A reverse phase high performance liquid chromatography (RPHPLC) method was developed and validated for the simultaneousestimationofatenololandchlorthalidone in marketed formulation. The determination was carried out on an Xterra RP8 (150 x 4.6 mm, 5 µm) column using a mobile phase of potassium dihydrogen phosphate buffer solution: methanol (50:50v/v, pH 3.6) with flow rate 0.5ml/min (UVdetection at 240 nm). The retention time for atenolol was 3.2 min and for chlorthalidone 5.0 min. Atenololandchlorthalidone showed a linear response in the concentration range of 50-150 µg/ml. The correlation co- efficient (' r ' value) for atenololandchlorthalidone was 0.9996. The developed method was validated with regard to linearity, accuracy, precision, selectivity and robustness and the method was found to be precise, accurate, linear and specific. The method was validated as per ICH guidelines. The RSD for intra-day and inter-day precision were found to be less than 2 %. The percentage recoveries obtained for atenololandchlorthalidone ranges from 100.54-103.32% and 98.03-102.77% respectively which was in good agreement with the labeled amount in the pharmaceutical formulations.
A simple, highly sensitive, isocratic reversed phase high performance liquid chromatography (RP-HPLC) was developed for the determination of efavirenz in the bulk drug and tablet dosage forms. Elution medium consisting of a mixture of methanol and water in the ratio of (89:11, v/v) at flow rate 1ml/min was employed in this study. The retention time of efavirenz was found 2.58 min. The calibration curves were linear with regression coefficient (r2) of 0.9999. The proposed method was extensively validated for linearity, range, accuracy, precision and specificity. The proposed method is sensitive, specific and was successfully applied for the estimationof efavirenz in pharmaceutical formulations (bulk drug and tablet).
Amlodipine and Atorvastatin standards were obtained from Arene Life sciences, and DSM Sinochem Pharmaceutical India Pvt., Ltd., methanol, acetonitrile and potassium dihydrogen phosphate (HPLC grade) were obtained from RanChem. Atorvastatin and amlodipine tablets were purchased commercially. Milli-Q Water was used throughout the experiment. Other chemicals used were have analytical or HPLC grade.
Amlodipine besylate (AMLO) is chemically 3-ethyl 5-methyl (4RS)-2-[(2 aminoethoxy)methyl]-4-(2-chlorophenyl) 6- methyl-1,4 dihydropyridine-3,5-dicarboxylate benzenesulphonate 8 (Figure 1), is a Calcium channel blocker, used in the treatment of hypertension 9 . It is official in IP, BP and USP. IP 1 , BP 3 and USP 2 describe HPLCmethod for its estimation. Literature survey reveals UV spectrophotometry 4 , RP-HPLC 5 , spectrophotometric 6 method for simultaneous determination of AMLO with other drug andRP-HPLC 7 method for simultaneous determination of AMLO with other drug methods for determination of AMLO in pharmaceutical dosage forms as well as in biological fluids. Indapamide (INDA) is chemically 4-Chloro-N-[(2RS)-2-methyl-2,3- dihydro-1H-indol-1-yl]-3-sulphamoylbenzamide 18 (Figure 2), is a Thiazide diuretics for the treatment of hypertension 19 . Indapamide is official in IP 10 , BP 12 and USP 11 . IP, BP, and USP describe HPLCmethod for its estimation. Literature survey reveals LC-MS 13 , spectrophotometric 14 andHPLC 15 method for simultaneousestimationof INDA in whole human blood, RP-HPLC 16 method for simultaneousestimationof INDA, LC-ESI-MS 17 methods for the determination of INDA in human plasma. This combination is not official in any pharmacopoeia hence official and reported methods of analysis are not available for this combination. The present manuscript describes simple, sensitive, accurate, precise, rapid and economic First derivative spectrophotometric method for simultaneousestimationof AMLO and INDA in tablet dosage form. MATERIALS AND METHODS
Multi-drug antiretroviral therapy has resulted in a significant improvement of the health condition of acquired immunodeficiency syndrome (AIDS). The multi-drug combinations of nucleoside reverse transcriptase inhibitors and non-nucleoside reverse transcriptase inhibitors are effective in the therapy of human immunodeficiency virus (HIV) infection. It is used as a part of highly active anti retroviral therapy (HAART), for the treatment of HIV-1 1, 2 . Lamivudine (3TC) is 4-amino-1-{(2R, 5S)-2-( hydroxyl methyl )-1,3-oxathiolan - 5 - yl - 1 , 2 - dihydropyrimidin-2-one, is a nucleoside reverse transcriptase inhibitor that is active against HIV- 1, HIV-2 and Hepatitis B virus (HBV) 4 . Stavudine (2'-3'-didehydro-2'-3'-dideoxythymidine, d4T, brand name Zerit) is a nucleoside analog reverse transcriptase inhibitor (NARTI) active against HIV. The literature survey reveals that several analytical methods have been reported for the quantification of these drugs individually or in combination with other drugs in pharmaceutical dosage forms or in human plasma by high performance liquid chromatography(5,11) spectrophotometry, titrimetry, liquid chromatography/tandem mass spectrometry and high performance thin layer chromatography1 (8) . From literature , no liquid chromatographic method has been reported for the simultaneousestimationof lamivudine and Stavudine. Hence, a rapid, precise and accurate method for the quantification of Stavudine and lamivudine in pharmaceutical formulations is developed and validated.
