ETORICOXIB AND PREGABALIN OF METHOD DEVLOPMENT IN RPHPLC BY UPEXA BAVADIYA
- 1. GUIDED BY DHIRENDRA KUMAR TARAI PREPARED BY: UPEKSHA J. BAVADIYA Department of Pharmaceutical Quality Assurance GUJARAT TECHNOLOGICAL UNIVERSITY Chandkheda, Ahmadabad – 382424 - Gujarat “Development and Validation for Simultaneous Estimation of Etoricoxib and Pregabalin in Bulk and Tablet Dosage Form by RP-HPLC”
- 2. CONTENT • Introduction • Drug Profile • Review of Literature • Rational • Aim and Objectives • Material and Equipment • Identification of API • Method development and validation • Summary and conclusion • Comment sheet • References • Paper Publication 2
- 4. INTRODUCTION OF DRUG (ETORICOXIB)(1) 4 Etoricoxib, sold under the trade name Arcoxia, is a selective COX-2 inhibitor from McOLSON Research Laboratories. Currently it is approved in more than 80 countries worldwide but not in the US, where the Food and Drug Administration (FDA) has required additional safety and efficacy data for Etoricoxib before it will issue approval Etoricoxib is indicated for the treatment of rheumatoid arthritis, psoriatic arthritis, osteoarthritis, ankylosing spondylitis, chronic low back pain, acute pain, and gout. Approved indications differ by country. In the U.K., it is also "used for the short term treatment of moderate pain after dental surgery" of adults. MOA : Like any other COX-2 selective inhibitor Etoricoxib selectively inhibits isoform 2 of cyclo-oxigenase enzyme (COX-2), preventing production of prostaglandins (PGs) from arachidonic acid.
- 5. INTRODUCTION OF DRUG (PREGABALIN)(2) Marketed under the brand name Lyrica among others, is a medication used to treat epilepsy, neuropathic pain, fibromyalgia, restless leg syndrome, and generalized anxiety disorder Its use in epilepsy is as an add-on therapy for partial seizures. When used before surgery, it reduces pain but results in greater sedation and visual disturbances It is taken by mouth Common side effects include headache, dizziness, sleepiness, confusion, trouble with memory, poor coordination, dry mouth, problem with vision, and weight gain. Serious side effects may include angioedema, drug misuse, and an increased suicide risk. MOA : Act as a ligand of the alpha2-delta subunit of calcium channels. Decreased calcium entry into nerve endings. less glutamate released from nerve endings .than relief of neuropathic pain. 5
- 6. Introduction to Dosage form Brand Name Contents Manufacturer Formulation Etoshine NP Etoricoxib + Pregabalin Sun Pharma laboratory pvt. Ltd Tablet 6
- 7. TECHNIQUES OF METHOD DEVLOPMENT 7
- 8. INTRODUCTION OF ANALYTICAL METHOD (3) Analytical chemistry is divided into two branches: Qualitative: A qualitative analysis provides information about the identity of atomic or molecular species or functional groups in sample. Quantitative: A quantitative analysis provides numerical information as to the relative amount of one or more of these components. Analytical methods development and validation play important roles in the discovery, development and manufacture of pharmaceuticals with the objectives. To qualify and quantify the active pharmaceutical ingredients in bulk as well as dosage form To establish impurity profile and limit of allowable impurities in dosage form. 8
- 9. 9 INTRODUCTION OF HPLC METHOD (4) HPLC is an analytical technique widely used for identification, separation, detection and quantification of various drugs and its related degradents. High performance liquid chromatography (HPLC), is a separation technique based on a solid stationary phase and a liquid mobile phase. Most of the drugs in multi- component dosage forms can be analyzed by HPLC method because of the several advantages like rapidity, specificity, accuracy, precision and ease of automation in this method. HPLC method eliminates tedious extraction and isolation procedures. Principle of separation: The principle of separation in normal phase mode and reverse phase mode is adsorption. When mixtures of components are introduced in to a HPLC column, they travel according to their relative affinities towards the stationary phase. The component which has more affinity towards the adsorbent travels slower. The component which has less affinity towards the stationary phase travels faster. Since no two components have the same affinity towards the stationary phase, the components are separated.
- 10. CONT….. Different modes of separation in HPLC: 1) Normal phase mode. 2) Reverse phase ion pair chromatography. 3) Reversed phase mode. 4) Size exclusion chromatography. 10
- 11. Method development by RP-HPLC (5) 11 Reversed-phase chromatography is the mainly used in chromatographic mode, it is used to separate neutral molecules in solution based on their hydrophobicity. As the name suggested that, reversed-phase chromatography is the reverse of normal-phase chromatography in the intelligence that it involves the employ of a polar mobile phase and a non-polar stationary phase. It ensures that a decrease in the polarity of the mobile phase results in a decreases in solute retention.
- 12. 12 HPLC Techniques Normal phase Mode Stationary phase : Polar Mobile phase : Non polar Reverse phase Mode Stationary phase : Non polar Mobile phase : Polar
- 13. 13 Fig. A schematic diagram of HPLC equipment.
- 14. VALIDATION OF ANALYTICAL METHOD (14) 14 Validation Validation is a process of establishing documented evidence, which provides a high degree of assurance that a specific activity will consistently produce a desired result or product meeting its predetermined specifications and quality characteristics. Method Validation Method validation is the process used to confirm that the analytical procedure employed for a specific test is suitable for its intended use. Results from method validation can be used to judge the quality, reliability and consistency of analytical results.
