HPLC

Bioanalytical methods of Reverse Phase-High performance liquid chromatography (HPLC) was developed and validated for simultaneous estimation of Isoniazid (INH) and Ciprofloxacin Hydrochloride (CIP HCl) encapsulated in lipid polymeric hybrid nanoparticles (LPNs) in plasma and in organ homogenates of lung, liver, spleen and kidney of mice. Chromatographic separation was done using Agilent® C18 bonded silica column of dimensions 150 × 4.6 mm, 5μmwith flow rate 1.0 ml/min, injection volume 20 µland column temperature 40°C. The mobile phase consists of a mixture of 70 volumes of 0.1 percent v/v of trifluoroacetic acid(TFA) and 30 volumes of acetonitrile (ACN). The results indicated that the developed method was linear and selective for all matrices studied. Analysis of accuracy and precision showed adequate values, with relative standard deviation values lower than 5, which are in accordance with USFDA guidelines for bioanalytical method validation. Isoniazid (INH) and Ciprofloxacin Hydrochloride (CIP HCl) were stable in plasma and tissue homogenates under different storage and processing conditions. This method was applied to study the pharmacokinetic and biodistribution profile of both drugs in free form and in bound state with lipid nanoparticles. The results showed that polymeric nanoparticles showed higher drug accumulation in the target site i.e. lung as compared to non-target organs fulfilling our aim of developing a HPLC method for the simultaneous estimation of both drugs and their application in determination of pharmacokinetic and pharmacodynamic potential of the lipid nanoparticles.

A simple, sensitive, accurate and specific stability-indicating high-performance liquid chromatographic method was developed and validated for the estimation of clopidogrel bisulphate in bulk. In the present study, extensive testing of clopidogrel bisulphate in different stress conditions were carried out as per the ICH guidelines Q1A (R2). The system consisted of a pump (Shimadzu, prominence, LC20AD), with 20µl sample injector, along with a PDA (Shimadzu, prominence, SPDM20A) detector at a wavelength of 254nm. Data was compiled using Shimadzu LC Solution software. The degraded products formed under various stress conditions were separated successfully from the drug by using a PHENOMENEX C8 Column (150 x 4.6mm, 5µm) with binary gradient conditions. Acetonitrile: phosphate buffer of pH 2.0 was used as mobile phase at flow rate of 1.2ml/min. Clopidogrel bisulphate was exposed to various stress conditions like oxidation, hydrolysis, photolysis and neutral decomposition. Clopidogrel bisulphate, which was found to degrade considerably in acidic, photo and oxidative conditions, was found to be stable in alkaline and neutral conditions. Apart from the formation of minor degraded products under accelerated conditions, the drug was reasonably stable in solid state. A good linear relationship over the concentration range of 150-500µg/ml was shown. Validation of the method was carried out as per the ICH guidelines. The method developed was found to be precise, accurate, specific and selective. Clopidogrel bisulphate showed degradation in 5M Hydrochloric acid at 80oC, in 3% hydrogen peroxide for 5min the drug showed around 35% of degradation, when exposed to sunlight for 15 min, formed around 25-30% of degradation products. Statistical analysis shows that the method is reproducible and selective for the estimation of clopidogrel bisulphate in dosage form.

Clitora ternatea is a disease resistant plant used in ethno-pharmacological preparations for its high therapeutic value of primary and secondary metabolites. The aim of this study was to investigate the amount of quercetin in plant parts of Clitoria ternatea and its antifungal activity on dermatophytes. To obtain chemical pattern of flavonoids and quercetin in different plant parts, the extraction solvent was standardized and a finger printing profile of flavonoid was established by means of high performance liquid chromatography (HPLC). The greatest amount of flavonoid was observed in methanolic extracts of C.ternatea flowers while the lowest level was found in the C.ternatea stem extracts, whereas HPLC fingerprinting of flavonoids in flowers was significantly different from stem and leaf extracts. These extracts also differ in their fungicidal capacity against dermatophytes tested, although they all showed significant antifungal activity against Candida sp.. and Aspergillus sp.. flower extracts showed concentration dependent strong inhibition on growth of Microsporum gypseum and Epidermophyton floccosum, and weak inhibition on Trichophyton metagraphytes. The results of this study will be used to promote application of C.ternatea for improving human health by providing antidermatophytic effect on skin infections.

