International Journal of Innovative Pharmaceutical Sciences and Research
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1 International Journal of Innovative Pharmaceutical Sciences and Research A NEW ANALYTICAL METHOD DEVELOPMENT AND VALIDATION FOR SIMULTANEOUS ESTIMATION OF NALTREXONE AND OXYCODONE IN CAPSULE DOSAGE FORM BY RP-HPLC METHOD Nikhath Jahan, K.M.Noorulla* Department of Pharmaceutical Analysis, Deccan School of Pharmacy, Hyderabad , Telangana, INDIA Abstract A simple, precise and accurate Reverse Phase High Performance Liquid Chromatographic method was developed for simultaneous estimation of Naltrexone and Oxycodone in its capsule dosage form was developed and validated in present studies. Chromatographic separation was achieved on a c18 column using mobile phase consisting of a mixture of 55 volumes of mixed phosphate buffer and 45 volumes of Acetonitrile. This was found to give sharp peak of Naltrexone and Oxycodone at a retention time of 2.71min and 3.77 respectively. HPLC Analysis was carried out at a wavelength of 263nm with a flow rate of 1 ml/min. Linearity was observed in the range 20-60µg/ml for NALTREXONE (R 2 =0.998) and µg /ml for OXYCODONE (R 2 =0.997) for the amount of drugs estimated by the proposed methods was in good agreement with the label claim. The developed method was employed with a high degree of precision and accuracy for the simultaneous estimation of Naltrexone and Oxycodone. The method was validated for accuracy, precision, robustness, ruggedness, specificity. The accuracy of the methods was assessed by recovery studies at three different levels. Recovery experiments indicated the absence of interference from commonly encountered pharmaceutical additives. The method was found to be precise as indicated by the repeatability analysis, showing %RSD less than 2. All statistical data proves validity of the methods and can be used for routine analysis of pharmaceutical dosage form. The proposed method can be used for the determination of these drugs in combined dosage form. Keywords: RP-HPLC, Naltrexone, Oxycodone, Validation. Corresponding Author: Dr. K M Noorulla Associate Professor, Department of Pharmaceutical Analysis, Deccan School of Pharmacy, Dar-Us-Salam, Aghapura, Hyderabad , Telangana, INDIA nooruas@gmail.com Phone: Available online: August Issue 51
2 INTRODUCTION Derivative of noroxymorphone that is the N-cyclopropylmethyl congener of naloxone. It is a narcotic antagonist that is effective orally, longer lasting and more potent than naloxone, and has been proposed for the treatment of heroin addiction. The FDA has approved naltrexone for the treatment of alcohol dependence. Oxycodone is a semisynthetic derivative of codeine that acts as a narcotic analgesic more potent and addicting than codeine. An extended-release (ER) form of oxycodone (Xtampza ER) was approved for the management of daily, around-the-clock pain management in April, The survey revealed the need for a more, precise, economical and simple analytical method for the estimation of Naltrexone and oxycodone. The present study describes the simple, precise and economical RP-HPLC method for the simultaneous estimation of Naltrexone and Oxycodone in dosage form [1-3]. MATERIAL AND METHOD All the solvents are of HPLC Grade and all the reagents are of AR Grade. The drugs Naltrexone and Oxycodone are obtained as gift samples. The marketed formulation Troxyca has been obtained from local pharmacy. Chromatographic Conditions: Mobile phase : Mixed phosphate buffer: Acetonitrile. (55:45) Column : Inertsil ODS, ( µ) Wavelength Flow rate Mobile Phase : 263 nm : 1ml/min A mixture of Mixed Phosphate buffer (ph): ACN were prepared. The mobile phase was sonicated for 10min to remove gases and filtered through 0.45µ membrane filter for degassing of mobile phase [4,5]. RESULTS AND DISCUSSION Solubility Studies These studies are carried out at 25 0 C Naltrexone: Freely soluble in ethanol and methanol, and slightly soluble in acetone and isopropanol and very slightly soluble in water. Available online: August Issue 52
