Page 1 Chapter 1.
Introduction HPLC-METHOD DEVELOPMENT HPLC method is used to analyze several o drugs because of several advantages of this method used by specific, accurately, preciseing, rapid, automatically and eliminates tedious extraction and isolation procedures. Advantages: 1. Speed (analysis sample willmbe be accomplished in 20 mins or less) 2. Greater aquired sensitivity Role of the Column The HPLC colums are the total heart of the methods, its difficult in performing the sample separation. The columns should possess the selectively, efficiency, and reproducibility .All of these characteristic are dependent on the column manufactures release of better quality columns and packaging materials. . Commonly used reversed phases are C18 (octadecylsilane, USP L1), C8(octylsilane, USP L7), phenyl (USP L11), and cyano (USP L18) (24).
Initial Mobile Phase Selection: Mobile phase is the most important parameter in reversed-phase HPLC. Mobile phase selection is the 2nd final common step in the develop of the clearence method and first developed one is the selection and of the adsorbent. The main aim for thepreparation of solvents is that it has to dissolve the analytes up to the concentration suitable for the detection. Role of Temperature: While temperature is a variable that can affect selectivity, a, its effect is relatively small.
Also, the k ‘ generally decreases with an increase in temperature for neutral compounds but less dramatically for partially ionized analytes. Snyder et al. (29) reported that an increase of 1 o C will decrease the k ‘ by 1 to 2%, and both ionic and neutral samples. Role of pH:Page 2 pH is another factor in the resolution equation that will affect the selectivity of the separation. In reversed-phase HPLC, sample retention increase when the analyte is more hydrophobic BUFFERS IN REVERSED-PHASE LIQUID CHROMATOGRAPHY Selection of a buffered aqueous mobile phase for reversed-phase liquid chromatography (RPC) may seem intimidating, but with an understanding of the fundamental effect of pH on retention of ionic analytes and checking some properties of the buffer options, a logical and reasonable choice can be derived. METHOD DEVELOPMENT GUIDE Fig1.1 selection of validation methodPage 3 Table No1.1: Limits of HPLC parameters GUIDELINES FOR ANALYTICAL METHOD VALIDATION: METHOD VALIDATION: Method validation is the process by which it is established, through laboratory studies, that the performance characteristics of the method meet the requirements for its intended purpose.
Typical analytical characteristics used in method validation, commonly referred to as the “Eight steps of method of validation”Page 4 Fig1.2 Eight steps of method of validationPage 5 Table1.2: Performance characteristics to be considered during the validation. Method parameters Short description Precision Random error of the method Repeatability Precision measured under the best condition possible (short period, one analyst…) Intermediate Precision Precision measure of the within-laboratory variation due to different days, analysts, equipments, etc.
Robustness Capacity of a method to remain unaffected by small variations in the method parameters as could Possibly occur during the normal use of the method (pH, mobile phase composition,…) Reproducibility Precision measure determined by inter-laboratory studies Specificity Ability to determine the analyte in presence of other compounds Limit of detection Lowest sample concentration that can be detected Limit of quantitation Lowest sample concentration that can be quantified with suitable bias and precision Linearity Ability of the method to obtain test results which are proportional to the concentration in the sample Range Concentration interval within the method has a documented suitable performance Stability Absence of an influence of time on the concentration of the analyte in a samplePage 6 Chapter 2. METHOD DEVELOPMENT Method Development Using HPLC In method development, best chromatographic conditions like the best column, the best mobile phase, the detection wavelength etc. are used for analysis of any drug. For the method development by this method some information about the sample is very essential i.
e. number of components present in the sample, pKa values of different components, UV-Visible Spectra of each analyte, solubility in different solvents, concentration range of each component, nature of sample etc. Parameters Affecting Changes in Chromatograph The various parameters affecting the changes in chromatographic conditions are 1. Flow rate 2. Temperature 3. pH 4. Ion pair reagent 5. Column efficiency 6.
