling possibilities of a Pro-C-epT Mi-Pro 250 ml with a batch size of 40 g were investigated by down-scaling an alpha-lactose monohydrate wet granulation process from a Collette Gral 10 (8 l batch size) to a Pro-C-epT Mi-Pro with different bowl volumes of 5 l, and 1900, 900 and 250 ml. The wet granulation process was optimised in the Gral 10, next an octagonal design was build around the central point. Two process parameters, the impeller tip speed and the water content, were varied at three levels, which resulted in a two-factor three-level experimental design. alpha-Lactose monohydrate 200 M was granulated using a polyvinylpyrollidone K 30 (2.5% on dry material) binder solution. The granules were dried at 25 degrees C for 24 h, sieved and characterised. The granules were compressed to tablets. In all mixer volumes the used impeller tip speed range did not influence the granule or tablet properties. In all bowl volumes the influence of water concentration on actual yield, particle size distribution and granule friability was similar. All batch sizes resulted in tablets of similar quality. For the selected formulation the lab scale Mi-Pro high shear mixers with different bowl volumes could be used to determine the optimal process parameters and to scale up to the Collette Gral 10 pilot scale.

A reversed phase ion-pair liquid chromatographic method using a base deactivated column and pulsed electrochemical detection on a gold electrode is described. It allows the separation of a mixture of spectinomycin sulfate, lincomycin hydrochloride and their related substances. A step gradient was necessary to obtain a good separation together with a reasonable analysis time of 40 min. The mobile phases consisted of an aqueous solution of 3.3 or 0.55 g/l pentanesulfonic acid, 10 mM acetic acid and 20 ml/l tetrahydrofuran. Both mobile phases were adjusted to pH 4.0 with diluted sodium hydroxide. The influence of the different chromatographic parameters on the separation was investigated. Two commercial samples were analyzed using the described method. In total 12 components could be separated.

Until now, no LC method is described to determine the purity and content of spectinomycin without prior derivatization. A reversed-phase ion-pair LC method using a base deactivated column and pulsed electrochemical detection is described. The mobile phase consisted of an aqueous solution containing 5.8 g/l pentafluoropropionic acid, 1.25 g/l potassium dihydrogen phosphate and 5.5 ml/l tetrahydrofuran. The pH was adjusted to 6.25 using dilute NaOH solution. An experimental design was used to optimize the chromatographic parameters and to check the robustness. The quality of separation was investigated on different stationary phases. The method allows the separation of spectinomycin from its related substances as well as some other components of unknown identity. The total time of analysis is 65 min. A number of commercial samples were examined using this method.

Organic impurity determination is a critical aspect since it has a great impact on the assigned content of chemical reference standards (CRS). This is especially the case for CRS of natural products (CRSNPs) which are mostly obtained from medicinal plants or animals. Total amount of organic impurities of CRSNPs is generally determined by high performance liquid chromatography based on official or reported methods. Since the brand of column is not always prescribed or when the brand prescribed is not available in the laboratory, this can cause serious consequences, especially when the sample contains a basic or weak acid compound with p K a values ranged from 3 to 6. In this paper, columns were originally selected based on a peer-reviewed column characterization database. Firstly two columns with orthogonal selectivity (selectivity factor F ≥6) were selected from the database to determine the purity of the respective CRSNPs. The selection was performed to minimize the risk to obtain different values caused by use of various brands of columns. If there is no significant difference between their results, then the purity result is cross-validated. Otherwise, a third column has to be selected from the database and its selectivity should be similar to the above one which give better separation ( F ≤2). The approach has been illustrated by the purity determinations of N - trans - p -coumaroyltyramine and (-)-epicatechin-3- O -gallate CRSNPs, and it is most likely to be applied in more CRSNPs, especially those of basic or weak acid ones.

An intrinsic performance comparison is made of the reduction in analysis time that can be obtained when switching from HPLC to UHPLC column formats in HILIC and reversed-phase conditions. A detailed overview of the packing characteristics of both stationary phase types is given first. It is demonstrated that HILIC columns demonstrate higher external porosity values than their reversed-phase counterparts resulting in lower flow resistance values. Column total porosity values determined from the elution time of a small marker molecule are shown to depend strongly on the composition of the mobile phase. To omit errors that might arise from an over- or underestimation of the column void time, all plate height and kinetic plot data are therefore expressed as a function of the interstitial velocity. Although only a limited number of columns are evaluated in this study, it is shown that the column efficiency of the HILIC columns is lower than that of their reversed-phase counterparts, at least for the compounds evaluated here. Despite this lower efficiency, the kinetic performance of both stationary phase types is similar, due to the much lower viscosity of the mobile phases typically used in HILIC conditions. Finally, it is demonstrated that a similar, yet slightly larger reduction in analysis time can be obtained when switching from HPLC column formats to UHPLC formats in HILIC compared to reversed-phase conditions. (C) 2014 Elsevier B.V. All rights reserved.