- 15. Validation of Analytical method 15
- 16. 2. DRUG PROFILE (15-17) 16
- 17. 2. Drug profile of Etoricoxib (17) INTRODUCTION CAS Number 202409-33-4 Description Etoricoxib is a synthetic, nonsteroidal anti-inflammatory drug (NSAID) with antipyretic, analgesic, and potential antineoplastic properties. Etoricoxib specifically binds to and inhibits the enzyme cyclooxygenase-2 (COX-2), resulting in inhibition of the conversion of arachidonic acid into prostaglandins. Structure Chemical Formula C18H15ClN2O2S 17
- 18. Mol. Weight 358.84 gm/mol IUPAC Name 5-chloro-2-(6-methylpyridin-3-yl)-3-(4-methylsulfonyl) phenyl) pyridine. Categories COX-2 Inhibitors Anti-Inflammatory Agents Solubility Slightly Soluble in water, freely soluble in methanol Mechanism Of Action Like any other COX-2 selective inhibitor Etoricoxib selectively inhibits isoform 2 of cyclo-oxigenase enzyme (COX-2), preventing production of prostaglandins (PGs) from arachidonic acid. LogP 3.7 pKa 4.96 Melting point 134-135°C 18
- 19. 2. Drug profile of Pregabalin (15-16) INTRODUCTION CAS Number 148553-50-8 Description Pregabalin is structurally similar to gamma- aminobutyric acid (GABA) - an inhibitory neurotransmitter. It may be used to manage neuropathic pain, postherpetic neuralgia, and fibromyalgia among other conditions Structure Chemical Formula C8H17NO2 19
- 20. Mol. Weight 259.22 gm/mol IUPAC Name 3-isobutyl GABA, (S)-3-isobutyl-γ-aminobutyric acid Categories Anticonvulsants Analgesics Solubility Freely Soluble in water Mechanism Of Action Act as a ligand of the alpha2-delta subunit of calcium channels. Decreased calcium entry into nerve endings. less glutamate released from nerve endings .than relief of neuropathic pain. LogP -1.3 pKa 4.2 Melting point 176 - 178ºC 20
- 21. 3. REVIEW OF LITERATURE 21
- 22. 3. Review of Literature Official Drugs Method Year Abstract Summary Ref. No Etoricoxib Monograph IP 2010 Column: stainless steel column 30 cm x 3.9 mm, packed with phenyl silica gel Mobile phase : mixture of 26 volumes of acetonitrile and 74 volumes of a 0.272 per cent w/v solution of sodium acetate, Flow rate: 1.0 ml/min Wavelength: 256 nm 18 Pregabalin Monograph IP 2010 Column: stainless steel column 25 cm x 4.6 mm packed with octadecylsilane bonded to porous silica (5 μm) Mobile phase : mixture of62 volumes of a buffer solution prepared by diluting about 7.18 ml of triethylamine to 1000 ml water, adjusted to pH 3.0 with orthophosphoric acid and 38 volumes of acetonitrile, Flow rate: 2.0 ml/min Wavelength: 340 nm 19 22
- 23. Non - official Drugs Method Authors/year /article /journal Abstract Summary Ref. No Etoricoxib RP-HPLC Bhattacharya I Bhattacharya SP . Asian J. Research Chem 2009 297-299 Column: C18 column Mobile Phase: phosphate buffer ( pH-7.8) and methanol (90:10 v/v) Wavelength: 235 nm., Flow rate: 1 ml/min Retention times: 3.428 min Linearity range: Etoricoxib - 10-200 μg/ml 20 Etoricoxib HPLC Haque M , Shaema N. Am. J. Pharm Tech Res. 2012 275 – 283 Column: C18 column (250 mm × 4.6 mm. 5μm particle size) Mobile Phase: Ammonium Acetate Wavelength: 235 nm., Flow rate: 1 ml/min 21 23
- 24. Drugs Method s authors/ year /articals Abstract Summary Ref. No Etoricoxib RP- HPLC Venugopal S, Tripathi UM, Devanna N, E-Journal of Chemistry 2011 S119-S126 Column : Zorbax SB CN (250 × 4.6 mm , 5µm) Mobile phase : buffer : methanol 50:50 Flow rate : 0.8 ml/min Wavelength : 235 nm 22 Pregabalin RP- HPLC Kasawar GR, Faroogui MN . Indian Journal of Pharmaceutical Sciences. 2010 517-519 Column: Hypersil BDS(C8, 150×4.6 mm,5 μm) column Mobile Phase: phosphate buffer pH 6.9 and acetonitrile in the ratio of 95:05 Flow rate: 1 ml/min 23 Pregabalin RP- HPLC Prathima SSP. INDO AMERICAN JOURNAL OF PHARMACEUTICAL SCIENCES 2015 1038-1047 Column: AGILENT ZORBAX SB-ODS C18(250×4.6 mm)5 μm Mobile Phase: dipotassium hydrogen phosphate: methanol in the ratio of 60:40% v/v Wavelength: 247 nm. ,Flow rate: 1 ml/minRetention times: 2.00 min Linearity range: 50 μg/ml to 150 μg/ml LOQ: 0.53 𝜇g/ml LOD: 1.61 𝜇g/ml 24 24
- 25. Drugs Method Authors/year /article /journal Abstract Summary Ref. No Etoricoxib (ETR) and Thiocolchico side (THC) RP- HPLC Kumar US , Natraj D, Khan A INTERNATIONAL JOURNAL OF RESEARCH IN PHARMACY AND CHEMISTRY 2011 649-65 Column: C18 stainless steel column (InertSil ODS-3, 250 mm x 4.6 mm ID, particle size 5μm) Mobile Phase: phosphate buffer (PH6, adjusted with ortho phosphoric acid) and methanol (30:70 v/v) Wavelength: 255 nm. , Flow rate: 1.2 ml/min Retention times: Etoricoxib - 2.506 min Thiocolchicoside - 4.600 min Linearity range: Etoricoxib - 40-80 μg/ml Thiocolchicoside 2-6 μg/ml 25 Paracetamol (PCM) and Etoricoxib (ETO) HPLC Baheti KG Shaikh S International Journal of PharmTech Research vol -3 2011 1719-1727 Mobile Phase: phosphate buffer : acetonitrile (60:40v/v) Wavelength: 242 nm. Retention times: PCM – 1.51 min ETO – 4.31 min Linearity range: PCM - 5-30 μg/ml ETO 1-6 μg/ml 26 25
- 26. Drugs Method Authors/year /article /journal Abstract Summary Ref. No NSAIDs and their commonly prescribed combinatio n drugs RP-HPLC Gananadhamu S, Patel PN . Chromatography Research International 2013 1-13 Column: Kromasil C18 (250 × 4.6 mm, 5 𝜇m) Mobile Phase: phosphate buffer pH 3.25 and acetonitrile with gradient elution Wavelength: 230 nm. Flow rate: 1.1 ml/min LOQ: 0.64 to 3.24 𝜇g/Ml LOD: 0.04 to 0.97 𝜇g/mL 27 Etoricoxib and Paracetam ol RP-HPLC Rao KP, Damanu G . Journal of Advanced Studies in Agricultural, Biological and Environmental Sciences 2014 2394-2606 Column: Hypersil BDS C18 (250 x 4.6 mm i.d., 5μm particle size)Mobile Phase: 0.05 M sodium dihydrogen phosphate buffer (adjusted pH 3.2 with o-phosphoric acid): acetonitrile (35:65 v/v) Wavelength: 235 nm. Flow rate: 1 ml/min concentration range: Etoricoxib - 1000 to 3000μg/ml 40-80 μg/ml Paracetamol - 1200 to 3600μg/ml 28 26
