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Kottu P. K*., Gadad A.P. and Dandagi P. M.
(Received 01 June 2012) (Accepted 12 october 2012) ABSTRACT
Objective: The objective of the present work was to design a simple, accurate, economical and reproducible Uv spectrophotometric method for the simultaneous estimation of a two-component drug mixture of pioglitazone and glimepiride in the combined tablet dosage form. Methodology: Simultaneous estimation method that involves maximum absorbance (λ max) of Pioglitazone and Glimepiride at 279.0 nm and 238.0 nm, respectively was developed. The proposed method was validated as per ICH guidelines for accuracy, precision, linearity, limit of quantification (LoQ) and limit of detection (LoD). The calibration curves were linear in the concentration range for pioglitazone (r value) and for glimepiride (r value) and were found to obey beer’s law in the linear concentration ranges. Statistical analysis and drug recovery data showed that simultaneous estimation method was simple, rapid, economical, sensitive, precise and reproducible. Hence, the proposed method was recommended for routine analysis of pioglitazone and glimepiride in combined tablet dosage form.
Key words: Pioglitazone, glimepiride, simultaneous
solid phase extraction method in human serum and estimation, calibration curve and statistical in dog serum as well as HPLC and LC mS in human plasma have been reported for the estimation of pioglitazone. However the above mentioned methods INTRODUCTION
are sophisticated, expensive and time consuming Chemically, pioglitazone (PIo) is (±) - 5- (4- when compared to simple Uv spectrophotometric [2- (5- ethyl- 2- pyridinyl) ethoxy] phenyl methyl] - 2, 4- thiazolidinedione monohydrochloride Glimepiride (GLIm) is a medium to long acting (Fig. 1). It improves sensitivity to insulin in muscle and sulfonylurea anti-diabetic drug. Glimepiride is a adipose tissue and inhibits hepatic gluconeogenesis potent third generation sulfonylurea derivative and also improves glycemic control while reducing is chemically, 3-ethyl-N, N-bis (3-ethyl-4-methyl-2- circulating insulin levels. It is used for the treatment oxo-5H-pyrrol-2-yl) - 4-methyl-2-oxo-5H-pyrrole-1- of diabetes mellitus type 2 (previously known as carboxamide (Fig. 2). It acts by stimulating insulin non-insulin-dependent diabetes mellitus, NIDDm) in secretions from the beta cells of pancreas and is monotherapy and in combination with a sulfonylurea, also known to increase peripheral insulin sensitivity, metformin, or insulin. various analytical methods thereby decreasing insulin resistance. Literature like spectrophotometric method and HPLC and survey reveals that few spectrophotometric, HPLC, meCK method in tablet dosage form, HPLC and HPLC-eSI – mS-mS and LCmS methods have been reported for the estimation of glimepiride 2.
* For correspondence
Department of Pharmaceutics,
Drugs are available in tablet dosage form as KLE University’s College of Pharmacy
pioglitazone 30 mg and glimepiride 2 mg (Duetact) Belgaum, Karnataka, India – 590010.
in the market. Literature survey revealed that Email:
pioglitazone has been estimated with other drugs using HPLC 3-8 and glimepiride has been determined period of 15 min to remove any air bubbles. Similarly, along with other drugs by Uv 9, and HPLC 6, 5-8, 10-11. the standard stock solution of glimepiride (100 µg/ml) Since no spectrophotometric method has been was prepared. The stock solutions were individually reported yet for simultaneous estimation of PIo diluted with pH 2.0, HCl buffer containing 0.5% w/v and GLIm in pH 2.0 HCl buffer an attempt has been SLS to get final concentration of 20 µg/mL each made to develop Uv-spectrophotometric method and the diluted solutions were scanned in 200-400 for simultaneous estimation of pioglitazone and nm range to determine the maximum absorbance (λ max) of corresponding solutions 12. It was found that pioglitazone and glimepiride show maximum MATERIALS & METHODS
absorbance (λ max) at 279.0 nm and 238.0 nm, respectively. All the solutions were filtered through Instrument
a 0.45µ membrane filter before they were scanned A Shimadzu Uv/visible spectrophotometer, model No. Uv-1700 was used for the study. The instrument had a spectral band width of 2 nm and Preparation of standard solutions:
wavelength accuracy of ± 0.1 nm, with automatic From the above prepared stock solution different wavelength correction employing a pair of quartz cel s aliquots of various concentrations (0.2, 1, 10, 25, 50, of 1 cm patch length. A Denver electronic analytical 75 and 100µg/mL) were prepared using pH 2.0, HCl balance (Tb214) was used for weighing the sample. buffer containing 0.5 % w/v of SLS. The solutions The pH of solutions was measured by a pH meter were filtered through a 0.45µ membrane filter before they were scanned by Uv Spectrophotometer for the linearity range. The linearity of the solutions was in Reagents
the concentration range of 1-12 µg/ml for Pioglitazone Pioglitazone and glimipiride were kindly supplied and Glimepiride, respectively. The Coefficient of by Sun Pharma Limited, Jammu. Sodium lauryl correlation for Pioglitazone and Glimepiride was found sulphate (SLS) needles were obtained from S. D. to be 0.9915 and 0.9904, respectively.
