Capillary electrophoresis method for fexofenadine hydrochloride in capsules

A simple, accurate, and effective capillary electrophoresis method with ultraviolet absorbance detection was developed and validated for the quantitation of the antihistamine fexofenadine in capsules. The separation was performed with an uncoated fused-silica capillary (47 cm x 75 microm id) and was operated at 20 kV potential. Temperature was maintained at 25 degrees C. The run buffer was prepared with 20mM Na2B4O7 x 10 H2O. Software was used for system control, data acquisition, and analysis. Method validation was performed by evaluation of the analytical parameters linearity, precision, accuracy, limits of detection and quantitation, and specificity. The method was linear (r = 0.9999) at concentrations ranging from 20 to 100 microg/mL, precise (relative standard deviation intra-assay = 1.2, 1.6, and 1.8% and interassay = 1.5%); accurate (recovery = 98.1%); and specific. The limits of detection and quantitation were 0.69 and 2.09 microg/mL, respectively. The method was compared to the liquid chromatography method developed previously by the authors for the same drug, and no significant difference was found between the 2 methods in fexofenadine hydrochloride quantitation.     

Development and validation of a new high-performance liquid chromatographic method for the loperamid hydrochloride determination in drugs

A selective, precise and new high-performance liquid chromatographic method for the analysis of loperamid hydrochloride in pharmaceutical formulations was developed and validated. The mobile phase consisting buffer (sodium-octansulphonate, triethylamine and ammonium hydroxide) in water: acetonitriie (45: 55, v/v) (pH 3.2). The absorbance was monitored with a DAD detector at 226 nm. The flow rate was 1.5 cm³ min⁻¹. The linearity (r = 0.9947) and the recovery (98.58–100.42%) were found to be satisfactory. The detection and quantitation limits were found to be 0.95 and 3.12 μg cm⁻³. The results demonstrated that the procedure was accurate, precise and reproducible. It can be suitably applied for the estimation of lopera-mid hydrochloride in pharmaceutical formulations. The article is published in the original.     

Development of validated stability-indicating chromatographic method for the determination of fexofenadine hydrochloride and its related impurities in pharmaceutical tablets

ABSTRACT: A simple reversed phase high performance liquid chromatographic method with diode array detector (HPLC-DAD) has been developed and subsequently validated for the determination of fexofenadine hydrochloride (FEX) and its related compounds; keto fexofenadine (Impurity A), meta isomer of fexofenadine (Impurity B), methyl ester of fexofenadine (Impurity C) in addition to the methyl ester of ketofexofenadine (Impurity D). The separation was based on the use of a Hypersil BDS C-18 analytical column (250 × 4.6 mm, i.d., 5 μm). The mobile phase consisted of a mixture of phosphate buffer containing 0.1 gm% of 1-octane sulphonic acid sodium salt monohydrate and 1% (v/v) of triethylamine, pH 2.7 and methanol (60:40, v/v). The separation was carried out at ambient temperature with a flow rate of 1.5 ml/min. Quantitation was achieved with UV detection at 215 nm using lisinopril as internal standard, with linear calibration curves at concentration ranges 0.1-50 μg/ml for FEX and its related compounds. The optimized conditions were used to develop a stability-indicating HPLC-DAD method for the quantitative determination of FEX and its related compounds in tablet dosage forms. The drugs were subjected to oxidation, hydrolysis, photolysis and heat to apply stress conditions. Complete separation was achieved for the parent compounds and all degradation products. The method was validated according to ICH guidelines in terms of accuracy, precision, robustness, limits of detection and quantitation and other aspects of analytical validation.     

High-pressure liquid chromatographic determination of propoxyphene hydrochloride in tablets and capsules.

The determination of propoxyphene hydrochloride by gas chromatography (GC) has been examined. Under a number of operating conditions significant on-column decomposition has occurred. The amounts of decomposition have been found to be dependent on sample size, flow-rate, column temperature, liquid phase loading, and solid support material. In view of this work a high-pressure liquid chromatographic procedure for quantitating propoxyphene hydrochloride has been developed. This method has been found to be specific for propoxyphene hydrochloride in the presence of its heat decomposition products formed with and without acetaminophen. This method does not suffer from problems associated with GC procedures.     

Development and validation of a reversed-phase liquid chromatographic method for analysis of demeclocycline and related impurities

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.     

Development and validation of an improved liquid chromatographic method for the analysis of dirithromycin

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 °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.     

