RP-HPLC Method for Simultaneous Estimation of Atorvastatin Calcium Ezetimibe in Pharmaceutical Formulation.
MB Mundlik, CK Gadewar*, NA Chandekar, NM Mahajan, PD Telgote and AV Chandewar
Dept. of Pharmaceutical Chemistry, P. Wadhwani College of Pharmacy, Dhamangaon Road Yavatmal- 445001
*Corresponding Author E-mail: ckgadewar@rediffmail.com
ABSTRACT:
A simple, selective, rapid and precise reverse phase HPLC method has been developed for the simultaneous estimation of Atorvastatin calcium and Ezetimibe in pharmaceutical dosage form. A Hypersil BDS (250 mm X 4.6mm i.d 5m) column was used for Separation. The mobile phase was Acetonitrile: water: Methanol (350:550:100, v/v) and adjust Ph to 4.0 with Orthophosphoric acid. Flow rate 2.0ml/min with detection at 250nm.The retention time of Atorvastatin calcium and Ezetimibe was 21.712 and 10.414 min. respectively. The developed method was validated in terms of accuracy, precision, Linearity, specificity and forced degradation, robustness, solution stability, system suitability, limit of detection. The proposed method can be used for these drugs in combined dosage forms. The proposed RP-HPLC Method for the simultaneous estimation of Atorvastatin calcium and Ezetimibe in combined dosage form is accurate, precise, linear, rugged, robust, simple, rapid and selective. It can be easily adopted for routine quality control (QC) analysis of raw materials, formulation studies. pH of the mobile phase is 4, which is good to increase the shelf life of the column.
KEYWORDS: RP-HPLC; Atorvastatin calcium, Ezetimibe.
Atorvastatin calcium1-3 is a second generation potent inhibitor of HMG-CoA. Chemically it is [R-(R,R*)]-2-(4-flurophenyl)-b,d-dihydroxy-5(1-methylethyl)-3- phenyl-4- [phenyl amino) carbonyl]-1H-pyrrole-1-heptanoic acid, calcium salt (2:1) trihydrate. It’s molecular formula is C66H68CaF2N4O10, 3H2O with a molecular weight of 1209.42.Ezetimibe4 it is a first of the selective cholesterol absorption inhibitor. Chemically it is (3R,4S)-1(4-flurophenyl)-3-[(3S)-3-(4-fluorophenyl)-3-hydroxypropyl]-4-(4-hydroxyphenyl)-2-azetidinone. Its molecular formula is C24H21F2NO3 with a molecular weight of 409.4. Atorvastatin calcium is an official in Indian Pharmacopoeia, British Pharmacopoeia, United State Pharmacopoeia but Ezetimibe is not official in any pharmacopoeia. Fixed dose combination containing Atorvastatin calcium 10 mg and Ezetimibe 10 mg is available in tablet dosage form in the market. In literature there is a HPLC method for Ezetimibe5 and also for Atorvastatin calcium6,7. HPLC Method for simultaneous estimation of Atorvastatin calcium, Ezetimibe is reported8 .
Present paper Describe a simple, rapid accurate precise method for simultaneous estimation of Atorvastatin calcium and Ezetimibe by reverse phase HPLC. The present RP-HPLC Method was validated following the ICH guidelines.915.
MATERIAL AND METHODS:
Reagent and Chemicals:-
Acetonitrile HPLC grade, Methanol HPLC Grade, Orthophosphoric acid AR Grade were procured from Qualigens fine chemical Mumbai. HPLC Grade water was obtained from a Milli-QRO water purification system. Working standard of Atorvastatin calcium and Ezetimibe were gift sample from Concept Pharmaceutical Ltd. Aurangabad.
Instruments and chromatographic conditions:
Chromatographic separation was performed on an Agilent liquid chromatographic system with chromeleon software equipped with a SPD M-10AVP photo diode array detector, Class-M 10 data station was applied for data collecting and processing. Hypersil BDS (250mmx 4.6mm i.d, 5m) was used for the separation. Mobile phase was Acetonitrile: water: Methanol (350:550:100, v/v) and adjust PH to 4.0 with Orthophosphoric acid. Flow rate 2.0ml/min with detection at 250nm. Under these conditions retention times of Atorvastatin calcium and Ezetimibe were 21.712 and 10.414 min respectively.
Preparation of standard solution:
Weigh accurately about 55mg of Atorvastatin Calcium and 50mg of Ezetimibe working standard into a 100 ml volumetric flask .Add about 70ml of methanol, sonicate to dissolve and make up the volume to 100 ml with methanol. Further dilute 5 ml of this solution to 25 ml with mobile phase.