A HPLC method for simultaneous estimation of Ramipril and Hydrochlorothiazide in tablets was developed and validated. The developed method involves Purosphere® Star Rp18e, 5μm, 150×4.6mm column with mobile phase composition of acetonitrile and sodium perchlorate (pH 2.5) buffer in the ratio of 3:2, at a flow rate of 1.0 ml/min and UV detection at 316nm for first five minutes for Hydrochlorothiazide and 210nm for Ramipril. The method was validated as per ICH guidelines, Linearity was observed over concentration range of 17.5 to 32.5 µg/ml for Ramipril and 87.5 to 162.5 µg/ml for Hydrochlorothiazide. The Accuracy of the proposed method was determined by recovery studies and found to be 97.95-102.3% and 97.98-102.66% for Ramipril and Hydrochlorothiazide respectively. The proposed method was extended for estimation of Ramipril and Hydrochlorothiazide in marketed tablet formulation (Ramace-HTM) and it was found to be well within the acceptance limit. The developed and validated HPLC method for simultaneous estimation of Ramipril and Hydrochlorothiazide was found to be linear, accurate, precise, robust and rugged. Hence it can be used for routine analysis of Ramipril and Hydrochlorothiazide in tablets.

A simple, specific, accurate and precise reverse phase high performance liquid chromatographic method is developed and validated for the estimation of Dapsone in tablet dosage form. The expected separation and peak shapes were obtained on Luna C18, 15 cm x 4.6 mm (5 μm)  column. To have an ideal separation of the drug under isocratic conditions, mixtures of solvents like methanol and water with or without different buffers indifferent combinations were tested as mobile phases on a Luna C18, 15 cm x 4.6 mm (5 μm) column. A mixture of Methanol:Water in the ratio of 40:60 v/v was proved to be the most suitable of all the combinations since the chromatographic peak obtained was better defined and resolved and almost free from tailing.  The flow rate was 1.0ml/min and effluents were monitored at 260 nm. The retention time for Dapsone was ± 2.4 min. The method was validated for accurate, precise, simple, sensitive and rapid and can be applied successfully for the estimation of Dapsone in bulk and in pharmaceutical formulations without interference and with good sensitivity. And recovery of Dapsone from tablet formulation was found to be 93%. The proposed method was successfully applied for the quantitative determination of Dapsone in tablet formulation.

A simple, sensitive and specific HPLC method with UV detection was developed for the estimation of irbesartan in tablet dosage form. Separation was achieved by Lithosphere 100 RP-18e, 5µ column having 250x4.0 mm internal diameter with mobile phase containing phosphate buffer and acetonitrile in 50:50 (v/v) with an ambient temperature. The flow rate was 0.8mL min-1 and eluent was monitored at 220 nm. The proposed method was found to be rectilinear over theconcentration range of 20µg/ml to 60µg/ml. This method was validated for system suitability, Specificity, Linearity, Precision, Accuracy, Range, Stability studies, Ruggedness, Robustness and Filter validation.  The results of all the validation parameters were well within their acceptance values. Therefore the proposed method can be used for routine analysis of estimation of Irbesartan in its tablet formulation

A simultaneous estimation of RP-HPLC method was developed and validated for the estimation of Nimesulide, Cetrizine and Pseudo ephedrine HCl in tablet dosage form using C18 column (250mm 4.6mm, 5mm) with mobile phase consisting of Methanol: Water (65:35v/v) with a  flow rate of 1.0ml/min (UVdetection 220nm). Linearity was observed over the concentration range 0.1–5 µg/ml (R2=0.996) with regression equation y = 0.501x + 0.043 for Nimesulide and for Cetrizine 0.1–5 µg/mL (R2=0.994) with regression equation y = 0.4519x + 0.1656 and PseudoephidrineHcl 0.1–5 µg/mL (R2=0.997) with regression equation y = 0.3554x + 0.0088. The method was validated as per ICH guidelines.