Development and validation of an improved liquid chromatographic method for the analysis of oxytetracycline

Summary An improved LC method is described for the separation of oxytetracycline and its impurities. The separation is much better than that obtained with official pharmacopoeia methods. The method uses XTerra RP-18, 5 μm (25cm×4.6 mm I.D.), a silica-based stationary phase with methyl end-capping, claimed to reduce silanol activity. The column temperature is set at 30°C and a UV detection is performed at 280 nm. Mobile phase containing acetonitrile −0.25 M tetrabutylammonium hydrogen sulfate pH 7.5−0.25 M ethylenediaminetetraacetic acid pH 7.5-water (115:360:160:365,v/v/v/v) is used at a flow rate of 1.0 mL.min⁻¹, to separate the impurities present in oxytetracycline base. A central composite experimental design is used to optimize the separation. A second isocratic method with higher content of acetonitrile is needed to separate the more retained impurities present only in oxytetracycline hydrochloride. The method is robust and shows good selectivity, repeatability, linearity and sensitivity.     

High-performance liquid chromatographic method development and validation for the simultaneous quantitation of naproxen sodium and pseudoephedrine hydrochloride impurities

A reversed-phase high-performance liquid chromatographic procedure for the simultaneous determination of impurities associated with pseudoephedrine hydrochloride (PSEH) and naproxen sodium (NapNa) is developed and validated. The method is developed using a Waters Spherisorb cyano column (5 microm, 250 x 4.6 mm). An isocratic elution in a water-acetonitrile-methanol-triethylamine mixture (850:75:75:5) is adjusted to a pH of 3.7 +/- 0.02 with formic acid as the mobile phase. The UV detection was set at 260 nm, and the wavelength was switched to 235 nm before the elution of the last component, 2-ethyl-6-methoxy-naphthalene (EMN). The method is shown to be linear at a concentration range of 0.24 to 1.92 microg/mL for benzaldehyde, benzoic acid, and 2-(methylamino)-propiophenone hydrochloride, which are known impurities of PSEH. The NapNa impurities, 2-(6'-hydroxy-2'-naphthyl) propionic acid, 2-hydroxy-6-methoxy-naphthalene, 1-(6'-methoxy-2'-naphthyl) ethanol, 2-acetyl-6-methoxy-naphthalene, and EMN are also demonstrated to be linear at a concentration range of 0.44 to 3.52 microg/mL. Under the chromatographic conditions of the method, all impurities are resolved from the active components.     

Development and validation of a reversed-phase ultra-performance liquid chromatographic method for assay of lacidipine and related substances

A simple isocratic, RP-ultra-performance LC method was developed and validated for the determination of lacidipine, three process impurities formed during synthesis, and three degradation products present in drug substance and the drug product. An efficient chromatographic separation was achieved on an Acquity BEH C18 column using pH 4.5 ammonium acetate-acetic acid buffer-methanol (70 + 30, v/v) mobile phase. The monitoring wavelength was 240 nm, and the flow rate 0.25 mL/min. Forced degradation studies using acid, alkali, peroxide, water, heat, and light were conducted, and all impurities were separated. The method was validated successfully for specificity, precision, linearity, accuracy, LOD, LOQ, and robustness, according to International Conference on Harmonization guidelines. The linearity of the calibration curve for lacidipine and each impurity was found to be very good (r2 > 0.999). This method is shown to be suitable for analysis of lacidipine to evaluate the quality of drug substance and a drug product.     

Development and validation of a high-performance liquid chromatographic method for determination of eprosartan in bulk drug and tablets

A simple, precise, and accurate isocratic RP-HPLC method was developed and validated for determination of eprosartan in bulk drug and tablets. Isocratic RP-HPLC separation was achieved on a Phenomenex C18 column (250 x 4.6 mm id, 5 microm particle size) using the mobile phase 0.5% formic acid-methanol-acetonitrile (80 + 25 + 20, v/v/v, pH 2.80) at a flow rate of 1.0 mL/min. The retention time of eprosartan was 7.64 +/- 0.05 min. The detection was performed at 232 nm. The method was validated for linearity, precision, accuracy, robustness, solution stability, and specificity. The method was linear in the concentration range of 10-400 microg/mL with a correlation coefficient of 0.9999. The repeatability for six samples was 0.253% RSD; the intraday and interday precision were 0.21-0.57 and 0.33-0.71% RSD, respectively. The accuracy (recovery) was found to be in the range of 99.86-100.92%. The drug was subjected to the stress conditions hydrolysis, oxidation, photolysis, and heat. Degradation products produced as a result of the stress conditions did not interfere with detection of eprosartan; therefore, the proposed method can be considered stability-indicating.     