3 Oxycodone Freely soluble in methanol and water. DETERMINATION OF ABSORPTION MAXIMA: Standard Solution of Naltrexone 10 mg of NALTREXONE was weighed and transferred in to 100ml volumetric flask and dissolved in methanol and then make up to the mark with water and prepare 100µg /ml of solution by diluting 1ml to 10ml with methanol. Standard Solution of Oxycodone 10 mg of Oxycodone was weighed in to 100ml volumetric flask and dissolved in methanol and then dilute up to the mark with water and prepare 100 µg /ml of solution by diluting 1ml to 10ml with methanol. Fig.1: UV- Visible Overlay Spectrum of Naltrexone and Oxycodone The wavelength of maximum absorption (λ max ) of the drug, 10 μg/ml solution of the drugs in methanol were scanned using UV-Visible spectrophotometer within the wavelength region of nm against methanol as blank. The isobestic point was found to be 263 nm for the combination. Method Development of Naltrexone and Oxycodone Mobile phase : Mixed Phosphate Buffer: ACN ph : 4.0 Ratio : 55:45 Column : Inertsil ODS, ( µ) Wavelength : 263 nm Flow rate : 1ml/min Available online: August Issue 53
4 Preparation of Mixed Standard Solution Weigh accurately 10mg of Naltrexone and 10 mg of Oxycodone in 25 ml of volumetric flask and dissolve make up the volume with mobile phase. From above stock solution 40 µg/ml of Naltrexone and 20 µg/ml of Oxycodone is prepared by diluting 1.5 ml to 10ml with mobile phase. This solution is used for recording chromatogram. Fig.2: Optimized Chromatogram of Naltrexone and Oxycodone. Observation All the system suitability requirements were met. The peak Asymmetry factor was less than 2 for both Oxycodone and Naltrexone. The efficiency was more than 2000 Oxycodone and Naltrexone. Resolution between two peaks >1.5. Table 1: Optimized Chromatographic Conditions Mobile phase Mixed Phosphate Buffer:ACN (55:45) ph 4.0 Column Inertsil ODS 3V column,c18(150x4.6 ID) 5µm Flow rate 1.0 ml/min Column temperature Room temperature(20-25 o C) Sample temperature Room temperature(20-25 o C) Wavelength 263 Injection volume 20 µl Run time 6 min Retention time About min for Naltrexone and min for Oxycodone. Available online: August Issue 54
5 Assay Preparation of Samples for Assay Preparation of Mixed Standard Solution Weigh accurately 10mg of Naltrexone and 10 mg of Oxycodone in 25 ml of volumetric flask and dissolve in 25ml of mobile phase and make up the volume with mobile phase. From above stock solution 40 µg/ml of Naltrexone and 20 µg/ml of Oxycodone is prepared by diluting 1.5 ml to 10ml with mobile phase. This solution is used for recording chromatogram. Capsule Sample 10 capsule (each capsule contains Oxycodone-30mg mg Naltrexone-3.6 mg) were weighed and taken into a mortar and crushed to fine powder and uniformly mixed. Capsule stock solutions of Oxycodone and Naltrexone (μg/ml) were prepared by dissolving weight equivalent to 10 mg of Oxycodone and 10mg Naltrexone and dissolved in sufficient mobile phase. After that filtered the solution using 0.45-micron syringe filter and Sonicated for 5 min and dilute to 10ml with mobile phase. Further dilutions are prepared in 5 replicates of 20μg/ml of OXYCODONE and 40ug/ml of NALTREXONE was made by adding 1.5 ml of stock solution to 10 ml of mobile phase. Calculation The amount of Oxycodone and Naltrexone present in the formulation by using the formula given below, and results shown in above table: Naltrexone Table 2: Assay Results Oxycodone Standard Area Sample Area Standard Area Sample Area Injection Injection Injection Injection Injection Average Area Assay(%purity) Observation The amount of NALTREXONE and OXYCODONE present in the taken dosage form was found to be %and100.18% respectively. Available online: August Issue 55
