SECTION I: DEVELOPMENT AND VALIDATION OF A STABILITY INDICATING HPLC ASSAY METHOD FOR DETERMINATION OF TAPENTADOL HYDROCHLORIDE IN TABLET FORMULATION

Transcription

1 PART - A SECTION-I DEVELOPMENT AND VALIDATION OF A STABILITY INDICATING HPLC ASSAY METHOD FOR DETERMINATION OF TAPENTADOL HYDROCHLORIDE IN TABLET FORMULATION

2 SECTION I: DEVELOPMENT AND VALIDATION OF A STABILITY INDICATING HPLC ASSAY METHOD FOR DETERMINATION OF TAPENTADOL HYDROCHLORIDE IN TABLET FORMULATION 1. INTRODUCTION OF TAPENTADOL HYDROCHLORIDE Tapentadol is a novel centrally acting analgesic that was approved for use by the Food and Drug Administration. It has structural similarities to tramadol. It provides analgesia at similar levels of more potent narcotic analgesics such as hydrocodone, oxycodone and morphine, but with a more tolerable side effect profile. Tapentadol has been approved for use as immediate release oral tablets in dosage forms of 50 mg, 75 mg and 100 mg. Tapentadol has been placed in schedule II by the FDA, as having a high potential for abuse [1]. 1.1 Description Tapentadol is chemically (-)-(1R,2R)-3-(3-Dimethylamino-1-ethyl-2-methylpropyl)-phenol hydrochloride (Figure 1).Its molecular formula is C 15 H 15 NO 2 S. HCl having molecular weight gm/mole. Tapentadol is a µ-opioid receptor agonist and as a norepinephrine reuptake inhibitor [2]. HO N Figure: 1 (-)-(1R,2R)-3-(3-dimethylamino-1-ethyl-2-methyl-propyl)-phenol 1.2 Pharmacodynamics Tapentadol was characterized as an µ-opioid receptor agonist and a norepinephrine transporter inhibitor in receptor binding assays and in functional µ-opioid receptor and norepinephrine synaptosomal reuptake assays. Norepinephrine and µ-opioid receptor reuptake inhibitors have analgesic effects, although the pain conditions in which 39

3 these two drug classes are most efficacious may be different. For example, it appears that µ-opioid receptor agonists are mostly effective against acute moderate-to-severe pain, whereas norepinephrine reuptake inhibitors are particularly effect against chronic pain. This implies that a medication that combines both mechanisms of action may be effective a broad spectrum of pain conditions. The exact mechanism of action is unknown [3]. Tapentadol is a centrally acting oral analgesic with a dual mechanism of action, combining mu-opioid receptor agonist and norepinephrine reuptake inhibition in a single molecule. Norepinephrine plays a role in the endogenous descending pain inhibitory system, and the analgesic efficacy of norepinephrine reuptake inhibitors has been shown in neuropathic pain. Analgesic effect of tapentadol has been demonstrated in a wide range of animal models of pain with nociceptive and neuropathic components, and development of tolerance to its analgesic effect was twice as slow as that of morphine. Although Tapentadol has a 50-fold lower binding affinity to mu-opioid receptor, its analgesic potency is only 2 to 3 times lower than that of morphine, indicating that the dual mode of action may result in an opiates paring effect [4]. 1.3 Pharmacokinetics Absorption of Tapentadol is rapid with the C max occurring in the serum at between hours post dose. Tapentadol is primarily detected as conjugated metabolites in the serum; the C max of the conjugates is between hours post dose. Approximately 99% of a dose is accounted for in the urine and 1% in the feces. More than 50% of the dose is excreted after 4 hours (t 1/2 = 3.93 hours) and over 95% within 24 hours of dosing [5]. Tapentadol has no pharmacologically active metabolites. The primary metabolic pathway is via glucuronidation, with sulfation of the phenolic hydroxyl group occurring to a minor extent. Minor phase-i bio transformations include hydroxylation of the aromatic ring, as well as demethylation and subsequent conjugation. The results of in vitro studies show that these metabolites are either unable to bind to, or have a low affinity for the µ-opioid receptor, and therefore are not likely to contribute to any analgesic activity. 40