Development and validation of a reverse-phase liquid chromatographic method for assay and related substances of abacavir sulfate

A simple isocratic liquid chromatographic method was developed for the determination of abacavir from its related substances and assay for the first time. This method involves the usage of C18 (Intertsil octadecyl silane-3V, 150 mm x 4.6 mm, 5 microm) column. The method was validated over the range of 0.002-0.1 mg/mL for chloro impurity, 0.005-0.1 mg/mL for amino impurity and pyrimidine impurity, and 0.005-0.2 mg/mL for abacavir. The mobile phase consists of a mixture of 10 mM ammonium acetate buffer and ACN in the ratio of 90:10. The flow rate was set at 1.0 mL/min with UV detection monitored at 214 nm. The drug substance was subjected to stress conditions of hydrolysis, oxidation, photolysis, and thermal degradation. The developed method was validated for linearity, range, precision, accuracy, and specificity. This method can be conveniently used in a quality control laboratory for routine analysis of both related substances and assay.     

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

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

Development and validation of a high-performance liquid chromatographic method for the determination of clomazone residues in surface water

A method is described for the determination of clomazone residues in surface water by high-performance liquid chromatography with UV detection. The method involves solid-phase extraction with C18 extraction tubes. Clomazone was separated on a C18 column with a mobile phase of methanol-water (65:35, v/v) at pH 4.0 and a flow-rate of 1.0 ml/min. After optimization of the extraction and separation conditions, the method was validated. The method developed can be used for determination of clomazone in surface water, at the limit of 0.1 mcirog/l set by the European Union drinking water directive, with a 400-fold preconcentration.     

Development and validation of a hydrophilic interaction liquid chromatographic method for determination of aromatic amines in environmental water

A simple, precise, and accurate hydrophilic interaction liquid chromatographic (HILIC) method has been developed for the determination of five aromatic amines in environmental water samples. Chromatography was carried out on a bare silica column, using a mixture of acetonitrile and a buffer of NaH₂PO₄–H₃PO₄ (pH 1.5, containing 10 mM NaH₂PO₄) (85:15, v/v) as a mobile phase at a flow rate of 1 mL min⁻¹. Aromatic amines were detected by UV absorbance at 254 nm. The linear range of amines was good (r² > 0.998) and limit of detection (LOD) within 0.02–0.2 mg L⁻¹ (S/N = 3). The retention mechanism for the analytes under the optimum conditions was determined to be a combination of adsorption, partition and ionic interactions. The proposed method was applied to the environmental water samples. Aromatic amines were isolated from aqueous samples using solid-phase extraction (SPE) with Oasis HLB cartridges. Recoveries of greater than 75% with precision (RSD) less than 12% were obtained at amine concentrations of 5–50 μg L⁻¹ from 100 mL river water and influents from a wastewater treatment plant (WWTP). The present HILIC technique proved to be a viable method for the analysis of aromatic amines in the environmental water samples.     

Development and validation of a stability-indicating liquid chromatographic method for determination of emtricitabine and related impurities in drug substance

A novel stability-indicating high-performance liquid chromatographic (HPLC) method was developed and validated for assay and determination of impurities of emtricitabine in drug substance. Emtricitabine was found to be degraded under acidic, alkaline, and oxidative stress conditions and to be more labile under oxidative conditions. The drug proved to be stable to dry heat and photolytic degradation. Resolution of major and minor degradation impurities was achieved on an Intersil ODS-3V column utilizing 10 mM sodium phosphate buffer and methanol (85:15) as mobile phase. Detection was at 280 nm. Validation studies were performed as per ICH recommended conditions. The developed method was found to be linear, accurate, specific, selective, precise, and robust.     

Development and validation of a liquid chromatographic method for the analysis of capreomycin sulfate and its related substances

A gradient LC method for the analysis of capreomycin sulfate and its related substances was developed. The chromatographic conditions include the use of a Hypersil base deactivated C₁₈ (250 mm × 4.6 mm, 5 μm) column maintained at 25 °C, a mobile phase containing acetonitrile, phosphate buffer pH 2.3 and 0.025 M hexanesulfonate at a flow rate of 1.0 mL/min and UV detection performed at 268 nm. Good separation of the four active components of capreomycin and eleven unknown impurities was achieved. A system suitability test to check the quality of the separation is specified. The method shows good repeatability, linearity and robustness.     