Preparation of sample solution:
Weigh accurately a quantity of powder containing the equivalent of about 50 mg of Atorvastatin and 50 mg of Ezetimibe into a 100 ml volumetric flask. Add 70 ml of methanol sonicate for about 20 minutes to dissolve and make up the volume to 100 ml with methanol and filter through 0.45m Nylon filter. Further dilute 5 ml of this solution to 25 ml with mobile phase.
Assay Method:
Wash a Column initially with Acetonitrile: Water (30:70) at a flow rate of 1 ml /min for 30 minutes and then run mobile phase for 30 min. separately inject equal volume (20 ml) of the Standard preparation (Five replicates) and Sample preparation (duplicate) into the chromatograph. Record the chromatograms and measures the peak response for Atorvastatin and Ezetimibe. The system suitability parameters should be met. From the peak responses, calculate the content of Atorvastatin calcium and Ezetimibe in the sample.
AT Wstd 5 100 25 Avg.Wt P
Content of drug = --- X----X ---X-----X---X--------X----X 100
(%Label claim) AS 100 25 Wtest 5 L 100
Where,
AT = Average area of Atorvastatin peak for sample solution,
AS = Average area of Atorvastatin peak for standard solution,
L = Label claim in mg.
P = Potency.
RESULT AND DISCUSSION:
Estimation of Atorvastatin calcium and Ezetimibe in dosage forms:
Estimation of Atorvastatin calcium and Ezetimibe in dosage forms by RP-HPLC method was carried out using optimized chromatographic condition. The typical chromatogram of standard and sample solution is given in fig.1 and 2. Peak area ratio of standard and sample solution was calculated. Assay procedure was repeated for six times the mean peak area ratio and content of drug is calculated. The percentage of individual drug found in formulation, mean, standard deviation in formulation were calculated and presented in Table I. The result of analysis shows that the amount of drugs was in good agreement with label claim of the formulation.
Method validation:
Accuracy and precision:
The accuracy of method was determined by recovery experiments. The recovery studies were carried out using standard addition method at 40, 100 and 160 % level; known amount of standards was added to preanalyzed sample and subjected them to the proposed HPLC method. Percentage recovery was calculated from the amount found and actual amount added result shows in Table II.
Fig.1.Typical chromatogram of sample solution
Table.I.Result of analysis of formulation
|
Drug |
Labelled amount |
%Assay (n=6) |
S.D |
% RSD |
|
Atorvastatine calcium |
10mg |
98.7 |
0.578 |
0.59 |
|
Ezetimibe |
10mg |
98.2 |
0.527 |
0.54 |
Fig.2.Typical chromatogram of standard solution
Fig.3 .Typical chromatogram for blank (mobile phase)
Precision of method was demonstrated by Repeatability, Reproducibility and Intermediate precision. Repeatability in which six replicate injections of standard solution were injected into HPLC System. The mean, SD and %RSD for peak areas of Atorvastatin calcium and Ezetimibe were calculated and result show in Table III, IV, V. Reproducibility in which six sample of a single batch were analysed as per test method and % assay for Atorvastatin Calcium and Ezetimibe in six samples were calculated. Intermediate precision in which six sample of the same batch used for method precision as per test method by different analyst using different instrument and different column on different day.
Table.II. Result of Accuracy
|
Spike level In % |
Actual Amount of ATC added in mg |
Amount of ATC Found In mg |
%Recovery of ATC |
Actual amount of EZT added in mg |
Amount of EZT Found In mg |
%Recovery of EZT |
|
Level-(40%) |
20.47 |
20.55 |
100.4 |
20.61 |
20.53 |
99.6 |
|
|
20.50 |
20.72 |
101.1 |
20.87 |
20.70 |
99.2 |
|
|
20.53 |
20.67 |
100.7 |
20.85 |
20.65 |
99.0 |
|
Level(100%) |
51.07 |
50.69 |
99.3 |
49.92 |
50.64 |
101.4 |
|
|
50.74 |
50.83 |
100.2 |
50.43 |
50.77 |
100.7 |
|
|
50.90 |
50.74 |
99.7 |
50.29 |
50.69 |
100.8 |
|
Level(160%) |
80.41 |
80.31 |
99.9 |
80.07 |
80.22 |
100.2 |
|
|
80.31 |
79.49 |
99.0 |
80.73 |
79.40 |
98.4 |
|
|
80.37 |
80.14 |
99.7 |
80.45 |
80.05 |
99.5 |
Fig.4 .Typical chromatogram for placebo
Fig.5. HPLC chromatogram for acid treated sample
Fig.6. HPLC chromatogram for Base treated sample
Fig.7. HPLC chromatogram for peroxide treated sample
Fig.8. HPLC chromatogram for heat treated sample
Fig.9. HPLC chromatogram for UV treated sample
The percentage assay of Atorvastatin calcium and Ezetimibe was determined. Calculated % RSD for assay of Atorvastatin calcium and Ezetimibe in six samples. From data obtained the developed HPLC method was found to be precise.
