Sravani and Haritha Indian Journal of Research in Pharmacy and Biotechnology ISSN: (Print) ISSN: (Online)

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1 A new development and validated RP-HPLC method for the assay and related substances of Itraconazole in capsule dosage form Sarvani Paruchuri*, Haritha Pavani K Nimra College of Pharmacy, Vijayawada, Andhra Pradesh, India *Corresponding author: sarvani4anu@gmail.com ABSTRACT The present investigation reveals about a simple, economic, selective, precise and accurate reverse phase high performance liquid chromatography method for analysis of Itraconazole and Related Substances of Itraconazole was developed and validated according to ICH guidelines. Itraconazole was well separated using ThermoHypersil BDS C18, 150mm Χ 4.6 mm, 5µm column for assay quantification in isocratic mode with mobile phase comprising of buffer: Acetonitrile (65:35) with a flow rate of 1.5ml/min and ThermoHypersil BDS C18, 100 mm x 4.6 mm, 3 µm column for Related substances quantification in gradient mode with mobile phase comprising of 0.08M tetra butyl ammonium hydrogen sulphate: Acetonitrile with a flow rate of 1.5ml/min. the retention time was found to be 6.2min and % assay was found to be 99.9%. The percentage recovery was found to be 99.6 to 101.2%. Proposed method was validated for precision, accuracy, linearity, range, specificity and robustness. The drug was subjected to forced degradation and stability studies. The drug was found to be stable for 4 days in Assay and for 3 days in Related compounds determination. This method was appropriate for the purpose of quantitating Itraconazole in Drug Product and the Related Substances of Itraconazole in the Finished Product and has been successfully validated. Key words: Itraconazole, HPLC, Method development, Method validation, Analysis INTRODUCTION Itraconazole is a (1-(butan-2-yl)-4-{4-[4-(4- {[2R,4S)-2-(2,4-dichlorophenyl)-2-(1H-1,2,4 triazole- 1- ylmethyl) 1, 3 dioxolan 4 -yl] methoxy}phenyl) piperazin -1-yl ] phenyl} 4, 4-dihydro-1H-1,2,4- triazole-5-one) is member of the drug class known as anti-fungal. It is used for the inhibition of fungal cytochrome p450 enzyme lanosterol 4 demethylase, used in the conversion of lanosterol to ergosterol, which is a main sterol in fungal cell membrane, thus inhibits replication and promotes cell death in case of the yeast cells transformation into hypothetically invasive hyphae. Literature survey revealed that very few methods have been reported for the analysis of Itraconazole which include UV spectroscopy, Reverse Phase High Performance Liquid Chromatography, Ultra Pressure Liquid Chromatography, LCMS, HPTLC methods. The present study illustrate development and validation of simple, economical, selective, accurate, precise RP-HPLC method for the determination of Itraconazole and Related Substances of Itraconazole in pharmaceutical dosage forms as per ICH guidelines. The goal of the present study is to develop rapid, economical HPLC method for the analysis of Itraconazole in pharmaceutical dosage form using most commonly employed column (C18) and simple mobile phase preparation. In the present work a successful attempt had been made to develop a method for the determination of Itraconazole in pharmaceutical dosage form and validate it. The method would help in estimation of the drug in single run which reduces the time of analysis and does not require separate method for the drug. Thus the paper reports an economical, simple and accurate RP-HPLC method for the above said pharmaceutical dosage form. MATERIALS AND METHODS Quantitative HPLC was performed on Schimadzu 2010 CHT system connected with UV Visible detector and LC solution software. The drug analysis data were acquired and processed using LC solution software running under windows XP on a Pentium PC and thermohypersil BDS C18 column of dimension 150 Χ 4.6, 5µm particle size. In addition an analytical balance (Mettler Toledo 0.1mg sensitivity), digital ph meter (Polmon), and an ultra sonicator were used in this study. Standards and Chemicals used: Itraconazole pure sample was taken as a gift sample from local labs and dosage forms Sporanox marketed by Janssen Pharmaceuticas was purchased from local pharmacy. Other chemicals all are of HPLC grade. Preparation of Mobile Phase: A suitable quantity of degassed mixture of buffer and acetonitrile in the ratio of 65:35 was prepared and filtered through 0.45µ filter under vaccum filtration. (27.2gm of tetra butyl ammonium hydrogen sulphate was prepared in 1000ml volumetric flask and is used as buffer) Preparation of Diluent: Prepare 99:1 mixture of Methanol and Hydrochloride (v/v) IJRPB 1(6) November December 2013 Page 857

