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International Journal of Analytical and Bioanalytical Chemistry Universal Research Publications. All rights reserved

ISSN-2231-5012 Original Article Development of oral immediate release and Sustained release dosage form of Simvastatin and its Pharmacokinetic evaluation 1

BasuvanBabu* 1Subramania Nainar Meyyanathan, 1ByranGowramma, 3 Selvadurai Muralidharan 1Kannan Elango, 2Bhojraj Suresh 1 Department of Pharmaceutical Analysis, JSS College of Pharmacy, Rocklands, Udhagamandalam – 643 001, India. 2 (JSS University, Mysore) 3 Faculty of Pharmacy, AIMST University, Semeling, Bedong, Malaysia E-mail:thammaba[email protected] Received 08 October 2012; accepted 24 November 2012

Abstract To examine newly developed single-unit of oral sustained release dosage form Simvastatin (SS) have been prepared by the wet granulation method. The hydrophilic matrix was prepared with xanthan gum with additives MCC PH101. On the in vitro drug release was studied. The studies indicated that the drug release can be modulated by varying the concentration of the polymer and fillers. Various pharmacokinetic parameters including AUC 0–t, AUC0–∞, Cmax, Tmax, T1/2, and elimination rate constant (Kel) were determined from plasma concentration of both formulations of test (Simvastatin 0.7 mg tablets) and reference (Simvastatin 1.4 mg tablets). The extent of absorption of drug from the sustained release tablets was significantly higher than that for the marketed simvastatin tablet because of lower elimination and longer half-life. Various pharmacokinetic parameters including AUC0-t, AUC0-∞, Cmax, Tmax, T1/2, and Keli were determined from plasma concentration of both Sustained and Immediate release tablets. © 2012 Universal Research Publications. All rights reserved Keywords: Simvastatin; Sustained release; Xanthan Gum; Release Kinetics; Pharmacokinetic parameters. 1. Introduction Statins are a group of 3-hydroxyl–3- methylglutaroyl-coenzyme A (HMG-CoA) reductase inhibitors used in heterozygotic hypercholesteraemia and hyperlipidemia [13]. Simvastatin, lovastatin and atrovastatin are the most used but only the first one is a prodrug [3,4] Prodrug form is better absorbed in comparison to non-modified form. The chemical structure of simvastatin, (1S,3R,7S,8S, 8aR)-8-[2[(2R,4R)-4-hydroxy-2H-pyran-2-yl]ethyl]-3,7-dimethyl1,2,3,7,8,8a hexahydronaphthalen-1-yl 2,2 dimethyl-butanoate [5]. Biotransformation into an active form of simvastatin (β-hydroxyacid) takes place in the liver by ringopening reaction of the lacton. The inhibition of the HMGCoA causes a decrease in LDL, lowdensity lipoprotein (20– 40 %), triglycerides (10–20 %), while it increases HDL, high-density lipoprotein (5–15%) and LDL receptor expression [3,6]. Due to this fact these compounds are the most commonly prescribed drugs for the prevention of atherosclerosis and heart disease, both as a prodrug or nonmodified form. The fact that they can be used after heart attack and in coexistence of diabetes as well as in kidney dysfunction gives statins the status of a first choice drug.


However, overdose of statins causes an increase of aminotransferases concentration which can lead to myopathy. The aim of this work was to prepare matrix tablets containing simvastatin, used as a model drug, and xanthan gum or karaya gum as the hydrophilic matrix to retard drug release. Hydroxypropyl methylcellulose (HPMC) was also used as a hydrophilic matrix for comparative purposes. The in vitro dissolution studies were performed on the formulation for drug release from hydrophilic matrices is known to be a complex interaction between dissolution, diffusion and erosion mechanisms. This work was an attempt to determine the relative contribution of the various drug release mechanisms from these matrix formulations. The LOD and LOQ of the present method is 2.0 ng/ml and 5.0 ng/ml and linearity was in the range of 10-120 ng/ml. No method reported previsouly estimation of simvastatin in plasma by using the protein precipitation (PPT) in the literature. The present work was aimed at developing a sensitive High Performance Liquid Chromatographic (HPLC) for determination of simvastatin in animal plasma. The advantages of present method include single step optimized