- 27. Drugs Method Authors/ year/ artical Abstract Summary Ref. No Thiocolchic oside and Etoricoxib RP-HPLC Padmavati k, Rao MS. World Journal of Pharmaceutic al Sciences. 2016 76-79 Column: Hypersil BDS C18 (250 x 4.6 mm i.d., 5μm particle size)Mobile Phase: phosphate buffer(pH-3.4) and acetonitrile in the ratio of 35:65 v/v Wavelength: 260 nm., Flow rate: 1 ml/min Retention times: Etoricoxib – 6.92 min Thiocolchicoside – 2.83 min 29 Pregabalin And Methalcob alamine RP-HPLC Bhatt KK, Patelia EM, Mori A, Journal of Analytical & Bioanalytical Techniques 2018 1-4 Column : C18 ( 250× 4.6 mm) 5µm Mobile phase : Methanol : water 40:60 Wavelength : 218 nm Retension time : 6.4 PH :6.5 30 27
- 28. Drugs Method Authors / year / artical Abstract Summary Ref. No Epalrestat and Pregabalin RP- HPLC Parmeshweri SA ,Anunam G. International Journal of Pharmaceutica l Sciences and Research 2018 1844-1850 Column: C18 column 250 × 4.6 mm Mobile Phase: 0.1% ortho phosphoric acid buffer and acetonitrile ( ratio of 45: 55) Wavelength: 244 nm. , Flow rate: 1 ml/min Detector: PDA Linearity range: Epalrestat - 37.5 – 225 μg/ml Pregabalin - 18.75 - 112.5 μg/ml Retention times: Epalrestat – 2.407 min Pregabalin – 3.272 min 31 Nortriptyli ne and Pregabalin RP- HPLC Potkuri H, Rao SB. Journal of the Chilean Chemical Society.62 2017 62 Column: C18 column BDS (250mm x 4.6 mm, 5m) Mobile Phase: Perchloric acid (0.1%) and acetonitrile in the ratio of 55:45 Wavelength: 210 nm. , Flow rate: 1 ml/min concentration range: nortriptyline – 37.5 - 22.5 μg/ml Pregabalin - 37.5 – 22.5 μg/ml Retention times: nortriptyline – 2.407 min Pregabalin – 3.272 min 32 28
- 29. Drugs Method Authors /year/art. Abstract Summary Ref. No Epalrestat and Pregabalin RP-HPLC Goday S, Ragaman A., Asian journal of pharmaceuti cal and clinical reaserch, vol -112018 319-324 Precision : epalrestat - 0.2% Pregabalin – 0.3% LOQ : Epalrestat - 0.65 μg/ml Pregabalin – 0.25 μg/ml LOD : Epalrestat - 0.21 μg/ml Pregabalin – 0.08 μg/ml Linearity range: Epalrestat - 30–180 ppm Pregabalin - 15–90 ppm 33 Pregabalin and Celecoxib RP-HPLC Swapna G Merugu M, World Journal of Pharmaceuti cal Research, 2017 1354-1360 Column: Hypersil BDS(150 mm x 4.6 mm, 5m) Mobile Phase: potassium di hydrogen orthophosphate buffer of pH 6.5 and acetonitrile in the ratio of (70:30) Wavelength: 238 nm. Flow rate: 1 ml/min Linearity range: Celecoxib - 100 μg/ml -750 μg/ml Pregabalin-37.5μg/ml-281.25 μg/ml 34 29
- 30. Drugs Method Author/year / artical Abstract Summary Ref. No Pregabalin and Aeclofenac stability RP-HPLC Suchitta TJ Gurupadoyy a B M . Acta scientific Pharmaceuti cal sciences. 2018 20-26 Wavelength: Aeclofenac - 276 nm., Pregabalin - 406 nm concentration range: Aeclofenac – 10 - 50μg/ml μg/ml Pregabalin - 50 - 500μg/ml μg/ml LOQ: Aeclofenac - 2μg/ml , Pregabalin - 4μg/ml LOD: Aeclofenac – 0.50 μg/ml ,Pregabalin – 0.6 μg/ml 35 Epalrestat and Pregabalin stability- indicating RP-HPLC Prasad R , Madhuharik a MY. International journal of pharmacy 2017 157-164 Column: C18 (150x4.6mm, 5μ) column Mobile Phase: Ammonium acetate buffer (pH 10): ACN 70:30 % v/v , Flow rate: 1 ml/min LOQ : Epalrestat - 0.57 μg/ml Pregabalin – 1.51 μg/ml , LOD : Epalrestat - 0.19 μg/ml Pregabalin – 0.50 μg/ml Linearity range: Epalrestat - 37.5 – 225 μg/ml Pregabalin - 18.75 - 112.5 μg/ml Retention times: Epalrestat – 2.516 min Pregabalin – 3.132 min 36 30
- 31. Drugs Method Authors /year/ articals/ journal Abstract Summary Ref. No Etoricoxib and Drotaveri-ne RP- HPLC Syal PK, Sahoo M, Ingale KD, Ingale SS, Choudhari VP And Kuchekar BS, Scholars Research Library 2010 93-102 Column: kramosil C18 ( 250 × 4.6 mm, 5µm) Mobile phase : methanol: buffer 51:49 Flow rate : 0.9 ml/min Wavelength : 244 nm 37 Pregabalin And Nor- triptiline RP- HPLC Mewada NA, Patel BR, Patel JG, Vegad KL and Patel VS, International Journal for Pharmaceutical Research Scholars 2017 1-7 Column : BDS Hypersil C18 column (250×4.6 mm ,5 µm) Mobile Phase : buffer : methanol 70:30 Flow rate : 1 ml /min Wavelength : 210nm 38 31
- 32. Drugs Method Authors /year/ articals/ journal Abstract Summary Ref. No Epalrestat and Pregabalin RP-HPLC Sivagami I, Kavyalalitha S, INTERNATIONAL RESEARCH JOURNAL OF PHARMACY 2018 9 Column: C8 (250 x 4.6 mm, 5μm) Column. Mobile Phase: Potassium Dihydrogen Phosphate Buffer: Acetonitrile taken in the ratio 45:55 Flow rate: 1 ml/min, Wavelength: 274 nm LOQ : Epalrestat - 0.73 μg/ml Pregabalin – 0.07 μg/ml LOD : Epalrestat - 0.24 μg/ml Pregabalin – 0.02 μg/ml Retention times: Epalrestat – 2.373 min Pregabalin – 2.967 min 39 32
- 33. 4.RATIONAL 33
- 34. 34 The drug selected for the present study is Etoricoxib and Pregabalin in tablet from combination dosage form. A review of literature reveals that only few chromatographic and spectrophotometric methods have been reported for the estimation of Etoricoxib and Pregabalin separately but not combined in simultaneous method and RP-HPLC method.
