INTRODUCTION:
Nepafenac is designated chemically as 2-amino-3-benzoylbenzeneacetamide with an empirical formula of C15H14N2O2 and molecular weight of 254.28g/mol. Nepafenac ophthalmic suspension 0.1% was recently made available for ophthalmic use and supplied as a sterile drug with a pH of approximately 7.4 and an osmolality of 305 mOsmol/kg. As a prodrug, nepafenac is a less active form of the drug, which is converted to the more active form, amfenac, after metabolic conversion through intraocular enzymatic hydrolysis. Nepafenac is a member of the new class of NSAID prodrugs for ophthalmic use, providing a novel drug delivery mechanism.
The analgesic and anti-inflammatory effect of nepafenac is the result of its fast penetration through the cornea in addition to conversion to amfenac. The superior corneal permeability of nepafenac is likely due to its molecular structure; it is an uncharged molecule whereas the other NSAIDs have acidic structures. Primary use non-steroidal anti-inflammatory agents are used in the management of inflammation associated with cataract surgery.
This paper describes a simple linear gradient reverse phase UPLC method which separates two of impurities reported in United States Pharmacopeia (Pharmacopeial Forum) as well as its possible impurities and degradants during its synthesis process developed in our laboratory. The structure of Nepafenac and its impurities are illustrated in Figure 1. Organic impurities can arise during the manufacturing process and storage of the drug substances, the criteria for their acceptance up to certain limits are based on the pharmaceutical studies or known safety data defined in International Conferences on Harmonization (ICH) (1) (ICH guidance on impurities in New Drug Substances Q3A(R2).2006). Most of analytical methods for Nepafenac are not LC-MS compatible but the analytical method discussed in this study is compatible to LC-MS and with elution time of about 6.5 minutes and equilibration time of 1.5 minutes with total runtime of around 8.0 mins. The accuracy, precision, limit of detection (LOD), limit of quantification (LOQ) and robustness of the method were determined in accordance with ICH guidelines (2) (International Conferences on Harmonization (ICH), Validation of analytical procedures: text and methodology, Q2 (R1), (2005).). This article reports, for the first time a new, rapid, efficient, LCMS compatible, simple and validated stability indicating UPLC method for separation of six potential impurities and degradation products as ‘one shot’ analysis.
MATERIAL AND METHODS:
Reagents and materials:
Ammonium formate, Formic acid purchased from Sigma-Aldrich, Hydrogen peroxide (30%) was bought from Thermo Fisher scientific. Methanol and acetonitrile were HPLC grade and others were analytical grade. HPLC-grade water was purified by a Milli-Q Reagent Water system (Millipore, Bedford, MA) and in preparing the aqueous solutions and the mobile phase throughout the experiments.
Sample of Nepafenac and its six impurities A-F (Figure 1) were synthesized and characterized by using MS, IR and NMR.
Instrumentation:
Chromatographic analysis was performed on an AcquityTM UPLC system (Waters Corp., Milford, MA, USA), equipped with a binary pump solvent management system, micro degasser, an autoplate-sampler, and thermostatic column compartment. Chromatographic separation was carried out on a Waters Acquity UPLC CSH C18, 100mm x 2.1mm, 1.7µ particle size column with an in-line filter (0.22μm) prior to the column. pH meter model 744Metrohm AG Switzerland, FS110D water bath were equipped with MV controller from Fisher Scientific (US), electro-thermostatic blast oven (Heratherm model, Thermo Scientific, US), Hundred Thousandth Balance XPE205DR, Mettler Toledo, Switzerland. A 50-W clear xenon lamp was employed as the light source for estimating the photolytic experiment (Newtronics photostability chamber (model NLPS4SI).
Stress degradation studies:
Stress degradation studies of Nepafenac were carried out under hydrolysis (acid and base), oxidation, photolytic and thermal forced conditions. For each degradation study, 10mg of Nepafenac sample in 10mL volumetric flask, dissolved in 4ml of Methanol. The tests of acidic and basic hydrolysis were carried out in 1ml of 0.01N hydrochloric acid (0.01mol·L-1), 1mL of 0.05N sodium hydroxide (0.05mol·L-1) respectively and the hydrolysis process was conducted at room temperature for 30 minutes for acid hydrolysis, 1 hour for base hydrolysis. Oxidative hydrolysis was carried out by adding 0.3mL of 30% Hydrogen peroxide at 25°C for 30 minutes and made up to volume with dissolving solvent. In the tests of photolytic and thermal studies, few milligrams of Nepafenac was put on the watch glasses and kept at 105°C for 24 Hrs in hot air oven, 1mg ml-1 sample solution was kept at 80°C for 8 hrs in shaking water bath the thermal experiment. Similarly Nepafenac was subjected to Photolytic exposure ICH guidelines (3) (International Conference on Harmonisation (ICH), Photo stability testing of new drug substances and products, Q1B, (1996)) to light providing an overall illumination of not less than 1.2 million lux hours and an integrated near ultraviolet energy of not less than 200 watt hours/square meter radiation in photostability chamber. Humidity degradation was carried out at 95% humidity/25°C/8 Hrs. The blank samples were prepared without adding the analytes in each stress condition.
Preparation of sample solutions:
The solutions obtained in the acidic and basic hydrolysis tests were neutralized with sodium hydroxide solution (0.01mol∙L-1) and hydrochloric acid solution (0.05 mol∙L -1), respectively and then diluted to the mark with dissolving solvent. The solutions obtained in the test of oxidative degradation were diluted to the volume with dissolving solvent and similarly for photolytic exposure and thermal degradation. All the solutions were filtered by 0.2μm membrane filters and kept at room temperature before UPLC analysis.
Chromatographic conditions:
Analysis was performed on a Waters Acquity UPLC equipped with diode array detector (DAD). Analysis was carried out at 245nm. Separation was achieved using Waters Acuity UPLC CSH C18 100mm x 2.1mm, 1.7µ particle size fast LC column. The data acquired via Waters Empower3 software. Mobile phase-A was 10 mM Ammonium formate in water pH adjusted to 4.0 with formic acid.
Mobile phase-B consists of a mixture of acetonitrile, methanol in the ratio 70:30. The gradient time program was (T min/A: B; T0.01/80:20; T4.5/40:60; T6.5/20:80 ;) thus the analysis time is 6.01 min and the initial eluent composition was restored at 8.0 min (80:20) and maintained further for 1.5 mins. The flow rate was set at 0.40mL min-1, the column temperature was maintained at 35°C and the injection volume was 1.00µL. A mixture of water: acetonitrile (50:50) was used as a dissolving solvent for the preparation of standard and sample solutions. Both mobile phase and diluent were filtered through a nylon membrane filter (pore size 0.2µm). A standard consisting of 0.0015mg/mL of all impurities along with Nepafenac was prepared. A sample solution consisting of Nepafenac 1.0mg/mL was prepared.
Experimental:
System suitability:
Standard solution containing Nepafenac and mixture of impurities at specification limit concentration (0.0015 mg/ml) was injected in six replicate and RSD for the area of all impurities and Nepafenac peaks were calculated. The resolution between impurity E and Impurity C was calculated.
Specificity:
During specificity study, Nepafenac and impurity-A to impurity-E were injected separately. Nepafenac sample preparation (1.0mg/mL) spiked with impurities at 0.15% level (mixture of all impurities at 0.0015mg/mL) were also injected. The spectra and purity plots were extracted through diode array detector for each ingredient in the spiked sample.
Furthermore, forced degradation studies were conducted in order to prove the stability indicating nature of the method. Sample solution was subjected to acid and base hydrolysis, oxidation using 30% H2O2, exposure to photolytic degradation, humidity (95%) and thermal (105°C). Peak purity was determined using PDA detector.
