1.0 INTRODUCTION:

Amlodipine Besylate is Amlodipine belongs to the dihydropyridine (DHP) class of calcium channel blockers (CCBs), the most widely used class of CCBs, with the chemical name of 3-ethyl 5-methyl 2-[(2-aminoethoxy)methyl]-4-(2-chlorophenyl)-6-methyl-1,4-dihydro pyridine-3,5-dicarboxylate (Figure-1a). Amlodipine is used to treat hypertension and chronic stable angina. It lowers blood pressure by relaxing the blood vessels so the heart does not have to pump as hard. It controls chest pain by increasing the supply of blood to the heart. Atorvastatin Calcium is chemically named (3R, 5R)-7-[2-(4-fluorophenyl)-3-phenyl-4-(phenylcarbamoyl)-5-(propan-2-yl)-1H-pyrrol-1-yl]-3,5-dihydroxyheptanoic acid (Figure-2b). It is used for lowering cholesterol.

The combination of both Amlodipine Besylate and Atorvastatin Calcium is used to treat the patients suffering from both high blood pressure and high cholesterol.

Figure-1a: Structure of Amlodipine Besylate

Figure-1b: Structure of Atorvastatin Calcium

Amlodipine Besylate and Atorvastatin Calcium tablets are available in the combination of 5+5 mg, 5+10mg, 10+5mg, 10+10 mg, 5+20 mg, 10+20 mg, 5+40 mg.

Literature survey revealed that only fewer spectrophotometric methods are available for simultaneous estimation of Amlodipine Besylate and Atorvastatin Calcium [1-4]. UV detection method is one of the methods for simultaneous estimation of Amlodipine Besylate and Atorvastatin Calcium [5]. Amlodipine in combination with other drug is reported by RP-HPLC method [6]. Atorvastatin Calcium in combination with other drug is reported by RP-HPLC method [7]. RP-HPLC method has been used for the simultaneous determination of Amlodipine Besylate and Atorvastatin Calcium in tablets by using mobile phase constituting of three solvents [8] and detection on absorbance mode [9]. The present work describes a validated reverse phase HPLC method for simultaneous determination of Amlodipine Besylate and Atorvastatin Calcium in tablets with high precise and sophisticated compared to earlier methods. The proposed method is validated as per ICH guidelines [10].

2.0 MATERIALS AND METHODS:

2.1 Reagents and Chemicals:

Sodium acetate (AR grade, SD Fine chem limited), methanol (HPLC grade, Merck limited), Milli–Q water, Amlodipine Besylate (99.8% w/w procured from Mankind chemicals) and Atorvastatin Calcium (99.8% w/w procured from Mankind chemicals). All other chemicals are of the highest grade commercially available unless otherwise specified.

2.2 Instrumentation:

The chromatographic system consisted of a Shimadzu class VP Binary pump LC-10ATvp, SIL-10ADvp Auto sampler, CTO-10Avp Column Temperature Oven, SPD-10Avp UV-Visible Detector. All the components of the system are controlled using SCL-10Avp System Controller. Data acquisition was done using LC Solutions software.

The mobile phase consisted of 70:30 % (v/v) of methanol and 10mM sodium acetate buffered to pH 4.0 operated on isocratic mode. The flow rate is 1.0mL/min. Chromatographic determination of Amlodipine Besylate and Atorvastatin Calcium was performed on Waters X-Terra, C18 (150mm × 4.6mm, 5µm) column. The wavelength of detection is 240 nm. The injection volume is 20µL.

2.3Preparation of standard solutions, Calibration Standards and Quality Control Samples:

Stock solutions of Amlodipine Besylate (2.38mg/mL), and Atorvastatin Calcium (10mg/mL) were prepared separately in a volumetric flask using methanol and labeled accordingly. Suitable dilutions were then prepared using 50:50 % (v/v) methanol and Milli-Q water as diluent solution. For the linear calibration curve, eight non-zero standards were prepared using diluent solution in the concentration range of 2.51 to 50.18 µg/mL for Amlodipine Besylate and 5.02 to 100.33 µg/mL for Atorvastatin Calcium. The calibration standard sample is then transferred into the auto sampler for analysis. Samples for specificity (sample with Amlodipine Besylate alone, sample with Atorvastatin Calcium alone, blank sample and sample containing both the drugs) were also prepared accordingly.