2 Preparation of Standard stock solution: About 25mg of itraconazole working standard was taken into a 50ml volumetric flask and add 30ml of diluent and sonicate to dissolve. Make up the volume with diluent to get a concentration of 500µg/ml. From this solution 5ml was taken in to 25ml volumetric flask and make up the volume with diluent to get a concentration of 200 µg/ml. Preparation of Sample Solution: The average fill weight was determined for 20 capsules was added weigh 25mg equivalent of itraconazole was taken and transferred in to 50ml volumetric flask. Add 30ml of diluent and sonicate to dissolve and make up the volume with diluent. From this solution 5ml was taken in to 25ml volumetric flask and make up the volume with diluent to get a concentration of 200 µg/ml. Method optimisation: The chromatographic separation was performed using Hypersil BDS C18 ( mm, 5µm) column. For selection of mobile phase, various mobile phase compositions were observed for efficient elution and good resolution. The mobile phase consisting of Tetra Butyl Ammonium Hydrogen Sulphate and acetonitrile in the ratio of 65:35 was found to be the optimum composition for efficient elution of analyte. The mobile phase was injected to the column at a flow rate of 1.5 ml/min for 12min. The column temperature was maintained at 35 ± 1 0 C. The analyte was monitored at 225nmusing UVdetector. The retention time of the drugs was found to be 6.2min. 99:1% of methanol and HCl was used as diluent. Figure.1.Structure of Itraconazole RESULTS AND DISCUSSION Method validation: System suitability:system suitability of the HPLC was conducted to find out the system performance and the results we are getting were accurate. Linearity: Linearity is the ability of the method to produce results that is directly proportional to the concentration of the analyte in samples with given range. The linearity of Itraconazole was in the concentration range of µg/ml. From the linearity studies calibration curve was plotted and concentrations were subjected to least square regression analysis to calculate regression equation. The regression coefficient was found to be and shows good linearity for the drug. Precision: Precision is the degree of closeness of agreement among individual test results when the method is applied to multiple sampling of a homogeneous sample. Study was carried out by injecting six replicates of the same sample preparations at a concentration of 200ppm/ml. The results were tabulated in table No 3 Accuracy: Accuracy is the closeness of results obtained by a method to the true value. It is the measure of exactness of the method. Accuracy of the method was evaluated by standard addition method. Recovery of the method was determined by spiking an amount of the pure drug (50%,100%,150%) at three different concentration levels in its solution has been added to the pre analyzed working standard solution of the drug. Forced degradation of Itraconazole: Acid/base hydrolysis: Weigh 25mg equivalent of Itraconazole and transfer into 50ml volumetric flask. To this add 2.5ml of 5N HCl/NaOH and place in a water bath at about 60 0 c for about 1hr with occasional shaking. After 1hr, add about 2.5ml of 5N NaOH/HCl to neutralize, to this add 10ml of Diluent. Sonicate for 30min and allow the solution to equilibrate to room temperature and dilute with diluent and mix well. Pipette 5.0ml of the above solution into a 25ml volumetric flask, dilute to volume with diluent. The results were tabulated in table