Method Development and Validation of Stability indicating HPLC method used for the estimation of Degradation Products of Atorvastatin Calcium

 

Ashish R. Deshpande, Ganesh Ramachandran and Ramesh S. Yamgar*

Chemistry Research Laboratory, Patkar-Varde College, Goregaon (W), Mumbai-400 062, Maharashtra, India.

*Corresponding Author E-mail: rsy_chem@rediffmail.com

 

ABSTRACT:

A simple, fast and precise gradient reversed phase high performance liquid chromatographic method was developed for the estimation of impurities and degradation products of the Atorvastatin Calcium and validated as per ICH guidelines. The drug substance, its impurities and degradation products were found well separated with gradient conditions having short run time of 35 mins by using Symmetry C18 column from Water’s (250 x 4.6 mm, 5µm). The flow rate was kept 1 mL/min. The gradient mobile phase consisted of A= 0.05% Trifluoroacetic acid in water and B= 0.05% Trifluoroacetic acid in Acetonitrile. Detection was performed at 249 nm using PDA detector.

 

The method was validated for Specificity, LOD, LOQ, Linearity and Accuracy as per ICH guidelines [1-2]. The stability indicating capability of the method was established by performing forced degradation study. The proposed method was found to be accurate, precise and fast for the estimation of Atorvastatin, its impurities and degradation products.

 

KEYWORDS: Atorvastatin, HPLC, Validation, degradation products.

 


 

INTRODUCTION:

Atorvastatin calcium is an antihyperlipoproteinemic drug, used for the treatment of hypercholesterolemia. Chemically it is (bR,dR)-2-(4-fluorophenyl)-b, d-dihyroxy-5-(1-methyl ethyl)-3-phenyl-4-[(phenyl amino)carboxyl]-1H-pyrrole-1-heptanoic acid.

 

Literature survey3-11 reveals that few HPLC methods are reported for the determination of Atorovastatin individually and in combination with other drugs in tablets form. However many methods involved tedious mobile phase preparation, but our study reports a simple, precise and rapid gradient HPLC method for the separation of Atorvastatin, possible impurities and its degradation product in Active pharmaceutical ingredient (API).

 

EXPERIMENTAL:

Working standard and Chemicals:

Trifluoroacetic acid was obtained from Acros (USA), Acetonitrile and HPLC grade water were obtained from Rankem (India). Atorvastatin standard was provided by Vitalife lab.

 

Gurgaon, Haryana, India. Atorvastatin sample, Impurity B [4-fluoro-a-(2-methyl-1-oxopropyl)-g-N-bdiphenylbenzene butaneamide] and Impurity C [(4R-cis)-1,1-dimethylethyl-6-[2-[2-(4-fluorophenyl)-5-(1-isopropyl)-3-phenyl-4-[(phenylamino)carbonyl]-1H-pyrrol-1-yl]ethyl]-2,2-dimethyl-1,3-dioxane-4-acetate] were provided by Nicholas Piramal India ltd., Mumbai, Maharashtra, India.

 

Instrument:

The analysis was carried out on Shimadzu LC-2010C HT separation module equipped with PDA detector and Class VP software.

 

Optimized Chromatographic conditions:

Analytical column                    :   Water’s Symmetry C18,                                            250 x 4.6 mm, 5mm.

Detection wavelength              :   249 nm

Flow rate                                  :   1 mL/min

Column oven temperature       :   25°C

Injection Volume                     :   10 ml             

Run time                                      : 35 min

Diluent                                     :  Methanol

Mobile Phase                           :  A: - 0.05% Trifluoroacetic                                         acid (TFA) in water.

                                                     B:- 0.05% Trifluoroacetic                           acid (TFA) in Acetonitrile.

 

Gradient program                 :

Time (Min)

%A

%B

0.0

50

50

10.0

40

60

15.0

40

60

18.0

10

90

30.0

10

90

31.0

50

50

35.0

50

50

Retention time: Atorvastatin ~ 10.5 min,                                                       Impurity B ~ 18.9 min and                                           Impurity C ~ 26.1 min.

 

Reference and System suitability solution preparation:

A reference solution of Atorvastatin, Impurity B and Impurity C was prepared by dissolving 5 mg each separately in a 50 mL Volumetric flask with diluent (0.1 mg/mL). Pipette out 10 mL of this solution into 100 mL volumetric flask and dilute with diluent (10 mg/mL). Further dilute 10 mL of the above solution (10 mg/mL) to 100 mL with the diluent (1.0 mg/mL). The reference solution was used as system suitability solution.

 

Test preparation:

Test solution was prepared by weighing 25 mg of the substance in a 25 mL of volumetric flask, dissolving and diluting upto the mark with diluent (1000 mg/mL).

 

Validation:

The developed HPLC method was validated for the following parameters.

 

Specificity:

Specificity was done in two parts,

1) By injecting diluent, Atorvastatin and related impurities solution of Impurity B and Impurity C (0.1 mg/mL each ) individually and in combination (1.0 mg/mL each) into the chromatograph. The diluent blank did not interfere with our peaks of interest and all the peaks were found to be well separated and resolved from each other.