4 2. LITERATURE REVIEW The literature reviews regarding Tapentadol hydrochloride suggest that various analytical methods were reported for its determination as drug, in pharmaceutical formulation and in various biological fluids. The literature reviews for analysis of Tapentadol hydrochloride are as under: 1. Sherikar, Omkar D.; Mehta, Priti J. have developed and validated three methods for determination of Tapentadol hydrochloride in bulk and laboratory tablet sample. In RP-HPLC method, elution was achieved in isocratic mode using combination of 50 mm phosphate buffer ph 3.62 and acetonitrile in ratio of 70:30 (% v/v) with 0.1% triethylamine and using HiQSil C8 column having specification, mm and 5 mm particle size. The flow rate was 1 ml/min and detection was done at 285 nm. UVspectrophotometric determination of Tapentadol was carried out at 272 nm. Third method consists of quantification of Tapentadol using Folin-Ciocalteu reagent in presence of 20% sodium carbonate solution. The blue color chromogen formed is measured at wavelength of maximum absorption 750 nm for Tapentadol against reagent blank. All 3 developed methods were validated according to ICH guidelines [7]. 2. Dousa, Michal; Lehnert, Petr; Adamusova, Hana; Bosakova, Zuzana have developed and validated a sensitive and specific high performance liquid chromatographic method for the seperation and determination of tapentadol enantiomers. Ten different chiral columns were tested in a normal phase system. Excellent enantioseparation with the resolution more than 2.5 for all enantiomers was achieved on Chiralpak AD-H using mixture of heptanepropan-2-ol-diethylamine (980:20:1, v/v/v). The detection was carried out using fluorescence detector at excitation wavelength of 295 nm and emission wavelength of 273 nm. The influence of mobile phase composition, mainly organic modifiers, additives, aliphatic alkanes and water content in mobile phase, on retention and enantio-separation was studied. This method is suitable for routine determination of chiral purity of (R,R)-Tapentadol in enantiopure active pharmaceutical ingredient [8]. 3. Kathirvel, Singaram; Satyanarayana, SuggalaVenkata; Devalarao, Garikapati have documented simple, rapid, selective, and isocratic RP-LC method for the quantitation and determination of tapentadol and its related substances in bulk samples and pharmaceutical dosage forms in the presence of its 2 process-related impurities. Chromatographic seperation was achieved on the reversed phase, Enable column (C18 (5-mm, mm, i.d.)) at ambient temperature using a mobile phase 41