No 4 and the chromatogram was shown in fig no7,8 Oxidative degradation: Weigh 25mg equivalent of Itraconazole and transfer into 50ml volumetric flask. To this add 2.5ml of 1%H 2 O 2 and place in dark for about 1 day. After 1 day add about 10ml of diluent, sonicate for 30min and allow the solution to equilibrate to room temperature and then dilute with Diluent and mix well. Pipette 5.0ml of the above solution into a 25ml volumetric flask, dilute to volume with diluent. The results were tabulated in table No 4 and the chromatogram was shown in fig no 9 Thermal degradation: Weigh 25mg equivalent of Itraconazole and transfer into 50ml volumetric flask. Place it in oven at C and let stand for 24hrs. After 24hrs, add 30ml of diluent and sonicate with occasional shaking for 30min and allow the solution to equilibrate to room temperature and then dilute with diluent and mix well. Pipette 5.0ml of the above solution into a 25ml volumetric flask, dilute to volume with diluent. Photolytic degradtion: (UV/LUX): Itraconazole samples were prepared and the solutions were exposed to light to determine the irradiation of light on the stability of the solution. Approximately 200mg of the drug powder was taken and is spread as a thin layer IJRPB 1(6) November December 2013 Page 858

3 and is placed in a photo stability chamber at 200watt hr/sq mt. After attaining 200 watt hours/sq mt/1.2million LUX hrs, quantitatively transfer a portion of the pellets equivalent to about 25mg of itraconazole into a 50ml volumetric flask and add 30ml of diluent and sonicate, and then dilute to volume with diluent and mix well. Pipette 5.0ml of the above solution into a 25ml volumetric flask, dilute to volume with diluent. Stability: Analyse the stored samples and standards. Evaluate the stability of the stored stability solutions by quantitating the amount of itraconazole found in the stored solutions against freshly prepared standard after one to seven days of storage and test atleast 3 days. Filter study: Make one injection of the filtered diluent, the filtered standard fractions, the unfiltered standards, the filtered sample fractions and the centrifuged sample fractions. Calculate, as area percentage of the average area from system suitability working standard injections, any peaks found in the retention time window of itraconazole from the filtered diluent blank injections. The percentage recovery for the filtered standards was calculated, the unfiltered standards, and the filtered sample and centrifuged samples. Discussions: Several trials has made until getting good peak resolution, acceptable plate count and tailing factor. Method was optimized and the retention time was reported as 6.12 minutes. System suitability: For the system suitability studies it was found that the %RSD was found to be 0.8%, theoretical plates 5623, and tailing factor 1.1. Linearity: From the linearity data it was observed that linearity concentration range was 500µg/ml. Correlation coefficient was found to be Precision: The percentage RSD for sample was found to be 0.2 Accuracy: The recovery of pure drug from the analysed solution of formulation was found to be 100.2%. Specificity: The chromatograms of standard and sample are identical with nearly same Retention time. No interference due to Placebo and Sample at the retention time of analyte which shows that the method was specific Robustness: The %RSD of retention time was within the limits for variation in flow rate (1.3ml and 1.7ml). Hence the flow rate should be between 1.3ml to 1.7ml. The % RSD of retention time was within the limits for variation in ph (1.3 and 1.7) hence the ph should be with in 1.3 and 1.7. The %RSD of retention time was within the limits for variation in Buffer composition (63:37 and 67:33). The system suitability requirements were met for the method specified parameters. The % itraconazole found was between 97.0% and 103.0%. Filter Study: The mean recovery from the filtered standards was within of the mean from the unfiltered standards. The mean recovery from the filtered samples was with in of the mean from the centrifuged