- 36. 5. AIM AND OBJECTIVES 36 Aim My prime aim is to develop a simple, rapid, efficient, accurate, reproducible, less time consuming and reliable new validated RP-HPLC chromatographic method of analysis for Etoricoxib and Pregabalin in pharmaceutical dosage form. Objectives Development of rapid, sensitive and accurate RP-HPLC method for the simultaneous estimation of Etoricoxib and Pregabalin To estimate Etoricoxib and Pregabalin simultaneously from tablet dosage form by developed method and validation of proposed method.
- 37. 6. MATERIALS & EQUIPMENTS 37
- 38. 6. MATERIAL AND EQUIPMENT 38 Equipment Manufacturer UV Visible Spectrophotometer Shimadzu 1800 HPLC CYBERLAB LC100 FTIR Shimadzu Ultrasonicator HINTRON CD 4820 Measuring cylinders 50, 100, 1000 ml pH meter LABTRONICS LT-14 ISO:2008 Volumetric flask (graduated) 10, 25, 50, 100 ml Pipettes 1, 2, 5,10 ml Beakers 250, 500, 1000 ml Equipment used in method :
- 39. 39 Materials used in method : S.NO. NAME MANUFACTURER 1. Etoricoxib SUN PHARMA PVT.LID 2. Pregabalin SUN PHARMA PVT.LID 3. Potassium dihydrogen phosphate Merck 4. Sodium perchlorate Merck 5. Perchloric acid Merck 6. Ortho phosphoric acid Merck 7. Methanol Merck 8. Acetontrile Merck 9. Water Merck 10. 0.45 µm Nylon filter Axivia 11. 0.45µm PVDF filter Rankem
- 40. 40 Melting point of Etoricoxib and Pregabalin have been determined by using open capillary method. Sr. No. APIs Melting point (˚C) Reported Measured 1 Pregabalin 176 - 178°C 176-177 ˚C 2 Etoricoxib 134-135 °C 134-135˚C 7. Melting Point Determination of Etoricoxib and Pregabalin
- 41. 41 IR Spectra of Etoricoxib: 8. Identification by FTIR Spectroscopy
- 42. 42 Sr. No. Functional Group Frequency 1 N-H 3058 2 C-H 2917 3 C=C 1599 4 S=O 1297 5 C-N 1143 6 C-Cl 840 7 C-H 772 IR Spectral interpretation of Etoricoxib : Structure of etoricoxib
- 43. 43 IR Spectra of Pregabalin :
- 44. 44 Sr. No. Functional Group Practical Peaks 1 N-H 2964 2 C-H 2666 3 C=O 2206 4 C-N 1333 5 C-O 1278 6 OH 1469 IR Spectral interpretation of Pregabalin Structure of Pregabalin
- 45. 45 The solubility of Etoricoxib and Pregabalin practically determined separately by taking 100 mg of both the drugs in 100 ml volumetric flasks, adding required quantity of solvent at room temperature and shaken for few minutes. Solubility data for each study was observed and recorded. Description Terms Relative Quantities of solvent for 1prts of solute Very Soluble Less than 1 part Freely Soluble From 1 to 10 parts Soluble From 10 to 30 parts Sparingly Soluble From 30 to 100 parts Slightly Soluble From 100 to 1000 parts Very Slightly Soluble From 1000 to 10000 parts Practically Insoluble More than 10000 parts Solubility table as per IP 2010 specification 9. Solubility Study
- 46. 46 Solubility data of Etoricoxib and Pregabalin Solvent Etoricoxib Pregabalin Water Soluble Freely Soluble 0.1 N HCl soluble soluble 0.1 N NaOH Very soluble Soluble Methanol Freely soluble Freely soluble
- 47. 47 Selection of Wavelength: • 60µg/ml solution of Etoricoxib and 75µg/ml solution Pregabalin was prepared using methanol as solvent. • The above mentioned solutions were scanned individually from 190 to 400 nm in UV-Visible spectrophotometer. • The optimal response for the overlain spectrum of Etoricoxib and Pregabalin was obtained at 234 nm. • Hence the complete method was processed at the wavelength of 234 nm. • Spectrums are shown in Fig. 10. DEVELOPMENT AND OPTIMIZATION OF RP-HPLC METHOD
- 48. 48 UV spectrum of Etoricoxib and Pregabalin for detection of wave.