This paper presents a general overview of the application of hydrophilic interaction chromatography (HILIC) in the analysis of antibiotics in different sample matrices including pharmaceutical, plasma, serum, fermentation broths, environmental water, animal origin, plant origin, etc. Specific applications of HILIC for analysis of aminoglycosides, β-lactams, tetracyclines and other antibiotics are reviewed. HILIC can be used as a valuable alternative LC mode for separating small polar compounds. Polar samples usually show good solubility in the mobile phase containing some water used in HILIC, which overcomes the drawbacks of the poor solubility often encountered in normal phase LC. HILIC is suitable for analyzing compounds in complex systems that elute near the void in reversed-phase chromatography. Ion-pair reagents are not required in HILIC which makes it convenient to couple with MS hence its increased popularity in recent years. In this review, the retention mechanism in HILIC is briefly discussed and a list of important applications is provided including main experimental conditions and a brief summary of the results. The references provide a comprehensive overview and insight into the application of HILIC in antibiotics analysis.

This paper describes the separation of the main component micronomicin from its related substances using a new established liquid chromatographic method with pulsed electrochemical detection (LC-PED). The mobile phase consists of 1 volume of acetonitrile and 99 volumes of an aqueous solution containing 1.25% (v/v) trifluoroacetic acid, 0.025% (v/v) pentafluoropropionic acid and 0.85% (v/v) of 50% sodium hydroxide. The pH of the aqueous solution is adjusted to 2.6 with 0.5 M NaOH. The influence of the different chromatographic parameters on the separation was investigated. A quadruple-potential waveform was used as detection waveform. 0.5 M NaOH was added post column at a flow rate of 0.3 mL/min to raise the pH of detection to at least 12. The LOD and LOQ of micronomicin are 0.08 μg/mL (1.6 ng injected) and 0.25 μg/mL (5 ng injected), respectively. The linearity of micronomicin ranges from 0.25 to 60 μg/mL with a correlation coefficient of 0.9978. Intra-day RSD and inter-day RSD of micronomicin are 0.89% and 0.55%, respectively. This method proved to be robust and is also applicable to a wider number of C18 columns. A number of commercial samples of micronomicin sulfate were analyzed using this method and 18 peaks can be separated from the main component and from each other in one sample. Seven peaks could be identified using reference substances. The chemical structure of two unknown impurities could be characterized by LC-MS based on comparison of their fragmentation patterns with those of available reference substances.

This work describes two separate liquid chromatographic (LC) methods which were developed to control gentamicin sulphate and its preservatives methylparaben and propylparaben in an injectable formulation for veterinary purposes. Owing to the absence of a UV absorbing chromophore in the gentamicin molecule, LC combined with pulsed electrochemical detection (LC-PED) was found to be suitable for the determination of this drug in the formulation. The LC-PED methods from edition 7.0 (valid till 06/2012) and 7.5 (supplement valid from 07/2012) of the European Pharmacopoeia (Ph. Eur.) were compared. The currently recommended LC-PED method allowed good separation between the main gentamicin components and their impurities without interference from additives. Because of its better performance the actual method was used to investigate the degradation profile of the gentamicin sulphate injection. Results obtained from the forced stability study showed that the formulation was stable to oxidative and thermal stress. For the control of the preservatives, a LC-UV method was applied. Both methods were found to be specific, linear and precise. Hence, these two LC methods can be used for routine analysis of the gentamicin sulphate injection. © 2013 The Royal Society of Chemistry. ispartof: Analytical Methods vol:5 issue:10 pages:2491-2496 status: published