samples Methods for related substances validation: Preparation of mobile phase: Mobile phase was introduced in gradient mode. Mobile phase A: 0.08M tetra butyl ammonium hydrogen sulfate (27.2 g of tetra butyl ammonium hydrogen sulfate per 1000 ml volume of preparation. Add DI Water and stir to mix. No ph adjustment is required.) Mobile phase B: Acetonitrile Gradient programme Time Mobile phase Mobile Phase (min) A (%) B (%) Preparation of diluent: In a suitable flask, prepare a 1:1 mixture of methanol and tetrahydrofuran (v/v). Preparation of Standard Solution: 50mg of Itraconazole working standard was transferred into 25ml volumetric flask. From this take 5ml of the solution and transferred to 100ml volumetric flask. From the above solution transfer 10ml of the solution in to 100ml volumetric flask to make a concentration of 10µg/ml Preparation of sample Solution: Determine the average fill weight of 20 capsules and weigh 500mg equivalent of itraconazole and transfer into 100ml volumetric flask and add 70ml of diluent and sonicate and dilute to volume with diluent and mix well (Concentration: 5000µg/ml Itraconazole). Method optimisation: The chromatographic separation was performed using Hypersil BDS C18 ( mm, 3µm) column. For selection of mobile phase, various mobile phase compositions were observed for efficient elution and good resolution. The mobile phase consisting of 0.08MTetra Butyl Ammonium Hydrogen Sulphate and acetonitrile in gradient phase was found to be the optimum composition for efficient elution of analyte. The mobile phase was injected to the column at a flow rate of 1.5 ml/min for 35min. The column temperature was maintained at 30 ± 1 0 C. The analyte was monitored at IJRPB 1(6) November December 2013 Page 859

4 225nmusing UV-detector. The retention time of the drugs was found to be 12.2min. 99:1% of methanol and HCl was used as diluent. The optimised chromatographic conditions were mentioned in Table No 1 and the chromatograms were shown in Fig 10, 11 and 12 RESULTS AND DISCUSSION Method validation: RRT and RRF: (Relative retention time, relative retention factor): Impurity B and Impurity G Stock Standard Solution: 25mg of itraconazole WRS was transferred into 50ml volumetric flask. From this take 5ml of the solution and transferred to 100ml volumetric flask. Working Itraconazole standard solutions with impurities: Prepared 50, 100, 200% of proposed specification level concentration by preparing aliquot solutions of 2.5, 5 and 10ml of impurity standard stock solution into 100ml volumetric flask individually and dilute to volume with diluent and mix well. Linearity: Linearity is the ability of the method to produce results that is directly proportional to the concentration of the analyte in samples with given range. The linearity of Itraconazole was in the concentration range of µg/ml. From the linearity studies calibration curve was plotted and concentrations were subjected to least square regression analysis to calculate regression equation. The regression coefficient was found to be and shows good linearity for the drug. Precision: Precision is the degree of closeness of agreement among individual test results when the method is applied to multiple sampling of a homogeneous sample. Study was carried out by injecting six replicates of the same sample preparations at a concentration of 5000µg/ml. Accuracy: Accuracy is the closeness of results obtained by a method to the true value. It is the measure of exactness of the method. Accuracy of the method was evaluated by standard addition method. Recovery of the method was determined by spiking an amount of the pure drug (50%,100%,200%) at three different concentration levels in its solution has been added to the pre analyzed working standard solution of the drug. Forced degradation of Itraconazole: Acid/base hydrolysis: Weigh 500mg equivalent of Itraconazole and transfer into 100ml volumetric flask. To this add 2.5ml of 5N HCl/NAOH and place in a water bath at about 60 0 c for about 1hr with occasional shaking. After 1hr, add about 2.5ml of 5N NAOH/HCl to neutralize, to this