- 49. 49 Preparation of Phosphate buffer Weighed 2.950 grams of potassium dihydrogen phosphate and 540mg of dipotassium hydrogen ortho phosphate into a 1000 ml beaker, dissolved and diluted to 1000 ml with HPLC water, the pH was found to be 6.26. Preparation of mobile phase Mixed the above buffer 250 ml (25%) and 750 ml of Acetonitrile HPLC (75%) and degassed in ultrasonic water bath for 5 minutes. Filtered through 0.45 µ under vacuum filtration. Diluent preparation Mixed the above buffer 250 ml (25%) and 750 ml of Acetonitrile HPLC (75%) and degassed in ultrasonic water bath for 5 minutes. Filtered through 0.45 µ under vacuum filtration. Preparation of solution
- 50. 50 Standard Solution Preparation : Accurately weighed and transferred 60 mg each of Etoricoxib & 75 mg of Pregabalin working standard into a 10ml clean dry volumetric flask added about 7ml of Diluent and sonicated to dissolve it completely and made volume up to the mark with the same solvent (Stock solution). Further pipetted 1ml of Etoricoxib & 1 ml of Pregabalin the above stock solution into a 10ml volumetric flask and diluted up to the mark with diluents. The solution contains 60µg/ ml and 75 µg/ ml concentration of Etoricoxib and of Pregabalin respectively Sample Solution Preparation : Accurately weighed and transferred equivalent to 60mg of Etoricoxib/ 75 mg of Pregabalin sample into a 10ml clean dry volumetric flask added about 7ml of Diluent and sonicated to dissolve it completely and made volume up to the mark with the same solvent (Stock solution). Further pipetted 1.0ml of Etoricoxib & 1.0 ml of Pregabalin of the above stock solution into a 10ml volumetric flask and diluted up to the mark with Diluent. The solution contains 60µg/ ml and 75 µg/ ml concentration of Etoricoxib and of Pregabalin respectively.
- 51. 51 Sr no Mobile phase Ratio Remarks 1 Phosphate buffer : methanol 45:55 Retension time are very low 2 Phosphate buffer : methanol 50:50 Peack are not clear 3 Phosphate buffer : methanol 40:60 No clear peak observed 4 Phosphate buffer : methanol 30:70 Peak shape are not clear 5 Phosphate buffer: ACN 50:50 No clear peak observed 6 Phosphate buffer : ACN 25 :75 Clear peak observed
- 52. 52 TRAIL-1 Preparation of Mobile phase The mobile phase is prepared by mixing Phosphate buffer (pH-6.26) and methanol in the ratio of 45:55. The mobile phase is then sonicated using Ultra-Sonicator to remove the impurities and dissolved gases, as they may lead to unwanted peaks in the chromatogram. Mobile phase: Phosphate buffer : methanol (45:55) Chromatographic Parameters Equipment High performance liquid chromatography equipped with UV detector Column C18 column Flow rate 1.0ml/min Wavelength 234nm Injection volume 20 µl Column oven Ambient Run time 9min
- 53. 53 Chromatogram-1 Conclusion The retention time was very low for Pregabalin which has been tried for many concentrations.
- 54. 54 TRAIL-2 Preparation of Mobile phase : The mobile phase is prepared by mixing Phosphate buffer (pH-6.26) and methanol in the ratio of 50:50. The mobile phase is then sonicated using Ultra- Sonicator to remove the impurities and dissolved gases, as they may lead to unwanted peaks in the chromatogram. Mobile phase: Phosphate buffer: methanol (50:50) Chromatographic Parameters Equipment High performance liquid chromatography equipped with UV detector Column C18 column Flow rate 1.0ml/min Wavelength 234nm Injection volume 20 µl Column oven Ambient Run time 6min
- 55. 55 Conclusion The plate count was found to be less and the retention time was found to be less for Etoricoxib with an less tailing which was found unsatisfactory. Chromatogram-2
- 56. 56 TRAIL-3 The mobile phase is prepared by mixing Phosphate buffer (pH-6.26) and methanol in the ratio of 40:60. The mobile phase is then sonicated using Ultra- Sonicator to remove the impurities and dissolved gases, as they may lead to unwanted peaks in the chromatogram. Mobile phase: Phosphate buffer: methanol (40:60) Chromatographic Parameters: Equipment High performance liquid chromatography equipped with UV detector Column C18 column Flow rate 0.9 ml/min Wavelength 234nm Injection volume 20 µl Column oven Ambient Run time 9min
- 57. 57 Chromatogram-3 Conclusion The small hump was seen in chromatogram with less tailing and plate count which was carried for many times hence found unsatisfactory.
- 58. 58 TRAIL-4 The mobile phase is prepared by mixing Phosphate buffer (pH-6.26) and methanol in the ratio of 30:70. The mobile phase is then sonicated using Ultra-Sonicator to remove the impurities and dissolved gases, as they may lead to unwanted peaks in the chromatogram. Mobile phase: Phosphate buffer: methanol (30:70) Chromatographic Parameters: Equipment High performance liquid chromatography equipped with UV detector Column C18 column Flow rate 1.0ml/min Wavelength 234nm Injection volume 20 µl Column oven Ambient Run time 6min
- 59. 59 Conclusion The peak shape was not clear for Pregabalin resolution was found to be less with less plate count hence tried by changing the concentration which gave rise to the optimized method. Chromatogram-4
- 60. 60 TRAIL : 5 Preparation of mobile phase : The mobile phase is prepared by mixing Phosphate buffer (pH-6.26) and Acetonitrile in the ratio of 30:70. The mobile phase is then sonicated using Ultra- Sonicator to remove the impurities and dissolved gases, as they may lead to unwanted peaks in the chromatogram. Mobile phase: Phosphate buffer: ACN (50:50) Chromatographic Parameters : Equipment High performance liquid chromatography equipped with UV detector Column C18 column Flow rate 1.0ml/min Wavelength 234nm Injection volume 20 µl Column oven Ambient Run time 6min
- 61. 61 Chromatography 5 Conclusion The peak shape was not clear for Pregabalin resolution was found to be less with less plate count hence tried by changing the concentration which gave rise.
