Rapid Estimation of Tadalafil by Reverse-phase High-performance Liquid Chromatography Method in Bulk and Tablet Formulation

The simple, selective, precise and accurate reverse-phase high-performance liquid chromatography method was developed and validated for analysis of tadalafil in bulk and tablet dosage form. The column was Inertsil C18 (150×4.6 mm; 5 μm) in isocratic mode. The mobile phase used was phosphate buffer (10 mM, pH 3.2) and acetonitrile (50:50% v/v) at the flow rate of 1.0 ml/min with ultraviolet detection at 295 nm at ambient temperature. The retention time for tadalafil was found to be 4.01 min. Linearity was observed in the concentration range from 60 to 140 μg/ml for tadalafil with a correlation coefficient of (r²) 0.9998. The method was validated according to International Conference on Harmonisation guidelines in terms of linearity, accuracy, precision and specificity. Hence, the proposed method can be utilized for routine quality control of tadalafil in bulk and tablet dosage form.     

Validated Stability-indicating Reverse-phase Ultra-performance Liquid Chromatography Method for Simultaneous Determination of Sodium Methylparaben,Sodium Propylparaben and Ketorolac Tromethamine in Topical Dosage Forms

A sensitive, fast, and stability-indicating isocratic reverse-phase ultra-performance liquid chromatography method was developed and validated for quantitative simultaneous determination of sodium methylparaben, sodium propylparaben and ketorolac tromethamine in topical dosage forms. Separation of all peaks was achieved by using acquity ethylene bridged hybrid C18 (50×2.1 mm, 1.7 μ) as stationary phase, mobile phase used was triethylamine buffer (pH 2.5):tetrahydrofuran:methanol (665:35:300, v/v/v) with isocratic mode at a flow rate of 0.40 ml/min. All component were detected at 252 nm with 10 min run time. The described method was found to be linear in the concentration range of 248-744 μg/ml for ketorolac tromethamine, 20.8-62.4 μg/ml for sodium methylparaben and 2.38-7.13 μg/ml for sodium propylparaben with correlation coefficients more than 0.999. Method was validated in terms of specificity, linearity, accuracy, precision, solution stability, filter equivalency, and robustness as per International Conference on Harmonization guideline. Formulation was exposed to the stress conditions of peroxide, acid, base, thermal, and photolytic degradation and proven all components were well separated in the presence of degradants.     

Development and Validation of Reverse-phase High-performance Liquid Chromatography Method for Estimation of Citicoline Sodium in Bulk and Dosage Form

A simple, accurate, precise and sensitive reverse-phase high-performance liquid chromatography method for the determination of citicoline sodium has been developed and validated. Drug was resolved on a C18 column (Phenomenex Luna, 250×4.6 mm, 10μ), utilizing mobile phase of potassium dihydrogen phosphate buffer and acetonitrile in a ratio of 30:70. Mobile phase was delivered at the flow rate of 1.0 ml/min and detection was carried out at 272 nm. Separation was completed within 2.22 min. Calibration curve was linear with good correlation coefficient (R²=0.999) over a concentration range 10-60 μg/ml. Recovery was between 98.84 and 101.76%. Method was found to be reproducible with relative standard deviation for intra and interday precision of <2.0% over the said concentration range. The method was successfully applied to the determination of the citicoline sodium, it can be very useful and an alternate to performing the stability studies.     

Development and Validation of a Reversed-Phase High-performance Liquid Chromatography Method for Determination of Desmopressin in Chitosan Nanoparticles

A simple isocratic reversed-phase high performance liquid chromatographic method was developed for determination of released desmopressin from chitosan nanoparticles in the in vitro media. The chromatographic separation was achieved with acetonitrile/water (25:75, v/v), in which water contained 0.1% v/v trifluoroacetic acid with pH=2.5 as mobile phase, a Chromolith^® Performance RP-18e column (150×4.6 mm; 5 μm) kept at 40° and ultraviolet detection at 220 nm. The compound was eluted isocritically at a constant flow rate of 1.6 ml/min. The method was validated according to the International Conference on Harmonisation guidelines. The validation characteristics included accuracy, precision, linearity rang, selectivity, limit of detection, limit of quantitation and robustness. The calibration curve was linear (r>0.9999) over the concentration rang 0.5-100 μg/ml. The limit of detection and limit of quantitation in the release media were 0.05 and 0.5 μg/ml, respectively. The proposed method had an accuracy of and intra- and inter-day precision <4.2. Furthermore, to evaluate the performance of the proposed method, it was used in the analysis of desmopressin level in real samples containing chitosan nanoparticles in the in vitro media.     

A Sensitive Method for Quantitation of Rifabutin and Its Desacetyl Metabolite in Human Biological Fluids by High-Performance Liquid Chromatography (HPLC)

Sensitive HPLC-UV methodology has been developed and validated for quantitating rifabutin, an antimycobacterial, and its 25-desacetyl metabolite, LM-565, in human plasma and urine. The HPLC separation for both plasma and urine samples was performed on an ODS, 5-µm, reverse-phase column (25 cm × 4.6-cm ID) using a mobile phase of acetonitrile/0.05 M potassium phosphate, pH 4.2, with triethylamine, (38:61.5:0.5, v/v), at a flow rate of 1.0 ml/min. The separation eluate was monitored by absorbance at 275 nm. Plasma samples (1 ml) were spiked with an internal standard (medazepam), buffered at pH 7.4 and extracted with 80:20 (v/v) hexane:ethyl acetate, and then back extracted with acidified water (0.05 M H3PO4). Linearity was established between 5.0–800 and 2.5–400 ng/ml for rifabutin and LM-565, respectively. Intraday imprecision for rifabutin and LM-565 plasma quality controls prepared at 7.3 and 3.2 ng/ml, respectively, was <15% relative standard deviation (RSD). Absolute recovery for parent drug and metabolite, from plasma, was >90% throughout the respective dynamic ranges and >70% for medazepam. Urine samples (1 ml) were acidified with 50 µl of 3.6 M H2SO4 and diluted with 0.1 M ammonium acetate. Linearity was established between 100 and 5000 ng/ml for both rifabutin and LM-565. Intraday imprecision for a urine control at 200 ng/ml was 12% RSD for either component. The method is currently being used to support Phase I kinetics program for rifabutin in prophylaxis of MAC infection of AIDS patients. Application of this method to a bioavailability assessment is presented.     

Development and Validation of RP-HPLC Method for Simultaneous Estimation of Cefpodoxime Proxetil and Dicloxacillin Sodium in Tablets

A simple, accurate, rapid and precise reversed-phase high-performance liquid chromatographic method has been developed and validated for simultaneous determination of cefpodoxime proxetil and dicloxacillin sodium in tablet. The chromatographic separation was carried out on kromasil C₁₈ analytical column (250×4.6 mm; 5 μm) with a mixture of acetonitrile:methanol:trifloroacetic acid (0.001%) with pH 6.5 (30:50:20, v/v/v) as mobile phase; at a flow rate of 1.0 ml/min. UV detection was performed at 235 nm. The dicloxacillin sodium and cefpodoxime proxetil were eluted at 1.92 and 3.35 min, respectively. The peaks were eluted with better resolution. Calibration plots were linear over the concentration range 0.5-20 μg/ml for cefpodoxime proxetil (r²=0.9996) and 5-50 μg/ml for dicloxacillin sodium (r²=0.9987). The method was validated for accuracy, precision, linearity and specificity. The method was very sensitive with limit of detection 0.0726, 0.3685 μg/ml and limit of quantification 0.220, 1.116 μg/ml for cefpodoxime proxetil and dicloxacillin sodium, respectively. The high recovery and low relative standard deviation confirm the suitability of the method for routine determination of cefpodoxime proxetil and dicloxacillin sodium in bulk drug and tablet dosage form.     

Stability-indicating Method for the Estimation of Riluzole in Tablets

A stability-indicating reverse-phase high-pressure liquid chromatography method with photodiode array detector was developed and validated for estimation of riluzole in the bulk and tablet dosage forms. Riluzole was subjected to stress conditions (light, heat, humidity, acid/base hydrolysis and oxidation) and the stressed samples were analyzed by developed method. Degradation was observed in acidic, basic, oxidative and thermal conditions. The degradation products were well resolved from riluzole peak. An inertsil-ods column (250×4.6 mm, 5 μ) with a mobile phase comprising 0.02% v/v formic acid:acetonitrile(35:65 v/v) at a flow rate of 1.0 ml/min was used and eluents were monitored at 260 nm. The retention time of riluzole was 5.7 min. Complete validation for the method was carried out according to Internation Conference on Harmonization guidelines. Linearity was achieved in the range 10-50 μg/ml with a correlation coefficient (r) 0.9998. The percent assay was 100.92 and mean percentage recovery was found to be 101.10.     

Development and Validation of RP-HPLC Method for the Simultaneous Estimation of Montelukast Sodium and Ebastine in Tablet Dosage Form

A rapid and sensitive RP-HPLC method with UV detection (244 nm) for routine analysis of montelukast sodium and ebastine in a pharmaceutical formulation (Ebast-M) was developed. Chromatography was performed with mobile phase containing a mixture of methanol:acetonitrile:ammonium acetate (80:10:10, % v/v/v), pH of mobile phase was adjusted 5.5 using glacial acetic acid and flow rate was 1.2 ml/min. The method was validated for linearity, accuracy, robustness and intermediate precision. The linearity was established over the concentration range of 0.01−0.06 mg/ml for both drugs. The correlation coefficients (r²) for ebastine and montelukast were 0.9989 and 0.9955, respectively. Statistical analysis of the data showed that the method was precise, accurate, reproducible and selective for the analysis of ebastine and montelukast drugs. The method was successfully employed for the determination of ebastine and montelukast in commercially available tablet dosage form.     

A Sensitive and Specific Procedure for Quantitation of ADR-529 in Biological Fluids by High-Performance Liquid Chromatography (HPLC) with Column Switching and Amperometric Detection

An HPLC method using electrochemical detection (ED) has been validated for the determination of ADR-529 in plasma and urine using ICRF-192 as an internal standard (IS). Prior to storage and quantitation, both plasma and urine samples require acid stabilization. Acidified plasma samples were prepared for HPLC using a two column solid-phase extraction (SPE). An aliquot of buffered plasma (i.e., pH 6-7) was first deproteinated and desalted on a C-18 SPE column. The analytes were then eluted onto a C-8 SPE column where retention and selective cleanup were achieved in the cation-exchange mode via silanol interactions. Acidified urine samples were diluted in acetonitrile prior to injection. The HPLC system for plasma and urine samples employed two narrow-bore silica columns used in the weak cation-exchange mode and separated by a switching valve. To prohibit late-eluting peaks from passivating the glassy carbon working electrode, a heart-cut containing ADR-529 and the IS was vented from the first silica column to the second using an automated switching valve. Amperometric detection at an oxidation potential of +1050 mV vs a Ag/AgNO₃ reference electrode was used. Linearity was validated between 5 and 500 µg/ml in plasma and between 2 and 100 µg/ml in urine. Imprecision and percentage bias were typically <10% for both plasma and urine controls throughout their respective dynamic ranges. The absolute recoveries for ADR-529 and the IS from plasma were >95%. This method is being successfully applied to the pharmacokinetic/dynamic evaluation of ADR-529 in animals and humans.     

Reversed-phase High-performance Liquid Chromatography Method for Analysis of Curcuminoids and Curcuminoid-loaded Liposome Formulation

A simple, sensitive, precise and specific method for the determination of curcuminoids and curcuminoid-loaded liposome formulation was developed using reverse-phase high-performance liquid chromatography method. The analysis was performed isocratically on Zorbax Eclipse XDB-C18 column (150×4 mm, 5 μm), analytical column using UV detector and mobile phase consisting of 0.1% orthophosphoric acid and acetonitrile. The proposed method for curcuminoids was validated for linearity in the range from 50 to 300 ΅g/ml with correlation coefficient above 0.997. Intraday and interday precision studies showed the relative standard deviation less than 2%. The limit of detection and limit of quantitation values were 2.5 and 8.25 ΅g/ml, respectively. Forced degradation study for curcuminoids and liposomal curcuminoids sample was carried out and observed that proposed method was also suitable for finding degradation products in the sample. Proposed method was successfully applied to estimate curcuminoids content without any interference of other excipients from liposomal formulation. Therefore, the method developed is well suited for curcuminoids and its liposome estimation.     

Validated LC Method for the Estimation of Voriconazole in Bulk and Formulation

Reversed phase high performance liquid chromatographic method was developed and validated for the estimation of voriconazole in bulk and formulation using prominence diode array detector. Selected mobile phase was a combination of water:acetonitrile (35:65 % v/v) and wavelength selected was 256 nm. Retention time of voriconazole was 3.95 min. Linearity of the method was found to be 0.1 to 2 μg/ml, with the regression coefficient of 0.999. This method was validated according to ICH guidelines. Quantification was done by calculating area of the peak and the detection limit and quantitation limit ware 0.026 μg/ml and 0.1 μg/ml, respectively. There was no significant difference in the intra day and inter day analysis of voriconazole determined for three different concentrations using this method. Present method can be applied for the determination of voriconazole in quality control of formulation without interference of the excipients.     

A Novel and Rapid Method to Determine Doxycycline in Human Plasma by Liquid Chromatography Tandem Mass Spectrometry

A simple, rapid, specific and sensitive liquid chromatography tandem mass spectrometric method has been developed and validated for the determination of doxycycline from the human plasma. Doxycycline is extracted from human plasma by solid phase extraction. Demeclocycline was used as an internal standard. Detection was performed at transitions of 444.800→428.200 for doxycycline and 464.700→448.100 for demeclocycline using mass spectrometry. Chromatographic separation of analyte and internal standard were carried out using a reverse phase C18, column at 0.500 ml/min flow. The assay of doxycycline is linear over the range of 0.055-7.612 μg/ml, with a precision <14.83%, regression coefficient (r²)=0.9961 and the limit of quantification in plasma for doxycycline was 0.055 μg/ml. Mean extraction recovery obtained was 95.55%. Samples are stable at room temperature for 6 h, processed samples were stable at least for 30.20 h and also stable at three freeze-thaw cycles. The method has been used to perform pharmacokinetic and bioequivalence studies in human plasma.     

高效液相色谱法测定注射用头孢噻肟钠舒巴坦钠中2-巯基苯并噻唑的残留量

目的：建立高效液相色谱法测定注射用头孢噻肟钠舒巴坦钠中2-巯基苯并噻唑的残留量。方法：高效液相色谱法的色谱柱：用十八烷基硅烷键合硅胶为填充剂Kromasil 100-5-C18（250 mm × 4.6 mm,5 μm）;流动相：甲醇-10 mmol·L-1乙酸铵（55∶45）;柱温：30 ℃;检测波长：320 nm;流速：1.0 mL·min-1;进样量：10 μL。结果：定量限、检测限、线性关系、耐用性、重复性、进样精密度均良好。结论：本法可用于注射用头孢噻肟钠舒巴坦钠中2-巯基苯并噻唑限度的测定。     

A Validated High-performance Liquid Chromatograhy Method for Estimation of Ferulic Acid in Asafoetida and Polyherbal Preparation

A high-performance liquid chromatography method was developed for the estimation of ferulic acid from asafoetida and a polyherbal preparation. The separation was carried out on HiQSil ODS C-18 column with a mobile phase of acetonitrile: 10% acetic acid (20:80 v/v). The developed method was validated as per International Conference of Harmonization guidelines for various parameters such as accuracy, precision, linearity, limit of detection, limit of quantification and specificity; and found to be reliable. Linear regression analysis showed a good corelation between peak area and concentration with a corelation coefficient r²=0.996 in the range 200-7000 ng/ml. The developed method can be utilized for standardization of herbal formulation comprising asafoetida.     

Development and Validation of Stability-indicating High Performance Liquid Chromatographic Method for the Estimation of Everolimus in Tablets

The present study depicts the development of a validated reversed-phase high performance liquid chromatographic method for the determination of the everolimus in presence of degradation products or pharmaceutical excipients. Stress study was performed on everolimus and it was found that it degrade sufficiently in oxidizing and acidic conditions but less degradation was found in alkaline, neutral, thermal and photolytic conditions. The separation was carried out on Hypersil BDS C18 column (100×4.6 mm, 5 μ) column having particle size 5 μ using acetate buffer:acetonitrile (50:50 v/v) with pH 6.5 adjusted with orthophosphoric acid as mobile phase at flow rate of 1 ml/min. The wavelength of the detection was 280 nm. A retention time (R_t) nearly 3.110 min was observed. The calibration curve for everolimus was linear (r²=0.999) from range of 25-150 μg/ml with limit of detection and limit of quantification of 0.036 μg/ml and 0.109 μg/ml, respectively. Analytical validation parameters such as selectivity, specificity, linearity, accuracy and precision were evaluated and relative standard deviation value for all the key parameters were less than 2.0%. The recovery of the drug after standard addition was found to be 100.55%. Thus, the developed RP-HPLC method was found to be suitable for the determination of everolimus in tablets containing various excipients.     

Development and Validation of RP-HPLC Method for the Determination of Methamphetamine and Propranolol in Tablet Dosage Form

A new isocratic reversed-phase HPLC method with diode-array UV detection was developed and validated for the determination of methamphetamine and propranolol in tablet dosage forms. Chromatography was carried out on an XTerra RP18 (150×4.6 mm, 5 μm) column using 50 mM pyrrolidine (pH 11.5) - acetonitrile (50:50, v/v) as mobile phase at a flow rate of 1 ml/min. Spectrophotometric detection was performed at a wavelength of 214 nm. The linearity was established over the concentration range of 0.075-0.60 mg/ml for both drugs. The correlation coefficients (r(2)) were ≥0.9998 in each case. The relative standard deviation values for intermediate precision studies were <1%. Statistical analysis of the data showed that the method was precise, accurate, reproducible and selective for the analysis of methamphetamine and propranolol drugs. The method was successfully employed for the determination of propranolol and methamphetamine in commercially available tablet dosage form.     

Development and Validation of a Rapid RP-UPLC Method for the Simultaneous Estimation of Bambuterol Hydrochloride and Montelukast Sodium from Tablets

A rapid, simple, sensitive and selective analytical method was developed by using reverse phase ultra performance liquid chromatographic technique for the simultaneous estimation of bambuterol hydrochloride and montelukast sodium in combined tablet dosage form. The developed method is superior in technology to conventional high performance liquid chromatography with respect to speed, resolution, solvent consumption, time, and cost of analysis. Elution time for the separation was 6 min and ultra violet detection was carried out at 210 nm. Efficient separation was achieved on BEH C18 sub-2-μm Acquity UPLC column using 0.025% (v/v) trifluoro acetic acid in water and acetonitrile as organic solvent in a linear gradient program. Resolutions between bambuterol hydrochloride and montelukast sodium were found to be more than 31. The active pharmaceutical ingredient was extracted from tablet dosage from using a mixture of methanol, acetonitrile and water as diluent. The calibration graphs were linear for bambuterol hydrochloride and montelukast sodium in the range of 6.25-37.5 μg/ml. The percentage recoveries for bambuterol hydrochloride and montelukast sodium were found to be in the range of 99.1-100.0% and 98.0-101.6%, respectively. The test solution was found to be stable for 7 days when stored in the refrigerator between 2-8°. Developed UPLC method was validated as per International Conference on Harmonization specifications for method validation. This method can be successfully employed for simultaneous estimation of bambuterol hydrochloride and montelukast sodium in bulk drugs and formulations.     

Analysis of Diltiazem and Desacetyldiltiazem in Plasma Using Modified High-Performance Liquid Chromatography: Improved Sensitivity and Reproducibility

Pharmaceutical Research -     

A Validated RP-HPLC Method for the Estimation of Lapatinib in Tablet Dosage form using Gemcitabine Hydrochloride as an Internal Standard

A simple, selective, rapid, precise and economical reverse-phase high-performance liquid chromatography method has been developed for the determination of lapatinib in tablet using gemcitabine hydrochloride as an internal standard. Chromatography was carried out on an ODS C-18 RP column (4.6 mm i.d. ×250 mm) using a mixture of acetonitrile and water (50:50 v/v) as the mobile phase at a flow rate of 1.0 ml/min. The drug was monitored at 232 nm. The retention times for lapatinib and gemcitabine hydrochloride were found to be 4.25±0.05 and 6.10±0.05 min, respectively. The method produced linear responses in the concentration range of 2-60 μg/ml of lapatinib. The limit of detection and limit of quantitation were 0.265 and 0.884 μg/ml, respectively.     

A New Rapid and Sensitive Stability-Indicating UPLC Assay Method for Tolterodine Tartrate: Application in Pharmaceuticals, Human Plasma and Urine Samples

A new rapid, simple, sensitive, selective and accurate reversed-phase stability-indicating Ultra Performance Liquid Chromatography (RP-UPLC) technique was developed for the assay of Tolterodine Tartrate in pharmaceutical dosage form, human plasma and urine samples. The developed UPLC method is superior in technology to conventional HPLC with respect to speed, solvent consumption, resolution and cost of analysis. Chromatographic run time was 6 min in reversed-phase mode and ultraviolet detection was carried out at 220 nm for quantification. Efficient separation was achieved for all the degradants of Tolterodine Tartrate on BEH C18 sub-2-μm Acquity UPLC column using Trifluoroacetic acid and acetonitrile as organic solvent in a linear gradient program. The active pharmaceutical ingredient was extracted from tablet dosage form using a mixture of acetonitrile and water as diluent. The calibration graphs were linear and the method showed excellent recoveries for bulk and tablet dosage form. The test solution was found to be stable for 40 days when stored in the refrigerator between 2 and 8 °C. The developed UPLC method was validated and meets the requirements delineated by the International Conference on Harmonization (ICH) guidelines with respect to linearity, accuracy, precision, specificity and robustness. The intra-day and inter-day variation was found be less than 1%. The method was reproducible and selective for the estimation of Tolterodine Tartrate. Because the method could effectively separate the drug from its degradation products, it can be employed as a stability-indicating one.