add 10ml of diluent. Sonicate for 30min and allow the solution to equilibrate to room temperature and dilute with diluent and mix well. Pipette 5.0ml of the above solution into a 25ml volumetric flask, dilute to volume with diluent. Oxidative degradation: Weigh 500mg equivalent of Itraconazole and transfer into 100ml volumetric flask. To this add 10ml of 30%H 2 O 2 and place in water bath at 80 0 c for 4 hrs. After 4hrs add about 60ml of diluent, sonicate for 30min and allow the solution to equilibrate to room temperature and then dilute with Diluent and mix well. Pipette 5.0ml of the above solution into a 25ml volumetric flask, dilute to volume with diluent. Thermal degradation: Weigh 500mg equivalent of itraconazole and transfer into 100ml volumetric flask. Place it in oven at C and let stand for 24hrs. After 24hrs, add 70ml of diluent and sonicate with occasional shaking for 30min and allow the solution to equilibrate to room temperature and then dilute with Diluent and mix well. Pipette 5.0ml of the above solution into a 25ml volumetric flask, dilute to volume with diluent. The results were tabulated in table No 4 and the chromatogram was shown in fig no16 Photolytic degradtion: (uv/lux): Itraconazole samples were prepared and the solutions were exposed to light to determine the irradiation of light on the stability of the solution. Approximately 1000mg of the drug powder was taken and is spread as a thin layer and is placed in a photo stability chamber at 200watt hr/sq mt. After attaining 200 watt hours/sq mt/1.2million LUX hrs, quantitatively transfer a portion of the pellets equivalent to about 25mg of itraconazole into a 50ml volumetric flask and add 30ml of diluent and sonicate, and then dilute to volume with diluent and mix well. Pipette 5.0ml of the above solution into a 25ml volumetric flask, dilute to volume with diluent. Filter study: Three of the six 100% Accuracy Samples prepared in Intermediate Precision were used. Place an aliquot from the three preparations into three 15-mL volumetric flasks and spin at high speed for 5 minutes. Remove an aliquot of the supernatant and place in an auto sampler vial for analysis. Remove a second aliquot from each of the three preparations and individually filter and analyse Preparation of Filter Blanks: Filter the Diluent through a 0.45 m Nylon (Millipore Brand) filter, fill an auto sampler vial for analysis. Repeat this two more times using a new 0.45 m Nylon (Millipore Brand) filter each time. Stability of standard and sample solutions: Specificity known impurities: The method was shown to be specific with regards to the Known IJRPB 1(6) November December 2013 Page 860

5 Impurities of Itraconazole with a Resolution between adjacent peaks of 14.1, 4.5, 4.2, 6.8, 4.6, 3.9 and 6.7. The Acceptance Criteria for the determination of RRT and RRF was met for all the Known Impurities. Specificity placebo: The method was shown to have a 0.05% of Placebo interference, thus the method was shown to be specific with regards to the Placebo. Specificity forced degradation: The method was shown to be capable of resolving the Known and Unknown peaks from each other and from the Itraconazole peak. The Peak Purity data confirmed that all peaks detected were pure. Linearity and range: The method was shown to be Linear (Correlation Coefficient of ) over a Range of 20% to 300% of the Working Standard Concentration which corresponds to the 2.00 µg/ml to µg/ml of the Test concentration. Precision - injection repeatability: The method was shown to have a 0.8% RSD for 10 replicate working standard injections thus demonstrating acceptable Injection Reproducibility. Precision - analysis repeatability: The method was shown to have a 0.8% RSD for Impurity-B, 0.7% RSD for Impurity-G. Spiking/Recoveries at the proposed specification limit for each known impurity thus demonstrating acceptable analysis reproducibility. Intermediate precision ruggedness: A second Analyst on a different day, using a different HPLC and a different column serial number was able to reproduce Mean Recoveries and %RSD as the first Analyst thus