INTRODUCTION. safety and efficacy throughout all phases of its shelf life, including storage. In
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1 INTRODUCTION Analytically monitoring of a pharmaceutical product is necessary to ensure its safety and efficacy throughout all phases of its shelf life, including storage. In Simultaneous processing of sample and standard - better analytical precision and accuracy is obtained and there is less need for Internal Standard, which is possible through RP-HPLC and HPTLC NEED FOR STUDY A drug may be defined as a substance meant for diagnosis, cure, mitigation, prevention, or treatment of diseases in human beings or animals or for alternating any structure or function of the body of human being or animals. Pharmaceutical chemistry is a science that makes use of general laws of chemistry to study drugs i.e., their preparation, chemical natures, composition, structure, influence on an organism. Pharmaceutical chemistry also studies the physical and chemical properties of drugs, the methods of quality control and the conditions of their storage etc. The family of drugs may be broadly classified as 1. Pharmacodynamic agents and 2. Chemotherapeutic agents It is necessary to find the content of each drug either in bulk or in single or combined dosage forms for purity testing its purity. It is also essential to know the concentration of the drug and it s metabolites in biological fluids after taking the dosage form for treatment. KLE University s College of Pharmacy, Belgaum 1
2 The scope of developing and validating an analytical method is to ensure a suitable method for a particular analyte which is more specific, accurate and precise. The primary objective is to improve the conditions and parameters to be followed in the development and validation. PLAN OF WORK 1. Development and validation of Metformin Hydrochloride and Pioglitazone by HPTLC and RP-HPLC in pharmaceutical dosage form. 2. Development and validation of Atorvastatin, Glimepiride and Metformin Hydrochloride by HPTLC and RP-HPLC in pharmaceutical dosage form. 3. Development and validation of Atorvastatin, Ezetimibe and Fenofibrate by HPTLC RP-HPLC in pharmaceutical dosage form. 4. Development and validation of Atorvastatin, Losartan Potassium, Atenolol and Aspirin by HPTLC and RP-HPLC in pharmaceutical dosage form. SELECTED FORMULATION FOR STUDY:- Brand name Amount present (mg) Exermet P515 Metformin Hydrochloride Pioglitazone 15 Tripill Atorvastatin 10+ Glimepiride1+ Metformin Hydrochloride 500 Fibator EZ Atorvastatin 10 + Ezetimibe 10 + Fenofibrate 160 Starpill Atorvastatin 10+ Losartan Potassium 50+ Atenolol 50+ Aspirin 75 KLE University s College of Pharmacy, Belgaum 2
3 LITERATURE REVIEW Lakshmi et al., developed a simple, sensitive and rapid reverse phase high performance liquid chromatographic method for the estimation of Metformin HCl (MET) and Pioglitazone (PIO) in pure and in pharmaceutical dosage forms. Dhaneshwar et al., developed a new, simple, precise, and accurate HPTLC method for simultaneous estimation of Metformin hydrochloride (MET), Atorvastatin (ATV) and Glimepiride (GLM) as the bulk drug and in tablet dosage forms. Ajmera et al., developed a simple, rapid, precise and accurate gradient reversephase HPLC method and validated for the simultaneous