6 VALIDATION [6-10] Specificity Fig.3: Chromatogram for specificity of Fig.4: Chromatogram for specificity of naltrexone and oxycodone sample naltrexone and oxycodone standard Linearity and Range Preparation of Standard Stock Solution Standard stock solutions 40ug/ml of Naltrexone and 20ug/ml of Oxycodone (microgram/ml) were prepared by dissolving 10 mg of Naltrexone and 10 mg of Oxycodone dissolved in sufficient mobile phase and dilute 1.5 to 10 ml with mobile phase. Table 3: Linearity Preparations Preparations Volume from standard stock transferred in ml Volume made up in ml (with mobile phase) Concentration of solution(µg /ml) Naltrexone Oxycodone Preparation Preparation Preparation Preparation Preparation Available online: August Issue 56
7 Table 4: Linearity of Naltrexone S.No. Conc.(µg/ml ) Area Table 5: Linearity of Oxycodone S.No. Conc.(µg/ml ) Area Fig. 6: Linearity graph of naltrexone and oxycodone Acceptance Criteria The relationship between the concentration of Naltrexone and Oxycodone and area of Naltrexone and Oxycodone should be linear in the specified range and the correlation should not be less than Observation The correlation coefficient for linear curve obtained between concentration vs. Area for standard preparations of Naltrexone and Oxycodone is and The relationship between the concentration of Naltrexone and Oxycodone and area of Naltrexone and Oxycodone is linear in the range examined since all points lie in a straight line and the correlation coefficient is well within limits. ACCURACY Accuracy of the method was determined by Recovery studies. To the formulation (pre analyzed sample), the reference standards of the drugs were added at the level of 50%, 100%, 150%. The Available online: August Issue 57
8 recovery studies were carried out three times and the percentage recovery and percentage mean recovery were calculated for drug is shown in table. To check the accuracy of the method, recovery studies were carried out by addition of standard drug solution to pre-analyzed sample solution at three different levels 50%, 100%, 150%. Acceptance Criteria: The % recovery of Naltrexone and Oxycodone should lie between 98 % and 102 %. Observation Table 6: Recovery results for naltrexone Table 7: Recovery results for oxycodone Accuracy Oxycodone Amount taken(mcg/ml) Area %Recovery The percentage mean recovery of Naltrexone and Oxycodone is % and % respectively. Recovery level PRECISION Recovery level Method precision Accuracy Oxycodone Amount taken(mcg/ml) Area %Recovery Average % Recovery Average % Recovery Prepared sample preparations of Oxycodone and Naltrexone as per test method and injected 6 times in to the column. Available online: August Issue 58
9 Acceptance Criteria The % Relative Standard Deviation of Assay preparations of Oxycodone and Naltrexone should be not more than 2.0%. Observation Table 8: Results for method precision of naltrexone and oxycodone Naltrexone S.No. Rt Area Avg Stdev %RSD Oxycodone S.No. Rt Area Avg Stdev %RSD Test results for Oxycodone and Naltrexone are showing that the %RSD of Assay results are within limits. SYSTEM SUITABILITY Fig.7: Chromatogram for system suitability of naltrexone and oxycodone sample Available online: August Issue 59
10 ROBUSTNESS Chromatographic conditions variation Table: 9 Results for system suitability of naltrexone Table 10: Results for system suitability of oxycodone To demonstrate the robustness of the method, prepared solution as per test method and injected at different variable conditions like using different conditions like flow rate and wavelength. System suitability parameters were compared with that of method precision. Acceptance Criteria Naltrexone S.No. Rt Area Avg Stdev %RSD Oxycodone S.No. Rt Area Avg Stdev %RSD The system suitability should pass as per the test method at variable conditions. Fig.8: Chromatogram of naltrexone and oxycodone robustness (0.8 ml/min) Available online: August Issue 60
11 Fig.9: Chromatogram of naltrexone and oxycodone robustness (1.2 ml/min) Fig.10: Chromatogram of naltrexone and oxycodone robustness (261nm) Fig.11: Chromatogram of naltrexone and oxycodone for robustness (265nm) Observation From the observation it was found that the method is robust at variable condition. 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.12: Chromatogram of analyst 01 standard preparation Available online: August Issue 61
