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RLD JOURNAL OFWorld PHAJournal RMACofYPharmacy AND PHand ARPharmaceutical MACEUTICA L SCIENCES BaigWetOal. Sciences SJIF Impact Factor 5.210

Volume 4, Issue 08, 1965-1981.

Research Article

ISSN 2278 – 4357

DEVELOPMENT AND VALIDATION OF IN-VITRO DISSOLUTION STUDIES OF GEMIFLOXACIN MESYLATE AND AMBROXOL HYDROCHLORIDE FROM ITS COMBINED SUSTAIN RELEASE TABLET DOSAGE FORM WITH RESPECT TO ACCELERATED AGING CONDITIONS BY HPLC ASSAY METHODS. Mirza Shahed Baig* and M. H. G. Dehghan Y. B. Chavan College of Pharmacy, Dr. Rafiq Zakaria Campus, Rauza Bagh, Aurangabad – 431001. Maharashtra State. India.

Article Received on 25 June 2015, Revised on 15 July 2015, Accepted on 01 Aug 2015

ABSTRACT In-vitro dissolution studies for gemifloxacin mesylate and ambroxol hydrochloride from its combined SR tablet dosage form was developed and validated successfully as per ICH guidelines. Dissolution studies were performed using USP Type II apparatus for period of 12 h, first 2

*Correspondence for Author Mirza Shahed Baig

h in 0.1N HCl (Acid stage), then continued in phosphate buffer pH 6.8 (Buffer stage) at 50 RPM. After 10 h both the drugs shows complete

Y. B. Chavan College of

dissolution profile. Exposure of tablets to ICH accelerated stability

Pharmacy, Dr. Rafiq

condition 400C ± 20C/75% RH ± 5% for 6 months and photolytic

Zakaria Campus, Rauza

studies in an open dish to light for 30 days, using a combination of

Bagh, Aurangabad – 431001. Maharashtra State. India.

cool white and ultraviolet fluorescent lamps in a photostability chamber. Exposure energy was minimum of 1.2 million lux h fluorescent light and 200W h/m2 UV. Dissolution samples were

analyzed by newly developed HPLC method comprising of mobile phase as 0.02M potassium dihydrogen orthophosphate buffer: Acetonitrile (70:30 v/v) with 1% Triethylamine (2 ml/100 ml mobile phase) and its pH adjusted to 5.5 with ortho phosphoric acid and detection was executed on PDA detector at 244 nm. The retention time (RT) of gemifloxacin mesylate and ambroxol hydrochloride was found to be 2.34 ± 0.2 and 4.21 ± 0.2 min respectively. Results of analysis demonstrated that accelerated aging results in rapid increases in tablet hardness, and changes in water content upon storage leads to decrease in dissolution rate. The tablet matrix appears to rapidly absorb atmospheric moisture, as demonstrated by tablet weight

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gain, this shows changes in dissolution curves as compared to fresh marketed formulation indicating influence on drug product stability.

KEY WORDS: Reverse phase HPLC, dissolution studies, accelerated aging conditions, gemifloxacin mesylate and ambroxol hydrochloride. INTRODUCTION Gemifloxacin

mesylate

(GEM)

chemically

is

(R,

S)-7

[(4Z)-3-(aminomethyl)-4-

(methoxyimino)-1-pyrrolidinyl]-1-cyclopropyl-6-fluoro-1,4-dihydro-4-oxo1, 8-naphthyridine -3-carboxylic acid. Gemifloxacin forms a ternary complex with gyrase and topoisomerase IV, which blocks DNA replication, thus resulting in DNA release, chromosomal disruption and cell death.[1

– 4]

Ambroxol (AMB) chemically is 4 - [(2 – amino – 3,5 – dibromophenyl)

methylamino] cyclohexan – 1 – ol. Ambroxol is a mucolytic agent. It produces inhibition of NO-dependent activation of soluble guanylate cyclase can suppress the excessive mucus secretion, therefore it lowers the phlegm viscosity and improves the mucociliary transport of bronchial secretions. The structures of both the drugs are shown in Fig. 1 and 2. Combination drug product used to treat pulmonary obstructive acute bacterial exacerbation of chronic bronchitis and community-acquired pneumonia. It promotes mucus clearance, facilitates expectoration and eases productive cough, allowing patients to breathe freely and deeply.