2) By forced degradation.

The forced degradation study was carried out to know in advance likely degradation products that may be generated during stability study or shelf life. Atorvastatin was exposed to the following dry environmental and wet chemical conditions.

 

A] Dry environmental conditions-

1.           White fluorescent light (NLT 1.2 million lux hours)

2.           UV Radiation (NLT 200 watt hrs. m-2.)

3.           Thermal (at 80oC for 8 hrs.)

B] Wet chemical conditions-

1.           Aqueous (water)

2.           Basic (Sodium hydroxide, 1N)

3.           Acidic (Hydrochloric acid, 1N)

4.           Oxidation (Hydrogen peroxide - 1%v/v)

 

In the forced degradation study it was observed that there was no degradation found in dry conditions, but in wet chemical condition, degradation was found in Basic (5.7%), Acidic (78.4%) and Oxidation (3.5%) respectively, which is shown in Figure 1, Figure 2 and Figure 3. Though degradation was found in the wet chemical condition, peak purity taken on PDA detector showed that the peak of Atorvastatin in all condition was found to be pure and well separated from all the impurities and degradation products. The stability indicating assay was demonstrated by comparing assay of the degraded samples with that of control sample. The assay and purity of the degraded sample is presented in the Table 1.

 

Figure 1: Chromatogram of sample after degradation in 1N hydrochloric acid at 80°C for 1 hr.

 

Figure 2: Chromatogram of sample after degradation in 1N Sodium hydroxide at 80°C for 1 hr.

 

Figure 3: Chromatogram of sample after degradation in 1%V/V Hydrogen peroxide at 80°C for 1 hr.

 

 


Table 1: Assay and Purity of the degraded Atorvastatin samples.

Degradation condition

Purity (Area%)

% Assay with respect to control sample

RRT of the major degraded peaks

Control sample (No degradation)

99.48

99.94

-

White fluorescent light (NLT 1.2 million lux hours)

99.50

100.03

No major degradation peak observed

UV Radiation (NLT 200 watt hrs. m-2.)

99.50

100.15

No major degradation peak observed

Thermal (at 125oC for 24 hrs.)

99.46

99.50

No major degradation peak observed

Aqueous (water) (at 80°C for 1hr.)

99.44

99.59

No major degradation peak observed

Basic (1N NaOH) (at 80°C for 1hr.)

93.91

96.89

0.44,1.28,1.32,1.44

Acidic (1N HCl) (at 80°C for 1hr.)

20.37

22.15

1.32,1.44,1.91,2.09,2.17

Oxidation (3% v/v Hydrogen peroxide) (at 80°C for 1hr.)

96.08

96.54

1.32,1.75,1.88,1.97

 

Table 2: Limit of detection (LOD)  and Limit of quantitation (LOQ) of all components.

Sr. no.

Component

LOD

LOQ

Conc. (mg/mL)

Conc. (%)

S/N Ratio

Conc. (mg/mL)

Conc. (%)

S/N Ratio

1.

Impurity B

0.1

0.01

3.9

0.3

0.03

11.5

2.

Impurity C

0.1

0.01

2.1

0.3

0.03

10.4

3.

Atorvastatin

0.1

0.01

3.0

0.4

0.04

9.9

 

 


Limit of detection (LOD) and Limit of quantitation (LOQ):

The limit of detection (LOD) and quantitation (LOQ) was determined by injecting a series of diluted solutions of impurities having concentration close to theoretical limit of detection and quantitation. The signal to noise ratio was calculated. For limit of detection signal to noise ratio must be at least 3: 1 and for limit of quantitation it must be 10:1 as given in ICH guidelines. The LOD and LOQ for Impurity B, Impurity C and Atorvastatin (for unknown impurity) were thus obtained which are tabulated in Table 2. The LOQ concentration for Atorvastatin, Impurity B and Impurity C were injected six times and % RSD of area of the replicate injections were calculated and found well within the limit of 10 %.

 

Linearity:

Linearity is determined by serial dilution of Atorvastatin, Impurity B and Impurity C to a 7 different concentration (prepared in combination) in the range of LOQ to 150% of impurity limit. The response was recorded as peak areas and plotted against the concentration. A correlation coefficient, slope and intercept were calculated. For Atorvastatin, Impurity B and Impurity C the correlation coefficient was obtained as 1.000, 0.999 and 0.998 respectively showing that response is linear.

 

Accuracy:

The accuracy was carried out from 50% to 150% of the impurity limit. Each impurity was spiked into the sample at three levels, 50%, 100% and 150% of impurity limit and injected into the chromatograph. From the data obtained, % recovery for each sample and at each level is calculated against added amount. The recovery for Impurity B and Impurity C was found to be in the range of 82.0 % to 89.0% and 84.0 % to 97.3% respectively The RSD for recovery for Impurity B and Impurity C was found to be 2.40 % and 4.63 % respectively. The mean recovery obtained for all components was within the limit of 80.0% to 120.0% and RSD calculated for all levels was less than 10 % indicating accuracy of analytical method.

