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12. Patel, P. M. and Mashru, R. C., Indian J. Pharm. Sci., 2005, 67, 389. 13. Jain, P.S., Talele, G.S., Talele, S.G. and Surana, S.J., Indian J. Pharm. Sci., 2005, 67, 358. 14. The United State Pharmacopoeia, XXIII, National Formulary, XVIII, US Pharmacopoeial Convention, Inc., Rockville M.D., 1995, 1776.
Accepted 19 December 2006 Revised 1 March 2006 Received 14 October 2005 Indian J. Pharm. Sci., 2006, 68 (6): 790-793
Development and V alidation of a HPTLC Method for Validation the Simultaneous Estimation of Atorvastatin m Calcium o fr and Ezetimibe
d ns a lo tio n a w c i do ubl e P ). e A simple, precise, accurate and rapid high-performance thin-layer method has been developed fr wchromatographic m and validated for the estimation of atorvastatin calciumrand ezetimibe simultaneously in combined dosage forms. o o The stationary phase used was precoated silica gel f60F . The mobile phase used was a mixture of chloroform: o c n . benzene: methanol: acetic acid (6.0:3.0:1.0:0.1 v/v/v/v). The detection of spots was carried out at 250 nm. The k and specificity. e precision w l d method was validated in terms of linearity, accuracy, The calibration curve was found to o b e be linear between 0.8 and 4.0 µg/spot for atorvastatin calcium and 0.1 and 1.0 µg/spot for ezetimibe. The limit of nwere found to be 170 ng/spot and 570 laatorvastatin M k detection and the limit of quantification ifor calcium ng/spot d a y respectively, and for ezetimibe, 20 ng/spot and 70 ng/spot respectively. The proposed method can be successfully v bformulation. e used to determine the drug contentaof marketed is ted w.m sand ezetimibe F w o The combination of atorvastatin calcium determination of atorvastatin calcium and ezetimibe in D w h ( P has recently been introduced in the market. Atorvastatin dosage forms. The objective of the e agent which inhibits pharmaceutical t s lowering calcium is a synthetic ilipid present work was to develop an accurate, specific and i s h anda ezetimibe HMG CoA reductase, inhibits the reproducible method for the simultaneous estimation of T absorption of cholesterol, decreasing the delivery of atorvastatin calcium and ezetimibe in pharmaceutical B. G. CHAUDHARI, N. M. PATEL 1 *, P. B. SHAH AND K. P. MODI 1
Shri B. M. Shah College of Pharmacy, Modasa-383 315, 1Shri B. M. Shah College of Pharmaceutical Education & Research, Modasa-383 315, India.
254
1,2
3,4
intestinal cholesterol to the liver. Chemically, atorvastatin calcium is [R-(R*,R*)-2-(4-flurophenyl)-β-dihydroxy-5-(1 methylethyl)-3-phenyl-4-[(phenylamino)carbonyl]-1H pyrrole-1-heptonic acid-calcium salt (2:1) trihydrate5-7, and ezetimibe is (3R,4S)-1-(4-flurophenyl)-3-[(3S)-3-(4 flurophenyl)-3-hydroxypropyl]-4-(4-hydroxyphenyl)-2 azetidinone5,8-9. Atorvastatin calcium and ezetimibe are not official in IP, BP or USP. HPLC method has been reported for the determination of atorvastatin and its impurities in bulk drug and tablets10. No analytical method has so far been reported for the simultaneous *For correspondence E-mail:
[email protected] November - December 2006
dosage forms. Atorvastatin calcium and ezetimibe working standards were procured as gift samples from Torrent Research Centre, Ahmedabad. Silica gel 60F254 TLC plates (E. Merck, Mumbai) were used as a stationary phase. Tablets containing 10 mg each of atorvastatin calcium and ezetimibe were purchased from the local market (Zetistat-10, Torrent Pharmaceuticals Ltd.). A Camag HPTLC system comprising of Camag Linnomate V automatic sample applicator, Hamilton syringe, Camag TLC Scanner 3, Camag WinCATS software, Camag twin-trough chamber and ultrasonicator was used during the study.
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Working standards of atorvastatin calcium and ezetimibe (10 mg each) were weighed accurately and diluted with methanol to obtain a final concentration of 1 mg/ml for atorvastatin calcium and 100 µg/ml for ezetimibe. The contents of 20 tablets were ground to a fine powder. Weight equivalent to 25 mg each of atorvastatin calcium and ezetimibe was transferred to a conical flask and dissolved in methanol. The solution was sonicated for 15 min. The extract was filtered through Whatman filter paper No. 41, and the residue was washed with methanol. The extract and washing were pooled and transferred to a 25 ml volumetric flask, and volume was made with methanol. Required dilutions were made to obtain 1000 µg/ml of atorvastatin calcium and 100 µg/ml of ezetimibe in two different 10 ml volumetric flasks.
method was validated 11–13 by establishing linearity, accuracy, inter-day and intra-day precision, specificity, repeatability of measurement of peak, as well as repeatability of sample application. The limit of detection and limit of quantification were also determined. The related impurities were determined by spotting higher concentration of the drugs so as to detect and quantify them. For the analysis of the marketed formulations, 2 µl (for atorvastatin) and 5 µl (for ezetimibe) of filtered solutions of the marketed formulations were spotted onto the same plate, followed by development scanning. The analysis was repeated six times. The spots were resolved into two peaks in the chromatogram of drug samples extracted from the marketed formulations. The content of the drug was calculated from the peak areas recorded.
m rf o d ns a The chromatographic estimations were performed using lo tio stationary phase, precoated silica gel 60F aluminium n a would give dense and compact sheets (20 × 10 cm, prewashed with methanol and dried A solventw system c that i o appropriate l and significantly different R values in an oven at 50° for 5 min); mobile phase, chloroform: spots with d b benzene: methanol: acetic acid (6:3:1:0.1 v/vv/v); chamber was desired for quantification of atorvastatin calcium and e Pinupharmaceutical . and plate saturation time of 30 min. Migration distance ezetimibe formulations. The mobile e ) r allowed was 72 mm; wavelength scanning was done at 250 f phase consisting of chloroform: benzene: methanol: acetic w ov/v/v/v) m gave R values r o nm (fig. 1). acid (6:3:1:0.1 of 0.3 (±0.04) and c fo 0.53 n . for atorvastatin and ezetimibe respectively k (±0.04) e of d(fig. l Aliquots of 0.8, 0.9, 1, 2, 3 and 4 µl of standard solution 2). w Linearity range for atorvastatin calcium and o was found to be in the range of 0.8-4.0 µg/spot b atorvastatin calcium and 1, 3, 6, 8 and 10 µl ofastandard e ezetimibe n l M and k0.1-1.0 µg/spot, with a correlation coefficient of 0.9992 solution of ezetimibe were applied on the TLCi plate. The d a y TLC plate was dried, developed v and analyzed 0.9995, respectively. The LOD and LOQ for b meand photometrically as described earlier.aThe calibration atorvastatin calcium were found to be 170 ng/spot and 570 d . s e i curves were prepared by plotting peak tarea versus for ezetimibe, 20 ng/spot and 70 ng/spot w ng/spot s F concentration (µg/spot) corresponding to each spot. The respectively. w o PD te h (w is si h T a 254
f
f
Fig. 1: Spectrum of atorvastatin calcium and ezetimibe Spectra of atorvastatin calcium and ezetimibe in which peak 1 and 2 are spectrum of atorvastatin and ezetimibe respectively, and X-Axis indicates area under curve (AUC), while Y-Axis indicates wavelength (nm) 794
Fig. 2: Chromatogram of atorvastatin and ezetimibe Chromatogram of atorvastatin calcium and ezetimibe in which peak 1 and 2 are chromatograms of atorvastatin and ezetimibe respectively, and X-Axis indicates area under curve (AUC), while Y-Axis indicates Rf value
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TABLE 1: VALIDATION PARAMETERS OF ATORVASTATIN CALCIUM AND EZETIMIBE Parameter
TABLE 3: ANALYSIS OF ATORVASTATIN CALCIUM AND EZETIMIBE
Result
Linearity range (µg/spot) Correlation coefficient Precision (% CV) Intra day (n=6) Inter day (n=6) Repeatability of sample application (n=6) Repeatability of peak area (n=6) Limit of detection (ng/spot) Limit of quantification (ng/spot) Specificity
Atorvastatin calcium
Ezetimibe
0.8-4.0 0.9992 1.05-1.15 1.39-1.50 1.09 0.14 170 570 Specific
0.1-1.0 0.9995 1.12-1.32 1.45-1.89 1.17 0.07 20 70 Specific
Label claim (mg/tablet) Atorvastatin calcium 10 Ezetimibe 10
Amount found*
% of drug found*
% RSD
10.04 9.84
100.40 98.40
1.96 0.767
*Each value is mean of six determinations
Recovery studies of drugs were carried out for accuracy parameters. These studies were carried out at three levels, i.e., multiple level recovery studies. Sample stock solution from tablet formulation of 1 mg/ml and 100 µg/ml of atorvastatin and ezetimibe respectively was prepared. To the above prepared solution, 50%, 100%, 150% of the standard atorvastatin solution and 20%, 40% and 60% of the standard ezetimibe solution were added. Dilutions were made and recovery studies were performed. Percentage recovery was found to be within limits, as listed in Table 2. For the detection of the related impurities, atorvastatin calcium and ezetimibe (0.1 g each) were dissolved separately in 10 ml of methanol, and these solutions were termed as sample solutions (10 mg/ml). One millilitre of each sample solution was diluted to 10 ml with methanol, and these solutions were termed as standard solutions (1000 µg/ml). Aliquots of both the standard solutions (2 µl) and sample solutions (20 µl) were spotted on the plate and chromatography performed as described earlier. The spot other than the principal spot and the spot of the starting point from the sample solution were not intense than the spot from the standard solution. The sample solution of atorvastatin calcium showed three unknown additional spots at Rf of 0.06, 0.41 and 0.47. The sample solution of ezetimibe showed three unknown additional spots at Rf of 0.37, 0.70 and 0.76. However, the areas of these spots were found to be less than 0.04% as compared to the areas of standard solution spots.
m rf o The intra-day and inter-day precision (RSD) values were d ns a determined for standard atorvastatin calcium (0.8-4.0 µg/ lo tio spot) and ezetimibe (0.1-1.0 µg/spot) six times on the same n day and over a period of 1 w. The intra-day and intera w c i day coefficients of variation are given in Table 1. do ubl e P ). Repeatability of sample application was assessed by e spotting 2 µl of atorvastatin calcium and 5 µl of ezetimibe fr w om solution six times on a TLC plate, followed by r o o n .c development of plate and recording the peak area for f6 k spots. The % RSD for peak area values of atorvastatin le1.17 ed ow b calcium and ezetimibe was found to be 1.09 and la M dkn respectively. i a by e v Repeatability of measurement ofapeak area was d and 5.m s calcium determined by spotting 2 µl of atorvastatin e i t w µl of ezetimibe solution on a TLC plate and developing s F w o fivew times the plate. The separated spot was scanned hplate ( % RSD without changing the position and PDoftthe e s area for measurement of ipeak atorvastatin and siandof 0.072 ezetimibe was foundh to be 0.143 T ofa the proposed respectively. To confirm the specificity method, the The assay value for the marketed formulation was found A summary of validation parameters of atorvastatin calcium and ezetimibe
solution of the formulation was spotted on the TLC plate, developed and scanned. It was observed that the excipients present in the formulation did not interfere with the peaks of atorvastatin calcium and ezetimibe.
to be within the limits, as listed in Table 3. The low RSD value indicated the suitability of the method for routine analysis of atorvastatin and ezetimibe in pharmaceutical dosage forms.
TABLE 2: RECOVERY OF ATORVASTATIN CALCIUM AND EZETIMIBE Label claim mg/tablet
Amount added (%)
Total amount added (mg)
50 100 150 20 40 60
15 20 25 12 14 16
Atorvastatin calcium 10
Ezetimibe 10
Amount recovered* (mg) ± SD 15.36 19.60 25.68 11.98 14.37 16.20
± ± ± ± ± ±
0.20 0.33 0.29 0.12 0.22 0.16
% recovery ± SD
% RSD
102.4 ± 1.36 98.00 ± 1.63 102.7 ± 1.16 99.87 ± 1.02 102.65 ± 1.59 101.23 ± 0.72
1.36 1.63 1.16 1.02 1.59 0.72
Recovery study of atorvastatin calcium and ezetimibe. *indicates that each value is a mean ± standard deviation of three determinations
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The developed HPTLC technique is simple, precise, specific and accurate, and the statistical analysis proved that method is reproducible and selective for the analysis of atorvastatin and ezetimibe simultaneously in bulk drug and tablet formulations.
6. 7. 8. 9.
ACKNOWLEDGEMENTS 10.
Authors sincerely thank the Torrent Research Centre, Ahmedabad, for providing gift samples of atorvastatin and ezetimibe.
11.
12.
