INTRODUCTION:

Atorvastatin (Lipitor) (Figure 1) is a member of the drug class known as statins. It is used for lowering cholesterol. Atorvastatin acts primarily in the liver. Decreased hepatic cholesterol levels increases hepatic uptake of cholesterol and reduces plasma cholesterol levels.^1-3Atorvastatin is a white powder with a molecular weight of 558.65 g/mol. Amlodipine (Figure 2) is a long-acting 1,4-dihydropyridine calcium channel blocker. It acts primarily on vascular smooth muscle cells by stabilizing voltage-gated L-type calcium channels in their inactive conformation. It is used to treat hypertension and chronic stable angina. Amlodipine is a white powder with a molecular weight of 408.879 g/mol.

Figure 1: Chemical Structure of Atorvastatin (AT)

Figure 2: Chemical Structure of Amlodipine (AM)

METHOD DEVELOPMENT:

Appropriate selection of HPLC method development relies upon the nature of the sample, its molecular weight and solubility. For effective method development various Chromatographic parameters such as pH, mobile phase, its composition and proportion, detection wavelength and other factors were systematically studied.

Selection of Chromatographic method:

Proper selection of method depends up on the nature of the sample, molecular weight, and solubility. The drug selected for the present study was Non polar.

Non polar compounds can be separated by either normal phase or reverse phase chromatography.Reverse phase chromatography was selected for initial separations from the knowledge of properties of the compounds.

Selection of Diluent:

The nature of the drug reveals certain information about the drug such as solubility, pKa. Based on the solubility of the drug, the diluent is selected. The solvent in which the drug has maximum solubility is selected as the diluents. Water: ACN (50:50) is used as primary diluent for stock solution.

Selection of detection Wavelength:

Standard solutions of Atorvastatin and Amlodipine were injected separately as well as in combination into HPLC system then scanned over entire the UV range (190-400 nm). The spectra of Atorvastatin and Amlodipine was recorded for determination of ƛ max. Finally, 220 nm set for chromatographic elution.

Selection of column:

In reverse phase chromatography non polar stationary phase is used for separation. C8, C18 are the commonly used columns in reverse phase chromatography. Here, Acquity UPLC HSS C18 x 1.8m is used for the separation.

Selection of Mobile phase:

A Number of trails were made to find out the mobile phase for eluting the drug. Better peak resolution and adequate retention time were obtained by using Buffer: Acetonitrile: Methanol taken in the ratio (45:55)

Mode of separation:

Several trails were conducted by changing flow rate, injection volume and other parameters till satisfactory separation was achieved. The resulting chromatograms were recorded and the chromatographic parameters such as column efficiency, theoretical plates were calculated. Finally, the condition that gives best result was selected for estimation.

EXPERIMENTAL:

Preparation of standard stock solution

Accurately Weighed and transferred 25mg of Atorvastatin and 12.5 mg of Amlodipine working Standards into a 25ml clean dry volumetric flask, add 3/4^th volume of diluent, sonicated for 5 minutes and make up to the final volume with diluents. 1ml from the above two stock solutions was taken into a 10ml volumetric flask and made up to 10ml.

Optimized method

A new RP-UPLC method has been developed and validated for the determination of Atorvastatin and Amlodipine in tablets. Initially Acquity UPLC HSS C18 x 1.8 m was used with mobile phase composition 0.1% Formic acid: Methanol (45: 55, v/v) at the flow rate was changed to 0.5 mL/min and both Atorvastatin and Amlodipine were eluted at 0.6 mins and 0.9 min respectively with UV detection at 220 nm (Figure 3). The method optimized conditions were shown in Table 1.

Table 1: Optimized conditions for determination of Atorvastatin and Amlodipine

Parameter	Optimized chromatographic conditions
Mobile Phase	0.1% Formic acid : Acetonitrile : Methanol (0.1:45: 55, v/v)
Flow Rate	0.3 mL/min
Detection	220 nm
Retention time	Atorvastatin (0.607 ± 0.02 mins) and Amlodipine (0.915 ± 0.03 mins)

Figure 3: Typical chromatograms of Atorvastatin (Rt 0.607min) and Amlodipine (Rt 0.915 min)

Analytical method validation¹⁴

The following parameters were considered for validating the developed method as per ICH guidelines.

System suitability parameter:

General, working standard solutions were injected and chromatograms were recorded. The system suitability studies were carried out as specified in USP. These parameters include Column efficiency, Capacity factor, Resolution, Tailing factor and Theoretical plates.