[5–6]

Literature survey report discloses dissolution studies of each drug alone, but not in combination products. Dissolution studies of each drug in single were reported by UV spectroscopy but not by HPLC method. So, attempt has been made to develop new dissolution studies for combination product by novel RP – HPLC method. None of the work has been reported for accelerated aging effect on drug product with respect to dissolution studies so far in literature.[7–15] O

O

F OH H3C

O N

N

N

N

H2N

Fig. 1. Chemical Structure of Gemifloxacin..

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Br

NH Br NH2 OH

Fig. 2: Chemical Structure of Ambroxol. MATERIAL AND METHOD Chemicals and reagents Gemifloxacin Mesylate and Ambroxol Hydrochloride are acquired as a gift sample from Hetero Pharmaceutical Hyderabad, India and Aristo Pharmaceutical Mumbai, India. Tribasic sodium phosphate (AR Grade) was procured from Fischer Ltd, Mumbai. Potassium dihydrogen ortho phosphate (AR Grade) was procured from Fischer Ltd, Mumbai, Ortho phosphoric acid (AR Grade) from Finar chemical Ltd, Ahmadabad. Triethyl amine (AR Grade) acquired from Merck Mumbai, India. Acetonitrile (HPLC Grade) procured from Fischer Ltd, Mumbai. Tribasic Sodium Phosphate (AR Grade) was used from Merck Mumbai, India. Hydrochloric Acid (AR Grade) purchase from Merck Mumbai, India. Sodium Hydroxide (AR Grade) from Merck Mumbai, India. Double Distilled Water (Freshly prepared) Laboratory Made. Marketed formulation G – CIN –A SR tablets containing 320 mg GEM and 75 mg AMB, were procured from local market manufactured by Lupin pharmaceutical limited. Instruments used Dissolution studies is performed by using TDT-06L tablet dissolution tester Electrolab, Mumbai, India. Selection of analytical wavelength is done by using V630 UV visible spectrophotometer Jasco Corporation, Tokyo, Japan. An accelerated aging study is performed in TS90S Stability Chamber, Thermolab Scientific Equipments Pvt Ltd, Thane, India. A dissolution sample is analyzed by using Jasco HPLC PU2080 isocratic pump, Jasco Corporation, Tokyo, Japan. Accelerated aging studies as per ICH Guidelines. Dissolution Stability is the retention of the dissolution characteristics of a solid oral dosage form from the time of manufacture to its expiration date. It is considered a critical parameter

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not only from the standpoint of quality control, but also for the impact on the bioavailability of the product, because significant changes of the in vitro release profile during storage in different conditions may affect its bioavailability. During aging, the absence of dissolution changes provides some assurance that the bioavailability remains intact. Studies on the stability of drug formulations have mainly been concerned with chemical decomposition. Moreover, the different excipients of a formulation may interact during exposure to high temperatures or high humidity, reducing the in vitro dissolution, an important quality attribute of a solid oral dosage form. [16–18] Effect of Temperature and Humidity. Twenty G – CIN –A SR tablets was kept in clean and dry Petri dish and was subject to different controlled storage conditions with respect to temperature and humidity using a stability chamber. Accelerated studies were performed at 40 0C ± 20C/75% RH ± 5% for 6 months. Effect of exposure to Light. The photolytic studies was performed by exposing the twenty G – CIN –A SR tablets in clean and dry Petri dish to light for 30 days, using a combination of cool white and ultraviolet fluorescent lamps in a photostability chamber. Exposure energy was minimum of 1.2 million lux h fluorescent light and 200W h/m2 UV. This was protected by a transparent cover. The samples are positioned to provide maximum area of exposure to the light source. The protected samples (wrapped in aluminum foil) are used as dark controls to evaluate the contribution of thermally induced change to the total observed change these are placed alongside the authentic sample. Exposure to light initiates many side reactions affecting the surface of formulation, excipients interactions, drug uniformity. This affects dissolution rate profile and correlated with bioavailability of drugs. Preparation of standard stock solution 10 mg each of GEM and AMB were weighed accurately and separately transferred in two different 100 ml volumetric flasks. Both the drugs were first dissolved in 25ml of methanol solvent by vigorous shaking and then volume was made up to mark with methanol to obtain final concentration of 100 µg/ml of each component.