5 consisting of 0.02 M potassium dihydrogen orthophosphate (adjusted to ph 6 with 1 M KOH) and acetonitrile (80:20, v/v). Flow rate was 1 ml/min with UV detection at 215 nm [9]. 4. Goud, EdigaSasiKiran; Reddy, V. Krishna have determined sensitive, specific, precise, and linear reverse phase HPLC method for the analysis of related substances in Tapentadol in bulk and pharmaceutical dosage form. The known related substances are methoxy impurity [(2R,3R)-3-(3-methoxyphenyl)-N,N,2-trimethylpentanamine] and alcoholic impurity [(2S)-1-dimethylamino-3-(3-methoxyphenyl)-2-methylpentan-3-ol hydrochloride]. The method was carried out on a Zodiac C18 column (250 mm 4.6 mm; 5 µm) using a mobile phase mixture of phosphate buffer ph 7.0, acetonitrile and MeOH in a gradient elution at a flow rate of 1.0 ml/min at wavelength of 220 nm. The method can be used for the detection and quantitative estimation of known and unknown impurities in drug and pharmaceutical dosage form [10]. 5. Marin, Stephanie J.; Hughes, John M.; Lawlor, Bryan G.; Clark, Chantry J.; McMillin, Gwendolyn A. have developed a fast (7.5 min) liquid chromatography-timeof-flight mass spectrometry (LC-TOF-MS) method in which sixty-seven drugs and metabolites were separated in serum or plasma. This method was developed as a blood drug screen, with emphasis on the detection of common drugs of abuse and drugs used to manage chronic pain. Compound identification is based on chromatographic retention time, mass, isotope spacing and isotope abundance. Data analysis software (Agilent) generates a compound score based on how well these observed criteria matched theoretical and empirical values. The method was validated using fortified samples and 299 residual patient specimens. The accuracy of positive results was >90% for drugs and/or metabolites[11]. 6. Bhatasana, Purvi T.; Parmar, Ashok R. have investigated reversed phase highperformance liquid chromatography method for the quantification of Tapentadol hydrochloride in tablet dosage form. The mobile phase consisting of solvent A MeOH: Solvent B Acidic water (ph 3.8 adjusted by triethylamine and o-phosphoric acid) in ratio of (58:42% v/v) was delivered at the flow rate of 1.2 ml/min and UV detection was carried out at 271 nm. The validation of method carried out as per ICH guidelines. As per validation data it was found that method is specific, robust, and precise within the described concentration range [12]. 7. Jiang, Xue; Jin, Yi; Xu, Haiyan; Xu, Pingwei; Zhai, Nannan; Yuan, Bo have derived a method to identify the metabolites of Tapentadol in rat urine, the Tapentadol 42

6 and its metabolites in the rat urine were identified or confirmed through MRM and full scan MS2 by liquid chromatography tandem mass spectrometry. The parent drug and its fifteen kinds of metabolites were found in the urine. Among the metabolites, dehydro- Tapentadol and dehydro-tapentado-o-glucuronide were firstly discovered. The LC- MS/MS method is a simple and fast way for the analysis of the metabolites of Tapentadol in the rat urine [13]. 8. Ramanaiah, Ganji; Ramachandran, D.; Srinivas, G.; Jayapal, G.; Rao, Purnachanda; Srilakshmi, V. have documented simple, rapid, selective, precise, and accurate isocratic reverse phase high performance liquid Chromatography assay method for simultaneous estimation of Tapentadol and Paracetamol in tablet formulations. The seperation was achieved by C18 column (Hypersil BDS, mm i.d.); in mobile phase ph 6.8 Phosphate Buffer and MeOH in the ratio of 700:300 vol./vol. The flow rate was 1.0 ml/min and the sepd. drugs were detected using UV detector at the wavelength of 215 nm. The method was validated as per ICH guidelines [14]. 9. Jin, Yi; Jiang, Xue; Zhai, Nannan; Xu, Pingwei; Yuan, Bo; Xu, Haiyan have derived sensitive and rapid LC-MS/MS method to determine the concentration of Tapentadol in rat plasma. After the extraction from plasma by protein precipitation, analytes and internal standards were separated by a Diamonsil C18 column. Methanol-5 mmol/l ammonium acetate-acetic acid (58:42:0.5, v:v:v) were used as the mobile phase. The multiple reaction monitoring was used for quantitative. determination in positive mode. The transitions were m/z: for Tapentadol, m/z: for fluconazol. No significant interferences for the detection of Tapentadol and fluconazol from endogenous substances in plasma were observed in the present study [15]. 10. Giorgi, M.; Meizler, A.; Mills, P. C. have developed and validated a simple HPLC-FLOURECENCE based method to quantify TAP in plasma. Several parameters both in the extraction and detection method were evaluated. The applicability of the method was determined by administering TAP orally to two dogs; the protocol yielded the expected pharmacokinetic results and plasma collected by jugular venipuncture at regular intervals. The mobile phase consisted of acetonitrile (A):acetic acid (B) (33 mm), delivered in gradient mode (5-95% B [0-20 min], 95-5% B [20-25 min] and finally 5% B isocratically [25-32 min]) with a flow rate of 1 ml/ min. Excitation and emission wavelengths were of 273 and 298 nm, respectively. TAP was extracted from the plasma using a mixture of Et 2 O:CH 2 Cl 2 (7:3, v/v) [16]. 43