demonstrating method Ruggedness. The difference in the mean recovery between analysts was not greater than 10.0% for each Known Impurity. The method was found to be Rugged. Accuracy and recovery from placebo: The method was shown to be Accurate and Precise by the successful demonstration of spiking and recovering the Known Impurities at 50%, 100% and 200% Levels of the proposed specification limit concentrations. The mean recovery of replicate preparations at each spiking level was between 85.0% and 115.0%. The %RSD at each level was not greater than 10.0%. The Accuracy and Recovery of this Method was found to be acceptable. Robustness: The method was shown to be Robust with regards to Flow Rate and Organic Phase composition. Filter Study: The use of 0.45 µm Nylon Membrane Filters from Millipore was shown not to contribute additional components or to absorb the Known Impurities as long as at least 3-mL of filtrate was discarded. There were no new peaks found in the Filtered Diluent. Stability of standard and sample solutions: The Standards and Sample Solutions were shown to be stable for a period of 3 days at room temperature. Table.1.Optimised chromatographic conditions Parameters Assay Related substances Column Thermohypersil BDS C18 Thermohypersil BDS C18 100Χ4.6mm, 3µm 150Χ4.6mm, 5µm Mobile phase Buffer: ACN (65:35) M.P A: 0.08M tetra butyl ammonium hydrogen sulphate M.P B: ACN Flow rate 1.5ml/min 1.5ml/min Wave length UV at 225nm UV at 225nm Injection volume 10µl 10µl Column temperature 25 0 c 30 0 c Run time 12min 35min Table.2.Linearity data Assay Related substances Mcg/ml Area Rt Mcg/ml Area Rt Correlation coefficient = Correlation coefficient Intercept = 8150 Intercept = -16 IJRPB 1(6) November December 2013 Page 861

6 Table.3.Validation parameters Parameters Assay Related substances Linearity mcg/ml mcg/ml Precision (%RSD) ,1.5 Accuracy ,101.0 Assay Ruggedness(%RSD) ,1.8 Table.4.Forced degradation studies Parameter Assay Related substances Stress Time Rt Time Rt Imp B Imp G Drug As such 24hrs hrs Acid 2hrs hrs Base 2hrs hrs Oxidation 24hrs hrs Photolysis Until attaining of temperature 6.2(UV) 6.22(LUX) Until attaining of temperature 8.46(UV) 8.59 (LUX 16.6 (UV) (LUX) (UV) (LUX) Thermal 24hrs hrs Table.5.Filter study Parameter Name Assay Related substances Area Rt Area Rt Centrifuged Impurity B Impurity G Itraconazole Filtered Impurity B Impurity G Itraconazole Table.6.Solution stability parameter Assay Related substances Area Rt Name Area Rt Impurity B Day Impurity g Itraconazole Impurity B Day Impurity G Day Day Day Itraconazole Impurity B Impurity G Itraconazole Impurity B - - Impurity G - - Itraconazole - - Impurity B Impurity G Itraconazole Figure.1.Chromatogram of sample Figure.2.Chromatogram of standard IJRPB 1(6) November December 2013 Page 862

7 Figure.3.Linearity plot Figure.4. Photolytic degradation (UV) Figure.5. Photolytic degradation(lux) Figure.6. Thermal degradation Figure.7. Acid degradation Figure.8. Base degradation Figure.9.Oxidative degradation Figure.10.Chromatogram of blank IJRPB 1(6) November December 2013 Page 863

8 Figure.11.Chromatogram of std Figure.12. Chromatogram of sample Figure.13. Linearity plot Figure. 14. Photolytic degradation (UV) Figure.15. Photolytic degradation (LUX) Figure.16. Thermal degradation Figure.17. Acid degradation CONCLUSION finally it concludes that all the parameters are within the limits and meet the acceptance criteria of ICH guidelines for method validation. The proposed method was simple, accurate, specific, precise, robust, Figure.18.Base degradation rugged and economical. Hence this method is validated and can be used for routine and stability sample analysis. IJRPB 1(6) November December 2013 Page 864

9 REFERENCES Beckett and Stenlake, Chromatography, Beckett A.H, editor, Practical pharmaceutical chemistry, 4 th ed, New Delhi, CBS publishers; 2007, Guru deep R. Chatwal, Sham K Anand, High performance liquid chromatography, Arora M, editor, Instrumental Methods of Chemical Analysis, 5 th ed, Mumbai, Himalaya Publishing House Pvt ltd, 2009, IJRPB 1(6) November December 2013 Page 865