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Selection of Analytical wavelength The detection wavelength was measured by scanning 10 μg/ml solution of both GEM and AMB in methanol AR respectively, in UV- spectrophotometry, overlain spectra of both the drugs shows wavelength of maximum absorption and Isobestic point where both the drugs shows equal absorption. λmax of GEM was found to be 292 nm and λmax of AMB was found to be 245 nm. Isobestic point was detected at 244 nm.

Fig. 3. Overlain spectra of both GEM and AMB showing λmax and Isobestic point Determination of Solution Stability. Solution stability was analyzed over 48 h at room temperature. Sample solutions were prepared in the same dissolution media and at the same conditions as for dissolution test. Aliquots (1 ml) were collected initially and at 24 h intervals for 2 days and spiked in HPLC system. The drug concentrations observed in samples at 0, 24, and 48 h were compared. The absolute differences between the results at time zero and the time of analysis indicate solution stability. Preparation of 0.1N HCl. 0.1N HCl was prepared by accurately measuring 8.5 ml of hydrochloric acid and diluting it to 1000 ml with double distilled water. The acid solution was stored in clean glass bottle at a temperature not exceeding 300C. Preparation of Phosphate buffer of pH 6.8 Phosphate buffer pH 6.8, was prepared by mixing 0.1N hydrochloric acid with 0.20M tribasic sodium phosphate (3: 1) and adjusting, if necessary, with 2 N hydrochloric acid or 2N sodium

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hydroxide to a pH of 6.8 ± 0.05. The buffer solution was stored in clean glass bottle at a temperature not exceeding 300C. Dissolution procedure. Acid Stage. Freshly, 900 ml of 0.1 N HCl was prepared, sonicated for 15 min to remove dissolved gases (deaerated) and then filter through Whatman filter paper no. 41. Dissolution media was placed in the vessel, and apparatus were assembled. The medium was allowed to equilibrate to a temperature of 37 ± 0.50C. One dosage unit was placed in each of six dissolution vessels and started the dissolution test. The paddle speed was established at 50 RPM with the amount of drug dissolved recorded at intervals of every 15 min for 2 h. Each time 10 ml of aliquots were withdrawn from each dissolution vessel and replaced with equal volumes of the dissolution medium. The solutions were filtered through Whatman filter paper no. 41 and then immediately analyzed by HPLC assay method. After 2 h of operation in 0.1 N HCl, further instantly dissolution proceeded as directed under Buffer Stage. Buffer Stage. For this stage of the procedure, the acid was drain from the vessel containing the dosage unit, and added to the vessel freshly prepared, sonicated for 15 min (deaeration) and filtered 900 ml of pH 6.8 phosphate buffer that was previously been equilibrated to a temperature of 37 ± 0.50C. The apparatus was continued to operate in same conditions for further 10 h. Aliquots of fluid were withdrawn at every 1 h in buffer stage. Each time 10 ml of aliquots were withdrawn from each dissolution vessel and replaced with equal volumes of the dissolution medium. The solutions were filtered through Whatman filter paper no. 41 and then immediately analyzed by developed Stability indicating HPLC assay method. Assay of dissolution sample. Each 10 ml of dissolution samples at different time intervals of fresh marketed formulation, dissolution on tablets subjected to accelerated aging conditions of temperature and humidity, and dissolution of tablets subjected to exposure effect of light, are filtered through Whatman filter paper no. 41. Further 1ml filtrate is transferred to 10 ml volumetric flask and volume is made up to the mark with mobile phase. 20 μl of this final working dilution was injected in HPLC system and quantified with respect to RT and peak area. Percent drug release with respect to time was plotted. Dissolution shifts were calculated between the stability time