Fine Chem. Ltd, mumbai. Potassium dihydrogen phosphate was purchased from S. D. Fine Chem. Simultaneous equation method:
Ltd, mumbai and sodium hydroxide was purchased The simultaneous estimation of both pioglitazone from Qualigens Fine Chemicals, mumbai. All the other and glimepiride was done by simultaneous estimation chemicals were of analytical grade. Double-distilled method. Firstly, the absorptivity values of the both the water was used throughout the study.
drugs were determined at λ of pioglitazone (279 nm, λ ) and glimepiride (238 nm, λ ). The absorptivity both pioglitazone and glimipiride were tested for value of the drugs is the ratio of absorbance at selected purity by measuring its melting point and thin layer wavelengths with the concentration of drugs in µg/ml. chromatography for the presence of any impurities.
using the absorptivity values a set of two simultaneous equations were framed (eqs. 1 and 2).
The stock solution of the samples was further Preparation of stock solutions
diluted with pH 2.0 HCl buffer containing 0.5 % w/v of based on preliminary studies conducted for SLS to get standard solution of concentration 10 µg/ml. the solubilization of both the drugs, methanol was The absorbance of the solution was measured at the selected as suitable solvent for analysis. Firstly, selected wavelengths and absorptivity was determined standard stock solution (100 µg/ml) of pioglitazone as a mean of three independent determinations. was prepared by dissolving 10 mg of drug in 100 mL Concentration of the drug in the samples was obtained methanol. The solution was kept for sonication for a and the calibration curve was plotted between absorbance and concentration of the drug.
Inter-day and Intra-day Precision
Precision and accuracy was studied using solution of concentration 10 µg/mL. Absorbance of the solution was measured for three replicate samples. Intra-day Where, A1 and A2 represent absorbance of precision studies were run in triplicate on the same mixture at λ1 and λ2, respectively, ax1 and ax2 are day and inter-day on three consecutive days. the absorptivities of PIo at λ1 and λ2 respectively and ay1, ay2 are the absorptivities of GLIm at λ1 and Limit of Detection (LOD) and Limit of
λ2 respectively. Cx and Cy are the concentrations of Quantification (LOQ)
pioglitazone and glimepiride in µg/mL, respectively.