Inc., Whitehouse Station, NJ. 2001, 148, 693. Baumann, K. L., Butter, D. E., and Deering, C. F., Tetrahedron Lett., 1992, 33, 2283. Kearney, A. S., Crawford, L. F., Mehta, S. C., and Radebaugh, G. W., Pharm. Res., 1993, 10, 1461. Rosenblum, S. B., Hvynh, T., Afonso, A., and Yumibe, N., J. Med. Chem., 1998, 41, 973. Wu, G., Wong, Y., Chen, X., and Ding, Z., J. Org. Chem., 1999, 64, 3714. Erturk, S., Sevinc, Aktas, E., Ersoy, L., and Ficicioglu, S., J. Pharm. Biomed. Anal., 2003, 33, 1017. United States Pharmacopoeia XXIV, National Formulary XIX, Assian Edn., US Pharmacopoeial Convention, Inc; Rockville, MD 2000, 2149. Shethi, P. D., Eds., In; HPTLC Quantitative Analysis for Pharmaceutical Formulations, 1st Edn., CBS Publishers & Distributors, New Delhi, 1996, 3. ICH Guideline Q 2B, Validation of Analytical Procedures, Methodology, 1996, 1.
m rf o d ns a lo tio n a w c i do ubl e P ). e fr w m r fo kno .co w le ofedAtorvastatin o b RP -HPLC Determination Calcium and RP-HPLC li a M dkn Amlodipine Besylate in T ablets a Combination Tablets y e v b a si ted w.m s w F o PD te h (w is si h A simple, specific, T accurate a and precise reverse-phase high-performance liquid chromatographic method was developed for the simultaneous determination of atorvastatin calcium and amlodipine besylate in tablet dosage REFERENCES
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1. 2. 3. 4. 5.
Cilla, D. D., Gibson, B. S., and Posvar, M. D., Clin. Pharmacol. Ther., 1996, 60, 687.
Bakker-Arkema, R. G., Best, J., Fayyad, R., and Marais, A. D.,
Atherosclerosis, 1997, 131, 17.
VanHeek, M., France, C. F., and Compton, D. S., J. Pharmacol. Exp. Ther., 1997, 283, 157.
VanHeek, M., Farley, C., Compton, D. S., Hoos, L., and Alton, K. B.,
Brit. J. Pharmacol., 2000, 129, 1748. Budavari, S., Eds., In; The Merck Index, 13th Edn; Merck and Co.
Accepted 19 December 2006
Revised 2 March 2006 Received 25 June 2005 Indian J. Pharm. Sci., 2006, 68 (6):793-796
D. A. SHAH* 1 , K. K. BHATT, M. B. SHANKAR, R. S. MEHTA, T. R. GANDHI 1 AND S. L. BALDANIA 1
1 Anand Pharmacy College, Opp. Town Hall, Anand-388 001, A. R. College of Pharmacy, P. Box No. 19, Vallabh Vidyanagar-388 120, India.
forms. A phenomenex Luna C-18, 5 µm column having 250 × 4.6 mm i.d. in isocratic mode, with mobile phase containing methanol: acetonitrile: 50 mM KH2PO4 (20:50:30; pH 3.5) was used. The flow rate was 1.0 ml/min and effluent was monitored at 240 nm. The retention time of atorvastatin calcium and amlodipine besylate was 7.6 min and 3.2 min respectively. The linearity for atorvastatin calcium and amlodipine besylate was in the range of 5120 µg/ml and 5-100 µg/ml respectively. The proposed method is accurate, precise, specific and rapid for simultaneous estimation of atorvastatin calcium and amlodipine besylate in tablets.
Atorvastatin calcium (ATV) is chemically [R-(R*,R*)]-2-(4flurophenyl)-β,δ-dihydroxy-5-(1-methylethyl)-3-phenyl-4[(phenylamino)carbonyl]-1H-pyrrole-1-heptanoic acid, calcium salt trihydrate1-2. Extractive spectrophotometry3, *For correspondence E-mail:
[email protected] 796
HPLC4-8, GC-MS9, LC-MS10, HPLC-electrospray tandem mass spectrometry11–13 and HPTLC14 methods have been reported for the estimation of atorvastatin calcium. While for estimation of atorvastatin calcium and aspirin combination, RP-HPLC15 method has been reported. Amlodipine besylate (AML) is 3-ethyl-5-methyl (±)-2-[(2aminoethoxy)methyl]-4-(o-chlorophenyl)-1,4-dihydro-6-
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