the chromatograph. The system suitability parameters were within the limits as shown in Table 5 and 6 for the drug. Limit of detection and limit of quantification of the method were calculated basing on standard deviation of the response and the slope (s) of the calibration curve at approximate levels of the limit of detection and limit of quantification. The LOD for the drug was found to be 0.07µg/ml and LOQ for the Drug was found to be 0.2μg/mL .The drug content formulations were quantified by using the proposed analytical method. The low coefficient of variation in the recovery data indicates the reproducibility of the method in dosage forms. It can be concluded that the proposed HPLCmethod is sufficiently sensitive and reproducible for the analysis of Prasugral in the Tablet formulation dosage forms within a short analysis time.
Pure samples of GPZ and MET were provided by Supra Chemicals, Mumbai and Dr. Reddy’s, Hyderabad, India. The commercial pharmaceutical preparation Glynase- MF containing 5mg and 500mg of Glipizide and Metformin hydrochloride respectively (manufactured by USV Pvt. Ltd.) were procured from local pharmacy. Acetonitrile HPLC grade, potassium dihydrogen orthophosphate, sodium hydroxide were procured from Thermo fisher scientific India Pvt. Ltd, Mumbai, India. High purity deionised water was obtained from [Millipore, Milli-Q] purification system. HPLC instrumentation and conditions
Pure standard of Omeprazole and Cinitapride (Assigned purity 99.98%) was obtained as a gift sample from Shasun Chemicals Pvt Ltd Puducherry India. The gift samples were used as standard without further purification. HPLC grade water, methanol (Qualigens), potassium di hydrogen phosphate, di potassium hydrogen phosphate, phosphoric acid, sodiumperchloricacid and triethylamine (S.D. fine chemicals, Mumbai, India), were used throughout the experiment. Commercial pharmaceutical preparation (BURPEX), ZYDUS (CADILA)) which was claimed to contain 20 mg of Omeprazole and 3 mg of Cinitapride is used in analysis. The chemical structure and purity of the sample obtained was confirmed by TLC, IR, Melting point studies. HPLC grade Acetonitrile from Merck specialties Pvt Ltd, Mumbai. Water HPLC grade was obtained from Rankem laboratories.
System Suitability testing is an integral part of liquid chromatographic methodvalidation performed to check and ensure on-going performance of a chromatographic system. The System Suitability was estimated by five replicate injections standard solution at 100% of test concentration. The column efficiency as determined from Telmisartan and Hydrochlorothiazide peaks is not less than 2000 USP plate count; the USP Tailing for the same peaks is not more than 2.0. RSD for corresponding peak areas of five replicate injections of the standard solution should not be more than 2.0%.
A simple, accurate, isocratic stability indicating RP‐HPLCmethod was developed for the determination of cefepime and amikacin in Pure and its pharmaceutical formulations. The method consists of methanol: acetonitrile:acetate buffer 75:20:05 (v/v) mobile phase at pH 5.1 with C18 column as stationary phase. The flow rate and detection wave length were 1.0 mL/min and 212 nm respectively. The linearity range for the method was found to be 2.5-25 µg/mL for amikacin and 10-100 µg/mL cefepime respectively. The developed method was validated as per ICH guidelines and the results of all the validation parameters were well within their acceptance values. Also the forced degradation studies were conducted with standard drugs. Degradation products formed during the different stress conditions were separated from both drugs. This validated method was applied for the simultaneousestimationof cefepime and amikacin in commercially available formulation sample.
mixture of Water: Acetonitrile: Methanol containing 0.2 % Triethylamine PH adjusted to 3.3 as isocratic mobile phase at a flow rate of 1.0 ml per minute at detection wavelength of 280 nm. The method was validated for accuracy, precision, linearity, specificity and sensitivity in accordance with International conference on Harmonization guidelines. Validation revealed the method is specific, accurate, precise, reliable and reproducible. Good linear correlation coefficients (r 2 > 0.999) was obtained for calibration plots in the range of 10 – 80 µg/ml. Intraday and Interday RSD of retention times and peak areas were less than 2.0 %. Average Percent Recovery was 98.83 %. The method was successfully used for quantitative analysis of this marker compound in Polyherbal formulation.
Standard solution 1-5µl (400-2000 ng/spot) was applied on TLC plate with the help of microlitre syringe, using Linomat V sample Applicator. The plate was developed as per mentioned chromatographic conditions. Each concentration was spotted six times on the plate. Peak area was recorded for each concentration level of drug and calibration curve was obtained by plotting peak areas against concentration of HCTZ. The observations are given in Table 3. A typical calibration curve is shown in Fig. 3.