- 62. 62 TRAIL : 6 Preparation of mobile phase : Mixture of above buffer 250 ml (25%) and 750 ml of Acetonitrile HPLC (75%) are mixed and degassed in ultrasonic water bath for 5 minutes. Filtered through 0.45 µ under vacuum filtration. The mobile phase is sonicated using Ultra-Sonicator for 5min to remove the impurities and dissolved gases, as they may lead to unwanted peaks in the chromatogram. Mobile phase: phosphate buffer: Acetonitrile (25:75). Chromatographic Parameters : Equipment High performance liquid chromatography equipped with UV detector Column C18 Flow rate 0.8 ml/min Wavelength 234nm Injection volume 20µl Column oven Ambient Run time 8 min pH 6.26
- 64. 64 Conclusion : The retention time, shape, resolution were found to be good when compared to other methods in trails, hence this method was finalized for the estimation of Pregabalin and Etoricoxib. A simple and sensitive reverse phase HPLC method has been developed for simultaneous analysis of Pregabalin and Etoricoxib in combined dosage form. The method utilizes sample preparation followed by separation on a Cosmosil C18. Analytes were monitored by UV detection at 234 nm using an isocratic mode with phosphate buffer (pH-6.26): Acetonitrile in the ratio 25:75 as mobile phase. The flow rate was set at 0.8 ml/min and effluent was monitored at 234nm. The retention time was 2.475min and 6.528 min for Pregabalin and Etoricoxib respectively.
- 65. 65 Analyze the placebo, Etoricoxib and Pregabalin separately. A solution of placebo was spiked with the Etoricoxib and Pregabalin at its working concentration. The solution was analyzed as per the RP-HPLC method described. (i) Placebo solution: Weigh accurately about 135 mg of powdered placebo into a clean 100 ml volumetric flask. Add 25 ml of water and 0.2 ml of ammonia solution. Sonicate for 5 minute, and make up the volume with the water and filter (Concentration: 4.30 mg/ml of placebo). (ii) Standard solution (Etoricoxib): Weigh accurately about 60 mg of Etoricoxib working standard into a clean 100 ml volumetric flask. Dissolve in 25 ml of water and 0.2 ml of ammonia solution. Sonicate for 5 minute, and make up the volume with the water (Concentration: 1.0 mg/ml of Etoricoxib). (iii) Standard solution (Pregabalin): Weigh accurately about 75 mg of Pregabalin working standard into a clean 100ml volumetric flask. Dissolve in 25 ml of water and 0.2 ml of ammonia solution. Sonicate for 5 minute, and make up the volume with the water (Concentration: 0.10 mg/ml of Pregabalin). Preparation of Solution
- 66. 66 (iv) Standard solution (Etoricoxib + Pregabalin): Weigh accurately about 60 mg of Etoricoxib working standard and 75 mg of Pregabalin working standard into a clean 100 ml volumetric flask. Dissolve in 25 ml of water and 0.2 ml of ammonia solution. Sonicate for 5 minute, and make up the volume with the water (Concentration: 1.0 mg/ml of Etoricoxib and 0.10 mg/ml of Pregabalin). (v) Standard + placebo solution: Weigh accurately about 135 mg of placebo, 60 mg of Etoricoxib working standard and 75 mg of Pregabalin working standard into a clean 100 ml volumetric flask. Dissolve in 25 ml of water and 0.2 ml of ammonia solution. Sonicate for 5 minute, and make up the volume with the water (Concentration: 4.30 mg/ml of Placebo, 1.0 mg/ml of Etoricoxib and 0.10 mg/ml of Pregabalin). Acceptance criteria: The placebo chromatogram should not show any peak at the retention time of Etoricoxib and Pregabalin.
- 67. 67 Method Validation 1. Specificity: Placebo solution was prepared separately at a concentration of 4.3 mg/ml with matrix blend. Etoricoxib was prepared at its working concentration and analyzed as per the method. Pregabalin was prepared at its working concentration and analyzed as per the method. A typical chromatogram for specificity is shown in Figure. Summarizes the retention time and relative retention time values for placebopeaks, Etoricoxib and Pregabalin. Figure 7.1: Chromatogram of blank
- 68. 68 Figure 7.2: Chromatogram of etoricoxib Figure 7.3: Chromatogram of Pregabalin
- 69. 69 Figure 7.4: Chromatogram of standard Figure 7.5: Chromatogram of standard + placebo
- 70. 70 Peak name Retention time (minutes) Blank peak 3.43 Etoricoxib 4.53 Pregabalin 9.79 Table 7.1: Summary of retention time and relative retention time values for placebo peaks, Etoricoxib and Pregabalin. No peak was observed at the retention time of Etoricoxib and Pregabalin in the chromatogram of placebo.
- 71. 71 2. Linearity and Range: (a) Linearity of Etoricoxib The linearity of the RP-HPLC method was demonstrated for Etoricoxib ranging from 40 µg/ml to 80 µg/ml. Five solutions at the concentrations within the mentioned range were prepared and analyzed as per the method. The linearity results obtained are shown in Figure shows the line of best fit for peak area versus concentration of etoricoxib . (b) Linearity of Pregabalin The linearity of the RP-HPLC method was demonstrated for Pregabalin solutions ranging from 25 µg/ml to 125 µg/ml. solutions at the concentrations within the mentioned range were prepared and analyzed as per the method. The linearity results obtained are in Figure shows the line of best fit for peak area versus concentration of Pregabalin. A typicshownal chromatogram for linearity is shown in
- 72. 72 Figure 7.6: Overlay chromatogram of different concentration
- 73. 73 Sr no Conc (µg/ml) Peak area 1 40 3202601 2 50 4162205 3 60 5122685 4 70 5859779 5 80 7028796 Correlation coefficient 0.9976 Table 7.4: Linearity of Etoricoxib 0 3202601 4162205 5122685 5859779 7028796 y = 86815x - 111409 R² = 0.9976 -1000000 0 1000000 2000000 3000000 4000000 5000000 6000000 7000000 8000000 0 10 20 30 40 50 60 70 80 90 Area Con. Figure 7.13: Linearity graph for Etoricoxib
- 74. 74 Sr No Conc (µg/ml) Peak area 1 25 289460 2 50 501587 3 75 728768 4 100 929599 5 125 1154724 Correlation coefficient 0.9997 Table 7.5: Linearity of Pregabalin 0 289460 501587 728768 929599 1154724 y = 9075.7x + 43460 R² = 0.9997 0 200000 400000 600000 800000 1000000 1200000 1400000 0 20 40 60 80 100 120 140 Area Con. Figure7.6: Linearity graph for Pregabalin
- 75. 75 3. Accuracy: • The accuracy of the method was determined by using three solutions containing placebo spiked with Etoricoxib and Pregabalin at approximately 80%, 100% and 120% of its working strength. Each level was analyzed as per the method. Level Working strength Theoretical Conc ( µg/ml) Measured Conc (µg/ml) %Recovery 80% 60 48 48.1 101.43 60 48 48.2 101.09 60 48 48.0 101.44 100% 60 60 59.2 98.83 60 60 59.01 98.35 60 60 59.2 98.80 120% 60 72 72.1 101.75 60 72 72.0 101.71 60 72 72.1 101.38 Table 7.6: Accuracy of Etoricoxib The percentage recovery values were in the range of 98.35%- 101.75% which is within the acceptance criteria.