determination of Atorvastatin (AT), Ezetimibe (EZ) and Fenofibrate (FE) in commercial tablets. Bhatia et al., developed a rapid, simple, sensitive, and selective chromatographic method for the simultaneous estimation of Atorvastatin calcium, Losartan potassium, Atenolol, and Aspirin in marketed formulation. EXPERIMENTAL SECTION HPTLC ANALYSIS WAS CARRIED OUT BY FOLLOWING THE BELOW MENTIONED EXPERIMENTAL CONDITIONS STRICTLY Experimental Conditions: Stationary phase : Pre-coated silica gel 60F 254 on aluminum sheets. Chamber saturation : 20 minutes Migration distance : 80 mm Band width : 6 mm Slit dimension : mm Source of radiation : Deuterium lamp KLE University s College of Pharmacy, Belgaum 3
4 CHAPTER- I A) DEVELOPMENT AND VALIDATION OF HPTLC METHOD FOR THE ESTIMATION OF METFORMIN HYDROCHLORIDE AND PIOGLITAZONE IN PHARMACEUTICAL DOSAGE FORM Parameters used for the HPTLC study Mobile phase : Toluene: Methanol: Triethylamine (8:2:0.1% V/V/V) Wavelength scanning : 230 nm R f value Metformin Hydrochloride : 0.25±0.03 Pioglitazone : 0.47±0.04 S.No Validation Parameters Metformin Hydrochloride Pioglitazone 1 Limit of detection (LOD) Limit of quantification (LOQ) Linearity and range Correlation Coefficient Precision studies (%RSD) a) Intra day b) Inter day c) Repeatability Sample application Measurement KLE University s College of Pharmacy, Belgaum 4
5 Fig.1. Chromatograms of Metformin Hydrochloride (100 ng/spot) and Pioglitazone (3 ng/spot) Synopsis Active Pharmaceutical Ingredient Formulation KLE University s College of Pharmacy, Belgaum 5
6 B) DEVELOPMENT AND VALIDATION OF HPLC METHOD FOR THE ESTIMATION OF METFORMIN HYDROCHLORIDE AND PIOGLITAZONE IN PHARMACEUTICAL DOSAGE FORM Selection of chromatographic method for the separation Reverse phase chromatographic techniques was selected since both the drugs are polar in nature. Parameters used for RP-HPLC method. Stationary phase : Phenomenax C 18 column (250x4.6) mm i.d., 5µm, particle size Mobile phase : 25 mm Sodium dihydrogen Phosphate (PH 3.65 with Orthophosphoric acid): Acetonitrile Solvent ratio : 60: 40%v/v ph : 3.65 Detection wavelength Flow rate Operating pressure Temperature : 230 nm : 1.0 ml/ minute : 181 kgf : Room temperature KLE University s College of Pharmacy, Belgaum 6
7 S.No Validation parameters Metformin Hydrochloride Pioglitazone 1 Limit of detection (LOD) µg/ml 0.01 µg/ml 2 Limit of quantification (LOQ) µg/ml 0.03 µg/ml 3 Linearity and range Correlation Coefficient 4 Precision studies (%RSD) a) Intra day b) Inter day c) Repeatability of injections 5-45 µg/ml µg/ml Fig.2: Chromatograms of Metformin Hydrochloride (15 g/ml) + Pioglitazone (0.45 g/ml) Active Pharmaceutical Ingredient Formulation KLE University s College of Pharmacy, Belgaum 7
8 CHAPTER-II A) DEVELOPMENT AND VALIDATION OF HPTLC METHOD FOR THE ESTIMATION OF ATORVASTATIN, GLIMEPIRIDE AND METFORMIN HYDROCHLORIDE IN PHARMACEUTICAL DOSAGE FORM Parameters used for the HPTLC study Mobile phase : Water: Methanol: Ammonium sulphate (3.5:3.5:12.6% v/v/v) Wavelength scanning : 245 nm R F values of Atorvastatin : 0.50 ± 0.01 Glimepiride : 0.65 ± 0.01 Metformin Hydrochloride : 0.33 ± 0.01 S.No Validation parameters Atorvastatin Glimepiride Metformin Hydrochloride 1 Limit of detection (LOD) Limit of quantification (LOQ) Linearity and range Correlation Coefficient Precision studies (%RSD) a) Intra day b) Inter day c) Repeatability i. Sample application ii. Measurement KLE University s College of Pharmacy, Belgaum 8