Capacity factor 7. Resolution 8. Retention time 9. Peak asymmetry Method Validation (ICH Guidelines) 1. Accuracy, 2. Precision, ? Repeatability, ? Intermediate precision.
3. Specificity / Selectivity, 4. Limit of Detection, 5. Limit of Quantitation, 6. Linearity,Page 7 7.
Range, 8. Robustness, 9. System Suitability.
Page 8 3. DRUG PROFILE VALBENAZINE Valbenazine is used to treat Tardive dyskinesia in adults. Tardive dyskinesia is a neurological disorder characterized by involuntary movements.
Table 3.1 General Information of DrugPage 9 Chapter 4: REVIEW OF LITERATURE Uttam Prasad Panigrahy* et. al., A novel approach was used to develop and validate a rapid, accurate, precise, simple, efficient and reproducible isocratic Reversed Phase-High Performance Liquid Chromatographic (RP-HPLC-DAD) method for the estimation of Valbenazine in bulk and pharmaceutical dosage form. Valbenazine were separated using Kromasil C18 column (250mm×4.6 mm, 5mm particle size), Waters Alliance e2695 HPLC system with 2998 PDA detector and the mobile phase contained a mixture of 0.01M Ammonium acetate (pH adjusted to 3.5 with orthophosphoric acid) and Methanol (30:70, v/v).
The flow rate was set to 1ml/min with the responses measured at 309nm. The retention time of Valbenazine was found to be 3.733min.
Linearity was established for Valbenazine in the range of 10-125?g/ml with correlation coefficient (r2=0.999). The percentage recoveries were between 100.3% to 100.6%. Validation parameters such as specificity, linearity, precision, accuracy, robustness, limit of detection (LOD) and limit of quantitation (LOQ) were evaluated for the method according to the International Conference on Harmonization (ICH) Q2 R1 guidelines.
The developed method was successfully applied for the quantification and hyphenated instrumental analysis.Page 10 Chapter 5 .NEED FOR THE STUDY Literature review indicates, no method is reported for quantification of bulk drug and its capsule formation till date. Only few LC-MS/MS methods were reported.Page 11 6. AIM AND PLAN OF WORK AIM: To develop new HPLC method for the estimation of Valbenazine in dosage form. PLAN OF WORK: ? Solubility determination of Valbenazine various solvents and buffers. ? Determine the absorption maxima of the drug in UV–Visible region in different solvents/buffers and selecting the solvents for HPLC method development.
? Optimize the mobile phase and flow rates for proper resolution and retention times. ? Validate the developed method as per ICH guidelines.Page 12 7. MATERIALS AND METHODS Table7.1: Instruments used Table7.2: Reagents used Chemicals 7.2 METHODS Preparation of Mobile phase: About 60 volumes of Acetonitrile, 40 volumes of Water and 0.
5% Triethylamine buffer (60:40: 0.5%) were mixed and sonicated for 15 mins for degassing and the solution was filtered through 0.45 micron membrane filter.Page 13 Preparation of 0.5% Triethylamine: About 0.5 ml of Triethylamine was transferred into 100 ml volumetric flask and the volume was made up to mark with water.
Page 14 Chapter 8: RESULTS AND DISCUSSIONS 8.1 Solubility Studies These studies are carried out at 25 0C Table 8.1: solubility studies. 8.
2 Determination of Working Wavelength (?max) 8.2.1 Preparation of Standard solution 10 mg of Valbenazine was weighed and transferred in to 100 ml volumetric flask and dissolved in methanol and then make up to the mark with methanol and prepare 10 µg /ml of solution by diluting 1ml to 10ml with methanol. RESULTS: The wavelength of maximum absorption (? max) of the solution of the drug in mobile phase were scanned using UV-Visible spectrophotometer within the wavelength region of 200–400 nm against mobile phase as blank. The absorption curve shows characteristic absorption maxima at 264 nm for Valbenazine (Fig.8.1), 248 nm was selected as detector wavelength for the HPLC chromatographic method.