- 76. 76 Level Working strength Theoretical Conc (µg/ml) Measured Conc (µg/ml) % Recovery 80% 75 60 60.1 100.84 75 60 60.2 100.18 75 60 60.1 100.98 100% 75 75 75.0 100.70 75 75 75.0 100.01 75 75 74.6 99.58 120% 75 90 89.1 99.09 75 90 88.7 98.59 75 90 89.4 99.44 The percentage recovery values were in the range of 98.59% - 100.98% which is within the acceptance criteria. Table 7.7: Accuracy of Pregabalin
- 77. 77 4. Precision: 1. Repeatability The data for repeatability of Peak Area measurement for Etoricoxib (60μg/ml) and Pregabalin (75μg/ml) based on six measurements of same solution of Etoricoxib (60μg/ml) and Pregabalin (75μg/ml). The % RSD for Etoricoxib and Pregabalin was found to be 2. Intraday precision Standard solution containing (50, 60, 70μg/ml) of Etoricoxib and (50, 75, 100μg/ml) of Pregabalin were analyzed three times on the same day and % R.S.D was calculated. 3. Interday precision Standard solution containing (50, 60, 70μg/ml) of Etoricoxib and (50, 75, 100μg/ml) of Pregabalin were analyzed three times on the different day and % R.S.D was calculated.
- 78. 78 Precision of Etoricoxib Conc. (µg/ml) Mean peak area (mAu*sec) ±SD (n=3) % RSD Intraday precision 50 417566±1.78 0.789 60 532568±1.98 1.124 70 586578±2.45 0.362 Interday precision 50 425566±3.89 1.305 60 543568±2.67 0.950 70 596578±3.78 0.572 Repeatability Conc. (µg/ml) Mean peak area (mAu*sec) ±SD (n=6) % RSD 60 4985023±2.65 0.24
- 79. 79 Precision of Pregabalin Conc. (µg/ml) Mean peak area (mAu*sec) ±SD (n=3) % RSD Intraday precision 50 501564±2.69 0.547 75 728546±3.69 0.754 100 928512±2.65 0.369 Interday precision 50 565864±2.09 0.863 75 723546±3.87 0.968 100 935612±3.59 0.652 Repeatability Conc. (µg/ml) Mean peak area (mAu*sec) ±SD (n=6) % RSD 75 740081±4.23 0.60
- 80. 80 • The LOD and LOQ of the developed method were determined by analyzing progressively low concentration of the standard solution using the developed methods. • The LOD is the concentration of the analyte that gives a measurable response (signal to noise ratio 3.3). • The LOQ is the lowest concentration of the analyte, which gives a response that can be accurately quantified (signal to noise ratio of 10). 5. LOD and LOQ • Calibration curve was repeated for five times and the standard deviation of the intercept was calculated. Then LOD and LOQ were calculated as follows, Where, SD = Standard deviation of intercepts of 6 calibration curves Slope = Mean slope of the 5 calibration curves
- 81. 81 Etoricoxib Pregabalin LOD = 3.3 x (SD / Slope) =3.3 x (86815/111409) =2.5715 μg/ml LOD = 3.3 x (SD / Slope) =3.3 x (9075.7/43460) =0.6891μg/ml Etoricoxib Pregabalin LOQ = 10 x (SD / Slope) =10 x (86815/111409) =7.7924 μg/ml LOQ = 10 x (SD / Slope = 10 x (9075.7/43460) = 2.088 μg /ml Limit of Quantitation Limit of Detection
- 82. 82 5. Robustness: The following table (Table 7.11) shows the parameters of the method that were altered to test the robustness of the method. The robustness of the method is to be determined by analyzing the standard solution six times with varying RP-HPLC conditions as described below. A typical chromatogram for robustness is shown in Figure. Parameter Actual Low High Flow rate 1.00 ml/min 0.90 ml/min 1.10 ml/min Mobile phase ratio 75 : 25 78 : 22 73 :27 Buffer pH 2.20 2.10 2.30 Column oven temperature 30°C 28°C 32°C Table 7.11: Parameters altered for robustness test
- 83. 83 6.1 Chromatographic conditions (Actual): Column : C18, 250 X 4.6 mm, 5 μm or equivalent Flow rate : 1.0 ml / min Detection wavelength : 234 nm Injection volume : 20 ml Oven temperature : 30°C Mobile phase : phosphate buffer: Acetonitrile (25:75). Figure 7.20: Chromatogram of robustness- standard (Actual) Figure 7.21: Chromatogram of robustness- sample (Actual)
- 84. 84 6.2 Chromatographic conditions (Flow-Low): Column : C18, 250 X 4.6 mm, 5 μm or equivalent Flow rate : 0.9 ml / min Detection wavelength : 234 nm Injection volume : 20 ml Oven temperature : 30°C Mobile phase : phosphate buffer: Acetonitrile (78 : 22). Figure 7.22: Chromatogram of robustness- standard (Flow low) Figure 7.23: Chromatogram of robustness- sample (Flow low)
- 85. 85 6.3 Chromatographic conditions (Flow-High): Column : C18, 250 X 4.6 mm, 5 μm or equivalent Flow rate : 1.1 ml / min Detection wavelength : 234 nm Injection volume : 20 ml Oven temperature : 30°C Mobile phase : phosphate buffer: Acetonitrile (73 :27) Figure 7.24: Chromatogram of robustness- standard (Flow high) Figure 7.25: Chromatogram of robustness- sample (Flow high)
- 86. 86 Flow Actual Low High ET PRE ET PRE ET PRE RT (min) 4.30- 4.53 9.09- 9.27 4.22- 4.23 8.12- 8.16 4.57- 4.59 10.16- 10.43 Area 4966028- 4991449 745914- 779360 4836134- 4872558 764132- 771434 4940974- 5020412 782565- 791915 Tailing factor 1.19 1.05 1.21 1.03 1.21 1.01 Theoretical plates 8586 14487 8604 13715 8586 14487 Average (Area) 4978738 762637 4850959 772652 4967201 786025 %RSD (Area) 0.27 0.65 0.28 0.73 0.59 0.43 Table 7.12: Summary of robustness results of standard (Flow)
- 87. 87 Table 7.13: Summary of robustness results of sample (Flow) Flow Actual Low High ET PRE ET PRE ET PRE RT (min) 4.31- 4.40 9.09- 9.27 5.14- 5.22 11.29- 11.38 4.13- 4.15 8.99- 9.05 Area 5003737- 4966028 735393- 745914 5775729- 5640003 873982- 890362 4585191- 4469026 715423- 720801 Tailing factor 1.23 1.05 1.19 1.02 1.20 1.03 Theoretical plates 8638 14392 10171 16407 8501 14020 Average (Area) 4985023 740081 5705806 885693 4519688 718206 %RSD (Area) 0.24 0.60 0.98 0.73 1.14 0.38
- 88. 88 DISCUSSION • The working condition for the RP-HPLC method were established for Pregabalin and Etoricoxib then applied on pharmaceutical dosage form. • Mobile phase system were prepared and used to provide ratio of Phosphate Buffer: ACN (75:25) gave a better resolution and sensitivity. The retention time of Pregabalin and Etoricoxib were found to be 9.79 and 4.53 respectively. The asymmetry factor or tailing factor of Pregabalin and Etoricoxib is 1.05 and 1.23 respectively which indicates symmetrical nature of peak. • The number of theoretical plates of Pregabalin and Etoricoxib was 14392 and 8638 respectively which indicates efficient performance of column. The retention time of drugs were found to be within the limits of 0-10 minutes. These parameters represent the specificity of method.
- 89. 89 • From the linearity studies, specified concentration range was determined. It was observed that Pregabalin and Etoricoxib were in the range of 50% - 150% each for the target concentration. The linearity range is 40 – 80 µg/ml for Etoricoxib and 25 – 125 µg/ml for Pregabalin were found to obey linearity with correlation coefficient of 0.9976 for Etoricoxib and 0.9997 for Pregabalin. The validation for the proposed method was verified by system precision and method precision. There was %RSD of system precision for Pregabalin and Etoricoxib was 0.60 and 0.24 respectively. The method precision was conducted and the % label claim was obtained as 103.71% for Etoricoxib and 105.16% for Pregabalin. The % recovery value of 98.35% - 101.75% for Etoricoxib and 98.58% - 100.98% for Pregabalin indicates non-interferences of excipient in the formulation. • The validation of the proposed method was verified by recovery studies. The robustness studies were performed by changing the flow-rate, pH, column temperature and mobile phase concentration. The ruggedness studies were performed. All parameters including flow-rate, temperature, detector, wavelength and sensitivity were maintained constant throughout the procedure. The analytical method validation was carried as per the ICH guidelines.
- 90. 90 SUMMARY REPORT: S.No Parameters Observation Acceptance Criteria 1 Specificity Placebo Interference No peak was observed at the retention time of Etoricoxib and Pregabalin in the chromatogram of placebo The placebo chromatogram should not show any peak at the retention time of Etoricoxib and Pregabalin. 2 Linearity & Range (a) Etoricoxib 0.9976 Correlation coefficient: > 0.995 (b) Pregabalin 0.9997 3 Accuracy (a) Etoricoxib 98.35 – 101.75 % 98.0 – 102.0% (b) Pregabalin 98.58 – 100.98 % 4 Precision (ET) Intraday Precision (%RSD, n=3) 0.75 % RSD: < 2.0 Interday Precision (% RSD, n=3) 0.94 Repeatability (% RSD, n=6) 0.24
- 91. 91 Sr. No Parameters Observation Acceptance criteria Precision (PRE) Intraday Precision (%RSD, n=3) 0.55 % RSD: < 2.0 Interday Precision(% RSD, n=3) 0.82 Repeatability (% RSD, n=6) 0.60 5 LOD (a) Etoricoxib 2.57 >2 or 3 (b) Pregabalin 0.68 6 LOQ (a) Etoricoxib 7.79 <10 (b) Pregabalin 2.08 7 Robustness (a) Etoricoxib 1.19 – 1.23 Tailing factor: <2.0 8501 – 10171 Theoretical plates: > 2000 0.06 – 1.14 % RSD: < 2.0 (b) Pregabalin 1.00 – 1.05 Tailing factor: < 2.0 13715 –16403 Theoretical plates: > 2000 0.32 – 0.73 % RSD: < 2.0
- 92. 92 CONCLUSION • C18 column as stationary phase and phosphate buffer: Acetonitrile (25: 75) as a mobile phase and 234 nm is selected for the detection in this RP-HPLC. • The system precision shows RSD value obtained was below 1 which indicate precision and retention time for Etoricoxib and Pregabalin were 4.53 and 9.79 respectively. • Quantitative estimation of Etoricoxib and Pregabalin gives accuracy which between 98.35% – 101.75% for Etoricoxib and 98.58% - 100.98% for Pregabalin. • The percentage purity of Etoricoxib and Pregabalin lies in between 103.71% and 105.16% respectively. • The proposed RP-HPLC method was simple, precise, rapid & accurate and involves easy sample preparation. The linearity, reproducibility and recovery data confirms no major interferences in the assay determination. So, this method can be used for routine quality control analysis of this drug.
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- 100. 100