For the preparation of quality control samples, a separate stock containing approximately the same concentration of the Amlodipine Besylate and Atorvastatin Calcium were prepared and labeled as quality control stocks. From these stocks, quality control samples containing Amlodipine Besylate and Atorvastatin Calcium were prepared at three concentration levels namely LQC, MQC, HQC so as to obtain low, medium and high concentration quality control samples. The performance of the linear calibration curve is then evaluated using quality control samples.

2.4 Assay:

The assay of tablets containing Amlodipine Besylate and Atorvastatin Calcium is done using the procedure given in Indian Pharmacopoeia under tablets. The active ingredients in each of 10 dosage units is taken by random sampling and analyzed by the developed method. The tablets are said to be compliance if the each individual content is 90 – 110 % of the average content or labeled claim.

For the current assay ten tablets were randomly taken and transferred separately into 100ml volumetric flasks and dissolved in 20ml methanol. The solution was then ultrasonicated for 10 min and then made up to volume. Required amount of solution is then taken and filtered through 0.45μ nylon membrane and diluted with diluent solution so that the resultant concentrations are within the calibration range of the developed method. The samples are then analyzed by using the validated method. The sample is then injected in triplicate.

2.5 Method Validation:

2.5.1 System Suitability:

A sample containing mixture of Amlodipine Besylate (approximate concentration of 25.09μg/ml) and Atorvastatin Calcium (approximate concentration of 50.17μg/ml) was used as system suitability sample. System suitability was assessed by six replicate analyses. A percent coefficient of variation (% CV) less than 1 % for retention times for the drugs is taken as the acceptance criterion.

2.5.2 Detection and Quantification Limits (Sensitivity):

Limits of detection (LOD) and Limit of quantification (LOQ) (Figure-2) were estimated from both linearity calibration curve method and signal to noise ratio method. The detection limit was defined as the lowest concentration level resulting in a peak area of three times the baseline noise. The quantification limit was defined as the lowest concentration level that provided a peak area with signal to noise ratio higher than 5, with precision (%CV) and accuracy with (±) 20%.

Figure-2a. Chromatogram for LOD Sample

2.5.3 Linearity (Calibration Curve):

The Linearity of detector response to different concentrations of both the drugs was studied with a series of working standard solutions prepared by diluting the stock solution. The standard plots were then constructed between concentration Vs. Peak area using eight non-zero standards ranging from 2.51 to 50.18 μg/mL for Amlodipine Besylate and 5.02 to 100.33μg/mL for Atorvastatin Calcium. The linearity was evaluated by linear regression analysis, which was calculated by least square method. It is depicted in (Figure- 3).

Figure-2b. Chromatogram for LOQ Sample

2.5.4 Accuracy and Precision:

According to ICH guidelines, repeatability should be assessed by using a minimum of nine determinations covering the specified range for the procedures (i.e.three concentrations and three replicates of each concentration). Precision was studied to find out intra and inter day variations of the proposed method at three different levels. The %CV values less than 2% indicate that the method was precise.

2.5.5 Specificity:

For demonstration of specificity, four samples namely blank sample, sample containing Amlodipine Besylate alone, sample containing Atorvastatin Calcium alone and sample containing the mixture of Amlodipine Besylate and Atorvastatin Calcium were prepared separately. Specificity of the method was determined by comparing results of all the samples (Figure-4). The developed method is said to be specific if the % interference calculated as peak area (if any) at the retention time of each of the analytes in the blank sample is less than 20% of peak area at the corresponding retention times of each of the drugs in the lowest calibration standard. Sample specificity is also observed in the degradation study of the drug. None of the degraded products must interfere with the quantification of the drug.