Page 15 Fig. 8.1: UV-VIS Spectrum of Valbenazine (264 nm) 8.3 METHOD DEVELOPMENT OF VALBENAZINE Trial -1 Chromatographic conditions Column : Inertsil ODS 3V (250×4.
6× 5µ) Mobile phase : Methanol: Acetonitrile : Water Ratio : 50:10:40 Flow rate : 1.0 mL/min Detection wavelength : 264 nm Injection volume : 20µL Run time : 15 min Preparation of Standard solution 10 mg of Valbenazine was weighed and transferred in to 100 ml volumetric flask and dissolved in mobile phase and then make up to the mark with mobile phase and prepare 10 µg /ml of solution by diluting 1ml to 10ml with mobile phase.Page 16 Fig. 8.2: Chromatogram of Trail 1 Table 8.2: Results for Trail 1 Observation Peak shape was not good and efficiency was not within the acceptance criteria.
So this trial was not considered. Trial -2 Chromatographic conditions Column : Inertsil ODS 3V (250×4.6 ×5µ) Mobile phase : Methonol: ACN: Phosphate buffer pH : 4.5 Ratio : 50:30:20 Flow rate : 1.0mL/min Detection wavelength : 264 nm Injection volume : 20 µLPage 17 Run time : 10 min Preparation of Standard solution 10 mg of Valbenazine was weighed and transferred in to 100 ml volumetric flask and dissolved in mobile phase and then make up to the mark with mobile phase and prepare 10 µg /ml of solution by diluting 1ml to 10ml with mobile phase. Fig 8.3: Chromatogram of Trail Table 8.3: Results for Trail 2 Observation From the above trial, it was found that tailing factor was not with the system suitable limits.
So this trial was not considered. 8.4 Optimised trial Chromatographic conditions Column : Phenomenex C18 (250×4.6 ×5µ) Mobile phase : Acetonitrile: Water : Triethylamine buffer (60:40: 0.5%) v/v Flow rate : 1.0mL/min Detection wavelength : 264nmPage 18 Injection volume : 20µL Run time : 5min Preparation of Standard solution 10 mg of Valbenazine was weighed and transferred in to 100 ml volumetric flask and dissolved in mobile phase and then make up to the mark with mobile phase and prepare 10 µg /ml of solution by diluting 1ml to 10ml with mobile phase. Fig 8.4: Chromatogram of Optimised trial Table 8.
4: Results for Optimised Trial Observation: ? All the system suitability requirements were met. ? The efficiency was more than 2000 for Valbenazine ? Hence this method was optimized.Page 19 8.
5 OPTIMIZED CHROMATOGRAPHIC CONDITIONS FOR ASSAY Table 8.5: Optimised condition Preparation of samples for Assay Preparation of Standard solution 10 mg of Valbenazine was weighed and transferred in to 100 ml volumetric flask and dissolved in mobile phase and then make up to the mark with mobile phase and prepare 10 µg /ml of solution by diluting 1ml to 10ml with mobile phase. Preparation of Sample solution Sample name: Ingrezza 10 mg capsules Weigh 20 capsules by removing the shell then crush with mortar and pestle then weigh a quantity of powder equivalent to 10mg of Valbenazine and transferred in to 100 ml volumetric flask and dissolved in mobile phase and then make up to the mark with mobile phase and prepare 10 µg /ml of solution by diluting 1ml to 10ml with mobile phase.Page 20 Where, AS: Average peak area due to standard preparation AT: Peak area due to assay preparation WS: Weight of Valbenazine in mg WT: Weight of sample in assay preparation DT: Dilution of assay preparation DS: Dilution of standard preparation P: Purity of Valbenazine AV: Average weight of tablets in mg LC: Labelled claim of Valbenazine in capsules Fig. 8.5: Chromatogram of Assay Standard-01 Fig.
8.6: Chromatogram of Assay Standard-02 Fig. 8.
