Separation and determination of synthetic impurities of difloxacin by reversed-phase high-performance liquid chromatography

A simple and rapid reversed-phase high-performance liquid chromatographic method for separation and determination of process-related impurities of difloxacin (DFL) was developed. The separation was achieved on a reversed-phase C(18) column using methanol-water-acetic acid (78:21.9:0.1, v/v/v) as a mobile solvent at a flow rate of 1.0 ml/min at 28 degrees C using UV detection at 230 nm. It was linear over a range of 0.03 x 10(-6) to 1.60 x 10(-6)g for process related impurities and 0.05 x 10(-6) to 2.40 x 10(-6)g for difloxacin. The detection limits were 0.009 x 10(-6) to 0.024 x 10(-6)g for all the compounds examined. The recoveries were found to be in the range of 97.6-102.0% for impurities as well as difloxacin. The precision and robustness of the method were evaluated. It was used for not only quality assurance, but also monitoring the synthetic reactions involved in the process development work of difloxacin. The method was found to be specific, precise and reliable for the determination of unreacted levels of raw materials, intermediates in the reaction mixtures and the finished products of difloxacin.     

The determination of oxalic acid, oxamic acid, and oxamide in a drug substance by ion-exclusion chromatography

Oxalic acid, oxamic acid and oxamide are potential impurities in some active pharmaceutical ingredients (API). The retention and separation of oxalic and oxamic acids are particularly challenging using conventional reversed-phase HPLC due to their high polarity. An ion-exclusion chromatography (IEC) method has been shown to provide good separation and sensitivity for the three oxalate-related impurities in a hydrophobic API matrix. The method uses a Dionex IonPac ICE-ASI column with 95/5 (v/v) 0.1% sulfuric acid/acetonitrile as the mobile phase and UV detection at 205 nm. Development and validation of this method are described.     

高效液相色谱法分离分析链霉素及其杂质

用离子对反相高效液相色谱法可从国内外商品链霉素中分离出包括剧毒的、致敏的及色素原部份等17种杂质,并可定量测定链霉素、链霉胍及链胍双氢链糖。由于此法系分离后再测定,故不受其他物质的干扰,较微生物法及化学比色法专属性高,且可用于测定原料、成品、发酵液及分离纯化各阶段的液体样品。     

Characterization of impurities in semi-synthetic vinorelbine bitartrate by HPLC-MS with mass spectrometric shift technique

A simple and sensitive method of high-performance liquid chromatography coupled with electrospray ionization mass spectrometry (HPLC/ESI-MS) was developed to separate and identify impurities in semi-synthetic vinorelbine bitartrate sample. The analytical HPLC was carried out on a reversed-phase C8 column using 0.02 M ammonium formicate buffer (pH 4.2) and methanol (46:54, v/v) as mobile phase at a flow rate of 0.8 ml/min at room temperature and a UV detection at 267 nm. The on-line HPLC/ESI-MS/MS was performed using ion trap analyzer in positive ion mode. Applying mass spectrometric shift technique to HPLC/ESI-MS/MS analysis, four impurities were identified as 18'-O-demethylvinorelbine (impurity-1), 6'-N-methylvinorelbine (impurity-2), 23-O-demethylvinoreline (impurity-3) and 17-bromovinorelbine (impurity-4), respectively, in investigated vinorelbine bitartrate sample. The four impurities, in which the impurity-1 was not reported as the semi-synthetic process impurity for vinorelbine bitartrate elsewhere, were isolated by preparative high-performance liquid chromatography. Their structures were further confirmed by means of 1D and 2D NMR spectra. Structural elucidation by spectral data was discussed.     

Optimization and validation of conventional and micellar LC methods for the analysis of methyltestosterone in sugar-coated pills

Two isocratic liquid chromatographic methods (conventional and micellar) for the determination of methyltestosterone in sugar-coated pills using fluoxymesterone as internal standard have been developed and validated. In conventional liquid chromatography a mobile phase 45% water:acetonitrile 55% (v:v), a flow-rate 1 mlmin(-1) and a C(18) Hypersil ODS (250 x 4.6 mm, 5 microm) column (25 degrees C) were used. In micellar liquid chromatography the conditions were: mobile phase 40 mM sodium dodecyl sulfate: 10% propanol, flow-rate 0.5 mlmin(-1) and C(18) Hypersil ODS (150 x 3.0 mm, 5 microm) column (60 degrees C). For both methods, UV absorbance detection at 245 nm was used and a separation up to base line was achieved. Prior to HPLC analysis a simple sample preparation was required.     