Figure- 3a. Linear calibration curve of Amlodipine besylate

Figure-3b: Linear calibration curve of Atorvastatin calcium

Figure-4a: Blank Chromatogram

Figure-4b: Chromatogram of Amlodipine besylate alone

Figure-4c: Chromatogram of Atorvastatin calcium alone

Figure-4d: Chromatogram of both Amlodipine besylate and Atorvastatin calcium

Figure-4:Comparison of (a)Blank Chromatogram,(b) Amlodipine besylate alone,(c) Atorvastatin calcium alone and(d)sample containing both Amlodipine besylate and Atorvastatin calcium

2.5.6 Stability:

The stability of the drug is determined by placing the MQC samples for the short term stability at room temperature up to 12 hours and then comparing the obtained peak area with that of the similarly prepared fresh sample. Further, auto-sampler stability for up to 24 hrs was studied and established.

2.5.7 Stress Degradation Studies:

For Stress Degradation Analysis, 1 mL aliquots (in duplicate) of samples containing MQC level concentration are treated separately with 100 μL of 0.1N HCl (Acid stress), 0.1N NaOH (Alkaline stress), 5% (v/v) Hydrogen Peroxide (Oxidative Stress), for 24 hrs. Samples for Photolytic stress are placed in a transparent glass vial and placed in a UV chamber for 24 hrs. Samples are then injected for analysis. The results of analysis are then compared with similarly prepared fresh samples. The analysis is performed in triplicate.

3.0 RESULTS AND DISCUSSION:

3.1 Method Development and Validation:

The HPLC procedure was optimized with a view to develop a stability indicating assay method. Chromatographic behavior at different pH values ranging from pH 3.0 to pH 6.5 using various columns like Hypersil-BDS-C18, Symmetry C18, Ymc-pack C18, Ymc-pack pro, Spherisorb C18, Phenomenex C18 have been tried with different buffer salts such as ammonium Formate, orthophosphoric acid, di-potassium hydrogen orthophosphate, in combination with acetonitrile, methanol and tetrahydrofuran. However less tailing and high theoretical plates are obtained with Waters X-Terra, C18 (150mm × 4.6mm, 5µm) column. The final mobile phase composition consisted of (70:30 v/v) of Methanol and 10mM Sodium acetate buffered to pH 4.0 on isocratic mode. The flow rate of the method is 1.0 mL/min. Calibration standards were prepared in diluents solution containing 50:50 % v/v of Methanol and Milli-Q water. The wavelength of detection is 240nm. The column temperature is maintained at 25^OC. At the reported flow rate, peak shape was excellent; however increasing or decreasing the flow rate resulted in unacceptable tailing factor and poor peak shape. Hence 1.0 mL/min was optimized flow rate decreasing the consumption of the mobile phase, which in turn proves to be cost effective for long term routine quality control analysis. To evaluate the feasibility of the experiment under regular lab conditions, the assessment of the stability of Amlodipine Besylate and Atorvastatin Calcium under room temperature and under normal light conditions is done.

Method Validation:

3.2 System Suitability:

The % CV of the peak area for both drugs is within the acceptable criteria (Table-1). The efficiency of the column was expressed as the number of theoretical plates for the six replicate injections was around 11358±101.40 for Amlodipine Besylate and 16122±478.36 for Atorvastatin Calcium. The USP tailing factor was 1.3 ± 0.0 for Amlodipine Besylate while that of Atorvastatin Calcium is 1.042 ± 0.01.

Table 1a.System suitability for Amlodipine besylate

Sample ID	Peak Retention Time	Peak Area	Theoretical Plates	Tailing Factor
1	2.33	192739	11537	1.30
2	2.32	186444	11308	1.30
3	2.33	178783	11246	1.30
4	2.34	164923	11305	1.30
5	2.34	187992	11358	1.30
6	2.33	189970	11397	1.30
MEAN	2.332	183475.2	11358.5	1.300
STDEV	0.0075	10231.49	101.40	0.0000
%CV	0.32	5.58	0.89	0.00

Table 1b. System suitability for Atorvastatin Calcium
Sample ID	Peak Retention Time	Peak Area	Theoretical Plates	Tailing Factor
1	7.78	285757	16168	1.05
2	7.81	302057	15829	1.04
3	7.83	302526	15468	1.04
4	7.84	280053	16095	1.03
5	7.84	309733	16277	1.04
6	7.79	301582	16899	1.05
MEAN	7.815	296951.3	16122.7	1.042
STDEV	0.0259	11426.51	478.36	0.01
%CV	0.33	3.85	2.97	0.72

Determination and Quantification Limits (Sensitivity):

Figure-2 represents the chromatogram of limit of detection and limit of quantification. The method is found to be sensitive which can be determined from data obtained from the Table-2.