Linearity, limit of detection (LOD) and limit of quantitation (LOQ):
Six different concentrations of linearity standard solutions were prepared with Nepafenac and mixture of impurities from LOQ to 200% of the specification limit concentration. Each linearity standard solution was injected in triplicate and linear regression analysis for each ingredient was performed.
System precision-repeatability-standard solution:
The system precision was examined by analyzing standard solution containing Nepafenac and its impurities at 0.0015mg/mL concentration in six replicates.
Method precision-repeatability-sample solution:
Method precision was examined by analyzing Nepafenac sample in six preparations and calculated the RSD for the individual and total impurities value.
Ruggedness- intermediate precision:
Precision was repeated using different analyst, on different day, on different instrument and using column of different lot. Over all RSD was calculated for the individual and total impurities values.
Accuracy:
Triplicate sample preparation of Nepafenac spiked with impurities at 50% level, 100% level and 120% level were analysed.
Robustness:
Several below parameters of the method were purposely altered in order to determine the robustness of the method. Standard solution containing Nepafenac and mixture of impurities at specification limit concentration was injected in six replicate and RSD for the area of all impurities and Nepafenac peaks was found to be less than 10.0%. The resolution between impurity E and Impurity C was found to be more than 1.5.
i) Variation in flow rate ± 10%
ii) Variation in column oven temperature ± 5ºC
iii) Variation in wavelength ± 5 nm
Solution stability:
Sample solution was injected at different time intervals for about 24Hrs kept at 25±2°C by spiking impurities at 0.15% level. The cumulative RSD was calculated for the area of impurities and Nepafenac peak in the standard solution and area for individual and total impurities in sample solution.
METHOD VALIDATION RESULTS AND TABLES:
System suitability:
The criteria of resolution between impurity E and Impurity C peak from the system suitability preparation was more than 1.5. RSD for the area of Nepafenac peak and all the impurities from the replicate injections of standard preparations was less than 10.0%, all the parameters were met during entire validation (Table1).
Specificity:
As shown in the Figure 3, Nepafenac peak was well separated from each other impurities. No blank peak interference was observed at the retention time of known peaks. The purity angle was less than purity threshold for the Nepafenac peak in the spiked sample. Hence the method was selective and specific. Furthermore, specificity of the method was confirmed through forced degradation studies. Nepafenac showed degradation products during acid, alkali hydrolysis and oxidation. Since peak purity angle was less than the purity threshold for Nepafenac peak in all the above degradation samples, the method was stability indicating for the determination of impurities in Nepafenac. The results from forced degradation studies are summarized in Table 2.
Linearity, limit of detection (LOD) and limit of quantification for related substance method:
Linear regression analysis for each ingredient showed that the calibration curves were linear over the concentration range shown in Table 3. Limit of detection and quantification were also presented in the same table.
Precision-repeatability:
RSD for the individual and total impurities were found to be below the acceptance value (Table 4).
Intermediate precision-ruggedness:
The RSD of individual and total impurities were calculated and found to be less than 10.0%. The overall RSD between method precision and intermediate precision were less than 10.0%, which demonstrates good precision of the method. Data presented in Table 4.
Accuracy:
The recovery of three sample preparations at three different levels were examined, the range was from 102.3% to 108.1%. Results are summarized in Table 4.
Robustness:
The results obtained from the robustness study were well within the limit for related substance method (RSD NMT 10.0%). Data incorporated in Table 5.
Solution stability:
Cumulative RSD was calculated for the individual impurity and total impurities in the standard solution and was found to be less than 10.0%. Results are summarized in Table 6.
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Nepafenac |
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Impurity A |
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Impurity B |
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Impurity C(Amfenac) |
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Impurity D |
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Impurity E |
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Impurity F |
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Figure 1: Structure of Nepafenac and its impurities.