9 Fig.3: Atorvastatin (4 ng/spot), Glimepiride (0.4 ng/spot) and Metformin Hydrochloride (200 ng/spot) Active Pharmaceutical Ingredient Formulation KLE University s College of Pharmacy, Belgaum 9
10 B) DEVELOPMENT AND VALIDATION OF HPLC METHOD FOR THE ESTIMATION OF ATORVASTATIN, GLIMEPIRIDE AND METFORMIN HYDROCHLORIDE IN PHARMACEUTICAL DOSAGE FORM 1. Selection of chromatographic method for separation drugs are polar in nature. Reverse phase chromatographic techniques was selected since both the Parameters used for RP-HPLC method. Stationary phase :Phenomenax C 18 column (250x4.6) mm i.d., 5µm, particle size Mobile phase Solvent ratio Detection wavelength Flow rate Operating pressure Temperature : 20 mm Potassium dihydrogen Phosphate: Acetonitrile : 65: 35%v/v : 245 nm : 1.0 ml/ minute : 145 kgf : Room temperature Retention time Atorvastatin Glimepiride : 8.51± 0.51 min : ± 0.62 min Metformin Hydrochloride: ± 0.70 min KLE University s College of Pharmacy, Belgaum 10
11 S.No Validation parameters Atorvastatin Glimepiride Metformin Values 1 Limit of detection (LOD) 0.01 µg/ml µg/ml 0.1 µg/ml 2 Limit of quantification (LOQ) 0.3 µg/ml 0.03 µg/ml 1 µg/ml 3 Linearity and range 1-5µg/ml µg/ml µg/ml Correlation Coefficient Precision(%RSD) a) Intraday b) Inter day c) Repeatability of injection Fig. 4: Chromatograms of Atorvastatin 2 g/ml, Glimepiride 0.2 g/ml and Metformin Hydrochloride 100 g/ml Active Pharmaceutical Ingredient Formulation KLE University s College of Pharmacy, Belgaum 11
12 CHAPTER-III A) DEVELOPMENT AND VALIDATION OF HPTLC METHOD FOR THE ESTIMATION OF ATORVASTATIN, EZETIMIBE AND FENOFIBRATE IN PHARMACEUTICAL DOSAGE FORM Parameters used for the HPTLC study Mobile phase : Chloroform: Toluene: Methanol: Glacial Acetic Acid (5:4:1:0.1% V/V/V/V) Wavelength scanning : 254 nm R F values of Atorvastatin : 0.36±0.08 Ezetimibe : 0.51±0.06 Fenofibrate : 0.87±0.04 S.No Validation parameters Atorvastatin Ezetimibe Fenofibrate 1 Limit of detection (LOD) 2 Limit of quantification (LOQ) Linearity and range Correlation Coefficient Precision studies (%RSD) a) Intra day b) Inter day c) Repeatability i. Sample application ii.measurement KLE University s College of Pharmacy, Belgaum 12
13 Fig.5: Chromatograms of Atorvastatin 20 ng/spot, Ezetimibe 20 ng/spot and Fenofibrate 320 ng/spot Active Pharmaceutical Ingredient Formulation KLE University s College of Pharmacy, Belgaum 13
14 B) DEVELOPMENT AND VALIDATION OF HPLC METHOD FOR THE ESTIMATION OF ATORVASTATIN, EZETIMIBE AND FENOFIBRATE IN PHARMACEUTICAL DOSAGE FORM Selection of chromatographic method for separation Reverse phase chromatographic techniques was selected since both the drugs are polar in nature. Parameters used for RP-HPLC method. Stationary phase Mobile phase : RP18e, Hibar RT column (250x4.6 mm) : Potassium dihydrogen phosphate: Methanol: Acetonitrile Solvent ratio Detection wavelength Flow rate Operating pressure Temperature : 10: 60: 30 % v/v/v : 254 nm : 1.0 ml/ minute : 140 kgf : Room temperature Retention Time Atorvastatin Ezetimibe Fenofibrate : 2.67±0.23 min : 4.32± 0.43 min : 11.48±0.56 min KLE University s College of Pharmacy, Belgaum 14