SPE-HPLC method for the determination and pharmacokinetic studies on paeoniflorin in rat serum after oral administration of traditional Chinese medicinal preparation Guan-Xin-Er-Hao decoction

A new HPLC method for the determination of paeoniflorin in rat serum with solid-phase extraction (SPE) for preconcentration is introduced. Paeoniflorin and an internal standard (pentoxifylline) were extracted from serum by means of SPE using cartridges with octadecyl chemically bound phase. The HPLC separation was then performed on a reversed-phase C(18) column using acetonitrile-water (18:82, v/v) as eluting solvent system, and UV detection at 230 nm to measure the analyte with a limit of quantitation about 10 ng ml(-1). The calibration curve for paeoniflorin was linear (r=0.9938) in the concentration range of 10-1200 ng ml(-1), both intra- and inter-day precision of the paeoniflorin were determined and their coefficience of variation did not exceed 10%. The validated method has been successfully applied for pharmacokinetic studies of paeoniflorin from rat serum after oral administration of Guan-Xin-Er-Hao decoction.     

Application of monolithic column in quantification of gliclazide in human plasma by liquid chromatography

A simple, rapid and sensitive isocratic reversed phase HPLC method with UV detection using a monolithic column has been developed and validated for the determination of gliclazide in human plasma. The assay enables the measurement of gliclazide for therapeutic drug monitoring with a minimum quantification limit of 10ngml(-1). The method involves simple, one-step extraction procedure and analytical recovery was complete. The separation was carried out in reversed-phase conditions using a Chromolith Performance (RP-18e, 100mmx4.6mm) column with an isocratic mobile phase consisting of 0.01M disodium hydrogen phosphate buffer-acetonitrile (52:48, v/v) adjusted to pH 4.0. The wavelength was set at 230nm. The calibration curve was linear over the concentration range 10-5000ngml(-1). The coefficients of variation for inter-day and intra-day assay were found to be less than 6.0%.     

HPLC determination of salinomycin and related compounds in fermentation media of Streptomyces albus and premixes

A rapid, sensitive and selective HPLC method with post-column derivatization was proposed for the determination of salinomycin and related products in fermentation broths and premixes. The solvent extracts of samples were analysed on a reversed-phase monolithic type column. The mobile phase consisted of methanol/zinc acetate (0.05 M) adjusted to pH 4.0 with acetic acid (85/15, v/v). Post-column derivatization with vanillin at 85 degrees C was used for simultaneous, selective detection of salinomycin at 520 nm and related products at 460 nm. Optimal ratio of mobile phase/reagent flow rate was 2:1. Alternatively, pre-column derivatization of salinomycin and related products with three different reagents (2,4-dinitrophenylhydrazine, p-bromophenacyl bromide and p-nitrobenzoyl chloride) was examined. Suitable derivates for HPLC separation and UV detection were prepared using p-nitrobenzoyl chloride. Extraction ability of various solvents for extracting of salinomycin and co-products from premix samples was also tested. Acetone, ethanol and pyridine were found to be the best extraction solvents for these compounds.     

Improved LC of minocycline drug substance

An isocratic liquid chromatographic method is described for the separation of minocycline and its impurities. This method uses XTerra RP-18, 5 microm (25 cm x 4.6 mm I.D.), a silica-based stationary phase with reduced silanol activity. A mobile phase composed of acetonitrile-0.2 M tetrabutylammonium hydrogen sulphate pH 6.5-0.2 M ethylenediaminetetraacetic acid pH 6.5-water (20:20:20:40; v/v/v/v) was used at a flow rate of 1 ml/min. The column temperature was set at 35 degrees C. UV detection was performed at 280 nm. Optimisation of the separation method and a robustness study were performed by means of a central composite experimental design. The method allows to separate minocycline from known impurities. Some unidentified impurities are also separated. The total time of analysis is less than 20 min.     

Separation and determination of synthetic impurities of norfloxacin by reversed-phase high performance liquid chromatography

A simple and rapid high performance liquid chromatographic method for the separation and determination of synthetic impurities of norfloxacin was developed. The separation was achieved on a reversed-phase C₁₈ column using 0.01 M potassium dihydrogen orthophosphate and acetonitrile (60:40, v/v, pH 3.0) as mobile solvent at a flow rate of 1.0 ml/min at 40 °C and a UV detection at 260 nm. The method was used not only for quality assurance but also for monitoring the chemical reactions during the process development work in the laboratory. It was found to be specific, precise and reliable for determination of unreacted levels of raw materials, intermediates and the finished products of norfloxacin.     