Validation of method
concentration of the analyte in the sample which can be analysed by the instrument. Limit of quantification The method developed here was validated as per (LoQ) is the minimum concentration of the analyte that ICH guidelines 13-15 for its accuracy, linearity, precision, specificity, Robustness, limit of detection and limit of can be reliably quantified. The Limit of detection (LoD) and Limit of quantification (LoQ) were measured using following formula. Ten blank determinations Accuracy
The accuracy of the developed method was LoD = (3.3 * SD of analytical blank signals) / slope determined by finding out the amount of recoveries of pioglitazone and glimepiride. For the accuracy standard addition method was used where, as LoQ = (10 * SD of analytical blank signals) / slope known amounts of pioglitazone and glimepiride of calibration curve. ------------- eq. 4 were added to the known concentration (10 µg/mL) of commercial tablets. The amount recovered was Assay of tablet formulation by simultaneous
found by measuring the absorbance of the solution equation method
and was expressed as mean recovery of samples with upper and lower limits of percent relatives of containing both pioglitazone and glimepiride standard deviation. Recovery was done at three (Duetact) were used for the study. Twenty tablets different levels viz. 80%, 100% and 120%, within the were taken and crushed. Powder equivalent to 10mg of PIo which includes 0.667 mg of GLIm and 9.33 mg of pure GLIm was added by standard addition Linearity
method in order to bring both drugs in 1:1 ratio and The linearity of this method was evaluated by the stock solution of this was prepared in methanol, linear regression analysis and calculated by least sonicated for 15 min, was then filtered through 0.45µm square method and the drug shows linearity in the membrane filter and then volume was made up to concentration range of 1-12 µg/ml for both drugs. 100 ml with methanol. This stock solution contains Standard dilutions were prepared using the required 100 µg/ml of each drug. Then the appropriate dilution volume from the stock solution and then volume was of 10 µg/ml was made using pH 2.0, HCl buffer made up to 50 ml with pH 2.0, HCl buffer containing containing 0.5% w/v SLS solution. All determinations 0.5% w/v SLS solution to yield the concentrations. were carried out in triplicate. In simultaneous equation Absorbance of the resulting solutions was measured method, the absorbance of the prepared solutions Table I: Optical Characteristics
Table II: Analysis of tablet formulation
Label claim (mg/tab)
Amount found (mg)
% drug found ± SD
Table III: Intra-day and Inter-day precision Pioglitazone and Glimepiride
Concentration (µg/ml)
%Accuracy ±SD
Intra-day precision
Inter-day precision
Values expressed as mean of ± SD (n=3); SD: Standard deviation. Table IV: Recovery
Amount added (µg/ml)
Amount recovered (µg/ml)
%Recovery ± SD
Values expressed as mean± SD (n=3); SD: Standard deviation Table V: Limit of quantification (LOQ), Limit of detection (LOD)
Fig. 1: Chemical structure of Pioglitazone
Fig. 3: Overlain spectra of Pioglitazone and Glimepiride
Fig. 2: Chemical structure of Glimepiride
was observed at 279 nm and 238 nm and then the concentration of both the drugs was calculated using equation 1 and 2.
Fig. 4: Linearity graphs
drugs (Table I). Regression analysis was made for The overlain spectrum (Fig. 3) of pioglitazone the slope (m), intercept (c) and correlation coefficient and glimepiride was found to be appropriate for the (R) as shown in Table I. Higher values of correlation estimation of both the drugs. based on the point of coefficient (R) indicate good linearity of the calibration maximum absorbance the λ of pioglitazone and curve for both the drugs as shown in Fig. 4. glimepiride was found to be 279 and 238 nm. The response for Pioglitazone was found to be linear in the concentration range of 2 – 30 µg/mL at 279 determined by inter- and intra-day precision methods. nm with correlation coefficient of 0.9904 (Table 1). The results varied between 98.34 and 98.4 % in Similarly, the derivative response for the glimepiride case of inter-day precision for simultaneous equation was found to be linear in the concentration range of method while in case of intra-day precision it ranged 1 - 20 µg/mL at 238 nm with correlation coefficient between 98.12%and98.57%. %RSD calculated was found to be less than 2 indicating the accuracy and reproducibility of the method. Results are shown in Amount of pioglitazone and glimepiride in marketed formulation determined by the proposed method ranges between 98.80 and 99.20 % The accuracy of the method was proved by for simultaneous equation method as shown in performing recovery studies on the commercial formulation at 80, 100 and 120% level. Recovery range a from 99.50 to 100.83% in simultaneous Linearity for detector response was observed equation method (Table Iv). The results of recovery in the concentration range of 1 – 20 µg/ml for both study indicate that these drugs could be quantified simultaneously and that there was no interference of method forthe Determination of Pioglitazone in Rat Serum, the excipients present in the formulation. The limits International Journal of Pharmaceutical Sciences and
Drug Research. 2011, 3(1), 38-41.
of detection and limit of quantification for pioglitazone 4. Srinivasulu D, Sastry b.S. and omprakash G.: and glimepiride are shown in table v.
Development and validation of New Rp Hplc method for Determination of Pioglitazone HCl in Pharmaceutical DISCUSSION
Dosage Forms, Int J Chem Res. 2010, 1(1), 18-20.