15 S.No Validation parameters Atorvastatin Ezetimibe Fenofibrate Values 1 Limit of detection (LOD) 0.5 µg/ml 0.2 µg/ml 2 µg/ml 2 Limit of quantification (LOQ) 1 µg/ml 1 µg/ml 16 µg/ml 3 Linearity and range 1-5 µg/ml 1-5 µg/ml µg/ml Correlation Coefficient Precision (%RSD) Intraday Inter day Repeatability of injection Fig.6: Chromatograms of Atorvastatin 1 g/ml, Ezetimibe 1 g/ml and Fenofibrate 16 g/ml Active Pharmaceutical Ingredient Formulation KLE University s College of Pharmacy, Belgaum 15
16 SECTION-IV A) DEVELOPMENT AND VALIDATION OF HPTLC METHOD FOR THE ESTIMATION OF ATORVASTATIN, LOSARTAN POTASSIUM, ATENOLOL AND ASPIRIN IN PHARMACEUTICAL DOSAGE FORM Parameters used for the HPTLC study Mobile phase : Methanol: Hexane: Ethyl acetate: Chloroform: GAA 4.5: 1.5: 4: 1: 0.1 %v/v/v/v/v Wavelength scanning : 220 nm R F values of Atorvastatin : 0.63 ± 0.05 Losartan Potassium : 0.85 ± 0.03 Atenolol : 0.21 ± 0.02 Aspirin : 0.41 ± 0.01 S.No Validation parameters Atorvastatin Losartan Potassium Atenolol Aspirin 1 Limit of detection (LOD) Limit of quantification (LOQ) Linearity and range Correlation Coefficient Precision studies (%RSD) a) Intra day b) Inter day c) Repeatability i. Sample application Measurement KLE University s College of Pharmacy, Belgaum 16
17 Fig.7: Chromatograms of Atorvastatin (50 mg), Losartan Potassium (250 mg), Atenolol (250 mg) and Aspirin (375 mg) Active Pharmaceutical Ingredient Formulation KLE University s College of Pharmacy, Belgaum 17
18 B) DEVELOPMENT AND VALIDATION OF HPLC METHOD FOR THE ESTIMATION OF ATORVASTATIN, LOSARTAN POTASSIUM, ATENOLOL AND ASPIRIN IN PHARMACEUTICAL DOSAGE FORM Selection of chromatographic method for separation Reverse phase chromatographic techniques was selected since both the drugs are polar in nature. Parameters used for RP-HPLC method. Stationary phase Mobile phase : RP18e, Hibar RT column (250x4.6 mm) : 10 mm Potassium dihydrogen Phosphate (ph 2.5 with Orthophosphoric acid): Methanol Solvent ratio : 20: 80 % v/v ph : 2.5 Detection wavelength Flow rate Operating pressure Temperature : 220 nm : 1.0 ml/ minute : 191 kgf : Room temperature Retention Time Atorvastatin Losartan Potassium Atenolol Aspirin : ± min : ± min : ± min : ± min KLE University s College of Pharmacy, Belgaum 18
19 S.N o Validation parameters Atorvastatin Losartan Potassium Atenolol Aspirin 1 Limit of detection (LOD) 0.5 µg/ml 2.0 µg/ml 2.5 µg/ml 4.5 µg/ml 2 Limit of quantification (LOQ) 1 µg/ml 20 µg/ml 25 µg/ml 10 µg/ml 3 Linearity and range µg/ml µg/ml µg/ml µg/ml Correlation Coefficient Precision studies (%RSD) a) Intra day b) Inter day c) Repeatability of injections Fig. 8: Chromatograms of Atorvastatin (10 mg), Losartan Potassium (50 mg), Atenolol (50 mg) and Aspirin (75 mg). Active Pharmaceutical Ingredient Formulation KLE University s College of Pharmacy, Belgaum 19
20 ANALYSIS OF METFORMIN HYDROCHLORIDE AND PIOGLITAZONE BY HPTLC AND RP-HPLC METHODS Labelled HPTLC RP-HPLC Drug amount (mg/tab) Linearity (ng/spot Estimated Amount (mg/tab) % Label claim 100% Recovery Linearity (µg/ml) Estimated Amount (mg/tab) % Label claim 100% Recovery MET PIO MET- Metformin Hydrochloride and PIO- Pioglitazone KLE University s College of Pharmacy, Belgaum 20