LC determination of glimepiride and its related impurities

Five impurities in glimepiride drug substance were detected and quantified using a simple isocratic reverse phase HPLC method. For the identification and characterization purpose these impurities were isolated from a crude reaction mixture of glimepiride using a normal phase HPLC system. Based on the spectroscopic data like NMR, FTIR, UV and MS these impurities were characterized and used as impurity standards for determining the relative response factor during the validation of the proposed isocratic reverse phase HPLC method. The chromatographic separation was achieved on a Phenomenex Luna C8 (2) 100 Å, 5 μm, 250 mm × 4.6 mm using a mobile phase consisting of phosphate buffer (pH 7.0)–acetonitrile–tetrahydrofuran (73:18:09, v/v/v) with UV detection at 228 nm and a flow rate of 1 ml/min. The column temperature was maintained at 35 °C through out the analysis. The method has been validated as per international guidelines on method validation and can be used for the routine quality control analysis of glimepiride as active pharmaceutical ingredient (API).     

HPLC method for the determination of nystatin in saliva for application in clinical studies

An isocratic high-performance liquid chromatographic method was developed, optimized and validated for the determination of nystatin in human saliva (UV and fluorescence detection). A reversed-phase Luna C18 column (25 degrees C), with a mobile phase of MeOH, H2O, and DMF (70:20:10, v/v/v), and a flow-rate of 0.8 ml/min were used. The elution time for nystatin was 5.8+/-0.2 min. Calibration curves in human saliva were linear from 0.78 to 50 microg/ml. Limits of quantification were 0.78 microg/ml and 0.75 microg/ml for UV and fluorescence detection, respectively. The accuracy and precision values of intra- and inter-day variation studies were within acceptable limits, according to FDA guidelines. The described method has proved to be useful to give accurate measurements of nystatin in real samples.     

Quantitative determination of piroxicam in a new formulation (piroxicam-beta-cyclodextrin) by derivative UV spectrophotometric method and HPLC

A derivative ultraviolet (UV) spectrophotometric method for the determination of piroxicam in piroxicam--beta-cyclodextrin tablets was developed. Phosphate buffer (pH 7.8, 0.1 M) and ethanol were used as a solvent system throughout the study. In this study, determination of piroxicam was conducted by using first order derivative amplitudes at 261.4 nm (n=4). Standards for the calibration graph ranging from 2.40 to 20.0 microg/ml were prepared from working standard. The proposed method is accurate with 99.70%+/-0.50 recovery value and precise with coefficient of variation (CV) of 1.29. The results were compared with those obtained using a high-performance liquid chromatography (HPLC) procedure. A reversed-phase C(18) column with aqueous phosphate buffer:methanol, 60:40, v/v, mobile phase was used. UV detector was set at 254 nm. Calibration solutions used in HPLC were ranging from 5 to 20 microg/ml. Results obtained in HPLC were comparable to those obtained by derivative UV spectrophotometric method.     

白芍的高效液相色谱指纹图谱研究

利用HPLC-DAD方法，研究白芍的高效液相色谱指纹图谱，为科学评价及有效控制其质量提供可靠方法。测定了28批白芍药材样品，并应用LC-MS，MS/MS技术指认指纹图谱共有模式中共有峰的归属。结果表明，28批白芍样品得到的色谱指纹图谱有11个共有峰，并指认了9个峰，分别为(+)-儿茶素，没食子酸甲酯，芍药内酯苷，芍药苷，四没食子酰葡萄糖，没食子酰芍药苷，五没食子酰葡萄糖，牡丹皮苷I和苯甲酰芍药苷。可见，白芍的指纹图谱特征性及专属性强，可结合含量测定用于全面控制白芍的质量。     

Conventional and micellar liquid chromatography method development for danazol and validation in capsules

Two isocratic liquid chromatographic methods (conventional and micellar) for the determination of danazol (DZ) in capsules using canrenone (CAN) as internal standard have been developed and validated. In conventional liquid chromatography a mobile phase 35% water:acetonitrile 65%, v:v, a flow-rate 1 ml min(-1) and a C18 Hypersil ODS (250 x 4.6 mm, 5 microm) column (25 degrees C) were used. In micellar liquid chromatography (MLC) the conditions were: mobile phase 40 mM sodium dodecyl sulfate:2% pentanol, flow-rate 0.5 ml min(-1) and C18 Hypersil ODS (150 x 3.0 mm, 5 microm) column (60 degrees C). For both methods. UV absorbance detection at 280 nm was used and a separation up to base line was achieved. Prior to HPLC analysis a simple sample preparation was required. The recoveries found in the accuracy test were 99 +/- 10 and 101 +/- 8%, in conventional liquid chromatography (CLC) and MLC, respectively. Repeatability and intermediate precision expressed as R.S.D. were lower than 5% for both methods. Detection limits obtained were 2.4 and 3.0 ng g(-1) in CLC and CLM, respectively.     