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extraction procedure using PPT economic and short run time. The optimization of extraction procedure was carried out by comparing protein precipitation, and liquid-liquid extraction for recovery and interference. PPT was selected because it had obvious advantages such as shorter processing time (8 mins); lesser organic solvent consumption, fewer steps, and good plasma sample clean up. Only limited analytical methods have been developed for the determination of simvastatin in biological methods [7-17]. There were no simple, rapid and reproducible methods so far reported for the estimation of by using high performance liquid chromatography of simvastatin in plasma. The outcome of a study depends upon the reliability, reproducibility and sensitivity of the analytical methodology employed. Therefore, the bioanalytical method was validated in accordance with USFDA guidelines prior to the initiation of the study. 2. Materials and methods 2.1. Materials Acetonitrile of HPLC grade and Methanol of HPLC grade were supplied by Merck Limited, Mumbai. Water HPLC grade obtained from Milli-Q RO system was used.Ammonium actate and sodium hydroxide was procured from merck. Working Standards of Simvastatin and Toltrazuril were obtained from the shasun

pharmaceuticals and drugs limited, kanchipuram, Tamil Nadu, India and sun labs mumbai. Xanthan gum , Polyvinyl pyrrolidine (PVP-K-30) was a gift sample from Anshul Agencies (Mumbai, India), Magnesium stearate, lactose and Talc were procured from S.D.Fine Chemicals (Mumbai, India), micro crystalline cellulose Ph 101 and cross carmellose sodium. 2.2. Preparation of SS (SR) Tablets All the batches showed uniform thickness. The average percentage deviation of 20 tablets of each formula was less than ± 5% and hence all formulations passed the test for uniformity of weight as per official requirements (Pharmacopoeia of India 1996). Good uniformity content was found among IR and different batches of SR tablets. Another measure of tablets strength is friability. In the present study, the percentage friability for all the formulations was below 1%, indicating that the friability is within the prescribed limits. All the tablets formulations showed acceptable pharmacotechnical properties and complied with the specifications for weight variation, drug content, hardness and friability. For the preliminary work, batches of 100 tablets. Batches were prepared for each formulation and the compositions of different batches of SS SR tablets are given in Table 1.

Table 1 Granule properties of the different formulations of Simvastatin


Code of formulations, bLoose Bulk Density, cTapped Bulk Density. *Fa – Code of formulations **All values represent mean ±SD (n=3) 2.3. Development of Simvastatin Immediate Release (IR) tablets Simvastatin IR tablets were prepared by the wet granulation method. All the composition, with the exception of magnesium stearate, cross carmellose sodium and talc were thoroughly mixed in a tumbling mixer for 5 min and wetted in a mortar with isopropyl alcohol. The wet mass was sieved (16 mesh) and granules were dried at 60°C for 2 hours. The dried granules were sieved (22 mesh) and these granules were lubricated with a mixture of magnesium stearate and talc (2:1). The Simvastatin tablets were prepared using an electrically operated punching machine. Compression was performed after granulation process with a single punch press applying a compression force of a 9 KN (preliminary work) or 12 KN (experimental design), equipped with a 5 mm concave punch. For the preliminary work, batches of 50 tablets were prepared. Each batch of experimental design consisted of 50 tablets (drug content in the tablet was 0.7 mg). The compressed tablets were evaluated for average weight and weight variation, thickness, diameter, drug content & content uniformity, hardness, friability and in vitro drug release. 2.4. Granule properties The granulation properties examined included the following: loss on drying, particle size distribution (after