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intervals and across multiple drug release time points. The dissolution profiles and dissolution shifts were analyzed as response variables for the stability of drug product. Validation of dissolution method. The developed dissolution method was validated according to International Conference on Harmonization (ICH) guidelines. Parameters including linearity, range, LOD, LOQ, accuracy studies, precision, specificity, ruggedness, and robustness. RESULTS AND DISCUSSION The in – vitro dissolution method procedure was optimized with a view to study the effects of accelerated aging condition on percent release of drug from its tablet dosage form. For G – CIN – A SR tablet, dissolution was selected as acid phase (0.1N HCl) for 2 h, followed by buffer phase (pH 6.8 phosphate buffer) for further 10 h. Stability indicating HPLC method was used to resolve the degraded products from the drug during the course of dissolution studies. Composition of the mobile phase was determined to be 0.02M potassium dihydrogen orthophosphate buffer: Acetonitrile (70:30 v/v) with 1% triethylamine (2ml/100 ml mobile phase) and its pH adjusted to 5.5 with ortho phosphoric acid and detection was executed on PDA detector at 244 nm. The results of analysis shows well separated and good quality peaks for the drug of interest since such result at the outset provide better confidence because of the unknown nature of the products formed during stressing and dissolutions studies.

Fig. 4. Typical HPLC chromatogram of combination of GEM and AMB of dissolution studies

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Stability of Sample and Standard Solutions. The results of analysis of solution stability of the drug substance and drug product (50 µg/ml of each drug) ensure that they are stable up to 48 h at 20C – 80C and ambient temperature. This indicates that there is no degradation occurring due to hydrolysis, photolysis, or adhesion to glass ware over the course of the run period and can be utilize for HPLC analysis for the specified time period. Table 1. Evaluation data of solution stability study for Dissolution studies by HPLC. Intervals (hours) Initial (0) 12 24 48 Average Mean SD % RSD

% Assay for Drug sample solution stored at 20C -80C GEM AMB ---------100.21 99.99 99.87 99.72 99.56 100.02 99.92 99.95 0.18 0.29 0.1845 0.2965

% Assay for Drug sample solution stored at ambient temperature GEM AMB 99.23 99.60 99.78 99.89 99.67 100.09 99.95 99.45 99.86 99.89 0.26 0.28 0.2633 0.2821

In vitro dissolution studies. Tablet dissolution was performed with respect to the three samples as fresh marketed formulation, dissolution on tablets subjected to accelerated aging conditions of temperature and humidity, and dissolution of tablets subjected to exposure effect of light. All tablets of conditions mentioned above are subjected to dissolution test in two stages as Acid stage and buffer stage.[19–24] In – vitro percent drug release during dissolution stability studies of GEM

Table 2: and AMB. Sr. No. 1 2 3 4 5 6 7 8

Time (min) 0 30 60 90 120 180 240 300

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% Drug Release (Marketed Formulation) GEM AMB 0 0 15.36 10.54 28.66 20.56 41.96 32.12 55.26 44.56 68.56 60.43 78.56 72.45 85.87 82.36

% Drug Release (Temperature and Humidity) GEM AMB 0 0 20.56 25.42 38.45 36.58 52.75 48.32 63.14 60.14 78.54 77.25 83.11 89.22 89.77 95.69

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% Drug Release (Photolytic studies) GEM 0 25.96 42.36 57.39 68.45 82.35 88.56 94.51

AMB 0 20.14 30.56 42.13 55.85 71.66 83.41 93.54

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360 420 480 540 600 660 720

91.45 96.23 97.65 98.45 99.56 99.05 98.12

92.45 98.45 99.12 99.76 100.05 99.23 98.42

92.15 97.22 100.03 98.87 97.44 94.78 92.32

97.41 98.47 99.36 97.85 96.45 95.33 94.54

98.53 99.25 100.12 98.23 97.55 96.12 95.09

98.45 99.86 100.23 98.22 96.41 94.11 92.36

Fig. 5. Percent drug release profile of G – CIN – A Tablet.

Fig. 6. Accelerated aging effect on GEM percent release profile.

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Fig. 7. Accelerated aging effect on AMB percent release profile. Validation parameters as per ICH guidelines. [25–26] Linearity. Linearity was performed in the range of 80 – 120 % of the specification limits. The linearity was found to be in the range of 10 – 60 μg/ml for both GEM and AMB respectively, The response of both the drugs was found to be linear in the investigation concentration range Table 38. The linear regression equation (y = mx + c), for GEM was Y = 21038*X + 32.4 with correlation coefficient 0.9999 and for AMB was Y= 1572*X + 75.13 with correlation coefficient 0.9997.

