RP-HPLC METHOD DEVELOPMENT AND VALIDATION FOR DETERMINATION OF DARUNAVIR IN BULK AND DOSAGE FORM.

Transcription

1 WORLD JOURNAL OF PHARMACY AND PHARMACEUTICAL SCIENCES Bhuse et al. SJIF Impact Factor Volume 6, Issue 7, Research Article ISSN RP-HPLC METHOD DEVELOPMENT AND VALIDATION FOR DETERMINATION OF DARUNAVIR IN BULK AND DOSAGE FORM. Kamlesh Valuba Bhuse* and Asst. Prof. Mrs. Jadhav P.B. Department of Quality Assurance Technique, SND College of Pharmacy, Babhulgaon, Yeola , Maharashtra, India. Article Received on 15 May 2017, Revised on 05 June 2017, Accepted on 26 June 2017, DOI: /wjpps *Corresponding Author Kamlesh Valuba Bhuse Department of Quality Assurance Technique, SND College of Pharmacy, Babhulgaon, Yeola , Maharashtra, India. ABSTRACT Background and objective: Objective of the present analytical research work was to developed and validate RP-HPLC method for the estimation Darunavir in bulk and dosage form. Method & Results: The RP-HPLC method for Darunavir was developed using column Phenomenex C18 column (5µm, 250mm 4.6mm) as stationary phase and Acetonitrile : water (80:20 v/v) as mobile phase. The mobile phase was maintained at a flow rate of 1.0 ml/min and volume of injection is 1 μl detection was carried out at 267 nm. The method was validated in accordance with ICH guidelines. Darunavir were found to be linear in the concentration rang of 5, 10, 15, 20, 25, 30 and 35 µg/ml respectively. The result of % assay of marketed formulation was found as % for Darunavir respectively. Accuracy of the method was determined by performing recovery study and the result were found in the range of %w/w and for Darunavir respectively. % RSD of precision study of these drugs were found less than 2 % which indicated good precision of the developed method. Conclusion: The developed HPLC method was simple, rapid, easy, accurate and precise. So, the method can be applied successfully for the routine analysis of Darunavir in pharmaceutical industry. KEYWORDS: Darunavir, Acetonitrile, RP-HPLC, Method Development, Validation. Vol 6, Issue 7,

2 INTRODUCTION Drug Darunavir Structure Description IUPAC Name Darunavir is a protease inhibitor used to treat HIV. It acts on the HIV aspartyl protease which the virus needs to cleave the HIV polyprotein into its functional fragments [(3aS,4R,6aR)-2,3,3a,4,5,6a-hexahydrofuro[2,3-b]furan-4-yl] N-[(2S,3R)-4- [(4-aminophenyl)sulfonyl-(2-methylpropyl)amino]-3-hydroxy-1- phenylbutan-2-yl]carbamate C 27 H 37 N 3 O 7 S Chemical Formula Molecular g/mol Mass Physical Sate White amorphous solid Storage Store Darunavir at 77 degree F (25degree c).brief storage at temp.between 59&86 degree f (15-30 degree celcius),store away from heat, moisture and light. Melting Point 101 C Solubility Acetonitrile, Methanol, Water etc. pka & 2.39 as strongest acidic and basic respectively EXPERIMENTAL WORK Raw material characterization Characterization of Darunavir ethanolate Determination of melting point Melting point was determined using digital melting point apparatus. The reference melting point of Darunavir ethanolate is 101 C. Determination λ max by UV 100 µg/ml solution of Darunavir ethanolate (DRV) was prepared by accurately weighing 10mg of DRV. It was then transferred to 100ml volumetric flask containing few ml of acetonitrile. Finally the volume was made up to the mark using water. The resultant solution Vol 6, Issue 7,