7 3. AIM OF PRESENT WORK As per discussion in the literature review UV, LC-MS and HPLC methods for the determination of Tapentadol hydrochloride in pharmaceutical dosage forms or in metabolite and plasma are reported. HPLC is the most commonly used method for analysis of Tapentadol hydrochloride. An extensive literature survey reveals few HPLC methods for estimation of Tapentadol hydrochloride in pharmaceutical dosage forms as well as biological fluids; however, not all of these are stability indicating. Most of the reported methods either do not include stress degradation studies or are not completely optimized and validated, and they are cumbersome, time-consuming and expensive. Method validation is an essential step in drug analysis. The process confirms that the analytical procedure employed for the analysis is suitable for its intended use and shows reliability of the results produced by any method. The primary objective of the present work was thus to develop and validate a stability indicating HPLC method for the assay of Tapentadol hydrochloride from its dosage form (tablets). This work also deals with the forced degradation of Tapentadol hydrochloride under stress condition like acid hydrolysis, base hydrolysis, and oxidation, thermal and photolytic stress. Hence, the method is useful for routine quality control analysis and also for determination of stability. The aim and scope of the proposed work are as under: To develop suitable HPLC method for Tapentadol hydrochloride. Forced degradation study of Tapentadol hydrochloride under stress condition. To resolve all major impurities generated during the force degradation studies of Tapentadol hydrochloride. Perform the validation for the developed method. 44

8 4. EXPERIMENTAL 4.1 Materials Tapentadol hydrochloride standard was provided by Ami Life sciences Laboratories Ltd., Baroda (India). Tapentadol tablets containing 50 mg Tapentadol hydrochloride and the inactive ingredient used in drug matrix were obtained from market. HPLC grade methanol was purchased from Spectrochem Pvt. Ltd., Mumbai (India). HPLC grade water was produced in-house by Milli Q (Millipore, Millford, USA) system. Membrane filters of 0.45µm (Millipore) were used. Analytical grade ortho-phosphoric acid, hydrochloric acid, sodium hydroxide pellets and 30% v/v hydrogen peroxide solution were obtained from Ranbaxy Fine Chemicals, New Delhi (India). 4.2 Instrumentation The chromatographic system used to perform development and validation of this assay method was comprised of a LC-10ATvp binary pump, a SPD-M10Avp photodiodearray detector and a rheodyne manual injector model 7725i with 20μl loop (Shimadzu, Kyoto, Japan) connected to a multi-instrument data acquisition and data processing system (Class-VP 6.13 SP2, Shimadzu). 4.3 Mobile phase preparation The mobile phase consisted of methanol M potassium dihydrogen phosphate buffer ph 3.0(60: 40 v/v). To prepare the buffer solution, g potassium dihydrogen phosphate were weighed and dissolved in 1000 ml HPLC grade water and then adjusted to ph 3.0 with ortho-phosphoric acid. Mobile phase was filtered through a 0.45 μm nylon membrane (Millipore Pvt. Ltd. Bangalore, India) and degassed in an ultrasonic bath (Spincotech Pvt. Ltd., Mumbai). 4.4 Diluent Preparation HPLC grade water was used as diluent. 4.5 Standard Preparation Tapentadol hydrochloride standard stock solution containing 500µg/ml was prepared in a 100 ml volumetric flask by dissolving 50.00mg of Tapentadol and then diluted to volume with water as a diluent. Further take 10 ml of this stock solution in 50 ml volumetric flask and make up to mark with diluents. The concentration obtained was 100 µg/ml of Tapentadol hydrochloride. 45