21 ANALYSIS OF ATORVASTATIN, GLIMEPIRIDE AND METFORMIN HYDROCHLORIDE BY HPTLC AND RP-HPLC Drug Labelled amount (mg/tab) Linearity Estimated Amount (mg/tab) HPTLC % Label claim 100% Recovery Linearity (µg/ml) Estimated Amount (mg/tab) RP-HPLC % Label claim 100% Recovery ATO GLI MET ATO- Atorvastatin, GLI- Glimepiride and MET- Metformin Hydrochloride KLE University s College of Pharmacy, Belgaum 21
22 ANALYSIS OF ATORVASTATIN, EZETIMIBE AND FENOFIBRATE PIOGLITAZONE BY HPTLC AND RP-HPLC METHODS HPTLC RP-HPLC Drug Labelled amount (mg/tab) Linearity Estimated Amount (mg/tab) % Label claim 100% Recovery Linearity (µg/ml) Estimated Amount (mg/tab) % Label claim 100% Recovery ATO EZE FEN ATO- Atorvastatin, EZE- Ezetimibe and FEN- Fenofibrate KLE University s College of Pharmacy, Belgaum 22
23 ANALYSIS OF ATORVASTATIN, LOSARTAN POTASSIUM, ATENOLOL AND ASPIRIN BY HPTLC AND RP-HPLC METHODS Drugs Labeled amount (mg/tab) Linearity HPTLC Estimated % Label Amount claim (mg/tab) 100% Recovery Linearity (µg/ml) RP-HPLC Estimated % Label Amount claim (mg/tab) 100% Recovery ATO LOS ATE ASP ATO - Atorvastatin, LOS - Losartan Potassium, ATE - Atenolol and ASP - Aspirin KLE University s College of Pharmacy, Belgaum 23
24 Conclusion: The proposed HPTLC and HPLC methods provide simple, accurate and reproducible quantitative analysis for simultaneous determination of drugs in tablets. Both the methods were validated as per ICH guidelines. The developed methods are free from interference due to the excipients present in tablets and can be used for routine simultaneous quantitative estimation of drugs in tablets. The results have shown that both HPLC and HPTLC are best methods for a simultaneous quantification of drugs in tablets. Bibliography 1. Lakshmi KS, Rajesh T, Sharma. Simultaneous determination of Metformin and Pioglitazone by reversed phase HPLC in pharmaceutical dosage forms, Int J Pharm Pharm Sci. 2009; 1(2): Dhaneshwar SR, Salunkhe JV, Bhusari VK. Validated HPTLC method for simultaneous estimation of Metformin hydrochloride, Atorvastatin and Glimepiride in bulk drug and formulation. J Anal Bioanal Tech.2010; 1: Ajmera A, Deshpande S, Patel P, Patel K, Solanki S, Rathod K. Reverse phase high performance liquid chromatographic (HPLC) method for simultaneous determination of Atorvastatin, Ezetimibe and Fenofibrate in commercial tablets. Int J Pharm Pharm Sci, 2011; 4(1): Bhatia NM, Gurav SB, Jadhav SD, Bhatia MS. RP-HPLC method for simultaneous estimation of Atorvastatin calcium, Losartan potassium, Atenolol, and Aspirin from tablet dosage form and plasma. J Liq Chromatogra Related Techn. 2012; 35(3): KLE University s College of Pharmacy, Belgaum 24
25 publications SL. NO. TITLE OF THE RESEARCH PAPER NAME OF THE JOURNAL 1. RP-HPLC METHOD FOR THE SIMULTANEOUS ESTIMATION OF METFORMIN HYDROCHLORIDE AND PIOGLITAZONE IN COMBINED DOSAGE FORM JOURNAL OF CELL AND TISSUE RESEARCH 2. RP-HPLC METHOD FOR THE SIMULTANEOUS ESTIMATION OF ATORVASTATIN, LOSARTAN POTASSIUM, ATENOLOL AND ASPIRIN IN COMBINED DOSAGE FORM TROPICAL JOURNAL OF PHARMACEUTICAL RESEARCH 3. HPTLC METHOD FOR THE ESTIMATION OF ATORVASTATIN, LOSARTAN POTASSIUM, ATENOLOL AND ASPIRIN IN COMBINED DOSAGE FORM INTERNATIONAL JOURNAL OF PHARMACEUTICAL RESEARCH KLE University s College of Pharmacy, Belgaum 25
Department of Pharmaceutical Analysis, KMCH College of Pharmacy, Coimbatore-35, India
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