Acceptance criteria:

The % RSD for the peak area responses of principal peak from 6 replicate injections of each standard solution should be not more than 2.0%. The number of theoretical plates (N) for the drug peak is not less than 2500. The tailing factor (T) for the drug peak is not more than 2.0

Linearity

Preparation of standard stock solution:

Table 2: Linearity of Atorvastatin and Amlodipine

Conc. (μg/mL)		*Mean peak area
AT	AM	AT	AM
25	12.5	486707	169557
50	25	905328	328458
75	37.5	1341683	487005
100	50	1728782	658537
125	62.5	2159639	820950
150	75	2557970	967314

Atorvastatin and Amlodipine obey Beer- Lamberts law 25-150 and 12.5-75 µg/mL (Table 2) respectively. Atorvastatin and Amlodipine have shown linear regression equations, y = 16919x + 42527 (R² = 0.9992) and y = 12957x + 4388 (R² = 0.9997) respectively (Figure 4).

Acceptance criteria: r² value should not be less than 0.98.

Figure 4: Calibration curve of A) Atorvastatin B) Amlodipine

Accuracy:

The closeness of agreement between the true values which is accepted either conventional new value or an accepted reference value and the value found. Inject the standard solution of accuracy 50%, 100%, 150% solutions. Calculate the amount found for Atorvastatin and Amlodipine and calculate the individual recovery and mean recovery values (Table 3).

Acceptance criteria:

The % Recovery for each level should be between 92.0 to 102.0%

Table 3: Accuracy study of Atorvastatin and Amlodipine

Drugs	Conc. added (μg/mL)	Conc. found (μg/mL)± SD*	% Recovery	RSD
AT	50(50%)	50.79±0.18	101.59	0.36
	100(100%)	98.92±0.46	98.92	0.46
	150(150%)	147.32±2.075	98.21	1.4
AM	25 (50%)	24.93±0.074	99.75	0.29
	50(100%)	50±0.306	100.26	0.61
	75(150%)	74±0.076	99.09	0.1

*Mean of three replicates

Precision:

The standard solution was injected for nine times and measured the area for all injections in HPLC. The %RSD for the area of nine replicate injections was found to be within the specified limits.

Acceptance criteria:

The % RSD for the area of six standard injections results should not be more than 2% (Table 4)

Table 4: Precision studies of Atorvastatin and Amlodipine

Drugs	Method Precision		System Precision
Drugs	*Conc. (μg/mL) ± SD	%RSD	* Conc. (μg/mL) ± SD	%RSD
AT	1783802± 19094.19	1.07	1738805± 18954.9	1.09
AM	658437.8± 2691.606	0.40	655437.8± 9542.932	1.45

Assay:

Assay of different formulations available in the market was carried by injecting sample corresponding to equivalent weight into HPLC system and percentage purity was found out by following formulae. Recovery studies were carried out. Calculate the percentage purity of Atorvastatin and Amlodipine present in the tablet using the calibration curve. Inject the sample solutions in triplicate and take the average area then calculate the value by using linearity calibration curve equation.

Concentration of tablet = dilution factor × sample concentration

Robustness:

(a) Effect of variation of flow rate:

A study was conducted to determine the effect of variation in flow rate by injecting 0.9 and 1.1 mL/min. Standard solution was prepared and injected into the HPLC system.

Acceptance criteria:

1. Tailing factor should not be more than 2.0 for variation in flow rate.

2. The %RSD should not be more than 2.0 for variation in flow rate.

(b) Effect of variation of Mobile phase composition:

A study was conducted to determine the effect of variation in mobile phase composition. Standard solution was prepared and injected into the HPLC system at 40:60 and 50:50. The effect of variation in mobile phase composition was evaluated.

Acceptance criteria:

1. Tailing factor should not be more than 2.0 for variation in mobile phase composition.

2. The %RSD should not be more than 2.0 for variation in mobile phase composition.

(c) Column to Column variation:

Procedure:

The standard solution is injected for by using different columns and the area for injections in UPLC was measured. The %RSD for the area of replicate injections was found to be within the specified limits

Acceptance criteria:

The %RSD should not be more than 2.0

Ruggedness:

Ruggedness is the degree of reproducibility of results obtained by the analysis of the same sample under a variety of normal test conditions ie different analysts, laboratories,columns, instruments, reagents, assay temperatures, different days etc. (i.e., from laboratory to laboratory, from analyst to analyst).

Acceptance Criteria

Overall %RSD should not be more than 2.0

LOD and LOQ

LOD is the lowest concentration in a sample that can be detected, but not necessarily quantitated, under the stated experimental conditions. The limit of detection is important for impurity tests and the assays of dosages containing low drug levels and placebos. lt is quoted as the concentration yielding a signal-to-noise ratio. From the prepared solution, take 5 µL of solution and make it up to 1 ml with the diluent. This is called 1ml of LOD sample and from this sample, 3 injections are injected into the HPLC system. Limit of detection was found to be 0.299 µg/mL and0.08 µg/mL for Atorvastatin and Amlodipine respectively.