3 was scanned using UV visible spectrophotometer in the range of nm. The reference λmax of DRV is 267 nm. Characterization of reagents and chemicals Sr. No Chemical Grade Make Characterization As HPLC grade no further characterization 1 Water HPLC -- performed As HPLC grade no further characterization 2 Acetonitrile HPLC Merck performed Experimental Work From results obtained from trial runs Acetonitrile: Water in the proportion of 80:20 was selected as mobile phase for HPLC method development. Preparation of standard stock solution DRV Accurately weighed 10mg of DRV and transferred to 100ml volumetric flask containing a mixture of acetonitrile: water (80:20). The volume was made up to the mark using same mixture of mobile phase. The resulting stock solution was filtered through 0.45µ membrane filter and sonicated for three cycles each of 10 min. Preparation of working solution for trial runs of DRV The 0.5ml of standard stock solution of DRV was pipetted out in 10ml volumetric flask and volume was made up to the mark to prepare the resultant solution of 5μg/ml. The later solution was filtered through 0.45μ membrane filter and ultra sonicated to degas. The same solution was used to optimize the chromatographic system. System suitability testing Preparation of working solution 0.5ml standard stock solution (refer section 6.2.1) was pipetted out and diluted up to 10ml to obtain consequential solution of 5µg/ml. The resulting solution was filtered through 0.45µ membrane filter and sonicated for three cycles each of 10 min. Six replicates of this solution were injected and results were recorded for RT, area, tailing factor (symmetry factor) and theoretical plates. Mean, SD and %RSD were calculated for the results obtained as well as other parameters were also verified for acceptability level. The column efficiency for DRV peak should not less than 2000 theoretical plates. The tailing factor for peak, should not more than 2.0. % CV for area shall NMT 1.5 and for RT NMT 0.5% HPLC method validation Vol 6, Issue 7,

4 Linearity From stock solution 0.5, 1.0, 1.5, 2.0, 2.5, 3.0 and 3.5 ml were pipetted out and diluted up to 10 ml to obtain 5, 10, 15, 20, 25, 30 and 35 µg/ml resultant solutions respectively. Calibration curve was constructed between concentrations versus peak area. Results were recorded for equation of line, correlation coefficient and intercept were determined. Where, Y- area X- Unknown concentration m- Slope of graph c- Intercept Precision From the calibration range three QC standard decided viz. 8, 18 and 28 µg/ml as LQC, MQC and NQC respectively. The solutions for QC standards were prepared by diluting stock solution of 0.8, 1.8, and 2.8ml solutions up to 10ml. Area of each QC standard were recorded for intraday and inter day precision in seven replicates as per ICH guidelines Q2R1. Results were recorded to calculate mean, SD, %RSD. % Accuracy % Accuracy was determined from the observations of precision study using following formula. Limit for % accuracy is NMT 5% RSD. Robustness 5µg/ml solution was selected for robustness study for the parameters like mobile phase ratio, flow rate, etc. Seven replicates for parameters given in table were injected and area for each of the parameter was recorded. The variation should not be more than 5% RSD. One factor was changed at time to estimate the effect. Deliberate variations for robustness experiment Parameter Standard Variation 1 Variation 2 Mobile phase proportion Acetonitrile: Water with (80:20) 81:19 79:21 Flow Rate (ml/min) 1.0 ml/min Vol 6, Issue 7,

5 %Recovery Preparation of stock from API Accurately weighed 10mg of DRV (API) was added in volumetric flask containing some amount of mobile phase and volume was made up to the mark using mobile phase. The resulting solution was filtered through 0.45µ membrane filter and sonicated for three cycles each of 10 min. From the stock solution 1.0ml of stock was pipetted out in triplicate and kept in three different volumetric flasks, cleaned previously and diluted up to 10ml by using mobile phase to obtain resultant solution of 10µg/ml. This solution was injected for given chromatographic system in triplicate and mean area was determined. Preparation of stock from dosage form Twenty tablets (Label claim 300mg of DRV, Cipla Ltd.) were weighted, average weight was determined and powdered. Powder equivalent to 10mg (21.4mg) was transferred to 100 ml of mobile phase. The resulting solution was filtered through 0.45µ membrane filter and sonicated for three cycles each of 10 min. From the stock 0.8, 1.0, 1.2ml solutions were pipetted out and diluted up to 10ml using mobile phase to obtain resultant solution of 8, 10 and 12µg/ml. Preparation of test solution for % recovery by spike method 10µg/ml solution of DRV (API) was spiked into each of above dilutions of 8, 10 and 12µg/ml to obtain solutions at 80%, 100% and 120% respectively. Each of these three levels were injected in triplicate and mean area for each level was determined. The mean area obtained on API injection was subtracted from the mean area of each of these three levels to obtain area corresponding to test solutions. % recovery was determined from the test and standard area using following formula. Determination LOD and LOQ LOD and LOQ were determined from the following formulae, LOD = 3.3 x SD/ S LOQ = 10 x SD/ S SD is standard deviation S is the slope of calibration curve. Vol 6, Issue 7,