drying and final blend), geometric mean diameter, bulk and tap densities and percent compres suability (Carr’s index). 2.5. Standard physical test of tablets The physical testing of tablets was performed after a relaxation period of at least 24 h stored at the same RH conditions than powders. The tablet average weight and the standard deviation (SD) were obtained from 20 individually weighed (Sartorius analytical balance, Mumbai) tablets according to USP. The thickness of ten tablets was measured individually using an electronic micrometer (Mitutoyo MDC-M293, Tokyo, Japan). The resistance to crushing of ten tablets was determined by diametral loading with a Monsanto hardness tester. Tablet friability was calculated as the percentage weight loss on using 20 tablets, 4 min at 25 r.p.m. in an (Electrolab Mumbai) friability tester. 2.6. Drug Content Five tablets were weighed individually and crushed in mortar. An accurately weighed quantity of powered tablets (1.4 mg) was extracted with phosphate buffer and the solution was filtered through 0.45µ membrane. The drug content was estimated by HPLC under suitable optimized condition after suitable dilution. 2.7. In vitro release studies The release characteristics of test and reference

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formulations of Simvastatin was determined using USP XXIII dissolution apparatus (type II, paddle), at 50 rpm. The dissolution media used for Simvastatin was 0.1 N hydrochloric acid ph 1.2 and phosphate buffer pH 7.4 containing for 24 hours maintained at 37°C. Dissolution tests were performed on six tablets. Five ml of the samples were withdrawn at 0.0, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 4.0, 6.0, 8.0,10, 12.0, 18.0 and 24.0 hours time intervals. Equal quantity of the dissolution medium was replaced to the dissolution jar after each sampling. The amount of the drug released was estimated by optimized and validated HPLC methods as described in section 5.2.1 .Percentage drug release and cumulative release at various time intervals were calculated and compared (Fig. 1).

Fig. 1. In vitro release profile of Simvastatin from Zanthan gum matrices (IR and F-1 to F-5) 2.8. Kinetics of drug release The cumulative amount of simvastatin released from matrix tablets at different time intervals was fitted to zero order kinetics using Least-Squares Method of analysis to find out whether the drug release from the formulations is providing a constant drug release. The correlation coefficient between the time and the cumulative amount of drug released was also calculated to find the fitness of the data to zero order kinetics. The fitness of the data to first order kinetics was assessed by determining the correlation coefficient between the time and the amount of drug to be released from the formulations. The data were also fitted to the model developed by Korsmeyer (1983) in order to find out the drug release mechanism from the formulations. The cumulative percent of drug released from the formulations was plotted against time on log–log scale, and analyzed for linearity using Least-Squares Method. Calculating correlation coefficients between time and the cumulative percent of drug released on log–log scale tested the fitness of the data (fig. 2 and 3).

