SIMULTANEOUS ESTIMATION OF AMANTADINE HYDROCHLORIDE AND OSELTAMIVIR PHOSPHATE USING PRECOLUMN DERIVATIZATION TECHNIQUEHTML Full Text
SIMULTANEOUS ESTIMATION OF AMANTADINE HYDROCHLORIDE AND OSELTAMIVIR PHOSPHATE USING PRECOLUMN DERIVATIZATION TECHNIQUE
V. Ramya 1, Zaranappa * 1 and Y. N. Munisham Gowda 2
Department of Pharmaceutical Chemistry 1, Government College of Pharmacy, Subbaiah Circle, Bengaluru - 560027, Karnataka, India.
Department of Drugs Control 2, Drug Testing Laboratory, Palace Road, Bengaluru - 560001, Karnataka, India.
ABSTRACT: A simple, efficient HPLC method has been developed and validated for simultaneous determination of Amantadine hydrochloride and Oseltamivir phosphate using pre-column derivatization with phenyl isothiocyanate (PITC). The chromatography separation was achieved on Phenomenex C18 column of 250 × 4.6 mm 5 µm particle size, mobile phase used for chromatographic run consisted of acetonitrile and water (60: 40% v/v) with a flow rate of 2 mL/min. The analysis was performed at ambient temperature, and the eluent was monitored at 250 nm using a UV detector. The retention time of Amantadine hydrochloride and Oseltamivir phosphate was found to be 7.1 min and 3.2 min. The limit of detection (LOD) for Amantadine hydrochloride and Oseltamivir phosphate was observed to be 0.1µg/mL and 0.25 µg/mL, respectively. Limit of quantification (LOQ) was found to be 0.5 µg/mL and 0.25 µg/mL. The developed method was validated as per ICH guidelines using parameters like linearity, specificity, precision, linearity, accuracy, ruggedness, robustness, LOD, and LOQ. All the validation parameters were found to be well within the acceptance criteria. Hence, the proposed method can be used for the routine analysis of Amantadine hydrochloride and Oseltamivir phosphate in bulk and tablet dosage forms.
Amantadine hydrochloride, Oseltamivir phosphate, Precolumn derivatization, RP-HPLC, Simultaneous estimation, ICH guidelines
INTRODUCTION: Amantadine is a synthetic tricyclic amine with antiviral, antiparkinsonian, and antihyperalgesic activities. Amantadine appears to exert its antiviral effect against the influenza A virus by interfering with the function of the transmembrane domain of viral M2 protein, thereby preventing the release of infectious viral nucleic acids into host cells.
Amantadine exerts its antiparkinsonian effects by stimulating the release of dopamine from striatal dopaminergic nerve terminals and inhibiting its presynaptic reuptake and also exerts an anticholinergic effect through inhibition of N methyl-D-aspartic acid (NMDA) receptor-mediated stimulation of acetylcholine, resulting in anti-hyperalgesia 1. Since Amantadine lacks useful chromophores in its structure and cannot be readily quantified by either UV or fluorescence detection techniques, consequently, Amantadine has to be derivatized before analysis.
Oseltamivir phosphate is an antiviral drug, a neuraminidase inhibitor used in the treatment and prophylaxis of both influenza A and influenza B 2. This can be used prophylactically to prevent influenza during epidemics. Oseltamivir is a drug of choice for bird flu (currently strain causing a pandemic is H5N1) as well as swine flu (H1N1). Oseltamivir carboxylate has an antiviral spectrum and potency similar to that of Zanamivir. It inhibits amantadine -resistant influenza A viruses 3. Combination chemotherapy provided a survival advantage over the single-agent treatment of mice inoculated with neurotropic H5N1 influenza virus. This strategy might be an option for the control of pandemic influenza viruses that are sensitive to Amantadine. Combination treatment with Amantadine and Oseltamivir provided greater protection against lethal infection with Amantadine sensitive H5N1 virus than did monotherapy. Moreover, the spread of the virus to the brain was prevented by both combination regimens. The efficacy of the drug combinations against Amantadine-resistant H5N1 virus was comparable to that of Oseltamivir alone 4.