Table 2: Sensitivity of Amlodipine besylate and Atorvastatin calcium:

	LOD Amlodipine besylate
	SR NO		Drug Retention Time		Peak Area
	1		2.51		34299
	2		2.51		35822
	3		2.51		34290
	MEAN		2.5		34803.7
	ST DEV		0.00		881.91
	% CV		0.00		2.53
LOD Atorvastatin Calcium
SR NO		Drug Retention Time		Peak Area
1		8.77		95161
2		8.78		98709
3		8.77		98721
MEAN		8.8		97530.3
ST DEV		0.01		2051.91
% CV		0.07		2.10

3.3 Linearity:

The linearity was demonstrated in triplicate. The results of the best fit line (y = mx + c) for the triplicate analysis is given in Table-3. The accuracy of the calibration standards was evaluated from the back calculated concentrations (Table-4). All the standards were found to be within the range of 97 – 103 %.

	LOQ Amlodipine besylate
	SR NO		Drug Retention Time		Peak Area
	1		2.50		68022
	2		2.50		65661
	3		2.48		71609
	MEAN		2.5		68430.7
	ST DEV		0.01		2994.98
	% CV		0.46		4.38
LOQ Atorvastatin Calcium
SR NO		Drug Retention Time		Peak Area
1		8.75		170303
2		8.69		176675
3		8.65		185084
MEAN		8.7		177354.0
ST DEV		0.05		7413.86
% CV		0.58		4.18

Table-3: Results of best-fit line for triplicate analysis for Amlodipine besylate (above) and Atorvastatin calcium (below)

Amlodipine besylate
curve	Slope	Intercept	r²
1	41001	39729	0.997
2	41101	38678	0.998
3	41238	39628	0.996
Mean	41113	39345	0.997
Atorvastatin calcium
Curve	Slope	Intercept	r²
1	52986	12518	0.997
2	52970	12524	0.998
3	52981	12420	0.996
Mean	52979	12487	0.997

3.4 Accuracy and Precision:

Accuracy and precision calculated for the QC samples during the intra- and inter –day run are given in the Table-5.The intra-day (day-1) and inter-day accuracy for Amlodipine Besylate ranged from 97.20 – 100.40 % while that of Atorvastatin Calcium ranged from 97.23 – 103.19 %. The results obtained from intermediate precision (inter-day) also indicated a good method precision. All the data were within the acceptance criteria.

Amlodipine besylate
Sample ID	Concentration (Microgram/mL)	Retention Time	Peak Area	Back Calc Concentration	% Accuracy
Blank	Blank	NA	0	NA	NA
CC - 01	2.51	2.49	129384	2.19	87.12
CC - 02	5.02	2.49	251529	5.17	102.90
CC - 03	10.04	2.44	448052	9.96	99.19
CC - 04	15.05	2.41	687942	15.81	105.05
CC - 05	20.07	2.43	882909	20.56	102.47
CC - 06	35.13	2.43	1398864	33.15	94.36
CC - 07	40.15	2.43	1687330	40.18	100.09
CC - 08	50.18	2.41	2136220	51.13	101.90
Blank	Blank	NA	0	NA	NA

Atorvastatin calcium
Sample ID	Concentration (Microgram/mL)	Retention Time	Peak Area	Back Calc Concentration	% Accuracy
Blank	0.00	NA	0	NA	NA
CC - 01	5.02	8.68	292458	5.28	105.24
CC - 02	10.03	8.72	577028	10.65	106.22
CC - 03	20.07	8.37	1022068	19.05	94.93
CC - 04	30.10	8.38	1676554	31.41	104.34
CC - 05	40.13	8.42	2155272	40.44	100.77
CC - 06	70.23	8.42	3558337	66.92	95.29
CC - 07	80.26	8.43	4237141	79.73	99.34
CC - 08	100.33	8.45	5453139	102.68	102.34
Blank	Blank	NA	0	NA	NA