Table.III.Precision study (Repeatability)
|
Peak Area(ATC) |
Mean±%RSD |
Peak Area(EZT) |
Mean±%RSD |
|
2487609 |
2487522± 0.15 |
2194317 |
2194936±0.15 |
|
2484737 |
2192151 |
||
|
2487395 |
2195389 |
||
|
2484180 |
2192043 |
||
|
2494409 |
2200954 |
||
|
2486799 |
2194761 |
* % RSD is a % relative standard deviation
Table.IV.Precision study (Reproducibility)
|
%Assay (ATC) |
Mean±%RSD |
%Assay(EZT) |
Mean±%RSD |
|
98.0 |
98.7± 0.59
|
97.6 |
98.2± 0.54 |
|
99.5 |
98.8 |
||
|
98.7 |
98.2 |
||
|
99.3 |
98.9 |
||
|
98.3 |
97.8 |
||
|
98.6 |
98.2 |
Table V. Precision study (Intermediate precision)
|
% Assay of ATC (n=6) |
% Assay of EZT(n=6) |
||
|
Analyst –I |
Analyst –II |
Analyst –I |
Analyst-II |
|
98.7 |
99.2 |
98.2 |
98.5 |
|
Over all Mean=99.0 |
Over all Mean=98.04 |
||
|
% RSD of ATC (n=6) |
% RSD of EZT (n=6) |
||
|
Analyst –I |
Analyst –II |
Analyst –I |
Analyst –II |
|
0.59 |
0.89 |
0.54 |
0.63 |
|
Over all %RSD =0.75 |
Over all %RSD = 0.57 |
||
Linearity and range:
Linearity of method was ranging from 41.59 mg/ml to 166.34 mg/ml for Atorvastatin calcium and 41.07 to 164.27mg/ml for Ezetimibe and is presented in Table VI. A graph was plotted with concentration on X axis and mean peak areas on Y-axis. The slope and intercept value for calibration curve was 23711, 14411 and R2 (0.99998) for Atorvastatin calcium and 21210, 11945 and R2 (0.99998) for Ezetimibe.The result show that an excellent correlation exist between concentration and mean peak areas within the concentration range.
Table VI. Linearity and range
|
ATC Concentration mg/ml |
Mean peak areas ATC |
EZT Concentration mg/ml |
Mean peak areas EZT |
|
41.59 |
1003936 |
41.07 |
886218 |
|
62.38 |
1494440 |
61.60 |
1319129 |
|
83.17 |
1981668 |
82.14 |
1749966 |
|
103.96 |
2477107 |
102.67 |
2187583 |
|
124.76 |
2966419 |
123.21 |
2619289 |
|
145.55 |
3477292 |
143.74 |
3072347 |
|
166.34 |
3955168 |
164.27 |
3492562 |
Specificity:
It is evaluated by injecting the blank, placebo and the control sample solution prepared as per the proposed method to check for the interference if any peak at the retention time of Atorvastatin calcium and Ezetimibe. As results are given in Table.VII.
Table.VII.Specificity
|
Sample type |
Peak Name |
Peak purity |
|
Control sample |
ATC |
999 |
|
EZT |
999 |
Table.VIII. Forced degradation
|
Condition |
%Assay ATC |
%Degradation w.r.t control for ATC |
%Assay EZT |
%Degradation w.r.t control For EZT |
Peak purity Data for ATC and EZT |
|
Control sample |
98.7 |
-------------- |
98.2 |
--------------- |
999 |
|
0.1N HCL |
86.6 |
12.28 |
69.9 |
28.83 |
999 |
|
0.01N NaOH |
97.0 |
1.79 |
96.2 |
2.08 |
999 |
|
3.0%v/v H2O2 |
96.9 |
1.84 |
97.1 |
1.17 |
999 |
|
800C |
96.2 |
2.54 |
96.4 |
1.84 |
999 |
|
UV-light at 254nm |
95.6 |
3.15 |
95.1 |
3.24 |
999 |
Forced degradation:
The Atorvastatin calcium and Ezetimibe Tablet sample was subjected to forced degradation under following stress condition. As results are given in Table.VIII.
Fig.10. Linearity graph for ATC
Limit of detection:
Limit of detection (LOD) of developed method were determine by injecting progressively low concentration of standard solution using developed RP-HPLC Method.LOD for Atorvastatin calcium and Ezetimibe was found to be 0.01 μg/mL and 0.002 μg/mL Respectively.
Fig.11. Linearity graph for EZT
Ruggedness and Robustness:
Ruggedness is a measure of the reproducibility of a result under expected operating condition from instrument to instrument and from analyst to analyst. The result of studies reveals that the developed method is rugged. Robustness is a measure of the capacity of a method to remains unaffected by small but deliberate variation in method condition. The result of the studies reveals that the developed method is robust.
Table.IX. System Suitability Test
|
System Suitability Parameters |
ATC |
EZT |
|
Retention Time (TR) Capacity Factor (K ’) Theoretical Plate (N) Tailing Factor (T) Resolution Factor (Rs) |
21.818 2.650 5092 1.34 2.19 |
10.586 5.61 6835 1.21 6.08 |
Solution stability:
In order to demonstrate the stability of standard and sample solution, prepare standard and sample inject into HPLC at initial and different time intervals up to 12hrs and determine cumulative % RSD for peak areas. The result show that for both solution retention time and peak area remains unchanged (%RSD. less than 2) and no significant degradation within the indicated period. So indicate that both solutions are stable.
System suitability studies:
Retention Time, Capacity Factor, Theoretical Plate, Tailing Factor, Resolution Factor Were calculated for the standard solution and result are presented in Table 8. The values obtained demonstrated the suitability of the system for analysis of this drug combination.
CONCLUSION:
The proposed RP-HPLC Method for the simultaneous estimation of Atorvastatin calcium and Ezetimibe in combined dosage form is accurate, precise, linear, rugged, robust, simple, rapid and selective. It can be easily adopted for routine quality control (QC) analysis of raw materials, formulation studies. pH of the mobile phase is 4, which is good to increase the shelf life of the column.
ACKNOWLEDGEMENT:
The Author thanks Concept pharmaceutical for providing gift sample.
REFERENCES:
1) The International Pharmacopoeia, WHO.2007, 4th Edn, Vol. 1-5.
2) USP 28, NF 23, the United State Pharmacopeial Convention, Asian Edn.2005;2389
3) Sonawane S.S. et al Application of UV - Spectrophotometry and RP-HPLC for Simultaneous determination of Atorvastatin calcium and Ezetimibe in Pharmaceutical dosage form, Eurasian Journal of Analytical Chemistry, 2006,1(1),31-41.
4) The CIMS. Mumbai: CMP Medica India private Limited; April-July 2005; 19.
5) Sistla R. et al J. Pharm. Biomed. Anal. 2005; 39: 517.
6) Bahrami G.et al, B Analyt Technol Biomed Life Sci. 2005; 826(1-2);41-50.
7) Unnam S and Kothapally C.B.,HPLC Analysis for Simultaneous Determination of Atorvastatin and ezetimibe in Pharmaceutical formulations, Journal of liquid chromatography and Related Technologies,2008;31(5);714-721.
8) Unnam S, Kothapally C.B., Journal of Liquid Chromatography & Related Technologies. 2008; 31(5); 714 – 721.
9) International Conference on Harmonization, Validation of Analytical Procedures: Methodology, Federal Register.1996;1.
10) International Conference on Harmonization, Draft Guideline on Validation of Analytical Procedure, Definitions and Terminology, Federal Register.1995;11260- 11262.
11) Mendum J. Denny, R.C. and Thomas M.N Vogel’s Text book of Quantitative Analysis, 6th Edn, Pearson Education Ltd.2004: 268
12) Skoog, West and Holler, Fundamental of Analytical Chemistry, 7th Edn, Saunders College Publishing. 1992:1
13) Gennaro A.R., Remington: The Science and Practice of Pharmacy, 19th Edn. Mack Publishing Company.1995: 534.
14) Beckett A.H. and Stanlake J.B., Practical Pharmaceutical Chemistry, 4th Edn. Part 2, CBS Publishers and Distributors. 2002:1
15) Corners K.A., Textbook of Pharmaceutical Analysis, 3rd Edn. A Wiley Interscience Publication. 1999: 616-622.
Received on 24.11.2009 Modified on 30.12.2009
Accepted on 27.01.2010 © AJRC All right reserved
Asian J. Research Chem. 3(2): April- June 2010; Page 485-490