6 RESULT AND DISCUSSION Raw Material Characterization Characterization of DRV Melting Point determination Melting point was determined using digital melting point apparatus (Labatronics) by capillary method and found to be 101º C. The observed melting point corresponding with reference value, as per USP (102 ºC). Determination of absorption maxima (λ max ) UV spectrum of DRV Absorbance values observed for DRV by UV Sr. No. Wavelength in (nm) Absorbance Absorption maximum for Darunavir was determined by recording absorbance of 30μg/ml solution prepared in mobile phase between UV ranges i.e nm. The UV spectrum obtained showed 267 nm as wavelength maximum for selected drug candidate as shown in Figure. This wavelength was used for quantification of Darunavir during HPLC method development and validation. Vol 6, Issue 7,

7 Characterization of reagents and chemicals All reagents and chemicals used in the prescribed study were of HPLC grade and for this reason no accompanying characterization was done. HPLC Method Development HPLC has become more vital technique for estimation of drugs in biological fluid. This helps analytical chemistry to study pharmacodynamics fate of drug molecule in vivo. This method also offers advantages of estimating the ingredients for the multi component system. Present studies include the use of this sophisticated technique for identification as well as quantification of Darunavir in bulk and pharmaceutical dosage form (Tablet). Selection of mobile phase The favored mobile phase for this analysis was proved to be Acetonitrile : Water ( with ortho phosphoric acid) in the ratio of 80:20.This mobile was set as the separation of Darunavir was found to be more promising with this mobile phase. The detector was set at 267 nm in order to obtain maximum response with in short period of time. Therefore, a foresaid mobile was selected to study various parameters as per ICH guideline Q2R1 in following sections. System suitability testing System suitability was studied to certify reliability of system for drug and chromatographic condition. System suitability is defined as examination of system, previous to or during analysis to make certain system performance. System critical issues comprise factors such as Theoretical plates, tailing factor and retention time. Chromatographic conditions Chromatographic Conditions Column Phenomenex C18 Mobile phase Acetonitrile: Water of ph (80:20 v/v) Detection Wavelength 267 nm Flow rate 1.0ml/min Temperature Ambient Sample size 1 μl Vol 6, Issue 7,

8 HPLC chromatogram for Darunavir observed for system suitability testing System suitability is a pharmacopoeial prerequisite and is used to confirm, whether the resolution and reproducibility of the chromatographic system are ample for analysis to be done. The test was performed by six replicate injections of standard solutions of DRV, however, the concentration of API was kept constant at 5ppm. This study results with best solved spectrum for DRV as shown in Figure. The results obtained at each replicate injection were assessed for parameter like area, retention time, theoretical plates and tailing factor and evaluated against standard pharmacopoeial limit and found within limit. The mean, SD and %RSD for area and retention time calculated as depicted in Table. The results were in conformity with the limit as per ICH guideline. Therefore, from system suitability experiment it was concluded that the system was found to be suitable for quantitative estimation of DRV with acetonitrile: water as mobile phase and at 267 nm wavelength. Results attained for system suitability testing Sr. No. System suitability Parameter *Mean study Limits Inference 01 Retention time (RT) in min 3.22 NLT 2.0 min Passed 02 Peak Area in mv NLT 2000 Passed 03 Theoretical plates 2967 NLT 2000 Passed 04 Tailing factor NMT 2.0 Passed 05 % RSD of RT 0.23 NMT 0.5% Passed 06 % RSD of Mean Area 0.63 NMT 2.0% Passed HPLC Method Validation Validation is defined as "documented evidence which gives a high degree of confidence that a process, system, facility will consistently produce a product meeting its predetermined Vol 6, Issue 7,

9 specifications and quality attributes.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; it is an integral part of any good analytical practice Linearity The linearity of a test procedure is its ability (within a given range) to obtain test results proportional to the concentration (amount) of analyte in the sample. Results obtained for Linearity experiment Linearity Sr. No. Concentration (μg/ml) Mean Area* From stock solution 0.5, 1.0, 1.5, 2.0, 2.5, 3.0 and 3.5 ml were pipetted out and diluted up to 10 ml to obtain 5, 10, 15, 20, 25, 30 and 35 µg/ml resultant solutions respectively. Calibration curve was constructed between concentrations versus peak area. Results were recorded for equation of line, correlation coefficient and intercept were determined. Where, Y- area X- Unknown concentration m- Slope of graph c- Intercept Linearity experiment of Darunavir (DRV) y = 2018x R² = Mean area* Linear (Mean area*) Vol 6, Issue 7,