RESULT AND DISCUSSION:

HPLC method development:

In the initial trials columns used were Hypersil BDS C18 (250 x 4.6 mm, 5mm), Water’s X-terra (50 x 4.6 mm, 3.5mm), Water’s atlantis d18 (150 x 4.6, 5mm) and Water’s symmetry C18 (250 x 4.6 mm, 5mm) as the stationary phase and 1) water : Acetonitrile and 2) 0.05% TFA in water and 0.05% TFA acid in Acetonitrile as the mobile phase in the gradient proportion. With water-Acetonitrile as the mobile phase peak elution, peak shape and resolution was not good. Also, with the columns such as Hypersil BDS C18 (250 x 4.6 mm, 5mm), Water’s X-terra (50 x 4.6 mm, 3.5mm) and Water’s atlantis d18 (150 x 4.6, 5mm) along with mobile phase containing 0.05% TFA in water and 0.05% TFA in Acetonitrile peak shape showed fronting and resolution was also not good with some degradants peak merging in the tailing of the main peak. After lot of trials, good peak shape and resolution could be achieved with the mobile phase containing 0.05% TFA in water and  0.05% TFA in Acetonitrile in gradient proportion and with Water’s symmetry C18 column. The TFA was added to the mobile phase as it act as ion-pairing agent and helps in improving peak shape and resolution.

 

CONCLUSION:

The method was found to be specific stability indicating as proved by injecting known components into the chromatograph and by forced degradation study. Limit of detection and limit of quantitation for known related impurities and unknown related impurities has been established. The Analytical method was found to be linear in the specified range and also found to be Accurate and Precise. From the above data, it is concluded that, the analytical method can be used for analysis of related substances of Atorvastatin Calcium and is capable of giving accurate and precise results.

 

REFERENCES:

1.       International Conference On Harmonization (ICH), Q2 (R1) (2005) Validation of analytical procedures: Text and Methodology.

2.       International Conference On Harmonization (ICH), Q3A (R2)(2006) Impurities in New Drug Substances.

3.       Amir G K, Alireza D, Kheirollah G and Neda J (2007) New high-performance liquid chromatographic method for serum analysis of oxazepam: Application to bioequivalence and pharmacokinetic study. Acta Poloniac Pharmaceutica-Drug Research 64 : 287.

4.       European Pharmacopoeia 6.0 (2008) 2 : 2577.

5.       British Pharmacopoeia (2008) II:1616.

6.       Snyder L R, Kirkland J J and Glajch J L (1997) Practical HPLC Method Development, 2nd Edition, John Wiley and sons Inc: New York,  p 685.

7.       Rajeswari KR, Sankar GG, Rao AL, Seshagirirao J. RP-HPLC method for the simultaneous determination of Atorvastatin and Amlodipine in tablet dosage form. Indian J Pharm Sci 2006;68:275-7.

8.       Chaudhari BG, Patel NM, Shah PB, Stability Indicating RP-HPLC Method for Simultaneous Determination of Atorvastatin and Amlodipine from Their Combination Drug Products. Chem Pharm Bull 2007;55(2):241-246.

9.       Shah D, Bhatt K, Mehta R, Shankar M, Baldania S. RP-HPLC method for the determination of atorvastatin calcium and nicotinic acid in combined tablet dosage form. India J Pharm sci 2007.

10.     Erturk S, Aktas ES, Ersoy L, Ficicioglu S. An HPLC method for the determination of atorvastatin and its impurities in bulk drug and tablets. J. Pharmaceutical and Biomedical analysis 2003; 33 (5):1017-1023.

11.     Fabio PG, Pedro LG, Joao MPA, Singh AK, Kedor-Hackmann ERM, Santoro MIRM. Development and validation of stability-indicating HPLC methods for quantitative determination of Pravastatin, Fluvastatin, Atorvastatin and Rosuvastatin in pharmaceuticals. Analytical letters 2009:42:1784-1804.

12.     Lincy J, Mathew G, Venkatarangarao B. Simultaneous estimation of Atorvastatin and Ramipril by RP-HPLC and Spectroscopy. Pak. J. Pharm. Sci., 2008:21(3):282-284.

13.     Ismail, Rajavel R, Ganesh M, Jagadeeswaran M, Srinivasan K, Valamathi J and Sivakumar T. RP-HPLC Method for the simultaneous determination of Aspirin, Atorvastatin and Pioglitazone in capsule dosage form. Asian J. Research Chem., 2008; 40-42.

14.     Vora DN, Kadav AA. Validated Ultra HPLC Method for the Simultaneous Determination of Atorvastatin, Aspirin, and their Degradation Products in Capsules. J. Liquid Chrom. & Related Technologies 2008;31(18):2821-2837.

15.     Stanisz B, Kania L. Validation of HPLC method for determination of Atorvastatin in tablets and for monitoring stability in solid phase. Acta Poloniac Pharm. Drug Research 2006; 63(6):471-476

 

 

 

 

Received on 02.06.2010        Modified on 22.06.2010

Accepted on 01.07.2010        © AJRC All right reserved

Asian J. Research Chem. 4(4): April 2011; Page 551-554