5. Lakshmi K.S, Rajesh T. and Sharma S.: Determination The developed method involves formation and of Pioglitazone and Glimepiride in Pharmaceutical solving of simultaneous equation which is further Formulations and Rat Plasma by Rp-Lc, International
based on absorptivity coefficients of two drugs at Journal of Pharm Tech Research. 2009, 1(3),
wavelength maxima of pioglitazone and glimepiride. After the measurement of absorbance of sample 6. Havaldar Fh. and vairal Dl.: Simultaneous estimation of Glimepiride, Rosiglitazone and Pioglitazone Hydrochloride solution at both the wavelengths the amount of in the Pharmaceutical Dosage Form, E-Journal of
drug in the sample can be found by substituting Chemistry. 2010, 7(4), 1326-1333.
the values in the two equations (eqs. 1 and 2). The 7. madhukar A, Naresh K, Kumar CH.N, Sandhya N. and framed equation was validated as per ICH guidelines. Prasanna P.: Rapid and Sensitive Rp-Hplc Analytical method Statistical analysis and drug recovery data showed that Development And validation of Pioglitazone Hydrochloride,
Der Pharmacia Lettre. 2011, 3(3), 128-132.
simultaneous estimation method was simple, rapid, 8. Karthik A, Subramanian G, mallikarjuna Rao C, economical, sensitive, precise and accurate and can bhat K, Ranjithkumar A, musmade P, Surulivelrajan thereby be easily adopted for routine quality control m, Karthikeyan K. and Udupa N.: Simultaneous analysis. Results of this analysis confirmed that the Determination of Pioglitazone and Glimepiride In bulk Drug and Pharmaceutical Dosage form by Rp-Hplc method, proposed method was suitable for the simultaneous Pak. J. Pharm. Sci. 2008, 21(4), 421-425.
determination of these drugs in pharmaceutical 9. Khedekar P.b, Dhole S.m. and bhusari K.P.: Application formulations with virtual y no interference of the of vierodt’s and Absorption Correction Spectrophotometric additives. Hence, the proposed method can be methods for estimation of Rosiglitazone maleate and successfully applied in simultaneous estimation of Glimepiride in Tablets, Digest Journal of Nanomaterials
and Biostructures. 2010, 5(1), 77 – 84.
PIo and GLIm in marketed formulations.
10. Samala S, Tatipamula S.R. and veeresham C.: Determination of Glimepiride in Rat Serum by Rp-Hplc 5. ACKNOWLEDGEMENTS:
method, American Journal of Analytical Chemistry.
The authors are thankful to KLe College of 11. Khan I.U, Aslam F, Ashfaq m. and Asghar m.N.: Pharmacy belgaum for providing al facilities to Determination of glimepiride in pharmaceutical formulations complete the work, Sun Pharma Limited, Jammu using HPLC and first-derivative spectrophotometric for providing pioglitazone and glimipiride, S. D. methods, Journal of Analytical Chemistry. 2009, 64(2),
Fine chem. Ltd, mumbai for providing sodium lauryl 12. Rubina R, bhagya Lakshmi J. and Ravi T.K.: Studies on Formulation and In vitro evaluation of Glimepiride Floating
Tablets, C. J. Chem. Pharm. Res. 2011, 3(3), 159-164.
13. ICH Topic Q 2 (R1) validation of Analytical Procedures: 1. Shakya P. and Singh K.: Determination of Pioglitazone Text and methodology, note for guidance on validation Hydrochloride in bulk and Pharmaceutical Formulations of analytical procedures: text and methodology (CPmP/ by Uv Spectrophotometric method, International Journal
of Pharmaceutical Sciences and Research. 2010,1(11),
14. International Conference on Harmonization; Draft Guidance on specifications: Test Procedures and 2. mishra K, Soni H, Nayak G, Patel S.S. and Singhai A.K.: Acceptance Criteria for New Drug Substances and Development and validation of A Rp-Hplc method for Products: Chemical Substances, Federal Register estimation of Glimepiride in Tablet Dosage Form, IJPI’s (Notices). 2000, 65(251), 83041–83063.
Journal of Analytical Chemistry. 2011,1(3), 1-8.
15. FDA, Analytical Procedures and methods validation: 3. Ravikanth CH, Kumar A.A, Kiran v.U, Prashanth S, Chemistry, manufacturing, and Controls, Federal Register madhu b. and Reddy Y.N.: Sensitive and Rapid Hplc (Notices). 2000, 65(169), 52,776– 52,777.


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