FIG 1: STRUCTURE OF AMANTADINE HYDROCHLORIDE
FIG 2: STRUCTURE OF OSELTAMIVIR PHOSPHATE
On literature survey that only few HPLC methods were reported on precolumn derivatization of Amantadine for individual drug 6, 7, 8, 14. Oseltamivir phosphate was estimated and validated by RP-HPLC 9, 10, 11, 12, 13 of the individual drug. But no method was found for simultaneous estimation of Amantadine hydrochloride and Oseltamivir phosphate by RP-HPLC. Hence, there is a need for the development of a newer, rapid, accurate, and reproducible analytical method for simultaneous estimation of Amantadine hydrochloride and Oseltamivir phosphate in bulk and pharmaceutical dosage forms.
MATERIALS AND METHODS:
Chemicals and Solvents: Pure standards (API) of Amantadine hydrochloride and Oseltamivir phosphate were procured from Apotex pharmaceuticals. Phenyl isothiocyanate (PITC 99%), sodium bicarbonate and sodium carbonate (Analytical grade) were purchased from Hi-media. Acetonitrile (HPLC grade) was purchased from E. Merck. Amantadine 100 mg and Oseltamivir 75 mg capsules were procured from local market Bengaluru, India.
Instrument and Chromatographic Conditions: The High-Performance Liquid Chromatography consisted of Shimadzu-SPD-20A prominence auto-sampler fitted with UV visible detector (SPD-20A), PDA detector (SPD-M20A) with Shimadzu-LC-20AT pump. The chromatographic separation was achieved by using Phenomenex C18 (250 mm × 4.6 mm, 5µ) stationary phase and mobile phase consists of acetonitrile and water (60:40 v/v with a flow rate of 2 mL/min. The chromatogram was recorded using LC solutions software. The analysis was performed at ambient temperature, and the eluent was monitored at 250 nm using a UV detector.
Preparation of Stock and Standard Solutions: Accurately 25 mg of Amantadine hydrochloride & 25 mg of Oseltamivir phosphate were weighed into a clean and dry 25 ml volumetric flask dissolved with sufficient volume of methanol. The final volume was made up to 25 ml with methanol to give the solution containing 1 μg/mL of Amantadine hydrochloride and 1 μg/mL of Oseltamivir phosphate.
Preparation of Phenyl Isothiocyanate Solution: 1 ml of phenyl isothiocyanate was transferred into 25 ml clean and dry volumetric flask. Sufficient volume of acetonitrile was added and vortexed for 1 min, and volume was made up to the mark with acetonitrile.
Preparation of Sodium Bicarbonate Solution: 1.25g of sodium bicarbonate was weighed and transferred into a clean and dry 25 ml volumetric flask. Sufficient volume of HPLC grade water was added, vortexed for 1 min. The volume was made up to the mark with HPLC grade water.
Preparation of Sodium Carbonate Solution: 0.25g of sodium carbonate was weighed and transferred into clean and dry 25 ml volumetric flask. Sufficient volume of HPLC grade water added, vortexed for 1 min. The volume was made up to the mark with HPLC grade water.
Derivatization Procedure for Standard and Sample Solutions: Derivatization of Amantadine hydrochloride and Oseltamivir phosphate with phenyl isothiocyanate was carried out. Transfer 1 ml of Amantadine hydrochloride and Oseltamivir phosphate, 0.5 ml of phenyl isothiocyanate, 0.5 ml of sodium bicarbonate solutions were added and heated at 40 °C water bath for 10 min. After 0.5 ml of sodium carbonate was added, cyclomixed and heated 40 °C again for 5 min and the solution was cooled down to room temperature, and final volume is made up to 25 ml with water and methanol (70:30% v/v) as a diluent and filtered with 0.45 µm nylon syringe filter. 20 µl of the derivatized solution was injected, and detection was carried out at 250 nm. All the solutions were stored at 4 °C until the analysis.