Osterode, Germany). The produced dry hydroalcoholic extract (85.83 g) was homogenized with 115.7 g of silica gel 60 (70-230 mesh, Merck, Darmstadt, Germany) in porcelain grade, and the adsorbent packed in a preparative glass column (3.0 × 35.0 cm) attached to a Kitasato flask and connected to a vacuum pump. So, the hydroalcoholic extract was fractionated being eluted from the column with 500 mL of increasing polarity solvents (hexane, dichloromethane, dichloromethane ethyl acetate (1:1, v/v), ethyl acetate and methanol). Each of the 5 resulting fractions was collected, the extract evaporated in rotavap to complete dryness and stored at −20 o C. All fractions were analyzed by thin-layer
For the validationof an analytical method to determine a drug in a pharmaceuticalformulation, the ability of this method to resist small and deliberate variations of an analytical parameters should be evaluated. Different values of pH, temperature and different manufacturers of the solvent used can be evaluated, as well as changes in sample preparation (ANVISA, 2003). The developed method was considered to be robust regarding the variation of the evaluated parameters, comparing manual stirring with magnetic stirring and the use of ethanol from diferent manufacturers. No signiicant diferences were observed during the preparation of the samples, with p values of 0.9601 and 0.28184, respectively (Table V). In addition, as a parameter for evaluating the robustness of the method, the stability of a PB sample in ME was demonstrated at 100% test concentration (12 µg/mL) in up to 2.5 hours (Figure 4). There was a signiicant loss of approximately 3.11% of drug content in the presence (p=0.0286) and 3.08% in the absence of light (p = 0.0260) when comparing 0 and 3.5 h times. There was no inluence of light on sample stability loss (p = 0.9189), comparing the 3.5 h time in both groups. Therefore, the use of samples prepared within 2 h is recommended considering that, although it does not exceed 10%, the loss is significant. Furthermore, the solution EtOH:NaOH used tends to interfere with the method 2 hours after preparation (data not shown).
Protease inhibitors were a major therapeutic advance in the mid-1990s for the treatment of HIV infection, which resulted in increased life expectancy for patients who had failed therapies in earlier. Darunavir, the newest protease inhibitors, is a non-peptide synthetic analogue of amprenavir [1], which was approved by the U.S.FDA in June 2006 and in February 2007 the European Commission approved its marketing [2]. It is effective in patients with experiments in antiretroviral treatments, such as those with HIV-1 strains that are resistant to more than one protease inhibitors [3].
The aim of the present work was to develop a new simple, precise, accurate and rapid method for the simultaneous determination of components having overlapping spectra in tablet formation. To prove the ability of the newly described method in resolving the overlapping spectral data andsimultaneous determination of each component, it was applied for the analysis of a mixture of Cefuroxime Axetil (CEF) and Linezolid (LIN) formulated together in the form of tablet widely used for the treatment of bacterial infection. [1-5]
interference of substances that are present in the synthetic medium. Although aldicarb peak come off at 13.16 min (Table 8), it was necessary to flush the column to avoid interference between samples, returning to the initial gradient of the mobile phase. Tests had been carried through to minimize the time, however without success, either for the variation of the retention times or for the appearance of ghost peaks.
Precision studies were carried out to ascertain the reproducibility of the proposed method. Repeatability was determined by preparing six replicates of same concentration of the sample and the absorbance was measured. Intraday precision study was carried out by preparing drug solution of same concentration and analyzing it at three different times in a day. The same procedure was followed for three different days to determine inter day precision. The results were reported as %RSD. The precision result showed a good reproducibility with percent relative standard deviation less
Res e at ol , , ’-t ih d o stil e e p ese ts a ioa ti e o pou d that as e t a ted as a hite po de f o the oots of Veratru gra difloru i [ ]. The , it as dete ted i o e tha se e t spe ies of pla ts, i ludi g g apes [ ], lue e ies, a e ies, ul e ies, pea uts, hite pi e a d o . Although the t o iso e i fo s of es e at ol a e fou d i the atu e Fig. , tra s- es e at ol is the ost o o l used iso e du i g i estigatio , e ause is-iso e is ot o e iall a aila le due to its sta ilit . Afte effe t of UV light o atu al da light, tra s-iso e is t a sfo ed to is-iso e [ ].
cratic RP-HPLCmethod with ultraviolet detection at 232 nm for the simultaneous determination of GFX and two diuretics i.e. HCT and FUR. Simultaneous determination of both drugs is desirable as this would allow more eficient generation of clinical data and could be performed at more modest cost than separate assays. The method is equally valid for the determination in bulk materials, pharmaceutical dosage formulations and human serum. This method can be used for the quantitative analysis of diuretics and gemiloxacin alone or in combination. The low LOD and LOQ values merit the method for the determination of these drugs in clinical samples.