. Fig. 8. Overlain HPLC linearity peaks of GEM.

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Fig. 9. Overlain HPLC linearity peaks of AMB. Table 3. HPLC Linearity results of GEM and AMB. Standard Sr. No. Concentration Peak Area of GEM (μg/ml) 1 10 21056 2 20 42563 3 30 62503 4 40 84235 5 50 105236 6 60 126358 Y = 21038*X + 32.4 Regression equation Correlation Coefficient 0.999 (r2 value)

Peak Area of AMB 16023 31458 46821 63254 78456 94587 Y= 1572*X + 75.13 R² = 0.999

Fig. 10. HPLC Linearity Calibration plot of GEM and AMB.

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LOD AND LOQ. The LOD for GEM and AMB was determined by injecting a series of dilute solutions of known concentration until the ratio analyte response signal became three times that of the noise and was found to 0.5μg/ml and 0.7μg/ml for GEM and AMB respectively. Whereas LOQ is the lowest concentration of an analyte which can be measured with an acceptable degree of confidence and verified by injecting six replicates at its concentration and recovery of spiked GEM and AMB in sample at the LOQ level and was found to be 1.6 μg/ml and 1.9μg/ml for GEM and AMB respectively. Specificity. The results for specificity are compared for placebo sample, matrix sample and the bulk drug at same concentration in five replicates with respect to peak area and RT of GEM and AMB respectively, after injecting into HPLC system. Specificity for an assay ensures that the signal measured comes from the substance of interest, and that there is no interference from excipients of the formulation and degradation products and/or impurities. Decrease in potency during analysis was explained by mass balance consideration. Hence it can be called as “Specific Stability Indicating HPLC Method”.

Fig. 11. Typical HPLC chromatogram for specificity studies of GEM and AMB during dissolution studies. Precision. The precision of the system was evaluated as repeatability by intra-day (within a day) precision and method precision was determined by intermediate precision (inter-day

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precisions). Each concentration was analyzed in triplicate; results are observed and calculated from corresponding peak area for GEM and AMB. Table 4: Results of Precision studies of GEM. Replicate Average Mean SD % RSD *n = 3

Intra – Day Precision *Final % release sample Sample 1 Sample 2 Sample 3 99.75 99.98 99.94 0.22 0.21 0.18 0.2278 0.2145 0.1923

Inter – Day Precision *Final % release sample Day 1 Day 2 Day 3 99.97 99.94 99.92 0.20 0.17 0.23 0.2088 0.1764 0.2356

Table 5: Results of Precision studies of AMB. Replicate Average Mean SD % RSD *n = 3

Intra – Day Precision *Final % release sample Sample 1 Sample 2 Sample 3 99.96 99.94 99.92 0.22 0.23 0.20 0.2301 0.2364 0.2133

Inter – Day Precision *Final % release sample Day 1 Day 2 Day 3 99.98 99.96 99.94 0.21 0.19 0.25 0.2287 0.1956 0.2674

Accuracy. GEM and AMB standard API was added to the dissolution vessels containing tablet dosage form in known amounts at 80%, 100%, and 120% levels of the tablet formulation. The recovered quantity of GEM and AMB was expressed in terms of average mean ± % RSD. Table 6: Accuracy studies results of dissolution studies of GEM and AMB. Level of % Recovery 80 100 120

Amount of Total Amount Standard added recovered (mg/tab) (mg) AMB GEM AMB GEM AMB 75 256 60 576.15 74.89 75 320 75 574.98 75.12 75 384 90 574.16 74.34 Average Mean SD % RSD

Label Claim (mg/tab) GEM 320 320 320

% Recovery GEM AMB 100.02 99.89 99.99 100.01 99.36 99.34 99.94 99.83 0.1459 0.2978 0.1460 0.2955

Ruggedness. The ruggedness was established through dissolution studies on tablet by different analysts on the same dissolution apparatus and chromatographic system. A study was also done on a different company dissolution apparatus by any one of the two analysts.

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Table 7: Ruggedness studies results of GEM and AMB in dissolution studies. Change in Analysts (Final percent release) Sr. No.