Table 5: Calculation of LOD and LOQ

Approach	LOD	LOQ
Visual Inspection	Minimum level detectable	Minimum level quantifiable
Signal-to- Noise Ratio	3:1	10:1
SD of response (σ) and slope (S)	{3.3×σ}/s	{10.0×σ}/s

LOQ is the lowest concentration of analyte in a sample that can be determined with acceptable precision and accuracy. lt is quoted as the concentration yielding a signal-to-noise ratio of 10:1. From this solution, taken 75µl and make it up to 1 ml with the diluent. This is called 1 ml of LOQ sample and from this sample, 3 injections are injected into the HPLC system. Limit of Quantification was found to be 0.907 µg/mL and 0.25µg/mL for Atorvastatin and Amlodipine respectively.

Specificity:

Specificity for an assay ensures that the signal measured comes from the substance of interest, and that there is no interference from excipient and/or degradation products and/or impurities. The specificity of the method was demonstrated by establishing a lack of interference from the diluent blank.

Stress degradation studies¹⁵

Generally, stress degradation studies were performed to verify specificity and stability indicating nature of the method. All solutions for degradation studies were prepared at initial concentration of 1 mg/mL Atorvastatin and Amlodipine was subjected to acidic, alkaline, oxidative and photolytic stress conditions (Table 6; Figure 5)

Acid hydrolysis: For acidic stress, 25 mL 0.1N HCl was added to 25 mL sample solution (1000 ppm). The mixture was refluxed for 8 h. similar procedure was repeated with 1 N (reflux, 12 h), 2 N (reflux, 24 h) and 5 N HCl (reflux, 24 h). The solutions were cooled, neutralized and diluted in mobile phase to give a concentration of 20 ppm. The solutions were filtered prior to injection.

Alkaline hydrolysis: For alkaline stress, 25 mL 0.1N NaOH was added to 25 mL sample solution (1000 ppm). The mixture was refluxed for 8 h. similar procedure was continued with 1 N (reflux, 12 h), 2 N (reflux, 24 h) and 5 N NaOH (reflux, 24 h). The solutions were cooled, neutralized and diluted in mobile phase to give a concentration of 20 ppm. The solutions were filtered prior to injection.

Oxidation: For oxidative stress, 25 mL 3% H₂O₂ was added to 25 mL sample solution (1000 ppm). The mixture was kept for 6h at room temperature. Similar procedure was repeated with 30% H₂O₂for 24 h. The solutions were diluted in mobile phase to give a concentration of 20 ppm. The solutions were filtered prior to injection.

Photolytic degradation: For photolysis, 25 mg of each sample was exposed to UV light (254 nm) for 10 days in UV chamber. The stressed drug was dissolved in appropriate volume of mobile phase and diluted to get 25 ppm solution that was filtered prior to injection.

Table 6: Stability studies of Atorvastatin (10 µg/mL) B) Amlodipine

Parameter	Retention time	Degradants
Acidic hydrolysis	No change	ü at 0.498 min
Alkaline hydrolysis	No change	ü at 0.497 min
Oxidation	No change	Nil
Photolysis	No change	Nil

RESULTS AND DISCUSSION:

In the present study numerous trails were carried out during the development of a method for the simultaneous estimation of Atorvastatin and Amlodipine HCl in bulk form. Method was optimized by using a mobile phase composition, 0.1% Formic acid: Acetonitrile: Methanol (0.1: 45: 55, v/v) at the flow rate was changed to 0.3 mL/min and both Atorvastatin and Amlodipine were eluted at 0.607 mins and 0.917 min respectively with UV detection at 220 nm. Atorvastatin and Amlodipine obey Beer- Lamberts law 25-150 and 12.5-75 µg/mL respectively. Atorvastatin and Amlodipine have shown linear regression equations, y = 16919x + 42527 (R² = 0.9992) and y = 12957x + 4388 (R² = 0.9997) respectively. The LOD and LOQ are found to be 0.299 µg/mL and 0.907 µg/mL for Atorvastatin and 0.08 µg/mL and 0.25 µg/mL for Amlodipine correspondingly. The significant parameters of the present method were compared with the previously published analytical methods in Table 7.

The method was validated as per ICH guidelines. The % RSD is 0.41 and % recovery 99.63-100.21 for Atorvastatin and 99.72-100.38 for Amlodipine) were found to be <2.0 %indicating that the method is precise and accurate. The theoretical plates were found to be 3466 and 13534 for Atorvastatin and Amlodipine (>2000) respectively whereas the tailing factor is found to be <1.5 in all the chromatographic observations. Atorvastatin calcium and amlodipine besylate stock solutions were subjected to acid and alkali hydrolysis, chemical oxidation and dry heat degradation. The degraded product peaks were well resolved from the pure drug peak with significant difference in their retention time values. Degradation peaks were noticed only in acidic and alkaline hydrolysis but in other degradation studies no degradation peaks were noticed and at the same time no variation in the retention time.