Development and validation of a liquid chromatographic method for the determination of hydroxymethylfurfural and alpha-ketoglutaric acid in human plasma

Hydroxymethylfurfural (HMF) and alpha-ketoglutaric acid (KG) have been recently investigated as potential cancer cell damaging agents. We herein report for the first time a validated quantitative assay for their simultaneous determination in human plasma which is amenable to be applied in the future screening of the target compounds in human probands in order to properly design a targeted chemotherapeutic regimen for certain types of malignant tumors.A simple liquid chromatographic method in conjunction to derivatization after a two-step optimized solid phase clean-up procedure is described. The method is based on the reaction of HMF and KG with 2-nitrophenylhydrazine or 2,4-dinitrophenylhydrazine in an aqueous environment. Reaction conditions were studied with respect to pH, reagent volume, reaction temperature and time. Exact testing of such parameters beside careful selection of the mobile phase composition rendered feasible the quantification of the chemically significantly differing analytes along a single chromatographic run. The formed derivatives could be separated isocratically by reversed-phase LC on a C₈-column. Detection in the UV and in the visible range is possible. Results showed good recovery and reproducibility with detection limits (S/N = 3) down to 2 picomoles analyte on column. Resolution of the syn and anti geometric isomers of the HMF and KG derivatives is possible. The isomeric ratio in relation to the reaction pH is discussed.     

Development and validation of a reversed-phase ion-pair high-performance liquid chromatographic method for the determination of risedronate in pharmaceutical preparations

A stability indicating, reversed-phase ion-pair high-performance liquid chromatographic method was developed and validated for the determination of risedronate in pharmaceutical dosage forms. The determination was performed on a BDS C₁₈ analytical column (250 mm × 4.6 mm i.d., 5 μm particle size); the mobile phase consisted of 0.005 M tetrabutylammonium hydroxide and 0.005 M pyrophosphate sodium (pH 7.0) mixed with acetonitrile in a ratio (78:22, v/v) and pumped at a flow rate 1.00 mL min⁻¹. The ultraviolet (UV) detector was operated at 262 nm. The retention times of magnesium ascorbyl phosphate, which was used as internal standard and risedronate were 4.94 and 5.95 min, respectively. The calibration graph was ranged from 2.50 to 20.00 μg mL⁻¹, while detection and quantitation limits were found to be 0.48 and 1.61 μg mL⁻¹, respectively. The intra- and inter-day percentage relative standard deviations, %R.S.D., were less than 5.9%, while the relative percentage error, %E_r, was less than 0.4%. The method was applied to the quality control of commercial tablets and content uniformity test and proved to be suitable for rapid and reliable quality control.     

Development and validation of a reversed-phase column liquid chromatographic method for the determination of five cephalosporins in pharmaceutical preparations

A new, simple, rapid, and precise RP-HPLC method has been developed and validated for the determination of five cephalosporins, namely, cefalexin, cefoperazone, ceftriaxone, ceftazidime, and cefepime. The method has been applied successfully for simultaneous determination of cefalexin in a binary mixture with sodium benzoate in a suspension, and cefoperazone in a binary mixture with sulbactam in vials. Chromatographic separation was achieved on a Waters microBondapak C18 column (250 x 4.6 mm id, 10 pm particle size) using the mobile phase monobasic potassium phosphate (50 mM, pH 4.6)-acetonitrile (80 + 20, v/v) with UV detection. A flow rate of 1 mL/min was applied. Linearity, accuracy, and precision were found to be acceptable over the concentration range of 30-300, 3-30, and 15-120 microg/mL for the studied cephalosporins, sodium benzoate, and sulbactam, respectively. The optimized method proved to be specific, robust, and accurate for QC of the cited drugs in their pharmaceutical preparations.     

Development and validation of a stability-indicating column high-performance liquid chromatographic assay method for determination of nebivolol in tablet formulation

A simple, precise, and accurate isocratic reversed-phase (RP) stability-indicating column high-performance liquid chromatographic (HPLC) assay method was developed and validated for determination of nebivolol in solid pharmaceutical dosage forms. Isocratic RP-HPLC separation was achieved on a Phenomenex Luna C8 (2) column (250 mm x 4.6 mm id, 5 microm particle size) using mobile phase composed of acetonitrile-pH 3.5 phosphate buffer (35 + 65, v/v) at a flow rate of 1.0 mL/min, and detection was performed at 280 nm using a photodiode array detector. The drug was subjected to oxidation, hydrolysis, photolysis, and heat to apply stress conditions. The method was validated for specificity, linearity, precision, accuracy, robustness, and solution stability. The method was linear in the drug concentration range of 40-160 microg/mL with a correlation coefficient of 0.9999. The repeatability relative standard deviation (RSD) for 6 samples was 0.69%, and the intermediate precision (RSD) for 6 samples was 1.39%. The accuracy (recovery) was between 98.57 and 99.55%. Degradation products produced as a result of stress studies did not interfere with detection of nebivolol, and the assay can thus be considered stability-indicating.