Development and Validation of Amlodipine Impurities in Amlodipine Tablets Using Design Space Computer Modeling

A rapid, sensitive, robust, rugged and linear HPLC method is developed using QbD approach and validated as per ICH for the estimation of amlodipine impurities in tablet dosage form. Phosphate buffer with triethyl amine adjusted to pH to 2.8 is used as the mobile phase and 3 μ particle size C18 column of 150 mm length and 4.6 mm internal diameter is used. Using photo diode array (PDA) detector, the compounds are monitored at 340 nm. All impurities are well separated and flow Gradient has been optimized to obtain the acceptable resolution between impurities and amlodipine. Diluent was chosen, based on the impurity peak shapes and recoveries. Test concentration and injection volume have been optimized to obtain limit of quantification (LOQ) values below the reporting threshold.


Introduction
is used to treat the high blood pressure by allowing the free flowing of blood through blood cells.Amlodipine belongs to calcium channel blocker group.Many pharma companies are formulating and marketing amlodipine as single or in combination with other active ingredients under different brand names like Asomex, Istin, Norvasc, Caduet and Twynsta.Stability studies provide us with information on the quality of the drug product.The studies must include the tests, which can monitor the quality of the drug product [1].Impurities can be generated by drug excipient interactions, storage conditions, hydrolysis etc.A sensitive, reproducible method is to be developed and validated to monitor the impurities in drug product.
Literature survey reveals that some analytical methods are available for the estimation of amlodipine alone or in combination with other drugs using HPLC, HPTLC, and LC-MS [2]- [13].Ph.Eur monograph method is also reported to estimate impurities in amlodipine besylate raw material.However, none of the analytical methods reported the estimation of all known and unknown impurities for amlodipine besylate.This paper describes the quantification of all impurities (IMP-A, IMP-B, IMP-D, IMP-E, IMP-F, IMP-G and IMP-H) of amlodipine besylate in amlodipine tablets.

Chemicals and Reagents
Amlodipine besylate tablets and all impurities were synthesized and supplied by Dr.
Reddy's Laboratories Ltd. Potassium dihydrogen phosphate, triethyl amine, orthophosphoric acid of AR grade and methanol and acetonitrile of gradient grade were purchased from Merck Chemicals Ltd.

Chromatographic Conditions
The mixed buffer was prepared by adding 7 ml of triethyl amine to 1000 ml of 50 mm monobasic phosphate buffer and then the pH was adjusted to 2.8 with orthophosphoric acid.Mobile phase-A was prepared by mixing the buffer with methanol in the ration of 60:40 (v/v) while Mobile phase-B was prepared by mixing the buffer with methanol and acetonitrile in the ratio of 20:40:40 (v/v/v).Mobile phase-A contains more buffer concentration to separate all impurities.Mobile phase-B contains more organic concentration to elute all the impurities.
A column with 150 mm length, 4.6 mm internal diameter and 3 µ particle sizes, C18 as stationary phase was used to separate all the impurities.Column temperature was maintained at 35˚C.All impurities were monitored at 340 nm except impurity-D.Impurity-D was monitored at 270 nm.Gradient mode flow was used to separate the impurities.Flow rate of the mobile phase was kept at 1.0 mL/min and 100 µL samples were injected into HPLC.Analysis was performed on Waters HPLC system with PDA detector.The diluent for extraction of impurities and amlodipine from formulation matrix was prepared by admixing Buffer, Methanol and acetonitrile in the ratio of 70:15:15 (v/v/v).

Standard Stock Preparation
70 mg of amlodipine besylate standard (potency-72%) was weighed and transferred into 250 mL of volumetric flask and it was dissolved and diluted to volume with methanol.5 mL of this solution was diluted to 100 mL with methanol.

Final Standard Stock Preparation
5 mL of the standard stock solution was diluted to 100 mL with diluent.

Sample Preparation
20 tablets were crushed into fine powder and then the powder equivalent to 25 mg of amlodipine was transferred into 100 ml volumetric flask and then 70 ml of diluent was added.The resulting solution was sonicated for 30 minutes with intermittent shaking and the temperature of the sonication was maintained below 25˚C.Then the solution was diluted to the volume with the diluent and was centrifuged the sample at 4000 RPM for 15 mins and then few ml of the supernatant solution was filtered through 0.45 µ membrane filter.

Impurity Stock Solution Preparation
2 mg of each impurity was weighed, transferred to 20 ml volumetric flask and then dissolved in methanol and the resulting solution was diluted to the mark.

Spiked Sample Preparation
Tablet powder equivalent to 25 mg of amlodipine was weighed and transferred into 100 mL volumetric flask.70 ml of the diluent was added.Then, 1.25 mL of impurity stock solution was added.The resulting solution was sonicated for 30 mins and then diluted to volume with the diluent.Then the solution was centrifuged at 4000 RPM for 15 mins and filtered the few ml of the supernatant solution through 0.45 µ membrane filter.

Analytical Method Validation
The developed method was validated as per ICH guidelines for specificity, linearity, precision, ruggedness and robustness.

Optimization of Chromatographic Conditions
Different trials were taken with mobile phases containing ammonium salts, phosphate and sodium perchlorate buffers to obtain optimum resolution between impurities.Final chromatographic conditions were finalized based on the DOE.Fractional design was used to perform DOE by considering the flow rate, pH of the buffer, % methanol and % acetonitrile in mobile phase-B as factors and resolution between the close eluting impurities (Impurity-B & H) as responses.A text plan was shown in Table 1        Design space was established and recommended parameters were near to the experimentally proposed values.The Proposed HPLC method was shown in Table 2.
Further, experimentally obtained data were used in setting the lower and upper bounds for each response variables.Using modelled data, visual inspections of interactive effects were performed from multiple overlay graphs plotting two parameters at a time.
Diluent was finalized based on recovery and peak shape.Injection volume and test concentrations were optimized to have LOQ value less than reporting threshold.Gradient was optimized to get optimal resolution between all impurities and main analyte.

Run time 70 minutes
Gradient 90% A upto 8 min (isocratic) with 1.0 mL Linear gradient to 75% A at 20 min with 1.0 mL Linear gradient to 70% A at 45 min with 1.0 mL Linear gradient to 20% A at 50 min with 1.2 mL Linear gradient to 0% A at 52 min with 1.2 mL 0% A from 52 min to 60 min with 1.2 mL Linear gradient to 90% A at 62 min with 1.0 mL 90% A from 62 min to 70 min with 1.0 mL

System Suitability
The authors prepared the standard at 0.2% level of the test concentration and injected it into HPLC system; and then calculated the % RSD for peak areas, USP plate count, tailing factor of amlodipine peak from replicate standard injections.% RSD from replicate injections was found to be 3.2.The tailing factor for main analyte is found to be 1.0 and plate count is found to be 43685.Results are tabulated in Table 3. Resolution between Impurity-B and Impurity-H was found to be 2.7.

Linearity and Range
Linearity was established over the range of 0.0001 mg/ml to 0.05 mg/ml for all the impurities and main analyte.Six different linearity solutions were prepared and injected into system.Results are tabulated in Table 4.

Specificity
Test samples were subjected to different stress conditions like acid, base, water hydrolysis, peroxide oxidation, thermal degradation, sun light/UV degradation and Humidity degradation.Sample was exposed to acidic (0.1 N HCl/5 mL/30 min reflux), alkaline (0.1 N NaOH/5 mL/60 min reflux), Oxidation (% 5H 2 O 2 /10 mL/30 min reflux), thermal (105˚C/24 hrs), water (10 mL/60˚C/30 min reflux) water conditions, sunlight (1.2 million lux hrs), UV light (200 watt hours) and Humidity (90% RH for 7 days).All the samples were injected into HPLC system with PDA detector to identify the purity of the known and main analyte peaks.Purity angles were less than purity threshold for all the known impurities and amlodipine peak.The results were tabulated in Table 5.On  perusal of the results, it may be concluded that all the unknown impurities generated in the degradation are well separated from the known and amlodipine peaks.Hence, the developed method is specific.

Precision and Accuracy
Recovery studies were performed for all the impurities from 0.000125 mg/ml to 0.001875 mg/ml and values were found to be between 85% -110%.Precision was performed by preparing six samples by spiking the impurities at 0.5% of the target test concentration.Results were tabulated in Table 4.The resultant % RSD values for the % impurities were found the below 5.0 (n = 6).Hence, it may be concluded that the method is precise and accurate.

LOD and LOQ
LOQ and LOD were established for the impurities and amlodipine using slope method.
LOQ values were found to be 0.01.The concentration with signal to noise ratio about three was taken as LOD and ten was taken as LOQ.Values were presented in Table 4.

Mobile Phase and Solution Stability
Mobile phase and solution stability were established over a period of 24 hours on bench top.System suitability parameters were evaluated and % impurities were calculated against fresh standard.From the results, it may be concluded that solution and mobile phase are stable up to 24 hours on bench top.

Conclusion
A novel HPLC method has been developed for the estimation of amlodipine impurities in amlodipine tablets formulation.Placebo interference was not observed at known and unknown impurities.Method is found to be linear, precise, accurate, robust and rugged.Hence, this method can be used for the estimation of amlodipine impurities in regular as well as stability sample analysis.
Figure 1 represents main effects plot for the resolution between Imp-B&H. Figure 2 represents interaction plot, Figure

Figure 1 .
Figure 1.The main effect chart for the resolution between Imp-B & H.

Figure 2 .
Figure 2. The interaction plot for the resolution between Imp-B & H.

Figure 3 .
Figure 3.The Pareto chart for standardized effects on the resolution between Imp-B & H.

Figure 4 .
Figure 4.The contour plot for the resolution between Imp-B & H.

Figure 5
Figure 5 represents the chromatogram for standard, Figure 6 represents chromatogram for spiked sample at 340 nm and Figure 7 represents chromatogram for spiked sample preparation at 270 nm.

Figure 5 .
Figure 5.The chromatogram for standard preparation.

Figure 6 .
Figure 6.The chromatogram for sample preparation at 340 nm.

Figure 7 .
Figure 7.The chromatogram for sample preparation at 270 nm.

Table 1 .
Design of experiments.

Table 5 .
Summary of peak purity and degradation data for amlodipine in stress study.