WOS: 000313807300017, PubMed ID: 23161341, A selective and low-cost CD-MEKC method under acidic conditions was developed for investigating the N-oxygenation of tamoxifen (TAM) by flavin-containing monooxygenases (FMOs). The inhibitory effects of methimazole (MMI), nicotine and 5,6-dimethylxanthenone-4-acetic acid (DMXAA) on the given FMO reaction were also evaluated; 100 mM phosphate buffer (pH 8.6) was used for performing the enzymatic reaction and the separation of TAM and its metabolite tamoxifen N-oxide (TNO) was obtained with a BGE consisting of 100 mM phosphoric acid solution adjusted to pH 2.5 with triethanolamine containing 50 mM sodium taurodeoxycholate, 20 mM carboxymethyl beta-CD and 20% ACN. The proposed method was applied for the kinetics study of FMO1 using TAM as a substrate probe. A MichaelisMenten constant (Km) of 164.1 mu M was estimated from the corrected peak area of the product, TNO. The calculated value of the maximum reaction velocity (Vmax) was 3.61 mu mol/min/mu mol FMO1; 50% inhibitory concentration and inhibition constant (Ki) of MMI, the most common alternate substrate FMO inhibitor, were evaluated and the inhibitory effects of two other important FMO substrates, nicotine and DMXAA, a novel anti-tumour agent, were investigated., Scientific and Technological Research Council of Turkey (TUBITAK), The authors are grateful to the Scientific and Technological Research Council of Turkey (TUBITAK) for the postdoctoral fellowship of D. Y. Also, the authors thank Dr. Jochen Pauwels for the help with enzymatic studies and for fruitful discussions.

Background: Physical fitness determines the level of human health. A good physical fitness can be achieved if conducted with a balance and active physical fitness. The aims of this study was to elucidate the effect of balanced physical exercise on physical fitness, endorphin levels, and malondialdehyde (MDA) levels. Methods: This study was a true experimental with pretest-posttest control group design using 24 students of IKIP PGRI Denpasar. Selected samples divided into two groups: the control group given conventional physical training (P0) and the treatment group given balanced physical training (P1). Physical fitness tests was performed using Cooper method and blood sampling was done to evaluate the level of endorphins and MDA before (pre test) and after (post test) treatment of 8 weeks. The data of endorphin and MDA levels were analyzed using independent T test. Whereas, the physical fitness was analyzed using Mann-Whitney U test. Results: Physical fitness of the group given a balanced physical training was significantly higher compare to the group given a conventional physical training (p < 0.05). Balanced physical training was proven to enhance physical fitness as measured by the Cooper method better than conventional physical training. In contrast, the levels of endorphins of the balanced physical training group did not different with the conventional physical training group (p > 0.05). Levels of MDA of balanced physical training group also did not different with the conventional physical training group (p > 0.05). Conclusions: balanced physical training can maintain physical fitness of people and improve the health and quality of life.

The determination of the relative amounts of the B and C components of neomycin sulphate by thin-layer chromatography using silica gel plates from Whatman as the stationary phase is described. The mobile phase consisted of methanol-20% (m/v) sodium chloride solution (15:85). Fluorescence detection was performed after derivatization with 4-chloro-7-nitrobenzo-2-oxa-1,3-diazole. The influence of different parameters on the separation was investigated. A number of commercial samples was analysed using this method and the results were compared with results obtained with ion-exchange chromatography and ninhydrin colorimetric detection, which is the official method prescribed by the European Pharmacopoeia. The described method is much easier to perform than the official method.

A simple, robust and fast high-performance liquid chromatographic method is described for the analysis of oxytetracycline and its related impurities. The principal peak and impurities are all baseline separated in 20 min using an Inertsil C₈ (150 mm × 4.6 mm, 5 μm) column kept at 50 °C. The mobile phase consists of a gradient mixture of mobile phases A (0.05% trifluoroacetic acid in water) and B (acetonitrile-methanol-tetrahydrofuran, 80:15:5, v/v/v) pumped at a flow rate of 1.3 ml/min. UV detection was performed at 254 nm. The developed method was validated for its robustness, sensitivity, precision and linearity in the range from limit of quantification (LOQ) to 120%. The limits of detection (LOD) and LOQ were found to be 0.08 μg/ml and 0.32 μg/ml, respectively. This method allows the separation of oxytetracycline from all known and 5 unknown impurities, which is better than previously reported in the literature. Moreover, the simple mobile phase composition devoid of non-volatile buffers made the method suitable to interface with mass spectrometry for further characterization of unknown impurities. The developed method has been applied for determination of related substances in oxytetracycline bulk samples available from four manufacturers. The validation results demonstrate that the method is reliable for quantification of oxytetracycline and its impurities.

Tobramycin is one of the aminoglycoside antibiotics that lack a UV absorbing chromophore. However, the application of pulsed electrochemical detection (PED) has been used successfully for the analysis of this and similar antibiotics. This work describes an improved liquid chromatographic (LC) method combined with PED, which is able to separate much more impurities than before. Using a Discovery C-18 RP column (250 mm×4.6 mm i.d., 5 μm), isocratic elution was carried out with a mobile phase, containing sodium sulfate (35 g/L), sodium octanesulphonic acid (1 g/L), tetrahydrofuran (14 mL/L) and 0.2 M phosphate buffer pH 3.0 (50 mL/L). Using these experimental conditions, the limit of quantification (LOQ, S/N=10) was 5 ng. The linearity was examined in the range LOQ-60 μg/mL and the coefficient of determination was 0.998. The method also proved to be repeatable and the recovery was close to 100%. The influence of the different chromatographic parameters on the separation was investigated by means of an experimental design. The proposed method is useful in quality control of tobramycin drug substances and drug products. ispartof: Journal of Pharmaceutical Analysis vol:3 issue:3 pages:161-167 ispartof: location:China status: published

Capillary electrophoresis has emerged as a small-scale analytical tool for enzyme assays. It is not only used to analyze and follow-up enzymatic reactions in an offline mode, but the reaction can also be performed online, inside the capillary, where the reaction products are formed and analyzed. In this way, an integrated setup is obtained which allows a higher degree of automation and miniaturization in analytical systems. This chapter presents an electrophoretically mediated microanalysis for in vitro characterization and screening of matrix metalloproteinase inhibitors.

A method to study Abl1 tyrosine kinase inhibitors (TKIs) by liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS) was developed and validated. Chromatographic separation was achieved on a Symmetry(®) C-18 column using a gradient. The detection was performed by selected ion monitoring (SIM) mode via positive ESI interface. The limit of quantification (LOQ) was 40.8 nM for p-Abltide [product, KKGEAIpYAAPFA-NH2] and 26.7 nM for Abltide (substrate, KKGEAIYAAPFA-NH2). The residual plot of linearity calibration curve indicated a good fit with a linear model. Intra- and inter-day precision was less than 10% and accuracy was from -6.93% to +0.15%. Matrix effect was not significant in this method. The validated method was applied to an Abl1 TKIs study. Imatinib mesylate (IM) and dasatinib were used to evaluate this method and the IC50 values were 202.1 nM and 925.1 pM, respectively. Two natural products (-)- epigallocatechingallate (EGCG) and caffeic acid were tested with this model. The IC50 value of EGCG was found at 64.03 nM and caffeic acid showed fluctuant inhibitory activity from 26% to 55% in the concentration range from 1 nM to 1mM. The IC50 value of a dimethylpyrrole hydroxylbenzoic acid derivative (MPB) was 1.915 μM.

Investigation of unknown impurities in a tylosin sample was performed using liquid chromatography coupled to mass spectrometry (LC/MS). Separation was performed according to the recently described LC-UV method of Ashenafi et al. (2011) [14]. This method was reported to have a good selectivity as it was able to separate the four main components of tylosin from the already known and 23 unknown impurities. However, as this method uses a mobile phase with non-volatile constituents, direct characterization of these impurities using LC/MS was not possible. The impurity fractions were therefore first collected and then desalted before sending them to the MS. Identification of the impurities in the tylosin sample was performed with a quadruple ion trap (IT) MS, with an electrospray ionization (ESI) source in the positive ion mode. The structure of the impurities was deduced by comparing their fragmentation pattern with those of the main components of tylosin. As several peaks in the LC-UV method contained multiple compounds, using this method in total 41 new impurities were (partly) characterized.

Capillary electrophoresis has emerged as a small-scale analytical tool for enzyme assays. It is not only used to analyze and follow-up enzymatic reactions in an offline mode, but the reaction can also be performed online, inside the capillary, where the reaction products are formed and analyzed. In this way, an integrated setup is obtained which allows a higher degree of automation and miniaturization in analytical systems. This chapter presents an electrophoretically mediated microanalysis for in vitro characterization and screening of matrix metalloproteinase inhibitors.

A rapid capillary zone electrophoresis method has been developed capable of quantifying 0.05% of R-enantiomer and assaying the main component in escitalopram formulations. Many parameters influencing enantioseparation were investigated, which include chiral selectors, buffer composition and pH, applied voltage, capillary length, temperature, and rinsing procedure. Optimal separation conditions were obtained by using a 25 mM phosphate buffer at pH 7.0, containing 1.6% (w/v) sulfated-β-cyclodextrin with short-end injection at 0.5 psi for 5 s. Online UV detection was performed at 205 nm. A voltage of -20 kV was applied and the capillary temperature was kept at 25°C. Separation was achieved in less than 2 min. The method was further validated, including robustness, stability of the solution, selectivity, linearity (escitalopram from 0.25 μg/mL to 600 μg/mL, y = 1528.3 × +1812.9; R² = 0.9999), LOD and LOQ (0.08 and 0.25 μg/mL, respectively), precision and accuracy. The proposed method was then applied to the quality control of the bulk sample and tablets of escitalopram (10 mg).

A simple, robust, and rapid reversedphase high-performance liquid chromatographic method for the analysis of demeclocycline and its impurities is described. Chromatographic separations were achieved on a Symmetry Shield RP8 (75 mm × 4.6 mm, 3.5 μm) column kept at 40°C. The mobile phase was a gradient mixture of acetonitrile, 0.06 M sodium edetate (pH 7.5), 0.06 M tetrapropylammonium hydrogen sulphate (pH 7.5) and water, A (2:35:35:28 v/v/v/v) and B (30:35:35:0 v/v/v/v) pumped at a flow rate of 1 mL/min. UV detection was performed at 280 nm. The developed method was validated according to the ICH guidelines for specificity, limit of detection, limit of quantification, linearity, precision, and robustness. An experimental design was applied for robustness study. Results show that the peak shape, chromatographic resolution between the impurities, and the total analysis time are satisfactory and better than previous methods. The method has been applied for the analysis of commercial demeclocycline bulk samples available on the market.

Sodium cysteamine phosphate is a prodrug derivative of cysteamine that can be used in cystinosis treatment. Although titrimetric assays are very well established and precise, iodimetric determination of sodium cysteamine phosphate requires considerably more carefulness and time from the analyst than usual. The possibility to assess sodium cysteamine phosphate by CE was evaluated by means of the quantification of its oxidation product, cystamine, which is a more suitable substance to be used as primary standard than sodium cysteamine phosphate. Apparently, this approach should be straightforward, but systematic differences between the results obtained with CE and titrimetric assays were noticed. MS and CE-MS were employed to aid in the investigation of the possible causes of imprecision of the sodium cysteamine phosphate titration and CE determination. For this purpose, a simple and inexpensive ESI source was constructed. It was observed that cystamine is not the final product of the cysteamine and/or sodium cysteamine phosphate iodine-oxidation and other species besides cystamine may be formed depending on the reaction conditions, which explains the difficulties observed in the sodium cysteamine phosphate quantification.

A reversed phase (RP)-LC method using a C 18 column resistant to basic pH and an alkaline (pH 10) aqueous mobile phase was developed and coupled to MS with an electrospray ionization (ESI) source in the positive ion mode which provides MS n capability. In total, 26 impurities were detected in a commercial sample. The structures of the impurities were proposed based on comparison of their fragmentation patterns with those of the available reference substances, the synthetic route and literature data. Starting material and its residual impurities, intermediates, synthetic by-products and degradation products were the main sources of those impurities. 14 impurities described in this work were newly identified.

The characterization of unknown (UNK) impurities in capreomycin (CMN) using liquid chromatography coupled to mass spectrometry (LC/MS) has been described. The ion-pair liquid chromatography method coupled to ultraviolet detection (LC-UV) described by Mallampati et al. was used for the separation of CMN from its related substances. As the method uses non-volatile reagents it could not be directly coupled to mass spectrometry (MS) for impurity characterization. So, these UNK impurities were collected and desalted before sending to MS for structural characterization. As no information about the fragmentation of the main components of CMN, except for CMN IB, was available in the literature, they were studied first. Next, the structures of the impurities were deduced by comparing their fragmentation to that of the main components of CMN. Fourteen UNK impurities that were never described before, were (partly) characterized.

A liquid chromatography–electrospray ionization–mass spectrometric (LC–ESI–MS) method was developed and validated to study Abl 1 tyrosine kinase. An online desalting system was adopted, and a transformation of the ratio of product to substrate instead of a deuterated internal standard was introduced to calculate the concentration of product. In this study, the substrate used was Abltide (KKGEAIYAAPFA-NH 2 ). The detection was performed by selected ion monitoring (SIM) mode via positive ESI interface. Chromatographic separation was achieved on a C 18 column using an isocratic mobile phase system. The limit of quantification (LOQ) was 10 nM for the product and 25 nM for the substrate. The simple ratios of product to substrate maintained a linear relationship ( R 2 = 0.9997) over the ratio of 0–50% product. Intra- and inter-day precision was less than 10% and accuracy was from −1.6 to +5.3%. The validated method was applied to the Abl 1 kinase kinetic study and the K m and V max constants obtained for Abltide were 34.78 μM and 5.563 μmol/mg/min and for adenosine triphosphate (ATP) were 43.61 μM and 5.906 μmol/mg/min. The enzymatic reaction of Abl 1 tyrosine kinase belongs to ternary-complex mechanism.

Cefpiramide is susceptible to many factors, including high temperature. Two solid states were observed in cefpiramide: a crystalline form and an amorphous one, and both the accelerated (40°C ± 2°C /75% ± 5% relative humidity (RH)) and stress (60°C ± 2°C / 75% ± 5% RH) stability study demonstrated that the crystalline form of cefpiramide was much more stable than its amorphous form, with related substances, high molecular mass polymers and assay as critical stability indicating parameters. Thermogravimetric analysis (TGA), differential scanning calorimetry (DSC) and water content results indicated that each molecule of crystalline cefpiramide may contain two molecules of crystal water, which can explain its high thermal stability. This study provides valuable information for cefpiramide and its drug products with respect to the improvement of its thermal stability through crystallization.

Reversed-phase liquid chromatography coupled with electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) was used to characterize the components of bitespiramycin, a group of 4"-acylated spiramycins produced by bioengineered strains. In total 38 components were characterized in commercial samples, including 12 impurities that had never been reported before and 12 other that were partially characterized. The structures of these unknown compounds were deduced by comparison of their fragmentation patterns with those of known major components. Their ultraviolet spectra were used to confirm the presence of an α-, β-, γ-, δ-unsaturated butadiene in the macrocyclic lactone. Compared with the classical method, LC/ESI-MS/MS is particularly advantageous in terms of sensitivity and efficiency to characterize minor components at trace levels in multi-component antibiotics.

The European Pharmacopoeia (Ph. Eur.) prescribes a selective and sensitive liquid chromatography/ultraviolet (LC–UV) method for the separation of the 16-membered ring macrolide josamycin and its related compounds. Since josamycin is obtained by fermentation, several closely related substances can be found in the sample. Several impurities have already been identified using reference substances. However, many peaks in the chromatogram cannot be correlated with known compounds or correspond to structures which were not described previously. The hyphenation of LC to mass spectrometry (MS) is a very useful tool for the characterization of impurities. The existing LC–UV method however uses non-volatile buffers, while for LC/MS a volatile mobile phase is required. In this study, each peak from the non-volatile system was collected separately and reinjected into a LC system using volatile mobile phase constituents. This way, the analyte could be separated from the buffer salts. Mass spectral data of this macrolide antibiotic were acquired on a LCQ ion trap mass spectrometer, equipped with an electrospray ionization (ESI) probe operating in the positive ion mode. The identity of the unknown compounds was deduced using the MS/MS and MS n collision-induced dissociation spectra of reference substances, combined with knowledge about the nature of functional group fragmentation behavior. The impurity profiling was done on 30 peaks in a josamycin bulk sample. This way, 12 compounds reported in the literature and Ph. Eur. were found in the bulk sample. Furthermore, 12 novel related substances were characterized and 18 compounds were partially characterized.

A commercial erythromycin formulation containing erythromycin A (EA) as the major compound showed the presence of an unknown degradation compound that was co-eluted with erythromycin E (EE) in the European Pharmacopoeia (Ph. Eur.) liquid chromatographic (LC) method. The amount of the degradation compound increased with respect to time. To separate this unknown (UNK1), investigation was performed with different LC methods coupled to ultraviolet detection (LC-UV). With the present Ph. Eur. method, the degradation compound could not be well separated. However, with the most selective LC-UV method (XTerra method), two more degradation products (UNK2 and UNK3) were found in the formulation which could not be observed using other methods because of their poor separation. By combining the results obtained with LC-UV, LC/MS and LC/NMR, the degradation products were identified as pseudoerythromycin A hemiketal (PsEAHK), erythromycin A enol ether carboxylic acid and erythromycin C enol ether carboxylic acid. PsEAHK is known to be a base-catalysed degradation product of EA, whereas the other two degradation products were newly identified.

A chiral capillary electrophoresis method has been developed for the quantification of 0.1% of the enantiomeric impurity (dextrocetirizine) in levocetirizine and determination of both in pharmaceuticals using sulfated-β-cyclodextrins (CDs) as chiral selector. Several parameters affecting the separation were studied such as the type and concentration of chiral selectors, buffer composition and pH, organic modifier, mixtures of two CDs in a dual system, voltage, and temperature. The optimal separation conditions were obtained using a 50 mM tetraborate buffer (pH 8.2) containing 1% (w/v) sulfated-β-CDs on a fused-silica capillary. Under these conditions, the resolution of two enantiomers was higher than 3. To validate the method, the stability of the solutions, robustness (two level half fraction factorial design for 5 factors using 19 experiments [2n−1+3]), precision, linearity (dextrocetirizine 0.25–2.5 μg/ml, R2 = 0.9994, y = 0.0375x + 0.0008; levocetirizine 15–100 μg/ml, R2 = 0.9996, y = 0.0213x + 0.0339), limit of detection (0.075 μg/ml, 0.03% m/m), limit of quantification (0.25 μg/ml, 0.1% m/m), accuracy (dextrocetirizine 84–109%, levocetirizine 97.3–103.1%), filter effect, and different CD batches were examined. The validated method was further applied to bulk drug and tablets of levocetirizine. Chirality, 2012. © 2012 Wiley Periodicals, Inc.

A simple method for the determination of nitrite and nitrate in human plasma has been developed using CZE with minimal sample preparation. Field-amplified sample stacking (FASS) was used to achieve submicromolar detection by dilution of the plasma sample with deionized water. In CZE, the separation of nitrite and nitrate was achieved within 10 min without adding EOF modifier. The optimal condition was achieved with 50 mM phosphate buffer at pH 9.3. The ninefold diluted plasma samples were injected hydrodynamically for 40 s into a 60 cm×75 μm id uncoated fused-silica capillary. The separation voltage was 20 kV (negative potential) and UV detection was performed at 214 nm. The linearity curves for nitrite and nitrate were obtained by the standard addition method. The estimated LODs for nitrite and nitrate in ninefold diluted plasma sample were 0.05 and 0.07 μM, respectively. The LODs for nitrite and nitrate in original plasma samples were 0.45 and 0.63 μM. The intra- and inter-day precisions for both analytes were

A selective reversed-phase (RP) liquid chromatographic (LC) method coupled with UV for the determination of tylosin and its related substances is described. The gradient method uses a Capcell pak C18 ACR column (25 cm×4.6 mm id, 5 μm) maintained at a temperature of 60°C. The mobile phases consist of acetonitrile, phosphate buffer pH 5.5 and water: (A; 27.5:10:62.5 v/v/v) and (B; 50:10:40 v/v/v). The flow rate is 1.0 mL/min and UV detection is performed at 280 nm. It allows the separation of all known and 22 other unknown related substances (≥0.02%) from the main compound and from one another. The method shows good precision, sensitivity, linearity (between 0.2 μg/mL and 1.25 mg/mL) and robustness. The limit of quantification is 0.2 μg/mL, corresponding to 0.020%. Seven bulk tylosin samples containing a large number of impurities were examined using this method.

A liquid chromatographic (LC) method was developed to analyze a formulation (mouthwash) containing lidocaine hydrochloride, hydrocortisone and nystatin. A single LC method with UV detection was developed. A Waters Symmetry C18 HPLC column (150 mm × 4.6 mm, 5 μm) was used as stationary phase and the assay was performed with gradient elution using mobile phases containing methanol – 0.1 M NaH 2 PO 4 with a pH that was previously adjusted to 4.5 with dilute phosphoric acid. The sample pretreatment was performed by treating the formulation with methanol followed by filtration. After method development, the influence of the different chromatographic parameters on the separation, the interference of other active compounds and excipients, linearity, accuracy, repeatability and intermediate precision were investigated. The method was shown to be selective, linear, accurate, precise and repeatable. Finally, the content of the compounds in the formulation was determined.

Gas chromatography-mass spectrometry is a well established analytical technique. However, mass spectrometers with electron ionization sources may suffer from signal drifts, hereby negatively influencing quantitative performance. To demonstrate this phenomenon for a real application, a static headspace-gas chromatography method in combination with electron ionization-quadrupole mass spectrometry was optimized for the determination of residual dichloromethane in coronary stent coatings. Validating the method, the quantitative performance of an original stainless steel ion source was compared to that of a modified ion source. Ion source modification included the application of a gold coating on the repeller and exit plate. Several validation aspects such as limit of detection, limit of quantification, linearity and precision were evaluated using both ion sources. It was found that, as expected, the stainless steel ion source suffered from signal drift. As a consequence, non-linearity and high RSD values for repeated analyses were obtained. An additional experiment was performed to check whether an internal standard compound would lead to better results. It was found that the signal drift patterns of the analyte and internal standard were different, consequently leading to high RSD values for the response factor. With the modified ion source however, a more stable signal was observed resulting in acceptable linearity and precision. Moreover, it was also found that sensitivity improved compared to the stainless steel ion source. Finally, the optimized method with the modified ion source was applied to determine residual dichloromethane in the coating of coronary stents. The solvent was detected but found to be below the limit of quantification.