Method Validation: The proposed method was validated in compliance with ICH guidelines for linearity, accuracy, precision, specificity, robustness, and system suitability parameters by the following procedures.
Linearity: Accurately 50 mg of Amantadine hydrochloride and 50 mg of Oseltamivir phosphate was weighed into a clean and dry 10 ml volumetric flasks separately, dissolved with sufficient volume of diluent. The volume was made up to 10 ml with diluent to get the concentration of 5000 μg/mL for Amantadine hydrochloride and 5000 µg/mL of Oseltamivir phosphate. From the above stock solutions, 2.5 ml of Amantadine hydrochloride and Oseltamivir phosphate were transferred into 25 ml volumetric flask and derivatized with PITC. The volume was made up to 25 ml with diluent to get the concentration of 500 μg/mL for Amantadine hydrochloride and 500 µg/mL of Oseltamivir phosphate.
Preparation of Working Standard Solutions: The various concentration of working derivatized solution of Amantadine hydrochloride and Oseltamivir phosphate was made by pipetting 0.1 ml, 0.2 ml, 0.5 ml, 1 ml, 1.2 ml and 1.5 ml from stock (II) into a series of 10 ml volumetric flask and diluted to 10 mL to get the final concentration of 5 µg/mL, 10 µg/mL, 25 µg/mL, 50 µg/mL, 75 µg/mL of Amantadine hydrochloride and 5 μg/mL, 10 μg/mL, 25 μg/mL, 50 μg/mL and 75 μg/mL of Oseltamivir phosphate solutions respectively.
Determination: The derivatized solutions of Amantadine hydrochloride from 5 µg/mL to 75 µg/mL and Oseltamivir phosphate from 5 µg/mL to 75 µg/mL were injected into a chromatograph at a flow rate of 2 ml/min.
Retention time and peak area obtained were recorded, and a standard calibration curve was plotted for Amantadine hydrochloride, and Oseltamivir phosphate and linearity equation was derived. The correlation coefficient, % curve fitting, were also calculated. The results obtained were shown in Table 1 & 2, Fig. 3.
TABLE 1: LINEARITY DATA OF AMANTADINE HYDROCHLORIDE AND OSELTAMIVIR PHOSPHATE
|Amantadine hydrochloride||Oseltamivir phosphate|
|Concentration (µg/mL)||Average Area||Concentration (µg/mL)||Average Area|
TABLE 2: LINEARITY REPORT OF AMANTADINE HYDROCHLORIDE AND OSELTAMIVIR PHOSPHATE
|Parameters||Amantadine hydrochloride||Oseltamivir phosphate||Acceptance criteria|
|Regression Equation||y = 18071×-36475||y = 47814×+47156||-|
|Correlation Coefficient||0.9991||0.999||More than 0.999|
FIG. 3: STANDARD CALIBRATION CURVES
Preparation of Sample Stock Solution: Twenty tablets, each containing 100 mg of Amantadine hydrochloride and 75 mg of Oseltamivir phosphate were weighed and finely powdered. Powder equivalent to 100 mg of Amantadine hydrochloride and 100 mg of Oseltamivir phosphate was taken and transferred into a clean, dry 100 ml volumetric flask. The powder was first dissolved in diluent and sonicated for 20 min. The resulting mixture was then filtered through Whatman filter no 0.45 μ. The final volume of the filtrate was made up to 100 ml with diluent
Preparation of Standard Stock Solution: Accurately weighed 10 mg of standard drug Amantadine hydrochloride and 10 mg of Oseltamivir phosphate was transferred into a clean, dry 10 ml volumetric flask and the volume was made up to 10 ml with diluent to get the concentration of 100 μg/mL of Amantadine hydrochloride and 100 μg/mL of Oseltamivir phosphate.