9 4.6 Test Preparation Twenty tablets were weighed and the average weight of tablet was determined. From these, five tablets were weighed and transfer into a 500 ml volumetric flask. About 50 ml of diluent was added and sonicated for a minimum 30 min. with intermittent shaking. Then content was brought back to room temperature and diluted to volume with diluent. The sample was filtered through 0.45µm nylon syringe filter. Further take 10 ml of this stock solution in 50 ml of volumetric flask and make up to mark with diluent. The concentration obtained was 100 µg/ml of Tapentadol hydrochloride. The degradation samples were prepared by transferring powdered tablets, equivalent to 50 mg of Tapentadol hydrochloride into a 250 ml round bottom flask. Then prepared samples were subjected to acidic, alkaline and oxidant media and also for thermal and photolytic conditions. After completing the degradation treatments, the stress content solutions were allowed to equilibrate to room temperature and diluted with mobile phase to attain 100 µg/ml concentrations of Tapentadol hydrochloride. Specific conditions were described as follows. 4.7 Chromatographic Conditions Chromatographic analysis was performed on a Phenomenex Luna C8 (150mm 4.6mm i.d., 5μm particle size) column applying an isocratic elution using methanol M potassium dihydrogen phosphate buffer ph 3.0 (60: 40 v/v) as a mobile phase. The mobile phase was filtered through 0.45μm membrane filter and degassed for 30 minute in an ultrasonic bath prior to its use. Flow rate of mobile phase was adjusted to 1.00 ml/min and injection volume was 20 μl. The chromatographic experiment was performed at ambient temperature and detection was carried out at 272 nm. The chromatographic run time was up to 10 minutes. In degradation study chromatography was done upto 30 minutes to observe whether any degradation product was eluted after specified run time (10 minutes) or not. 46

10 5. RESULT AND DISCUSSION 5.1 Development and Optimization of the HPLC Method Proper selection of the method depends upon the nature of the sample (ionic or ionisable or neutral molecule), its molecular weight and solubility. Tapentadol hydrochloride can be dissolved in polar solvent hence RP-HPLC was selected to estimate them. To develop a rugged and suitable HPLC method for the quantitative determination of Tapentadol hydrochloride, the analytical conditions were selected after testing the different parameters such as diluents, buffer, buffer concentration, organic solvents for mobile phase and mobile phase composition and other chromatographic conditions. Our preliminary trials using different composition of mobile phases consisting of water with methanol or acetonitrile, did not give good peak shape. The mobile phase consisted of methanol M potassium dihydrogen phosphate buffer ph 3.0 (60: 40 v/v). To prepare the buffer solution, g ammonium potassium dihydrogen phosphate was weighed and dissolved in 1000 ml HPLC grade water. Mobile phase was filtered through 0.45 μm nylon membrane (Millipore Pvt. Ltd. Bangalore, India) and degassed in an ultrasonic bath (Spincotech Pvt. Ltd., Mumbai). By using M potassium dihydrogen phosphate buffer in 1000 ml of HPLC water and keeping mobile phase composition as methanol M potassium dihydrogen phosphate buffer (60: 40, v/v), best peak shape was obtained. For the selection of organic constituent of mobile phase, methanol was chosen to resolve degradation peaks of drug from drug peak properly and to attain good peak shape. Figure 2 represents UV spectrum for wavelength selection. Figure 3 and Figure 4 represent the chromatograms of standard and test preparation respectively. Figure 2: Wavelength selection of standard preparation 47

11 Figure 3: Chromatogram of standard preparation Figure 4: Chromatogram of test preparation 5.2 Degradation Study The degradation samples were prepared by transferring powdered tablets, equivalent to 50.0 mg Tapentadol hydrochloride into a 250 ml round bottomed flask. Then drug content were employed for acidic, alkaline and oxidant media and also for thermal and photolytic stress conditions. After the degradation treatments were completed, the stress content solutions were allowed to equilibrate to room temperature and diluted with diluent to attain 100 µg/ ml Tapentadol hydrochloride concentrations. Specific degradation conditions were described as follows. 48