Analysts – I

Analysts – II

1 2 3 4 5 Average Mean SD % RSD

100.03 99.36 99.23 99.56 100.14 99.89 0.538 0.538

99.89 100.32 99.98 99.37 99.97 99.92 0.354 0.354

Change in Instrument (Final percent release) Electro Lab Electro Lab TDT-06L TDT-08L 100.09 99.38 99.59 99.70 99.98 99.98 100.32 99.89 99.49 100.04 99.98 99.87 0.410 0.523 0.410 0.523

Robustness studies. Table 8. Robustness studies results of GEM and AMB for dissolution studies. Robustness study of Gemifloxacin Chromatographic changes Final % Final % Factor Level release release Assay Time (h) Speed of Paddle (RPM) 25 -25 99.23 14 50 0 99.15 12 75 +25 100.5 10 Deviation at different paddle speed is 15 % pH 1.0, 6.6 -0.2 99.80 12.30 1.2, 6.8 0 99.95 12 1.4, 7.0 +0.2 99.55 11.30 Deviation at different pH is 5 % Change in Bath Temperature (0C) 34 -3 99.66 14 37 0 99.89 12 40 +3 98.95 10 Deviation at different bath temperature is 10 to 15 % Change in Filters Nylon 0.45µm 98.96 12 (N66) Whatman 20 µm 100.03 12 No. 41 Millipore Filter 0.45 µm 99.15 12 Paper Deviation at different filters is 0.2 %

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Robustness study of Ambroxol Chromatographic changes Final % Final % Factor Level release release Assay Time (h) Speed of Paddle (RPM) 25 -25 100.21 14 50 0 99.84 12 75 +25 99.89 10 Deviation at different paddle speed is 15 % pH 1.0, 6.6 -0.2 101.2 12.30 1.2, 6.8 0 99.94 12 1.4, 7.0 +0.2 99.85 11.30 Deviation at different pH is 5 % Change in Bath Temperature (0C) 34 -3 98.55 14 37 0 99.26 12 40 +3 99.35 10 Deviation at different bath temperature is 10 to 15 % Change in Filters Nylon 0.45µm 98.54 12 (N66) Whatman 20 µm 100.12 12 No. 41 Millipore Filter 0.45 µm 99.33 12 Paper Deviation at different filters is 0.2 %

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CONCLUSION Dissolution pattern gets affected by exposing the drug product to accelerated aging conditions which primarily contribute to atmospheric moisture absorption, as demonstrated by tablet weight gain, this shows changes in dissolution curves as compared to fresh marketed formulation indicating influence on drug product stability. A simple, precise, accurate and robust HPLC method has been developed for dissolution studies of combination drug product. This can be used for routine quality control test and dissolution stability of both the drugs in combination. ACKNOWLEDGEMENTS The authors are very much thankful to the Chairman, Mrs. Fatma Rafiq Zakaria, Maulana Azad Educational Trust, Dr Rafiq Zakaria Campus for providing necessary facilities for the project work. REFERENCES 1. Indian Pharmacopoeia, Indian Pharmacopoeial Commission, Gaziabad, 2010; 2: 707 – 709. 2. http://www.cimsasia.com/India/drug/info/G-CIN-A/G-CIN-A%20tab. (accessed on 5th march 2010). 3. http://www.rxlist.com/cgi/generic3/factive.htm. (accessed on 5th march 2010) 4. http://www.drugs.com/cdi/gemifloxacin.html (accessed on 5th march 2010) 5. Indian Pharmacopoeia, Indian Pharmacopoeial Commission, Gaziabad, 2010; 2: 91 – 92. 6. Kack, J., Koss, F.W., Schraven, E. and Beisenherz, G. US. Patent; 3,536,713; October 27: 1970; assigned to Boehringer lngelheim G.m.b.H. 7. S. Poongothai1, V. Balaji, A. Rajasekhar Reddy, P.Y. Naidu, C.M. Karrunakaran, R. Ilavarasan. In vitro-In vivo correlation (IVIVC) study of Gemifloxacin immediate release (IR) oral formulation. Der Pharmacia Lettre., 2010; 2(6): 166-175. 8. Khan Hajera. Development and Validation of a Dissolution Test with Spectrophotometric Analysis for Gemifloxacin in Tablet Dosage Form. Inter. J. Research in Pharma. and Biomedical Sci., 2012; 3(1): S990 – 995. 9. Khan Hajera, Zahid Zaheer. Development and Validation of a Dissolution Test with Spectrophotometric Analysis For gemifloxacin Mesylate and Ambroxol Hydrochloride in Tablet Dosage Form. Inter. J. Pharm. Tech. Research., 2012; 4(2): 661-668.