10 Linearity of method was assessed by diluting aliquots of standard stock solution of DRV to obtain 5 to 30µg/ml solutions. Each standard solution was injected to given chromatographic system in triplicates and mean area was recorded. A calibration curve was plotted with concentration on X axis and mean area on Y-axis and the same was as depicted in Fig. The calibration constructed for DRV showed linear relationship between concentration and mean area with regression coefficient of Other parameters like equation of straight line, slope, intercept etc. were determined. Furthermore, linearity experiment gave a range for the determination of DRV i.e. 5 to 30μg/ml. Precision Precision is the assessment of how close the records are to each other for a number of measurements under the same experimental conditions. From the calibration range three QC standard decided viz. 8, 18 and 28 µg/ml as LQC, MQC and NQC respectively. The solutions for QC standards were prepared by diluting stock solution of 0.8, 1.8, and 2.8ml solutions up to 10ml. These QC standards were 8, 18 and 28ppm concentrations of DRV standard solutions. Six repeated measurements for each QC standard were performed injected for given instrument setting. Precision of the method was established for intra-day (repeatability) and inter-day measurements. Results obtained for precision study Sr. No. Conc. (µg/ml) Intra day Inter day Mean Area*±SD %RSD Mean Area*±SD %RSD ± ± ± ± ± ± % Accuracy determination from the results obtained in precision experiment The accuracy of an analytical procedure expresses the closeness of agreement between the value which is accepted either as a conventional true value or an accepted reference value and the value found. This is sometimes termed trueness. Accuracy was calculated from the results of precision experiment and the results obtained for percent accuracy at three levels across range were as depicted in Table 6. According to ICH Vol 6, Issue 7,

11 guideline Q2R1, accuracy was determined at three concentration levels (QC standards) across the range. As per ICH guideline Q2R1 accuracy was determined at three concentration levels (QC standards) across the range. The mean area of three replicate injections was determined at said three dissimilar levels and equivalent concentration for each level was computed from calibration curve. From the investigational concentrations and correspondent nominal concentrations and percent accuracy was determined using formula stated in section Results of the same were as cited in. Results for % Accuracy determined from precision data Sr. No. Nominal Concentration (μg/ml) *Mean area Mean measured Concentration (μg/ml) % Accuracy (w/w) Inference Passed Passed Passed Robustness The robustness of an analytical method is a assessment of its capability to stay unaffected by small, but purposeful variations in method parameters and provides an indication of its consistency throughout customary usage. Experiments were performed for 5ppm concentration of DRV by altering circumstances such as mobile phase ratio (±1) and flow rate ratio (±0.05). The mean area and mean RT of 5ppm DRV solution was recorded in three replicates for purposeful change in method parameters viz, flow rate (0.95 and 1.05 ml, Table 12) and mobile phase ratio (79:21, and 81:19,Table 12). The percent assay values corresponding to experimental concentrations were determined. All values acquired for percent assay were in conformity with pharmacopoeial standard ( % w/w) for DRV. Consequently, the developed method was robust for deliberate rise and falls in mobile phase ratio and flow rate. Results observed for robustness study Sr. No. Conc. Robustness Mean % Limit (98- Variations (μg/ml) Parameter area* (mv) Assay w/w 102%) Mobile phase 81: Passed composition 79: Passed Flow Rate Passed (ml/min) Passed Vol 6, Issue 7,

12 Percent recovery study HPLC chromatogram resulted for % recovery experiment Percent recovery is determination of percent purity of given analyte finished product. The accuracy of the methods was determined by calculating recoveries of DRV by the standard addition method. Known amount of standard solutions of DRV (8, 10 and 12 µg/ml) were added to a pre-analysed standard solution of DRV (10µg/ml). Each solution was injected in triplicate and the percentage recovery was calculated by measuring the peak areas and fitting these values into the regression equations of the calibration curves. Data obtained was as represented in Table 13. The proposed method afforded the percent recovery of DRV as per pharmacopoeial limit ( % w/w). Data obtained for percent recovery experiment performed with Tablet dosage form Recovery Level Conc. Taken (μg/ml) Amount added (μg/ml) Mean Area* Amount Recovered (μg/ml) % Recovery (98-102% w/w) Inference 80 % Passed 100 % Passed 120 % Passed LOD and LOQ The detection limit is an attribute of limit tests. It is the smallest amount of analyte in a sample that can be detected, but not necessarily quantitated under the stated experimental conditions. The quantitation limit is a characteristic of quantitative assays for low levels of compounds in sample matrices like impurities in bulk drug substances and degradation products in finished pharmaceuticals. The sensitivity of the method was estimated in terms of Vol 6, Issue 7,