12 5.2.1 Acidic condition Acidic degradation study was performed by heating the drug content in 0.1 N HCl at 60 C for 30 min and mixture was neutralized. In acidic degradation, it was found that around 7% of the drug degraded. (Figure 5) Figure 5: Chromatogram of acidic forced degradation study Alkaline condition Alkaline degradation study was performed by ambient temperature in 0.05N NaOH for 30 min and mixture was neutralized. In alkali degradation, it was found that around 22 % of the drug degraded. (Figure 6) Figure 6: Chromatogram of alkali forced degradation study Oxidative condition Oxidation degradation study was performed by heating the drug content in 30% v/v H 2 O 2 at 80 C for 45 min. Major degradation was found in oxidative condition that product was degraded up to 12 %. (Figure 7) 49

13 Figure 7: Chromatogram of oxidative forced degradation study Thermal condition Thermal degradation was performed by exposing solid drug to dry heat at 80 C in a conventional oven for 72 hr. In thermal degradation, it was found that around 0.45 % of the drug degraded. (Figure 8) Figure 8: Chromatogram of thermal degradation study Photolytic condition Photolytic degradation study was performed by exposing the drug content in UVlight for 72 hours. In photolytic degradation, it was found that around 0.26% of the drug degraded. (Figure 9) 50

14 Figure 9: Chromatogram of UV-light degradation study 5.3 Method Validation Specificity The specificity of the method was determined by checking the interference of placebo with analyte and the proposed method was eluted by checking the peak purity of Tapentadol hydrochloride during the force degradation study. The peak purity of the Tapentadol hydrochloride peak was found satisfactory (0.999) under different stress condition. There was no interference of any peak of degradation product with drug peak Linearity Seven points calibration curve were obtained in a concentration range from μg/ml for Tapentadol hydrochloride. The response of the drug was found to be linear in the investigation concentration range and the linear regression equation was y = 3E+07X with correlation coefficient (Figure 10) Chromatograms obtained during linearity study were shown in figure Figure 10: Linearity curve for Tapentadol hydrochloride 51

15 Figure 11: Linearity study chromatogram of level-1 (40%) Figure 12: Linearity study chromatogram of level-2 (60%) Figure 13: Linearity study chromatogram of level-3 (80%) 52

16 Figure 14: Linearity study chromatogram of level-4 (100%) Figure 15: Linearity study chromatogram of level-5 (120%) Figure 16: Linearity study chromatogram of level-6 (140%) 53

17 Figure 17: Linearity study chromatogram of level-7 (160%) LOD and LOQ The limit of detection and limit of quantification were evaluated by serial dilutions of Tapentadol hydrochloride stock solution in order to obtain signal to noise ratio of 3:1 for LOD and 10:1 for LOQ. The LOD value for Tapentadol was found to be 0.6 ppm and the LOQ value 0.2 ppm. Chromatograms of LOD and LOQ study were shown in Figure Figure 18: Chromatogram of LOD Study of Tapentadol hydrochloride 54

18 Figure 19: Chromatogram of LOQ study of Tapentadol hydrochloride Precision The result of repeatability and intermediate precision study are shown in Table 1. The developed method was found to be precise as the %RSD values for the repeatability and intermediate precision studies were < 0.50 % and < 0.94 %, respectively, which confirm that method was precise. Table 1: Evaluation data of precision study Set Intraday (n = 6) Interday (n = 6) Mean Standard deviation % RSD Accuracy The HPLC area responses for accuracy determination are depicted in Table 2. The result shows that excellent recoveries ( %) of the spiked drug were 55