Fig. 3. Higuchi chart of optimized Simvastatin formulation (F-3) 2.9. HPLC analysis of simvastatin in dissolution fluids and matrix tablets The quantitative determination of simvastatin was performed by HPLC. A gradient HPLC (Shimadzu HPLC Class VP series) with two LC-2010A HT VP pumps, dual wave length programmable UV/VIS Detector SPD-10A VP, CTO-10AS VP Column oven (Shimadzu), SCL-10A VP system controller (Shimadzu), a disposable guard column LC-18 (Pelliguarde, LC-18, 2 cm, Supelco, Inc., Bellefonte, PA.) and RP C-18 (250 × 4.6 mm, particle size 5µ) was used. The HPLC system was equipped with the software ‘Class-VP series version 6.04 (Shimadzu)’.The mobile phase used was a mixture of .01mole ammonium acetate : acetonitrile (pH adjusted with formic acid to 4.40) in the ratio of 20:80. The filtered mobile phase components were pumped at a flow rate of 1.0 ml/min. The eluent was detected by UV detector at 238 nm, and the data were acquired, stored and analyzed with the software ‘Class-VP series version 6.04 (Shimadzu). 3. In vivo studies in animal volunteers A single dose complete cross over method was followed to carry out bio availability studies. [JSSCP/IAEC/M.Pharm/Ph.Anal/05/2010-11] Healthy albino rabbits (2.0-2.5 kg) were obtained and kept in individual cages for 30 days prior to the study in the departmental animal house for the purpose of acclimatization. They had free access to water and food. A constant day-night cycle was maintained and the temperature of the animal room was kept constant throughout the period. Healthy over night fasted animals were used for the experiments. Zero hour fasting blood samples were withdrawn early in the morning. The animals were then divided in to 3 groups. The dose for the rabbits was selected based on the surface area ratio of rabbit and man. Group 1 (3 animals) received sustained release formulation (3.5 mg), Group 2 (2 animals) received immediate release formulation (1.7 mg) and Group 3 (1 animal) was kept as control. Immediately after administration the animals were given 5 ml of water. Blood samples (0.5 ml) were withdrawn from the marginal ear vein at 0, 0.5, 1, 1.5, 2,2.5, 3, 4, 6, 8,10, 12, 18 and 24 hours period using a sterilized syringe. The blood samples collected in the ria vial containing the anti coagulant (50µl of sodium citrate) were centrifuged at 4000 rpm for 4 minutes and the plasma

Fig. 2. Zero order chart of optimized Simvastatin formulation (F-3) International Journal of Analytical and Bioanalytical Chemistry 2012; 2(4): 252-259


samples were separated and stored at -70°C. The plasma 3.2. Statistical Analysis samples were deproteinised by acetonitrile, and the For the purpose of bioequivalence analysis AUC0–t, AUC0– contents were vortexed for 2 minutes. It was then infand Cmax were considered as primary variables. centrifuged at 4000 rpm for 4 minutes and the supernatant Bioequivalence of two formulations was assessed by means liquid was separated and analysed. Estimation of plasma of an analysis of variance (ANOVA) for crossover design sample by HPLC was carried out using optimized and calculating 90% confidence interval of the ratio of chromatographic conditions. The standard and sample test/reference using log transformed data. The formulation chromatograms were recorded. was considered bioequivalent when the difference between 3.1. Pharmacokinetic analysis two compared parameters was found statistically [20, 21] The plasma concentration of simvastatin at different time insignificant (p > 0.05) and confidence interval for these intervals after oral administration of the tablet formulations parameters fell within 80-125%. to human volunteers was subjected to pharmacokinetic 4.0 Results and Discussion analysis to calculate various parameters such as maximum 4.1 Granulation properties plasma concentration Cmax time to reach maximum Granulation is the key process in the production of many concentration Tmax and area under the curve (AUC0-t). The dosage forms. The sustained release tablets were prepared values of Cmax and Tmax were directly read from the by wet granulation technique. Physical properties of arithmetic plot of time versus plasma concentration of granules such as specific surface area, shape, hardness, Simvastatin. The overall elimination rate constant Keli was surface characteristics and size can significantly affect the calculated from the slope of the terminal elimination phase rate of dissolution of drugs contained in a heterogenous of a semi-logarithmic plot of concentration versus time, formulation. The granules of different formulations were after subjecting it to linear regression analysis [18,19] The evaluated for angle of repose, loose bulk density, tapped relative bioavailability of simvastatin from matrix tablets in bulk density and Carr’s index as shown in Table 1. The comparison to reference formulation (immediate release results of angle of repose indicate good flow properties of dosage form) was calculated by dividing its AUC0–t with the granules. However, the granules had fair to poor Carr’s that of immediate release tablet dosage form after applying index values. Aerosil therefore was added to dried granules dosage correction. prior to compression to improve the flow. Table 2 Comparison of the physical properties of the matrix tablets containing Simvastatin


Code of formulations,b Results represents the mean of replicate determination with the standard deviation given in parenthesis *Fa – Code of formulations **All values represent mean ±SD(n=3) Table 3 In vitro release profile of Simvastatin from Xanthan gum matrices (IR and F-1 to F-5)

*All values represent mean (n=6) ±SD 4.2. Standard Physical test The physical properties of different batches of developed tablets are given in Table 2. All the batches showed uniform thickness. The average percentage deviation of 20 tablets of each formula was less than ±5% and hence


all formulations passed the test for uniformity of weight as per official requirements (Pharmacopoeia of India 1996). Good uniformity content was found among three different batches of tablets.

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Table 4 Mean pharmacokinetic profile (ng/ml) of Simvastatin immediate release and sustained release tablets Type of formulation Cmaxc Tmaxc AUC0-tc t1/2c kelc AUC0- c a IR 107.94±2.98 2±0.0 300.50±13.86 0.57±0.07 1.23±0.15 323.83±23.04 SRTb 107.35±8.31 5.33±1.15 1073.21±14.93 0.12±0.01 5.61±0.30 1178.32±102.76 a Immediate release tablets b Sustained release tablets c Results represents the mean of replicate determination with the standard deviation given in parenthesis Table 5 Precision study for Simvastatin

Table 6 Stability study of Simvastatin Freeze and Thaw Cycle 1 Cycle 2 Cycle 3 Mean S.D (+/-) C.V. (%) % Nominal Short Term Plasma at Room Temperature After 1 hr After 2 hr After 3 hr Mean S.D (+/-) C.V. (%) % Nominal

Nominal Concentration (ng/ml) LQC MQC 30 40 28.95 39.73 32.63 37.88 33.48 41.06 31.686 39.556 2.408 1.597 7.599 4.037 94.677 101.121 Nominal Concentration (ng/ml) LQC MQC 30 40 26.77 37.26 29.54 35.18 25.29 39.39 27.2 37.27 2.157 2.105 7.932 5.647 110.294 107.306

Another measure of tablets with less than 1 % w/w of their weight is generally considered acceptable. In the present study, the percentage friability for all the formulations was below 1%w/w, indicating that the friability is within the prescribed limits. All the tablet formulations showed acceptable pharmaceutical properties and complied with the specifications for variation, drug content, hardness and friability. 4.3. Friability test Friability values of all the batches were less than 1%. There was no accordance between hardness values and friability observations. Small values in friability imply much less friability during transportation.


HQC 100 96.01 102.37 99.25 99.21 3.180 3.206 100.796 HQC 100 102.54 96.11 97.28 98.64 3.425 3.472 101.375

4.4. Invitro dissolution studies A suitable in vitro dissolution method serves as a valuable quality control tool to assess batch to batch release performance and to assure the physiological availability of the drug. The in vitro dissolution test is also used to guide formulation development and to monitor manufacturing process. As a regulatory test, it is used to approve minor changes in formulation, changes in the site of manufacturing and also to assess the scale-up of the biobatch to the production batch. All the batches have shown that as the polymer concentration increases, the drug release rate decreases for simvastatin. The in vitro drug release characteristics of the developed sustained release

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(SR) and the marketed immediate release (IR) tablets were studied. Dissolution data for all the experiments were highly reproducible and hence only the average values were plotted. The dissolution of the marketed IR tablets indicated that more than 80% of the drug is released within 1h, which complies with the pharmacopoeial specifications. In all the batches, we observed that as the polymer concentration increases, the drug release rate decreases (Table 3). 4.5. Kinetics of drug release To know the mechanism of drug release from these formulations, the data were treated according to Zero-order, first order, Higuchi and Peppas equvations that are clearly revealed. The release profile the DXI, when plotted according to Higuchi’s equvation and Peppas equvation [22-25] confirm that drug release was diffusion control as evident by the values of correlation (r2). 4.6. Bioavailability studies The relative bioavailability of the SR 1.4 mg tablets given daily was compared with one dose of the marketed 0.7 mg IR tablet. The developed SR tablet produced a plasma concentration-time profile typical of the prolonged dissolution characteristic of a SR formulation, as evident from Fig. 6 and Table 4. The developed SR tablets demonstrated a longer time to reach a peak concentration than the marketed tablets and appeared to have more consistent over all performance. 4.7. Validation of HPLC methods Estimation of the drugs selected in plasma samples from the volunteers was carried out using optimized chromatographic conditions. The validation [26] parameters such as accuracy, precision (repeatability and reproducibility), linearity and range, sensitivity (limit of detection and limit of quantitation), robustness/ruggedness, stability, selectivity/specificity and system suitability were evaluated. The system suitability parameters are given in Table 8. 4.7.1. Specificity HPLC-UV analysis of the blank rabbit plasma samples showed the separation of Simvastatin and Toltrazuril (internal standard IS), no interference with either of these were observed. Hence the specificity of the method was established by comparison with rabbit plasma (control). Representative chromatograms of extracted blank plasma, is shown in Fig 4. indicating no interference in the blank plasma and in drug-free rabbit plasma at the retention time of 7.7 for the drug Simvastatin and at the retention time of 4.0 for the IS.

Fig. 5. Typical chromatogram of sample 4.7.2. Sensitivity The limit of reliable quantitation was set at the concentration of the LLOQ QC, 10 ng/ml for Simvastatin and lowest non-zero standard (Fig. 5). 4.7.3. Linearity A regression equation with a weighing factor of 1/concentration2 was judged to produce the best fit for the concentration/detector response relationship for Simvastatin in human plasma. The linearity range for Simvastatin was found to be 10, 20, 25, 30, 40, 80, 100 and 120 ng/ml. The results are given in Table 9 and is shown in Fig. 7 with correlation coefficient (r2) was greater than 0.99.

Fig. 6. Mean plasma concentrations (ng/ml) for immediate release and sustained release product of Simvastatin

Fig. 7 Calibration curve of Simvastatin

Fig. 4. Typical chromatogram of blank plasma


4.7.4. Precision The precision of the assay was measured by the percent coefficient of variation over the concentration range of

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Table 6 a Stability study of Simvastatin Long Term Plasma Sample at 70º After 1 week After 2week After 4 week Mean S.D (+/-) C.V. (%) % Nominal N Standard Stock solutions After 3 hr After 6 hr After 4 Week Mean S.D (+/-) C.V. (%) % Nominal N

Nominal Concentration (ng/ml) LQC 30 33.4 30.27 28.94 30.87 2.290 7.417 97.182 3 Nominal Concentration (ng/ml) LQC 30 29.93 27.54 25.09 27.52 2.420 8.794 109.012 3

Table 7 Accuracy (Recovery study) for Simvastatin Concentration of Amount of drug recovered Level drug added ng/ml (ng/ml) in plasma sample Level -I


Level -II


Level -III


27.84 ± 4.25

38.95 ± 4.19 102 ± 0.57

LLOQ, low, middle and high quality control sample of Simvastatin during the course of validation. The accuracy of the assay was defined as the absolute value of the ratio of the calculated mean values of the LLOQ, low, middle and high quality control samples to their respective nominal values, expressed as percent. The results are given in Table 5. 4.7.5. Stability studies The stability studies of plasma samples spiked with selected drugs were subjected to three freeze-thaw cycles, short term stability at room temperature for 3 hours and long term stability at – 70oC. In addition, stability of standard solutions was performed at room temperature for 6 hours and freeze condition for four weeks. The mean concentrations of the stability samples were compared to the theoretical concentrations. The results indicate that selected drugs in plasma samples can be stored for a month without degradation in frozen state. The results of short term storage at room temperature stability and freeze-thaw cycles indicate no degradation of selected drugs in plasma as well as in sample solution and hence plasma samples could be handled without special precautions. The results are given