3.5 Specificity:

Specificity was determined by comparison of the Blank chromatogram with that of the Standard chromatogram (Figure-4)

Table 5a: Results of inter and intra-day accuracy and precision for Amlodipine Besylate:

Amlodipine besylate	Nominal concentration(µg/mL)
	12.55(LQC)		25.09(MQC)		37.64(HQC)
DAY 1 (Intra day)
MEAN (n=6)	99.90		100.40		97.93
STDEV	2.80		0.43		3.10
% CV	2.80		0.42		3.17
DAY 2
MEAN (n=6)	99.80	97.48		98.34
STDEV	0.75	3.23		1.14
% CV	0.75	3.31		1.15
DAY 3
MEAN (n=6)	99.7	98.60		97.20
STDEV	0.50	0.98		0.25
% CV	0.50	0.99		0.25

Table 5b: Results of inter and intra-day accuracy and precision for Atorvastatin:

Atorvastatin calcium	Nominal concentration(µg/mL)
	25.08(LQC)		50.17(MQC)		75.25(HQC)
DAY 1 (Intra day)
MEAN (n=6)	103.19		99.90		98.61
STDEV	0.23		0.51		2.97
% CV	0.22		0.51		3.01
DAY 2
MEAN (n=6)	102.30	99.80		100.60
STDEV	0.25	0.60		0.52
% CV	0.24	0.60		0.51
DAY 3
MEAN (n=6)	101.62	100.95		97.23
STDEV	0.43	0.42		0.90
% CV	0.24	0.39		0.92

3.6 Room Temperature Stability:

Stability studies were done for short term stability up to 12 hrs on the bench top for the MQC levels conditions. Stability is calculated as the ratio of the mean peak area of the stability sample to the mean peak area of the fresh sample and expressed as the percentage (n = 6). The room temperature stability was found to be 96.76 % for Amlodipine besylate and 97.16 % for Atorvastatin Calcium. The results are tabulated in Table-6.

3.7 Stress Degradation:

Stress studies revealed that Amlodipine besylate is not susceptible to degradation under acid, oxidative stress, light (UV) stress conditions (Figure -5a). However, in alkaline conditions (0.1N NaOH), the drug was unstable and the degradation peak eluted later accompanied with a drastic peak distortion and increased tailing. Except for alkaline conditions, the drug content was within 99 –109 % for all stress conditions indicating the stability and specificity of the analytical method to differentiate the degradation peaks.

Stress studies on Atorvastatin calcium indicated the stability of drug under acid, oxidative stress and light (UV). However, Atorvastatin calcium has gone degradation under alkaline (0.1N NaOH) conditions (Figure-5b). This has been clearly demonstrated by the help of overlap spectra of all the stress samples as compared with that of freshly prepared sample of similar concentration (Figure-5b). Except for alkaline stress, for all the other stress conditions, the Atorvastatin Calcium content was within 99 –105 % indicating the stability and specificity of the analytical method to differentiate the degradation peaks.

Fig-5a: Overlap Chromatogram showing the influence of various stress conditions On Amlodipine besylate

Data 1-Fresh sample; Data 2-Acid stress; Data 3- Oxidative stress; Data 4-Photolytic stress; Data 5-Alkaline stress

Figure-5b: Overlap Chromatogram showing the influence of various stress Conditions on Atorvastatin Calcium

Data 1-Fresh sample; Data 2- Photolytic stress; Data 3- Oxidative stress; Data 4-acid stress; Data 5-Alkaline stress