19 obtained at each added concentration, indicating that the method was accurate. Chromatograms obtained during accuracy study were shown in Figure Table 2: Evaluation data of accuracy study Level (%) Amount added concentration a (mg/ml) Amount found concentration a (mg/ml) % Recovery % RSD a Each value corresponds to the mean of three determinations Figure 20: Accuracy study chromatogram of level-1 (50%) Figure 21: Accuracy study chromatogram of level-2 (100%) 56

20 Figure 22: Accuracy study chromatogram of level-3 (150%) Solution stability study Table 3 shows the results obtain in the solution stability study at different time intervals for test preparation. It was concluded that the test preparation solution was found stable up to 48 h at 2-8 C and ambient temperature, as during this time the result was not decreased below the minimum percentage. Table 3: Evaluation data of solution stability study Intervals % Assay for test preparation solution stored at 2-5 C % Assay for test preparation solution stored at ambient temperature Initial h h h h

21 5.3.7 Robustness The result of robustness study of the developed assay method was established in Table 4. The result shown that during all variance conditions, assay value of the test preparation solution was not affected and it was in accordance with that of actual. System suitability parameters were also found satisfactory; hence the analytical method would be concluded as robust. Chromatograms obtained during robustness study were shown in figure Table 4: Evaluation data of robustness study Robust conditions % Assay System suitability parameters Theoretical Asymmetry plates Flow 0.9 ml/min Flow 1.1 ml/min Buffer ph Buffer ph Methanol-Buffer (58: 42,v/v) Methanol-Buffer (62: 38,v/v) Column change Figure 23: Standard chromatogram (0.9 ml/min flow rate) 58

22 Figure 24: Standard chromatogram (1.1 ml/min flow rate) Figure 25: Standard chromatogram (Methanol-Buffer (62: 38, v/v)) Figure 26: Standard chromatogram (Methanol-Buffer (58: 42, v/v)) 59

23 Figure 27: Standard chromatogram (Column change) System suitability A system suitability test of the chromatographic system was performed before each validation run. Five replicate injections of standard preparation were injected and asymmetry, theoretical plate and % RSD of peak area were determined for same. Acceptance criteria for system suitability, asymmetry not more than 2.0, theoretical plate not less than 4000 and % RSD of peak area not more than 2.0 were fulfilled during all validation parameter. 60

24 6. CALCULATIONS AND DATA Calculation formula used 1. Calculation formula for % assay of Tapentadol hydrochloride Mean Test Area Standard Weight % Assay = Mean Standard Area Test Weight Mean Test Weight Potency Lable Claim of Standard 2. Relative standard deviation Standard Deviation of Measurments % RSD = 100 Mean Value of Measurments 3. Recovery % Recovery = Amount Amount found 100 Added 4. Amount found Mean Test Area Amount Found ( mg / ml) = Standard Mean Standard Area Concentration 5. Amount added Weight Amount Added (mg/ml) = Volume 61

25 Specificity Study for Analytical Method Validation of Tapentadol hydrochloride Tablets Standard weight (mg) 50.0 Standard dilution Standard potency 100% Standard concentration (mg/ml) Replicate Standard area Mean standard area Standard deviation %RSD Test Replicate area Mean test area Test weight (mg) Calculation: Label claim (mg) 50.0 Mean test weight (mg) % Assay Prototype calculation for one set: % Assay = = % 62

26 Linearity Study for Analytical Method Validation of Tapentadol hydrochloride Tablets Standard weight (mg) 50.0 Standard dilution Standard potency 100% Standard concentration (mg/ml) Concentration of linearity stock sol Replicate Standard area Mean standard area Standard deviation %RSD Concentration level % Volume of linearity stock solution taken (ml) Diluted to (ml) Final concentration (mg/ml) Mean area Correlation coefficient Slope Intercept