Recovery (%) Mean : 98.47 CV : 7.28 N : 6 Mean : 99.11 CV : 2.36 N : 6 Mean : 100.05 CV : 1.10 N:6

MQC 40 43.33 39.19 41.61 41.3767 2.080 5.027 96.673 3

HQC 100 96.55 101.42 105.39 101.12 4.428 4.379 98.892 3

MQC 40 44.02 39.74 41.87 41.8767 2.140 5.110 95.519 3

HQC 100 103.28 97.56 99.43 100.09 2.917 2.914 99.910 3

Amount of Drug recovered (%) in Mobile phase

Relative Recovery ( %)

Mean : 99.16 CV : 1.44 N :6


Mean : 99.57 CV : 0.25 N :6


Mean : 101.88 CV : 0.27 N :6


in Table 6 and 6a. 4.7.6. Accuracy (Recovery study) Analyte recovery from a sample matrix (extraction efficiency) is a comparison of the analytical response from an amount of analyte added to that determined from the sample matrix. The detailed results are presented in Table 7. The results indicate that the recovery of Simvastatin was consistent at all levels. 4.7.7. Ruggedness and robustness The ruggedness and robustness of the methods were studied by changing the experimental conditions. No significant changes in the chromatographic parameters were observed when changing the experimental conditions (operators, instruments, source of reagents and column of similar type) and optimized conditions (pH, mobile phase ratio and flow rate). 5. Conclusions The sustained release tablets of simvastatin were well absorbed and the extent of absorption was higher than that of the immediate release tablet. The sustained and efficient drug delivery system developed in the present study will maintain plasma simvastatin levels better, which will

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overcome the drawbacks associated with the conventional therapy. The statistical comparison of AUC and Cmax clearly indicated no significant difference in the two formulations of 5.0 mg of simvastatin tablets. Ninety percent confidence interval for the mean (T/R) of AUC0–t, AUC0–infand Cmax indicates that the reported values were entirely within the bioequivalence acceptance range of 80– 125% (using log transformed data). This was no significant difference in extent of absorption as assessed by measurement AUC0-t. However AUC0-∞ values for the SR tablets were higher than the marketed IR tablets indicating more efficient and controlled drug delivery, which would maintain plasma SS levels better. This was also evident by the lower elimination rate and higher t ½ Values. Hence it was concluded that formulation ‘Test’ and ‘Reference’ can be interchangeable in clinical practice. References [1] H. Yang, Y. Feng, Y. Luan. J. Chromatogr. B. 785 (2003) 369–375. [2] W. Kostowski, ZS. Herman. Farmakologia. Podstawy farmakologii. (2005) 3rd ed. PZWL, Warsaw. [3] D. Anantha Kumar, D.P. Sujan, V. Vijayasree, J.V.L.N. Seshagiri Rao. E.J.Chem. 6 (2009) 541–544. [4] B. Patel, N. Sharma, M. Sanyal, P.S. Shrivastav. J Sep Sci 31 (2008) 301–313. [5] A Melanovic, MMedenica, D Ivanovic, B Jancic. Chromatographia. 63 (2006) 95–100. [6] M.A, Veronin N.T. Nguyen Ann Pharmacother. 42 (2008) 613–620. [7] Seo-Ryung Kim, Jin-Ki Kim, Jeong-Sook Park, Chong-Kook Kim. Int. J. Pharmaceut. 404 (2011) 2301-2307. [8] G. Carcucci, P. Mazzeo, L. Biordi, M. Bologna J. Pharm. Bio. Med. Anal. 10 (1992) 693-697. [9] Carcucci G, Mazzeo P (1992) Farmaco 47: 817-823. [10] Lingeswara Rao Punati, Prasad Rao Srungapathi, Ganesh M, Saravanan VS, Siva Kumar T (2006) Proceedings of the 58th Indian Pharmaceutical Congress, Dec. 1-3 Mumbai, India, p.259.

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Source of support: Nil; Conflict of interest: None declared


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