3.8 Robustness study:

Robustness is the measure of method capacity to remain unaffected by deliberate small changes in the chromatographic conditions. The experimental conditions were deliberately altered to evaluate the robustness of the method. The impact of flow-rate (1.0± 0.1 mL/min), and effect of mobile-phase composition (± 5%) on chromatographic parameters such as retention time, theoretical plates, and tailing factor, were studied. At normal flow rate, the retention time of Amlodipine Besylate was 2.5± 0.01 minutes (n = 6) while that of Atorvastatin Calcium was 8.6 ± 0.01 minutes. At normal flow rate, the tailing factor for Amlodipine besylate is 1.32 ± 0.01 while that of Atorvastatin Calcium was 1.04 ± 0.0. At higher flow rate, tailing factor for Amlodipine Besylate is 2.2±0.02 and Atorvastatin Calcium is 7.4±0.04. At a lower flow rate of 0.9mL/min, Amlodipine Besylate and Atorvastatin Calcium eluted at 2.8±0.06 and 9.8± 0.19 minutes respectively. The tailing factor of Amlodipine Besylate and Atorvastatin Calcium were 1.33±0.01 and 1.04±0.0 respectively (n = 6). At mobile phase composition of 70: 30 % (v/v) of Methanol and 10mM Sodium acetate buffered to pH 4,the retention times of Amlodipine besylate and Atorvastatin calcium were 2.5 ± 0.01 and 8.6 ± 0.01 minutes (n = 6). At mobile phase composition of 65: 35 % (v/v) of Methanol and 10mM Sodium acetate, the retention times of Amlodipine Besylate and Atorvastatin Calcium were 2.5 ± 0.01 and 8.24± 0.07 minutes (n = 6).

3.9 Application of the method to dosage forms:

The HPLC method developed is sensitive and specific for the quantitative determination of Amlodipine Besylate and Atorvastatin Calcium. Also the method is validated for different parameters; hence it has been applied for the simultaneous estimation in pharmaceutical dosage forms. The amount of Amlodipine Besylate and Atorvastatin Calcium in the commercial tablet dosage form is within the pharmacopoeial specifications. None of the tablets ingredients interfered with the analyte peak. The spectrum of Amlodipine Besylate and Atorvastatin Calcium in the extracted tablet was matching with that of standard compounds indicating the purity of the compounds in the tablets.

4. CONCLUSIONS:

The method gave accurate and precise results in the concentration range of 2.51 to 50.18μg/mL for Amlodipine Besylate and 5.02 to 100.33 μg/mL for Atorvastatin Calcium. The mobile phase composition consists of 70:30 % v/v of Methanol and 10mM Sodium acetate buffered to pH 4. The retention time of Amlodipine Besylate is 2.5± 0.01 minutes and that of Atorvastatin Calcium is 8.6 ± 0.01minutes. The column is Waters X-Terra, C18 (150mm × 4.6mm) with the particle size of 5μm.A rapid sensitive and specific method for the simultaneous estimation of Amlodipine Besylate and Atorvastatin Calcium in the pharmaceutical tablet formulations has been developed and validated.

5. REFERENCES:

1. MR Khan, Deepti Jain, Simultaneous spectrophotometric determination of atorvastatin calcium and amlodipine besylate in tablets, Indian Journal of Pharmaceutical Sciences, Year 2006 , Volume 68 , Issue 4 , Pages 546-548.

2. R.Sahu,Vandana B Patel, Simultaneous spectrophotometric determination of amlodipine besylate and atorvastatin calcium in binary mixture, Indian Journal of Pharmaceutical Sciences, Year 2007, Volume 69,Issue 1, Pages 110-111.

3. Devi Ramesh, S Ramakrishna, New spectrophotometric methods for simultaneous determination Of Amlodipine besylate and Atorvastatin calcium in tablet dosage forms, International Journal of Pharmacy and Pharmaceutical Sciences Year 2010, Volume 2, Issue 4, Pages 215 -219.

4. Bharat G. Chaudhari, Ashok B. Patel, Simultaneous spectrophotometric estimation of Atorvastatin Calcium and Amlodipine besylate in tablet dosage forms, International Journal of ChemTech Research, Year 2010,Volume 2, Issue1, Pages 633-639.