A validated, sensitive HPLC method for the determination of trace impurities in acetaminophen drug substance

A high-performance liquid chromatography (HPLC) method has been developed and validated for the simultaneous determination of n-propionyl-p-aminophenol, 3-chloro-4-hydroxyacetanilide, 4'-hydroxyacetophenone, 4-hydroxyacetophenone oxime, 4-acetoxyacetanilide and 4'-chloroacetanilide, the main impurities in acetaminophen drug substance. The chromatographic separation was achieved on an Eclipse XDB-18 reversed-phase column using a gradient elution, being solvent A: 0.01 M phosphate buffer at pH 3.0 and solvent B: methanol. The limit of quantitation (S/N=10:1) was 0.1 microg/ml for each impurity. The coefficients of variation were less than 4% for intra-day and inter-day analyses. The individual recovery of acetaminophen spiked samples ranged from 94 to 104% and the mean recovery for each level from 99 to 103% in the 1-150 microg/ml range for all impurities. The proposed method was successfully applied to the analyses of different lots and different manufactures of acetaminophen drug substance. The proposed method can be used for the routine quality control of acetaminophen.     

Determination of diminazene aceturate in pharmaceutical formulations by HPLC and identification of related substances by LC/MS

A validated, reversed-phase, isocratic high-performance liquid chromatographic method for the simultaneous assay of diminazene aceturate, antipyrine (excipient) and diminazene impurities in pharmaceutical formulations is described. The chromatographic system consisted of a Lichrospher-60 RP-select B column with a mobile phase composition of acetonitrile-methanol-ammonium formate (pH 4.0, 20 mM) (10:10: 80 v/v/v) and UV detection at 254 nm. The method is specific, precise and accurate for the determination of diminazene in the presence of its manufacturing and degradation impurities with a limit of detection and quantification of 50 ng/ml and 10 microgram/ml (RSD<3.0%), respectively. The major manufacturing impurity [1-(4 amidino phenyl)3-(4 carbamoyl phenyl)-triazene] and a degradant (p-aminobenzamidine) of diminazene aceturate have been resolved and identified by liquid chromatography/electrospray ionization-mass spectrometry operated in a positive ion mode.     

Analysis of erythromycin estolate by liquid chromatography.

A method is described for the determination of erythromycin estolate by liquid chromatography. A C18 reversed-phase column (25 x 0.46 cm i.d.) was used with acetonitrile-tetrabutylammonium sulphate (pH 6.5, 0.2 M)-phosphate buffer (pH 6.5, 0.2 M)-water [x:5:5:(90-x), v/v/v/v] as mobile phase. The proportion of acetonitrile (x) has to be adapted to the type of stationary phase used. For RSil C18 LL 42.5% (v/v) was used. The column was heated at 35 degrees C, the flow rate was 1.5 ml min-1 and UV detection was performed at 215 nm. The main component, erythromycin A propionate, was separated from all other components which were present in commercial samples. The impurities most frequently observed were the propionate ester of erythromycin C and the amide N-propionyl-N-demethyl-erythromycin A. Erythromycin A was shown to be present in specialties.     

Simultaneous determination of zonisamide, carbamazepine and carbamazepine-10,11-epoxide in infant serum by high-performance liquid chromatography

This study developed a simple method for the simultaneous determination of zonisamide (ZNS), carbamazepine (CBZ) and its active metabolite, carbamazepine-10,11-epoxide (CBZE) in infant serum using reversed-phase high-performance liquid chromatograph (HPLC). The method involves a single-step protein precipitation procedure that uses no solid-phase or liquid-liquid extraction. The HPLC separation was carried out on a Cadenza CD-C18 column (3 microm, 4.6 mm x 150 mm) with potassium phosphate buffer (pH 4.6; 25 mM)-methanol-acetonitrile (65:20:15 (v/v/v)) as a mobile phase at a 1.0 ml/min flow rate: ZNS was detectable using a UV detector at 235 nm, and both CBZ and CBZE were at 215 nm. The quantification limits were established in accordance with each therapeutic range at 2.5 microg/ml for ZNS, 0.5 microg/ml for CBZ, and 0.25 microg/ml for CBZE. The respective coefficients of variation were 1.3-6.0% and 2.2-7.7% for the intra- and inter-assay.     

In-process control of midazolam synthesis by HPLC

A high-performance liquid chromatographic assay coupled with UV detection (239 nm) has been developed for the determination of midazolam and its synthesis precursors. The separation of the analytes was performed on a Kromasil C8 column (15 cm x 4.6 mm i.d., 5 microm) at 30 degrees C. The mobile phase [ammonium chloride (pH 5.5, 1 g l(-1))-methanolacetonitrile (45:22:33, v/v/v)] was pumped at a flow-rate of 1.5 ml min(-1). This method is rapid (less than 11 min), sensitive (limit of detection (LOD) ranged between 0.05 and 0.5 mg l(-1)) and selective for the determination of midazolam, and it could be used for monitoring different synthetic routes.