Embelin is the principal phytoconstituent of Embelia tsjeriam-cottam A. DC., commonly known as Baibidanga, belonging to the family Myrsinaceae. Embelin, obtained from E. tsjeriam-cottam (an alternative source to E. ribes),is having diverse biological activities and used as anti-inflammatory, anti-diabetic, antimicrobial, anticancer and antioxidant. Fruit-samples of E. tsjeriam-cottam were collected from five different geographical locations of Odisha. These fruits were extracted and assessed for embelin content through spectrophotometric and high performance liquid chromatographic (HPLC) methods utilizing different solvent systems. Significant variation in embelin content was observed among the samples. Embelin content in fruits of E. tsjeriam-cottam was ranged from 4.93 to 2.1% (dry wt.), when extracted with chloroform and 4.88 to 1.03% (dry wt.), when extracted with methanol. However, in case of HPLC analysis, embelin content in the selected fruits were varied from 2.72 to 1.32% (dry wt.), when extracted with chloroform and 1.75 to 0.5% (dry wt.), when extracted with methanol. In general, embelin content was found higher in the fruits collected from Chura Reserve Forest (AG3) followed by Sulia Reserve Forest (AG4), Bargarh (AG1), Ghana Reserve Forest (AG5) and Jajpur (AG2). Among the selected solvent systems, chloroform prevailed to be the excellent solvent for extraction of embelin.

A simple, precise and sensitive reverse-phase high performance liquid chromatographic method was developed and validated for the simultaneous estimation of Ramipril and Metoprolol tartarate in pharmaceutical formulations. Chromatographic separation was performed on a High performance liquid chromatography equipped with auto sampler and UV detector. Good sensitivity for all analyte was observed with UV detection at wavelength of 218 nm, Separation was performed on a BDS Hypersil C18 (250 X 4.6mm) 5µm, using a mixture of 0.1% Triethylamine buffer pH 3.5 and Acetonitrile in the ratio of (10:90, v/v). The method results in excellent separation with good resolution between the two analytes. The within day variation %RSD values between Ramipril and Metoprolol tartarate were 0.55 and 0.82. The recovery was greater than 98% with %RSD less than 1.00. The method was validated according to ICH guidelines by performing linearity, accuracy, precision, limits of quantitation and selectivity. The results show the method is suitable for its intended use.

This paper deals with the development and validation of stability indicating an isocratic high performance liquid chromatographic method for the quantitative determination of memantine hydrochloride. The method is simple, highly sensitive, selective and is capable of quantitative determination of memantine hydrochloride. The chromatographic separation is achieved by injecting 20µL standard solution of memantine hydrochloride into HPLC system with refractive index detector using a YMC ODS-AQ, 5µm (150 x4.6)mm column. The mobile phase consists of 10 mL Triethylamine in 1L water(pH 5.5 adjusted with glacial acetic acid):MeOH in the ratio of  40:60 v/v. The flow rate was set at 0.9 ml/min with column and cell temperatures at 40˚C and 50 ˚C respectively and runtime was optimized to 10 min. Forced degradation studies were performed on bulk sample of memantine hydrochloride using acid (5.0Nhydrochloric acid) ,alkali (1.0N sodium hydroxide),oxidation (30% hydrogen peroxide),thermal(105 ˚C),photolytic and humidity conditions. The peak purity was checked with LC-MS and LC-MS/MS. The developed LC method was validated with respect to specificity, precision, linearity, ruggedness, stability of analytical solution and robustness.