7: Chromatogram of Assay Standard-03Page 21 Fig. 8.8: Chromatogram of Assay Standard-04 Fig. 8.9: Chromatogram of Assay Standard-05 Fig. 8.10: Chromatogram of Assay Sample-01 Fig. 8.
11: Chromatogram of Assay Sample-02Page 22 Fig. 8.12: Chromatogram of Assay Sample-03 Fig. 8.
13: Chromatogram of Assay Sample-04 Fig. 8.14: Chromatogram of Assay Sample-05Page 23 Table 8.6: Results for Valbenazine Table 8.7: Results of assay Observation The amount of Valbenazine present in the taken dosage form was found to be 99.
35 %.Page 24 Chapter 9: VALIDATION 9.1 System Suitability& System precision To verify that the analytical system is working properly and can give accurate and precise results were evaluated by 10µg/mL of Valbenazine was injected six times and the chromatograms were recorded for the same. Table 9.1: Result for system suitability. Acceptance criteria 1. The % RSD for the retention time of valbenazine Peaks from 6 replicate injections of each Standard solution should be not more than 2.0 2.
The % RSD for the peak area responses of valbenazine peak from 6 replicate injections of each standard solution should be not more than 2.0%. 3. The number of theoretical plates (N) for the valbenazine peaks is not less than 2000. 4. The Tailing factor (TP) for the valbenazine peak is not more than 2.
0. Result The plate count and tailing factor results were found to be within the limits and the % RSD was found to be 0.1 so system is suitable and giving precise results.
Page 25 9.2 Method precision Method precision was determined by injecting sample solutions of concentration valbenazine (10?g/mL) for six timesare prepared separately. Fig. 9.1: Chromatogram of Method Precision-01 Fig. 9.2: Chromatogram of Method Precision-02 Fig. 9.
3: Chromatogram of Method Precision-03 Fig. 9.4: Chromatogram of Method Precision-04Page 26 Fig. 9.5: Chromatogram of Method Precision-05 Fig 9.
6: Chromatogram of Method Precision-06Page 27 Table 9.2: Method precision results Result The % RSD of Assay for 6 Samples determinations of valbenazine found to be within the acceptance criteria (less than 2.0%). Hence method is precise. 9.
3 Linearity and range Preparation of standard stock solution Standard stock solutions of Valbenazine were prepared by dissolving 100 mg of Valbenazine in 100 mL of mobile phase. After that filtered the solution using 0.45-micron syringe filter and Sonicated for 5 min further dilutions were given in the Table 9.3.Page 28 Table 9.3: Linearity Preparations. Fig 9.7: Chromatogram of linearity for preparation 1.
Page 29 Fig 9.8: Chromatogram of linearity for preparation 2 Fig 9.9: Chromatogram of linearity for preparation 3.
Fig 9.10: Chromatogram of linearity for preparation 4. Fig 9.11: Chromatogram of linearity for preparation 5.Page 30 A graph was plotted for valbenazine against the concentrations of the solutions and the peak areas (Table 9.9). The correlation coefficient R2 was determined and was found to be 0.
999 for valbenazine (Fig. 9.12) Table 9.4: Linearity data.
Page 31 Fig 9.12: Graph for Linearity data. Table 9.5: Linearity results.
Acceptance criteria The relationship between the concentration of valbenazine and area of valbenazine should be linear in the specified range and the correlation should not be less than 0.99. Result The correlation coefficient for linear curve obtained between concentration vs. Area for standard preparation was found to be 0.999 9.4 Specificity: Blank solution was injected and the chromatogram was recorded for the same as given in figure below y = 54703x – 45.96 R² = 0.999 0 0.
2 0.4 0.6 0.8 1 1.
2 0 0.2 0.4 0.6 0.
8 1 1.2 Series1 Linear (Series1)Page 32 Placebo solution was prepared and it was injected and the chromatogram was recorded for the same as given in Fig. 9.14 Fig. 9.13: Chromatogram of Placebo Table 9.14: Chromatogram of Blank Result Chromatograms of blank and placebo solutions had shown no peaks at the retention times of Valbenazine.
It was observed that diluent or excipient peaks do not interfere with the Valbenazine Peak. 9.5 Accuracy Accuracy of the method was determined by Recovery studies. To the formulation (preanalysed sample), the reference standards of the drugs (50µg/ml, 100µg/ml and 150µg/ml ) were added at the level of 50%, 100%, 150%. The recovery studies were carried out three times and the percentagePage 33 recovery and percentage mean recovery were calculated for drug is shown in Table 9.6 Fig 9.
15: Chromatogram of 50% recovery-1 Fig 9.16: Chromatogram of 100% recovery-1. Fig 9.17: Chromatogram of 150% Recovery-1Page 34 Fig 9.18: Chromatogram of 50% Recovery-2 Fig 9.19: Chromatogram of 100% Recovery-2 Fig 9.20: Chromatogram of 150% Recovery-2 Fig 9.21: Chromatogram of 50% Recovery-3Page 35 Fig 9.
22: Chromatogram of 100% Recovery-3 Fig 9.23: Chromatogram of 150% Recovery-3Page 36 Table 9.6: Results for Recovery Acceptance criteria The Average % recovery of Valbenazine should lie between 98% and 102%. Result The percentage mean recovery of Valbenazine was found between 99.0 to 102.
0 LIMIT OF DETECTION = 3.3 * (551277)/51766 = 35.14µg/ml (Valbenazine) Where, = the standard deviation of the response S = the slope of the calibration curve The slope S may be estimated from the calibration curve of the analyte.
Observation: The LOD for this method was found to be 35.14µg/ml (Valbenazine)Page 37 LIMIT OF QUANTIFICATION (LOQ) = 10* (551277)/51766 = 106.48µg/ml (Valbenazine) Where = the standard deviation of the response S = the slope of the calibration curve The slope S may be estimated from the calibration curve of the analyte. OBSERVATION: The LOQ for this method was found to be 106.
48µg/ml (Valbenazine) 9.6 Robustness The Robustness of the method was determined. The results obtained by deliberate variation in method parameters are summarized below in Table 9.
7 Fig. 9.24: Chromatogram of Valbenazine Robustness (0.8 ml/min) Fig.9.25: Chromatogram of Valbenazine Robustness (1.2 ml/min)Page 38 Fig.
9.26: Chromatogram of Temperature from 30 to 25°C. Fig. 9.
27: Chromatogram of Temperature from 30 to 35°CPage 39 Table 9.7: Results for Robustness Result The tailing factor was found to be within the limits on small variation of flow rate and wavelength. 9.7 Ruggedness The ruggedness of the method was studied by the determining the analyst to analyst variation by performing the Assay by two different analysts Acceptance criteria: The % Relative standard deviation of Assay values between two analysts should be not more than 2.0%. Fig.
9.28: Chromatogram of Analyst-01 standardFig. 9.29: Chromatogram of Analyst-01 samplePage 40 Fig.
9.30: Chromatogram of Analyst-02 standard Fig. 9.31: Chromatogram of Analyst-02 samplePage 41 Table 9.8: Results for Ruggedness Results: From the above results % Assay and %RSD obtained acceptance criteria 2% so method is ruggedPage 42 Chapter 9: CONCLUSION A new precise, accurate, rapid method has been developed for the estimation of Valbenazine pharmaceutical dosage form by HPLC. From the above experimental results and parameters it was concluded that, this newly developed method for the estimation Valbenazine was found to be simple, precise, accurate and high resolution and shorter retention time makes this method more acceptable and cost effective and it can be effectively applied for routine analysis in research institutions, quality control department in meant in industries, approved testing laboratories studies in near future From results the proposed method is highly sensitive, precise and accurate and it successfully applied for the quantification of API content in the commercial formulations of Valbenazine Educational institutions and Quality control laboratories.