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10. V. Balaji, S. Poongothai, B. Madhavi, Rajasekhar Reddy, P.Y. Naidu, C. M. Karrunakaran and R. Ilavarasan. Development and Validation of a Dissolution Test with Spectrophotometric Analysis for Gemifloxacin in Tablet Dosage Form. An Inter. J. Pharm. Sci., 2014; 2(2): S-132-145. 11. Paim CS, Araújo BV, Volpato NM, Steppe M, Schapoval EE. Gemifloxacin mesylate (GFM): dissolution test based on in vivo data. Drug Dev Ind Pharm., 2015; 41(4): 567-72. 12. T. Alighieri. S. S Avanessian S Berlini S G Bianchi P Deluigi R Valducci Pieter Guelen. Bioavailability of ambroxol sustained release preparations. Part I: In vitro dissolution studies. Arzneimittel Forschung., 1988; 38(1): 92-4. 13. Dewan Taslima Akhter, Riaz Uddin, Kumar Bishwajit Sutradhar and Md. Shohel Rana. In vitro release kinetic study of ambroxol hydrochloride sustained release matrix tablets using hydrophilic and hydrophobic polymers. J.. Chem. and Pharm. Res., 2012; 4(3): 1573-1579. 14. Khan Hajera, Zahid Zaheer. Development and Validation of a Dissolution Test with Spectrophotometric Analysis For gemifloxacin Mesylate and Ambroxol Hydrochloride in Tablet Dosage Form. Int. J. Pharm.Tech Res., 2012; 4(2): 661-668. 15. Prabha Singh, Amrita Bajaj, Pranjali Apsegaonkar. Design Development and Evaluation of Matrix Tablets of Ambroxol Hydrochloride: in vitro – in vivo Study. Int J Pharm Pharm Sci., 2014; 6(6): 445-449 16. Sumie Yoshioka and Valentino J. Stella. Stability of Drugs and Dosage Forms. Kluwer Academic Publishers, 2002; pp. 160 – 167. 17. Jens.T.Carstensen, C.T.Rhodes, Drug stability principles and practices, 3rd ed. New York, Marcel Dekker, 2000; 306 – 312. 18. Kim Huynh. BA.

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Regulations, Methodologies, and Best Practices, Springer Science Business Media, LLC, Spring Street, New York, NY 10013, USA., 2009; 139 – 163. 19. Chapter 711: Dissolution. In: United States Pharmacopeia 31 (USP 31), National Formulary., 2008; 26(NF 26): 267–274. 20. Chapter 724: Drug release. In: United States Pharmacopeia 31 (USP 31), National Formulary., 2008; 26(NF 26): 275 – 278. 21. Chapter 1092: The dissolution procedure. Development and validation. In: United States Pharmacopeia 31 (USP 31), National Formulary., 2008; 26(NF 26): 573– 678. 22. Azarmi S, Roa W, Lobenberg R. Current perspectives in dissolution testing of conventional and novel dosage forms. Int. J. Pharm, 2007; 328: 12–21.

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23. Om Anand, Lawrence X. Yu, Dale P. Conner, and Barbara M. Davit.

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Testing for Generic Drugs: An FDA Perspective. American Association of Pharm. Sci. J., 2011; 13(3): 328 – 335. 24. Abdou HM. Dissolution. Remington: the science and practice of pharmacy. 19th ed. Mack Publishing Company, 1995; 593–604. 25. ICH, Validation of Analytical Procedures: Text and Methodology, Q2 (R1), ICH Harmonised Tripartite Guideline, Nov. 2005. 26. Bhavesh Vaghela, Rajan Kayastha, Nayana Bhatt, Nimish Pathak and Dashrath Rathod. Development and validation of dissolution procedures. J. Applied Pharm. Sci., 2011; 1(3): 50 – 56.

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