Preparation of Standard and Sample Mixture:
Level I (80%): Volume of 0.8 ml sample stock solution, 1.0 ml of standard solution was transferred to 10 ml volumetric flask and derivatized with PITC separately, and volume was made up to mark with diluent (three replicates).
Level II (100%): Volume of 1.0 ml sample stock solution, 1.0 ml working standard stock solution was transferred to 10 ml volumetric flask and derivatized with PITC. Volume was made up to mark with diluent (three replicates).
Level III (120%): Volume of 1.2 ml sample stock solution, 1.0 ml of working standard stock solution was transferred to 10 ml volumetric flask and derivatized with PITC. Volume was made up to mark with diluent (three replicates).
Determination: The resulting mixture was injected repeatedly into the chromatograph, the peak area and chromatogram obtained were recorded, and the % recovery of standard Amantadine hydrochloride and Oseltamivir phosphate was calculated. The results obtained are presented in Table 3, 4 & 5.
Specificity: The specificity of the method was established by injecting the solutions of a placebo, standard individually to examine any interference. These results show that the peak of analyte was pure and excipients in the formulation did not interfere with the analysis.
TABLE 3: RECOVERY STUDY DATA FOR AMANTADINE HYDROCHLORIDE
|The actual amount added (µg/mL)||Amt of std. recovered (µg/m)||% Recovery|
TABLE 4: RECOVERY STUDY DATA FOR OSELTAMIVIR PHOSPHATE
|Actual amount added (µg/mL)||Amt of std. recovered (µg/m)||% Recovery|
TABLE 5: REPORT OF RECOVERY STUDIES FOR AMANTADINE HYDROCHLORIDE AND OSELTAMIVIR PHOSPHATE
|Level||Mean % Recovery of Amantadine hydrochloride||Mean % Recovery of Oseltamivir phosphate||Acceptance
FIG. 4: PLACEBO CHROMATOGRAM OF AMANTADINE HYDROCHLORIDE AND OSELTAMIVIR PHOSPHATE
The peak purity indices for sample and standard were found to be greater than 0.999, and this confirms the specificity of the method. There was no peak detected at a retention time of Amantadine hydrochloride 7.01 min and Oseltamivir phosphate 3.02 min. so, the proposed method is specific.
LOD and LOQ: LOD and LOQ for Amantadine hydrochloride and Oseltamivir phosphate by this method were evaluated based on signal-to-noise ratio method described in ICH guidelines. A signal-to-noise ratio between 3 or 2:1 is generally considered acceptable for estimating the detection limit. A typical signal-to-noise ratio required for LOQ is 10:1. Using the proposed HPLC method, the LOD and LOQ values were calculated and are given in Table 6.
TABLE 6: DATA FOR LOD AND LOQ OF AMANTADINE HYDROCHLORIDE AND OSELTAMIVIR PHOSPHATE
|Parameter||Amantadine hydrochloride||Oseltamivir phosphate|
|Peak Area||Concentration in µg/mL||Peak Area||Concentration in µg/mL|
Robustness: To evaluate the robustness of the developed RP-HPLC method, small, deliberate variations in the optimized parameters were made in chromatographic conditions like of flow rate, mobile phase ratio, and wavelength. The effect of change in flow rate, mobile phase ratio, and wavelength of detection on retention time and tailing factor were examined. The values obtained are mentioned in Table 7, 8 & 9. The method was found to be unaffected by the small changes like ± 0.2 mL/min in flow-rate of mobile phase and change in mobile phase ratio from 54:46 and 66:34 and ± 2 nm in detection wavelength.
System Suitability: Six replicates of Amantadine hydrochloride and Oseltamivir phosphate sample containing were given to evaluate equipment, electronics, analytical operations, and samples suitability.
Parameters calculated for system suitability were % RSD of retention time and area, number of theoretical plates and resolution. The results are given in Table 10.
TABLE 7: ROBUSTNESS DATA OF AMANTADINE HYDROCHLORIDE AND OSELTAMIVIR PHOSPHATE WITH CHANGE IN FLOW RATE
|Change in flow rate
|Peak area* of
|Peak area* of Oseltamivir phosphate||%
TABLE 8: ROBUSTNESS DATA OF AMANTADINE HYDROCHLORIDE AND OSELTAMIVIR PHOSPHATE WITH CHANGE IN MOBILE PHASE
|Change in Mobile phase ratio v/v||Peak area* of
|Peak area* of Oseltamivir phosphate||%
TABLE 9: ROBUSTNESS DATA OF AMANTADINE HYDROCHLORIDE AND OSELTAMIVIR PHOSPHATE WITH CHANGE IN WAVELENGTH
|Change in wavelength in nm||Peak area* of
|Peak area* of Oseltamivir phosphate||%
TABLE 10: DATA FOR SYSTEM SUITABILITY PARAMETER FOR AMANTADINE HYDROCHLORIDE AND OSELTAMIVIR PHOSPHATE
|System Suitability Parameters||Amantadine
Ruggedness: Intermediate precision expresses the variations within laboratories variations: (different days, different analysts, different equipment, etc.). The intermediate precision was performed for Amantadine hydrochloride and Oseltamivir phosphate by a different analyst on the different instrument using a different lot of column on a different day.
The % RSD for the same was calculated for Intermediate precision. The results are given in Table 11 and 12.
RESULTS AND DISCUSSION: Amantadine hydrochloride and Oseltamivir phosphate were derivatized with PITC successfully. The obtained derivatized solutions were analyzed.
TABLE 11: INTERMEDIATE PRECISION DATA OF ANALYST 1
|Amantadine Hydrochloride||Oseltamivir Phosphate|
|Replicates||Peak Area*||% Assay||Peak Area*||% Assay|
*Average of six determinations
TABLE 12: INTERMEDIATE PRECISION DATA OF ANALYST 2
|Amantadine Hydrochloride||Oseltamivir Phosphate|
|Replicates||Peak Area*||% Assay||Peak Area*||% Assay|
*Average of six determinations
Chromatographic conditions were screened for mobile phase composition, wavelength proportion and flow rate, mobile phase of acetonitrile: water (60:40 v/v) was optimized to give symmetric peak with short runtime at UV detection wavelength of 250 nm, and flow rate at 2 mL/min was found to be appropriate with adequate separation between the two drugs. Chromatogram of Amantadine hydrochloride and Oseltamivir phosphate at optimized chromatographic condition was recorded, the runtime was 10 min, and the retention times of Amantadine hydrochloride and Oseltamivir phosphate were found to be 7.1 and 3.2 min respectively.
CONCLUSION: The proposed HPLC method was found to be economical, sensitive, accurate, precise, specific and robust and can be used for the routine analysis of Amantadine hydrochloride and Oseltamivir phosphate in industry and academics. The results of linearity, accuracy, specificity, ruggedness, and robustness, proved to be within limits. The method provides selective quantification of Amantadine hydrochloride and Oseltamivir phosphate without interference from placebo.
ACKNOWLEDGEMENT: The authors are thankful to principal K. P. Channabasavaraj Government College of Pharmacy, Bengaluru for their guidance, support. We are thankful to drug testing laboratory Bengaluru for providing laboratory facilities.
CONFLICTS OF INTEREST: The authors declare no conflicts of interest.
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How to cite this article:
Ramya V, Zaranappa and Gowda YNM: Simultaneous estimation of Amantadine hydrochloride and Oseltamivir phosphate using precolumn derivatization technique. Int J Pharm Sci & Res 2019; 10(12): 5443-49. doi: 10.13040/IJPSR.0975-8232.10(12).5443-49.
All © 2013 are reserved by International Journal of Pharmaceutical Sciences and Research. This Journal licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 3.0 Unported License.
V. Ramya, Zaranappa * and Y. N. M. Gowda
Department of Pharmaceutical Chemistry, Government College of Pharmacy, Bengaluru, Karnataka, India.
20 March 2019
11 June 2019
17 July 2019
01 December 2019