12 Fig.13: Chromatogram of analyst 01 Fig.14: Chromatogram of analyst 02 sample preparation standard preparation Observation Fig.15: Chromatogram of analyst 02 sample preparation Table 11: Results for ruggedness Naltrexone %Assay Oxycodone %Assay Analyst Analyst Anaylst Anaylst The % deviation between analysts should not be more than 2%. CONCLUSION A simple, accurate, economic and robust RP-HPLC method has been developed, validated and reported for the Simultaneous estimation of Naltrexone and Oxycodone in pharmaceutical capsule dosage form. There was no interference from any excipient in the determination of drugs in the studied dosage form which indicates the method is specific. Reverse phase liquid chromatography was performed using Agilent LC C 18 ( mm, 5 µm) column and mixed phosphate buffer: acetonitrile (55:45) as mobile phase with flow rate 1 ml/min. The detection was carried out at 263 Available online: August Issue 62
13 nm. The retention times were found to be 2.71 min for naltrexone and 3.77 for oxycodone. Assay of naltrexone was found to be 99.90% and for oxycodone was found to be % respectively. The concentration range of 20-60µg/ml with (r 2 =0.998) for Naltrexone and in the concentration range of 10-30µg/ml with (r 2 =0.997) for the amount of drugs estimated by the proposed method was found to be in good agreement with the label claim. All the method validation parameters were within the acceptance criteria as per ICH guidelines. So we can conclude that this method is simple, linear, accurate and precise.hence, the method can be successfully transferred for the routine analysis of naltrexone and oxycodone in Pharmaceutical capsule dosage forms. ACKNOWLEDGEMENT Authors are thankful to the Deccan school of pharmacy for providing necessary facilities to complete this study. REFERENCES 1. Corner, S. D., Sullivan, M. A., Elmer, Y., Rothenberg, J.L., Herbert, D., Kyle, K., Dackis, C., Brien, C. (2006). Injectable, Sustained-Release Naltrexone for the Treatment of Opioid Dependence. Archives of general psychiatry, 63(2), Johansson, B.A., Berglund, M., Anna, L. (2006). Efficacy of maintenance treatment with naltrexone for opioid dependence. A meta-analytical review. 101 (4), Ray, L.A., Oslin, D.W., (2009). Naltrexone for the treatment of alcohol dependence among African Americans: Results from the combine Study. Drug and Alcohol Dependence. 105 (3): Gebauer, M.G., Mcclure, A. F., Vlahakis, T. L. (2001). Stability indicating HPLC method for the estimation of oxycodone and lidocaine in rectal gel. International Journal of Pharmaceutic, 2223 (1-2) David, P. N., John, A. I., Atkinson, R. L., David, K. S., Malcolm, R. J., Mitchell, J. E.,(1986). Naltrexone hydrochloride (Trexan): A review of serum transaminase elevations at high dosage. NIDA research monograph, 67, Sharma, B. K., High Performance Liquid Chromatography. Instrumental Methods of Chemical Analysis, 24th ed., Goel Publishers.: Meerut, 2005; page no: Minozzi, S., Amato, L., Simona, V., Simona, D., Ursula, K., Annette, V. (2011).Oral naltrexone maintenance treatment for opioid dependence. Systematic Reviews, 4, Cochrane Database of Available online: August Issue 63
14 8. Kranzler, H.R., Kirk, J., (2001).Efficacy of Naltrexone and Acamprosate for Alcoholism Treatment: A Meta-Analysis. Alcoholism Clinical and Experimental Research, 25 (9), Jafari, M., Barzin, J., Mobedi, H. (2016).A stability-indicating HPLC method for simultaneous determination of morphine and naltrexone. Journal of Chromatography, B. Analytical Technologies in the Biomedical and Life Science, 1011, Pfohl, D.N., Allen, J.I., Atkinson, R.L., Morley, J.E. (1986). Naltrexone hydrochloride (Trexan): A review of serum transaminase elevations at high dosage. NIDA research monograph, 67, Available online: August Issue 64
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