13 limit of detection and limit of quantification by using formulae viz. LOD = 3.3 δ/s and LOQ=10 δ/s (Where S was slope of calibration curve and δ is the standard deviation of area in calibration plot). LOD and LOQ were found to be 0.65 µg/ml and 1.98µg/ml respectively. As a result, the concentration of DRV as low as 0.48 µg/ml can be detected and 1.4 µg/ml can be successfully quantified devoid of any trouble of contamination. LOD and LOQ determined Drug LOD (μg/ml) LOQ (μg/ml) DRV SUMMARY AND CONCLUSION The projected study was aimed at develop a sensitive, precise and accurate HPLC method for the investigation of Darunavir Ethanolate (DRV) in bulk drug and in pharmaceutical dosage forms. In order to make it convenient the analysis of the component peaks of DRV, mixtures of Acetonitrile (ACN) with Water in different combinations were tested as mobile phase on a C18 stationary phase. A combination of Acetonitrile and Water in a proportion of 80:20 v/v was demonstrated to be the most appropriate of all amalgamation seeing as the chromatographic peaks were superior defined and resolved and approximately complimentary from tailing. The retention time obtained for DRV was 3.22min. Each of the sample was injected six/three (as per the requirements of ICH guideline Q2R1) times and the reproducible retention times were observed in all injections. System suitability parameters were studied with six replicate standard solutions of the drug and the calculated parameters were found inside the acceptance criteria. The tailing factor and the number of theoretical plates were in the acceptable confines. The peak areas of DRV were reproducible as indicated by low coefficient of variation. A good linear relationship (R 2 =0.997) was observed between the concentration of DRV and the respective mean peak area. The regression curve was constructed by linear regression fitting and its mathematical expression was Y =2018 X+4414 (where Y gives peak area and X is the concentration of the drug found). When DRV solutions containing 8, 18 and 28ppm was analysed by the proposed method for finding out intra and inter-day variations, low %RSD was observed. High recovery values obtained from the dosage form by the proposed method illustrated method accuracy. The nonappearance of supplementary peaks depicted non-interference of ordinary excipients used in the tablets manufacturing. The drug content in tablets was quantified using the proposed analytical method. The tablets were found to contain an Vol 6, Issue 7,

14 average of to % w/w of the labelled amount of the drug (DRV). The intentional changes in the method have inadequately exaggerated the peak tailing, theoretical plates and the percent assay. This illustrated that this method was robust. The smallest possible values of LOD and LOQ were obtained by this HPLC method indicated the method sensitivity. The standard solution of the drug was stable up to 24 hours as the difference in percent assay is within acceptable limit. Hence, the author proposed that the present HPLC method was sensitive and reproducible for the analysis of DRV in bulk as well as pharmaceutical dosage forms with small analysis time. Eventually, from above discussion it concluded that the projected method can be used for scheduled analysis of Darunavir Ethanolate in bulk as well as pharmaceutical dosage form (Tablet) over the period of its expiry. REFERENCES 1. Skoog D, Holler F, Principle of Instrumental Analysis, 1 st ed., Thomson Publications, India, 2007; 1-3: , Kasture A V, Wadodkar S G, Pharmaceutical Analysis, 7 th ed., Vol. 2, Nirali Prakashan, 2004; Sethi P D, High Performance Liquid Chromatography, Quantitative Analysis of Pharmaceutical Formulations, CBS Publishers and Distributors, New Delhi, 2001; 1(3-11): Daharwal S J, Journal on analytical method development, 2006; 4: Gennaro A, Karen B, The Science and Practice of Pharmacy, 19 th ed., The Mack Publishing Company, Pennsylvania, 1995; Willard H, Merritt L, Dean J, Instrumental Methods of Analysis, 7 th ed., CBS Publisher and Distributors, New Delhi, 2002; Michael D, Modern HPLC for practicing scientist, A John Wiley & Sons, 4 th ed., 2008; 194, Khopkar S., Basic concepts of analytical chemistry, 2 nd ed., New age International Ltd. Publishers, New Delhi, 1998; 2: Christian GD, Analytical Chemistry, John Wiley and Sons, 2003; 5: 35-42, Chatwal G R, Anand, S K, Instrumental Methods of Chemical Analysis, 5 th ed., Himalaya Publishing House, New Delhi, 2004; Vol 6, Issue 7,

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