27 Precision Study for Analytical Method Validation of Tapentadol hydrochloride Tablets Standard weight (mg) 50.2 Standard dilution Standard potency 100 Label claim (mg) 50 Mean test weight(mg) Standard concentration (mg/ml) Replicate Standard area Mean standard area Standard deviation %RSD Description Mean area Test weight (mg) % Assay Set Set Set Set Set Set Mean Standard deviation %RSD Calculation: Prototype calculation for one set: % Assay = = % 64

28 Intermediate precision study for Analytical Method Validation of Tapentadol hydrochloride Tablets Standard weight (mg) 49.8 Standard dilution Standard potency 100% Mean test weight(mg) Standard concentration (mg/ml) Replicate Standard area Mean standard area Standard deviation %RSD Description Mean area Test weight (mg) % Assay Set Set Set Set Set Set Mean Standard deviation %RSD Calculation: Prototype calculation for one set: % Assay = = % 65

29 Comparison for Precision and Intermediate Precision Study for Analytical Method Validation for Tapentadol hydrochloride Tablets Set %Assay Precision study Intermediate precision study Mean Standard deviation %RSD

30 Accuracy study for Analytical Method Validation of Tapentadol hydrochloride tablets Standard weight (mg) 50.0 Standard dilution Standard potency % Standard concentration (mg/ml) Replicate Standard area Mean standard area Standard deviation % RSD

31 Recovery Level 50% 100% 150% * S.D.= Standard deviation Mean area Weight (mg) Volume (ml) Amount added concentration (mg/ml) Amount Found concentration (mg/ml) % Recovery set set set set set set set set set Mean % Recovery *S.D. % RSD Calculation: Prototy pe calculation for one set: Amount Found ( mg / ml) = = mg/ml Amount Added ( mg / ml) = = mg/ml % Recovery = 100 = %

32 Robustness Study for Analytical Method Validation of Tapentadol hydrochloride Tablets Flow Rate at 0.9ml/min Flow Rate at 1.1ml/min Buffer ph =2.8 Buffer ph=3.2 Methanol-Buffer 62: 38 Methanol-Buffer 58: 42 Column Change Replicate Standard Area Standard Area Standard Area Standard Area Standard Area Standard Area Standard Area Mean S.D % RSD Replicate Test Area Test Area Test Area Test Area Test Area Test Area Test Area Mean Standard weight (mg) Test weight (mg) Label claim (mg) Mean test weight (mg) % Assay

33 Part-A (Section- I) Solution Stability Study for Analytical Method Validation of Tapentadol hydrochloride Tablets System suitability of standard preparation for solution stability Initial After 12 After 24 After 36 hours hours hours After 48 hours Standard Standard Standard Standard Standard Replicate Peak area Peak area Peak area Peak area Peak area Mean S.D %RSD Solution stability for standard preparation at 2-8 C After 12 hours After 24 hours After 36 hours After 48 hours Standard Standard Standard Standard Replicate Peak area Peak area Peak area Peak area Mean S.D %RSD

34 Part-A (Section- I) Solution stability for standard preparation at room temperature After 12 hours After 24 hours After 36 hours After 48 hours Standard Standard Standard Standard Replicate Peak area Peak area Peak area Peak area Mean S.D %RSD Solution stability for test preparation at 2-8 C Initial After 12 After 24 hours After 36 After 48 hours Standard Standard Standard Standard Standard Replicate Peak area Peak area Peak area Peak area Peak area Replicate Test Area Test Area Test Area Test Area Test Area Mean % Assay Standard weight (mg) Test weight % Difference

35 Solution stability for test preparation at room temperature Initial After 12 hours After 24 After 36 hours hours After 48 hours Standard Standard Standard Standard Standard Replicate Peak area Peak area Peak area Peak area Peak area Replicate Test Area Test Area Test Area Test Area Test Area Mean % Assay Standard weight (mg) Test weight (mg) % Difference compared to that of Initial Calculation: Prototype calculation for one set: % Assay = = % 72

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