Static headspace (HS) sampling has been commonly used to test for volatile organic chemicals, usually referred to as residual solvents (RS) in pharmaceuticals. If the sample is not soluble in water, organic solvents are used. However, these seriously reduce the sensitivity in the determination of some RS. Here, mixed aqueous dilution media (a mixture of water and an organic solvent like dimethyl formamide, dimethyl sulfoxide or dimethyl acetamide) were studied as alternative media for static HS-gas chromatographic analysis. Although it has been known that mixed aqueous dilution media can often improve sensitivity for many RS, this study used a systematic approach to investigate phase volumes and the organic content in the HS sampling media. Reference solutions using 18 different class 1, 2 and 3 RS were evaluated. The effect of salt addition was also studied in this work. A significant increase in the peak area was observed for all RS using mixed aqueous dilution media, when compared with organic solvents alone. Matrix effects related to the mixed aqueous dilution media were also investigated and reported. Repeatability and linearity obtained with mixed aqueous dilution media were found to be similar to those observed with pure organic solvents.

Enzymes are major drug targets in drug discovery and development processes in the pharmaceutical and biotechnology industry. A recent survey found that nearly half of all the marketed small-molecule drugs are inhibitors of enzymes. Matrix metalloproteinases (MMPs) are a family of 28 enzymes capable of degrading the constituents of the extracellular matrix (ECM) and the basement-membrane. MMPs play an essential role in several normal physiological processes including growth, wound healing and tissue repair. Over-expression and activation of MMPs has been linked to a range of diseases which include osteoarthritis, tumor metastasis, angiogenesis and cardiovascular diseases. The development of MMP inhibitors as therapeutic agents has kept an important place in drug discovery. Therefore, there is also an increasing need for robust analytical methods for evaluation of inhibitory potency and for the analysis of MMP inhibitors and their metabolites which can even play a more significant role than the parent drug. Modern analytical techniques and hyphenated instrumentations such as liquid chromatography-mass spectrometry with a function of structure elucidation can provide a profound insight into the research of MMP inhibitors and also serve as a complementary method to zymographic techniques for the analysis of biological samples. This review mainly summarizes bioanalytical methods, pharmacokinetics and related metabolites of MMP inhibitors over the last 12 years.

Liquid chromatography combined with pulsed electrochemical detection (LC-PED) is the method of choice in the European Pharmacopoeia for the determination of gentamicin and its related substances. A recently approved improved LC-PED method, with a reversed-phase C(18) column and a mobile phase consisting of trifluoroacetic acid (TFA), pentafluoropropionic acid (PFPA), sodium hydroxide and acetonitrile, showed better separation and more sensitive detection of the gentamicin components than the previous method using a polymer column. More unknown peaks can be separated from the main components and from each other. As the LC-PED method cannot be directly coupled to a mass spectrometer (MS), the unknown substances were collected after the LC column, desalted and analyzed by MS. The structures of the unknown compounds were deduced based on comparison of their fragmentation patterns with those of reference substances investigated by MS(n) experiments using an electrospray ion trap mass spectrometer. A comparison was also made with an already previously published LC-MS method using a volatile mobile phase.

The European Pharmacopoeia (Ph. Eur.) describes liquid chromatography-ultraviolet (LC-UV) methods using C(18) stationary phases for the analysis of polymyxin B and colistin. Several unknown impurities were detected in commercial samples of those polypeptide complexes. However, the Ph. Eur. does not specify any related substances for polymyxin B and colistin. Since both methods use non-volatile buffers, the mobile phases were incompatible with mass spectrometry (MS). For the identification of related substances in bulk samples by LC/MS, volatile mobile phase systems were developed. However, the LC/MS methods (with volatile additives) showed inferior chromatographic separation compared to the LC-UV method (with non-volatile additives). Moreover, previously identified impurities by LC/MS could not be assigned in LC-UV methods as the separation in both systems was different. In this study, known impurities were traced in the LC-UV methods and new impurities present in polymyxin B and colistin bulk samples were characterized. To achieve this, each peak from the non-volatile system was collected separately and reinjected into an LC system with a volatile mobile phase coupled to MS. This way, collected impurity peaks were rechromatographed on a reversed phase column in order to separate the analyte from the buffer salts. Using this method, out of 39 peaks, five novel related substances were characterized in a polymyxin B bulk sample. Fourteen impurities, which were already reported in the literature were traced as good as possible in the LC-UV method. In the case of colistin, a total of 36 peaks were investigated, among which four new compounds. Additionally, 30 known impurities were traced in the LC-UV method.