5. P Mishra, Alka Gupta, K Shah, Simultaneous estimation of atorvastatin calcium and amlodipine besylate from tablets, Indian Journal of Pharmaceutical Sciences, Year 2007, Volume 69, Issue 6, Pages 831-833.

6. SS Chitlange, Kiran Bagri and DM Sakarkar, Stability Indicating RP- HPLC method for simultaneous estimation of Valsartan And Amlodipine in Capsule Formulation. Asian J. Research Chem. ,Year 2008,Volume 1, Issue1, Pages 15-18.

7. N. Jain, R. Raghuwanshi, and Deepti Jain, Development of RP- HPLC Method for Simultaneous Estimation of Atorvastatin Calcium and Fenofbrate in tablet dosage forms, Indian Journal of Pharmaceutical Sciences, Year 2008,Volume 70, Issue 2, Pages 263-265.

8. DA Shah, KK Bhatt, MB Shankar, RS Mehta, TR Gandhi, SL Baldania, RP-HPLC determination of atorvastatin calcium and amlodipine besylate combination in tablets, Indian Journal of Pharmaceutical Sciences ,Year 2006, Volume 68, Issue 6 , Pages 796-799.

9. K Raja Rajeswari, GG Sankar, AL Rao, JVLN Seshagirirao, RP-HPLC method for the simultaneous determination of Atorvastatin and Amlodipine in tablet dosage form , Indian Journal of Pharmaceutical Sciences, Year 2006, Volume 68, Issue 2 , Pages 275-277.

10. International Conference on Harmonization(ICH Q2(R1)). Validation of Analytical procedures: Text and Methodology, IFPMA, Geneva, Switzerland, 2005.

Received on 11.03.2014 Modified on 30.03.2014

Asian J. Research Chem. 7(4): April 2014; Page 438-445

FRESH SAMPLE Amlodipine besylate				STABILITY SAMPLE Amlodipine besylate
SR NO	SAMPLE ID	DRUG RETENTION TIME	PEAK AREA	SR NO	SAMPLE ID	DRUG RETENTION TIME	PEAK AREA
1	FRESH SAMPLE	2.50	960154	1	STABILITY SAMPLE	2.46	1070148
2	FRESH SAMPLE	2.49	1057096	2	STABILITY SAMPLE	2.46	1027005
3	FRESH SAMPLE	2.47	1061114	3	STABILITY SAMPLE	2.44	1010513
4	FRESH SAMPLE	2.47	1028042	4	STABILITY SAMPLE	2.45	1042378
5	FRESH SAMPLE	2.47	1075515	5	STABILITY SAMPLE	2.44	858605
6	FRESH SAMPLE	2.46	1066363	6	STABILITY SAMPLE	2.44	1037674
MEAN			1041380.67	MEAN			1007720.50
STDEV			42898.34	STDEV			75644.79
% CV			4.12	% CV			7.51
					%Stability	96.77

FRESH SAMPLE Atorvastatin				STABILITY SAMPLE Atorvastatin
SR NO	SAMPLE ID	DRUG RETENTION TIME	PEAK AREA	SR NO	SAMPLE ID	DRUG RETENTION TIME	PEAK AREA
1	FRESH SAMPLE	8.48	2396538	1	STABILITY SAMPLE	8.49	2678333
2	FRESH SAMPLE	8.49	2649897	2	STABILITY SAMPLE	8.48	2571968
3	FRESH SAMPLE	8.46	2621944	3	STABILITY SAMPLE	8.46	2512961
4	FRESH SAMPLE	8.45	2532298	4	STABILITY SAMPLE	8.46	2587337
5	FRESH SAMPLE	8.42	2632951	5	STABILITY SAMPLE	8.39	2084116
6	FRESH SAMPLE	8.41	2577287	6	STABILITY SAMPLE	8.38	2539276
MEAN			2568485.83	MEAN			2495665.17
STDEV			94439.81	STDEV			209364.35
% CV			3.68	% CV			8.39
					% Stability	97.16484063

Method Development and Validation for Simultaneous Determination of Amlodipine Besylate and Atorvastatin Calcium by RP–HPLC Technique

1.0 INTRODUCTION: