Development and validation of an HPLC method for the quantitation of 1,3-dihydroxy-2-methylxanthone in biodegradable nanoparticles

A rapid and simple high-performance liquid chromatographic method for the analysis of 1,3-dihydroxy-2-methylxanthone (DHMXAN) in biodegradable poly(D,L-lactide-co-glycolide) (PLGA) nanosphere and nanocapsule formulations is developed and validated. The method does not require any complex sample extraction procedure. Chromatographic separation is made with a reversed-phase C18 column, using methanol-water (90:10, v/v) containing 1% (v/v) acetic acid as a mobile phase at a flow rate of 1 mL/min. Identification is made by UV detection at 237 nm. The isocratic system operates at ambient temperature and requires 7.5 min of chromatographic time. The developed method is statistically validated according to ICH guidelines and USP 29 for its specificity, linearity, accuracy, and precision. The assay method proposed in this study is specific for DHMXAN in the presence of nanosphere and nanocapsule excipients. Diode-array analyses confirm the purity of DHMXAN peak in stress conditions (> 99.0%). The method is shown to be linear (r > or = 0.999) over the concentration range of 0.25-3.0 microg/mL. Recovery ranges from 99.0% to 102.7% (RSD: 1.49%) and from 98.3% to 101.6% (RSD: 1.07%) for nanospheres and nanocapsules, respectively. Repeatability (intra-assay precision) and intermediate precision is acceptable with RSD values ranging from 0.6% to 1.9% and from 0.3% to 2.0%, respectively. The method is shown to be suitable for the evaluation of DHMXAN content entrapped in PLGA nanoparticles.     

Development and validation of an HPLC method to quantify camptothecin in polymeric nanocapsule suspensions

A simple, rapid, and sensitive reversed-phase column high-performance liquid chromatographic method was developed and validated to quantify camptothecin (CPT) in polymeric nanocapsule suspensions. The chromatographic separation was performed on a Supelcosil LC-18 column (15 cm x 4.6 mm id, 5 microm) using a mobile phase consisting of methanol-10 mM KH2PO4 (60 + 40, v/v; pH 2.8) at a flow rate of 1.0 mL/min and ultraviolet detection at 254 nm. The calibration graph was linear from 0.5 to 3.0 microg/mL with a correlation coefficient of 0.9979, and the limit of quantitation was 0.35 microg/mL. The assay recovery ranged from 97.3 to 105.0%. The intraday and interday relative standard deviation values were < 5.0%. The validation results confirmed that the developed method is specific, linear, accurate, and precise for its intended use. The current method was successfully applied to the evaluation of CPT entrapment efficiency and drug content in polymeric nanocapsule suspensions during the early stage of formulation development.     

Development and validation of a fast RP-HPLC method for determination of methotrexate entrapment efficiency in polymeric nanocapsules

A rapid and effective isocratic chromatographic procedure is successfully developed to determinate methotrexate (MTX) entrapment efficiency (EE) in polymeric nanocapsules using reversed-phase high-performance liquid chromatography. The method employed a RP-C(18) Shimadzu Shim-pack CLC-ODS (150 mm x 4.6 mm, 5 microm) column with mobile phase constituted by a mixture of water-acetonitrile-tetrahydrofuran (65:30:5 v/v/v; pH 3.0) at a flow rate of 0.8 mL/min. The eluate is monitored with a UV detector set at 313 nm. The parameters used in the validation process are: linearity, specificity, precision, accuracy, and limit of quantitation (LOQ). The linearity is evaluated by a calibration curve in the concentration range of 10-50 microg/mL and presented a correlation coefficient of 0.9998. The polymers (PLA or PLA-PEG), oil, and surfactants used in the nanocapsule formulation did not interfere with analysis and the recovery was quantitative. The intra and inter-day assay relative standard deviation were less than 0.72%. Results are satisfactory, and the method proved to be adequate for the determination of methotrexate in nanocapsules formulations.     

Development and validation of a rapid reversed-phase HPLC method for the determination of insulin from nanoparticulate systems

A reversed-phase high-performance liquid chromatographic (HPLC) method has been developed and validated for the determination of insulin in nanoparticulate dosage forms. Its application for the development and characterization of insulin-loaded nanoparticulates composed of polyelectrolytes has also been carried out. A reversed-phase (RP) C18 column and gradient elution with a mobile phase composed of acetonitrile (ACN) and 0.1% aqueous trifluoroacetic acid (TFA) solution at a flow rate of 1 mL/min was used. Protein identification was made by UV detection at 214 nm. The gradient changed from 30:70 (ACN:TFA, v/v) to 40:60 (v/v) in 5 min followed by isocratic elution at 40:60 (v/v) for a further five minutes. The method was linear in the range of 1-100 microg/mL (R2 = 0.9996), specific with a good inter-day and intra-day precision based on relative standard deviation values (less than 3.80%). The recovery was between 98.86 and 100.88% and the detection and quantitation limits were 0.24 and 0.72 microg/mL, respectively. The method was further tested for the determination of the association efficiency of insulin to nanoparticulate carriers composed of alginate and chitosan, as well as its loading capacity for this protein. Encapsulant release under simulated gastrointestinal fluids was evaluated. The method can be used for development and characterization of insulin-loaded nanoparticles made from cross-linked chitosan-alginate.     

Liquid chromatographic determination of cetirizine in oral formulations

The development and validation of a reversed-phase liquid chromatographic (LC) method for the determination of cetirizine dihydrochloride in oral formulations are described. An isocratic LC analysis was performed on a reversed-phase C18 column (250 x 4.6 mm id, 5 microm particle size). The mobile phase was 1% orthophosphoric acid solution, pH 3.0-acetonitrile (60 + 40, v/v), pumped at a constant flow rate of 1.0 mL/min. Measurements were made at a wavelength of 232 nm. The calibration curves were linear over the range of 10-30 microg/mL (r2 = 0.9999). The relative standard deviation (RSD) values for intraday precision were 0.94 and 1.43% for tablets and compounded capsules, respectively. The RSD values for interday precision were 0.13 and 0.82% for tablets and compounded capsules, respectively. Recoveries ranged from 97.7 to 101.8% for tablets and from 98.4 to 102% for compounded capsules. No interferences from the excipients were observed. Because of its simplicity and accuracy, the method is suitable for routine quality-control analysis for cetirizine in tablets and compounded capsules.     

Simultaneous liquid chromatographic determination of ezetimibe and simvastatin in pharmaceutical products

A reversed-phase liquid chromatographic (LC) method was developed and validated for the simultaneous determination of ezetimibe and simvastatin in pharmaceutical dosage forms. The LC method was carried out on a Synergi fusion C18 column (150 mm x 4.6 mm id) maintained at 45 degrees C. The mobile phase consisted of phosphate buffer 0.03 M, pH 4.5-acetonitrile (35 + 65, v/v) run at a flow rate of 0.6 mL/min, and detection was made using a photodiode array detector at 234 nm. The chromatographic separation was obtained within 15.0 min, and calibration graphs were linear in the concentration range of 0.5-200 microg/mL. Validation parameters such as specificity, linearity, precision, accuracy, and robustness were evaluated, giving results within the acceptable range for both compounds. Moreover, the proposed method was successfully applied for the routine quality control analysis of pharmaceutical products.     

A high-performance liquid chromatographic method for saikosaponin a quantification in rat plasma

A high-performance liquid chromatographic method with UV detection has been developed for the determination of saikosaponin a in rat plasma. Saikosaponin a and internal standard jujuboside A were isolated from plasma samples by solid-phase extraction. The chromatographic separation was achieved on a reversed-phase C(18) column with the mobile phase of acetonitrile-water (35:65, v/v) at a flow rate of 1 mL/min and UV detection was set at 205 nm. The standard curve for saikosaponin a was linear over the concentration range 0.25-10 microg/mL and the limit of detection was 0.05 microg/mL. The absolute recovery was greater than 82%. The precision and accuracy ranged from 3.05 to 9.59% and 95.61 to 110.00%, respectively. The validated method was used to determine saikosaponin a in plasma samples in a pharmacokinetic study of saikosaponin a administered to Sprague-Dawley rats.     

An improved liquid chromatographic method for the analysis of tylosin and its impurities

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.     

Determination of omeprazole in bulk and injectable preparations by liquid chromatography

An accurate, simple, reproducible, and sensitive liquid chromatographic method was developed and validated for the determination of omeprazole in powder for injection and in pellets. The analyses were performed at room temperature on a reversed-phase C18 column of 250 x 4.6 mm id, 5 microm particle size. The mobile phase, composed of methanol-water (90 + 10, v/v), was pumped at a constant flow rate of 1.5 mL/min. Detection was performed on a UV detector at 301 nm. The method was validated in terms of linearity, precision, accuracy, and ruggedness. The response was linear in the range 32-48 microg/mL (r2 = 0.9976). The relative standard deviation values for intra- and interday precision studies were 1.22 and 1.56% for injectable and 2.13 and 2.45% for pellets, respectively. Recoveries ranged between 95.81 and 100.48%.     

Determination of calycosin-7-O-beta-D-glucopyranoside in rat plasma and urine by HPLC

A reversed-phase high-performance liquid chromatographic (HPLC) assay for calycosin-7-O-beta-D-glucopyranoside in rat plasma and urine with solid-phase extraction (SPE) was developed. Rutin was employed as an internal standard. The mobile phase consisted of acetonitrile-water (16:84, v/v) at a flow rate of 1.0 mL/min. Detection was set at 280 nm. The limit of quantitation of calycosin-7-O-beta-D-glucopyranoside was 0.2 microg/mL in both plasma and urine. The standard curve was linear from 0.2 to 10.0 microg/mL in plasma, and 0.2 to 5.0 microg/mL in urine. Both intra- and inter-day precision of the calycosin-7-O-beta-d-glucopyranoside were determined and their RSD did not exceed 10%. The method was successfully applied to the analysis of samples obtained from a basic pharmacokinetic study, in which calycosin-7-O-beta-d-glucopyranoside was administered orally to rats.     

Determination of gatifloxacin in bulk and tablet preparations by high-performance liquid chromatography

A sensitive, precise, and specific high-performance liquid chromatographic (HPLC) method was developed for the assay of gatifloxacin (GATX) in raw material and tablets. The method validation parameters yielded good results and included the range, linearity, precision, accuracy, specificity, and recovery. It was also found that the excipients in the commercial tablet preparation did not interfere with the assay. The HPLC separation was carried out by reversed-phase chromatography on a C18 absorbosphere column (250 x 4.6 mm id, 5 microm particle size) with a mobile phase composed of acetic acid 5%-acetonitrile-methanol (70 + 15 + 15, v/v/v) pumped isocratically at a flow rate of 1.0 mL/min. The effluent was monitored at 287 nm. The calibration graph for GATX was linear from 4.0 to 14.0 microg/mL. The interday and intraday precisions (relative standard deviation) were less than 1.05%.     

An HPLC method for the determination of bisoprolol in human plasma and its application to a pharmacokinetic study

A new high-performance liquid chromatographic method is described for the determination of bisoprolol in human plasma. The proposed method was based on the derivatization of bisoprolol with 4-chloro-7-nitro-2,1,3-benzoxadiazole in borate buffer at pH 9.5 to yield a fluorescent product. Chromatographic separation of bisoprolol was achieved by using isocratic elution at a flow rate of 1.2 mL/min on a C18 reversed-phase column (Inertsil, 4 μm, 150 4.6 mm) at 40°C. The mobile phase used for the analysis was methanol-water (70:30, % v/v). Fluorescence detector was used at the excitation and emission wavelengths of 458 and 525 nm, respectively. The method was validated for linearity, limit of detection, limit of quantification, precision, accuracy, recovery and system suitability. The assay was linear over the concentration range of 10-2000 ng/mL. This method was applied in pharmacokinetic studies of bisoprolol preparations in healthy volunteers.     

Development and validation of a reversed-phase liquid chromatographic method for separation and simultaneous determination of COX-2 inhibitors in pharmaceuticals and its application to biological fluids

An isocratic reversed-phase high-performance liquid chromatographic method has been developed for separation and simultaneous determination of COX-2 inhibitors, viz., celecoxib, rofecoxib, valdecoxib, nimesulide and nabumetone, using 4-chloro-2-nitroaniline as internal standard. Good chromatographic separation was achieved using a reversed-phase Inertsil C(18) column with mobile phase consisting of methanol and 0.05% aqueous glacial acetic acid (68:32 v/v) using photodiode array (PDA) detector at 230 nm. It was validated with respect to accuracy, precision, linearity, limit of detection and quantification. The linearity range was found to be 1.0--20 microg/mL and the percentage recoveries were between 97.55 and 100.14. The method is suitable not only for the estimation of active ingredients in pharmaceutical dosage forms but also in vitro estimations in human plasma. It is simple, rapid, selective and capable of detecting and determining COX-2 inhibitors with a detection limit of 0.127--1.040 microg/mL simultaneously.     

Determination of aliskiren in tablet dosage forms by a validated stability-indicating RP-LC method

A reversed-phase liquid chromatography (RP-LC) method is validated for the determination of aliskiren in tablet dosage form. The LC method is carried out on a Waters XBridge C(18) column (150 × 4.6 mm i.d.), maintained at 25°C. The mobile phase consisted of acetonitrile:water (95:5, v/v)/phosphoric acid (25 mM, pH 3.0) (40:60, v/v), run at a flow rate of 1.0 mL/min, with photodiode array detector set at 229 nm. The chromatographic separation is obtained with aliskiren retention time of 3.68 min, and it is linear in the range of 10-300 μg/mL (r = 0.9999). The limits of detection and quantitation are 2.38 and 7.93 μg/mL, respectively. The specificity and stability-indicating capability of the method are proven through degradation studies, which also showed that there is no interference of the formulation excipients, showing that peak is free from any coeluting peak. The method showed adequate precision, with a relative standard deviation (RSD) values lower than 0.92%. Good values of accuracy were also obtained, with a mean value of 99.55%. Experimental design is used during validation to calculate method robustness. The proposed method is applied for the analysis of the tablet dosage forms, contributing to improve the quality control and to assure the therapeutic efficacy.     

Determination and pharmacokinetic study of chlorogenic acid in rat dosed with Yin-Huang granules by RP-HPLC

A reversed-phase high-performance liquid chromatographic (RP-HPLC) method was described for the determination of chlorogenic acid (CGA) in rat plasma using protocatechuic acid as internal standard (IS). CGA in plasma was extracted with acetonitrile, which also acted as deproteinization agent. Chromatographic separation was performed on a Kromasil C18 column with methanol-0.2 m acetic acid (pH 3.0, 25:75, v/v) as mobile phase at a flow-rate of 1.0 mL/min with an operating temperature of 30 degrees C and UV detection at 300 nm. The standard curve was found to be linear over the concentration ranges of 0.4-2.5 microg/mL and 2.5-40 microg/mL, and the limit of quantification (LOQ) was 0.4 microg/mL. The analytical precision and accuracy were validated by relative standard deviation (RSD) and relative error, which were in ranges 3.14-10.78% and -2.20-5.00%, respectively. The average recovery of CGA was 87.59%. The method was successfully applied to the pharmacokinetic study of CGA in Yin-Huang granules.     

Simultaneous determination of vincristine, vinblastine, catharanthine, and vindoline in leaves of catharanthus roseus by high-performance liquid chromatography

A simple reversed-phase liquid chromatographic method is developed for the simultaneous quantitation of the anticancerous drugs vincristine, vinblastine, and their precursors catharanthine and vindoline using a Merck Chromolith Performance reversed-phase high-performance liquid chromatography column. A better resolution is obtained in comparison with available particulate-type C18 columns. The column provides good reproducibility and peak symmetry. Chromatography is carried isocratically with a mobile phase of acetonitrile-0.1M phosphate buffer containing 0.5% glacial acetic acid (21:79, v/v; pH 3.5) at a flow rate of 1.2 mL/min and UV detection at 254 nm. Parameters such as linearity, limits of quantitation (LOQ) and detection (LOD), precision, accuracy, recovery, and robustness are studied. The method is selective and linear for alkaloid concentration in the range 0.25 microg-25 microg/mL. The LOQ and LOD are 25, 46, 56, and 32 microg/mL and 8, 14, 18, and 10 microg/mL, respectively. The results of accuracy studies are good. Values for coefficient of variation are 2.50, 1.82, 1.33, and 1.13, respectively. The percent recovery of the alkaloids was found to be 96%, 97%, 98%, and 98%, respectively. Peak purity and homogeneity of these compounds in plant extract is studied using a photodiode-array detector. This simple and rapid method of analysis is applied for the determination of these alkaloids in a large number of leaf extracts of Catharanthus roseus..     

Validation of an HPLC method for quantitation of MDMA in tablets

An isocratic reversed-phase high-performance liquid chromatography (HPLC) method is developed and validated for the quantitation of 3,4-methylenedioxymethamphetamine (MDMA) in tablets. The chromatographic separation is achieved with potassium phosphate buffer (pH 3.2)-acetonitrile (9:1, v/v) as mobile phase, a Chromspher B column, and UV detection at 210 nm. The calibration curve is linear from 1.4 to 111 microg/mL. The percent relative standard deviation for intra- and interday precision studies is 2.7% each. The measurement uncertainty is estimated to 9%. The method is specific and successfully used for routine quantitation of MDMA in tablets.     

Simultaneous determination of albiflorin and paeoniflorin in rat urine by solid-phase extraction and high-performance liquid chromatography following oral administration of Si-Wu decoction

A high performance liquid chromatographic method (HPLC), together with solid phase extraction (SPE), was developed for simultaneous determination of albiflorin and paeoniflorin in rat urine after oral administration of Si-Wu decoction. The samples were pretreated with solid phase extraction using Extract-Cleantrade mark cartridges. Analysis of the extract was performed on a reversed-phase C18 column and a mobile phase made up of acetonitrile and 0.03% formic acid (17:83, v/v). UV detection was set at 230 nm. The assay was linear over the range 2.625-52.50 mg/mL for albiflorin and 3.875-77.50 microg/mL for paeoniflorin. The average percentage recoveries of three spiked urines were 97.01 +/- 3.32 and 102.32 +/- 6.97 for albiflorin and paeoniflorin, respectively. The intra-day precision (RSD) ranged from 0.21 to 1.79% at concentrations of 4.20, 10.50, 26.25 and 39.375 microg/mL of albiflorin and 0.12 to 2.92% at concentrations of 3.875, 10.85, 23.25 and 58.125 microg/mL of paeoniflorin, and inter-day precision (RSD) was from 1.02 to 1.86% for albiflorin and 0.94 to 3.30% for paeoniflorin, at the same four concentrations. This method was applied in order to analyze albiflorin and paeoniflorin in rat urine following oral administration of traditional Chinese medicinal preparation of Si-Wu decoction.     

HPLC determination and pharmacokinetics of chlorogenic acid in rabbit plasma after an oral dose of Flos Lonicerae extract

A simple and sensitive high-performance liquid chromatography (HPLC) method has been developed for the determination of chlorogenic acid (3-O-caffeoyl-D-quinic acid) in plasma and applied to its pharmacokinetic study in rabbits after administration of Flos Lonicerae extract. Plasma samples are extracted with methanol. HPLC analysis of the extracts is performed on a C(18) reversed-phase column using acetonitrile-0.2% phosphate buffer (11:89, v/v) as the mobile phase. The UV detector is set at 327 nm. The standard curves are linear in the range 0.0500-1.00 microg/mL (r = 0.9987). The mean extraction recovery of 85.1% is obtained for chlorogenic acid. The interday precision (relative standard deviation) ranges from 5.0% to 7.5%, and the intraday precision is better than 9.0%. The limit of quantitation is 0.0500 microg/mL. The plasma concentration of chlorogenic acid shows a C(max) of 0.839 +/- 0.35 microg/mL at 34.7 +/- 1.1 min and a second one of 0.367 +/- 0.16 microg/mL at 273.4 +/- 39.6 min.     

Simultaneous determination of benzydamine hydrochloride and five impurities in an oral collutory as a pharmaceutical formulation by high-performance liquid chromatography

A reversed-phase high-performance liquid chromatographic method for the determination of benzydamine hydrochloride and its impurities 3-dimethylaminopropyl 2-benzylaminobenzoate, 3-dimethylaminopropyl-2-aminobenzoate,1-benzyl-1H-indazol-3-ol, 1-benzyl-2-(3-dimethylaminopropyl)-1,2-dihydro-3H-indazol-3-one, and 1-benzyl-3-(3-(3-dimethylaminopropyl)-3-methylamino)propoxy-(1)H-indazole in a collutory formulation is developed. The separation is carried out on a Gemini C(18) (250 × 4.6 mm, 5 μm) column using acetonitrile-methanol-ammonium carbonate buffer (10 mM; pH 10.5) (37.5:37.5:25, v/v/v) as mobile phase at a flow rate of 1.0 mL/min, column temperature 30°C, and UV detection at 218 nm. Famotidine is used as an internal standard. The total run-time is less than 15 min. The analytical curves present coefficients of correlation greater than 0.99, and detection limits are calculated for all analytes. Excellent accuracy and precision are obtained for benzydamine hydrochloride. Recoveries vary from 98.25 to 102.8%, and intra- and inter-day precisions, calculated as the percent relative standard deviation, are lower than 2.2%. Specificity and robustness for benzydamine hydrochloride are also determined. The method allows the quantitative determination of benzydamine hydrochloride and its impurities, and it is suitable for routine analysis in quality control laboratories.