Download RP-HPLC METHOD DEVELOPMENT AND

Subhashini Edla et al., IJSID, 2011, 1 (3), 433-440
ISSN:2249-5347
IJSID
International Journal of Science Innovations and Discoveries
Research Article
An International peer
Review Journal for Science
Available online through www.ijsidonline.info
RP-HPLC METHOD DEVELOPMENT AND VALIDATION FOR THE ANALYISIS OF CARVEDILOL IN
PHARMACEUTICAL DOSAGE FORMS
Subhashini.Edla*, B.Syama Sundhar
Dept of Chemistry, Acharya Nagarjuna University, Nagarjuna Nagar. Guntur, AP, India.
Received: 19.09.2011
Modified: 20.10.2011
Published: 29.12.2011
*Corresponding Author
ABSTRACT
A simple, selective, linear, precise and accurate RP-HPLC method
was developed and validated for rapid assay of carvedilol in tablet
dosage form. Isocratic elution at a flow rate of 1ml min-1 was employed
on a symmetry C18 column at ambient temperature. The mobile phase
consisted of Methanol: water: OPA 75:20:05 (v/v/v). The UV detection
wavelength was at 243nm.Linearity was observed in concentration
Name:
Subhashini Edla
Place:
Guntur, AP, India
E-mail:
[email protected]
range of 1-0.2mg/ml. The retention time for Carvedilol was 3.0 min. The
method was validated as per the ICH guidelines. The proposed method
can be successfully applied for the estimation of Carvedilol in
INTRODUCTION
pharmaceutical dosage forms.
Key words: Carvedilol, HPLC, Linearity, Precision, 243nm.
INTRODUCTION
International Journal of Science Innovations and Discoveries, Volume 1, Issue 3, November-December 2011
433
Subhashini Edla et al., IJSID, 2011, 1 (3), 433-440
INTRODUCTION
Carvedilol is a non-selective beta blocker/alpha-1 blocker indicated in the treatment of mild to moderate
congestive heart failure (CHF). It is marketed under various trade names including Carvil (Zydus Cadila), Coreg
(GSK), Dilatrend (Roche), Eucardic (Roche), and Carloc (Cipla) as a generic drug (as of September 5, 2007 in the
U.S.).,[1] and as a controlled-release formulation, marketed in the US as Coreg CR (GSK). Carvedilol was discovered
by Robert R. Ruffolo, Jr. It has had a significant role in the treatment of Congestive heart failure
Figure 1 : Stricture of Carvedilol
Carvedilol is both a beta blocker (β1, β2) and alpha blocker (α1) , Norepinephrine stimulates the nerves that
control the muscles of the heart by binding to the β1- and β2-adrenergic receptors. Carvedilol blocks the binding to
those receptors,[2] which both slows the heart rhythm and reduces the force of the heart's pumping. This lowers
blood pressure thus reducing the workload of the heart, which is particularly beneficial in heart failure patients.
Norepinephrine also binds to the α1-adrenergic receptors on blood vessels, causing them to constrict and raise
blood pressure. Carvedilol blocks this binding to the α1-adrenergic receptors too,[3] which also lowers blood
pressure.
Relative to other beta blockers, carvedilol has minimal inverse agonist activity.[4] This suggests that
carvedilol has a reduced negative chronotropic and inotropic effect compared to other beta blockers, which may
decrease its potential to worsen symptoms of heart failure. However, to date this theoretical benefit has not been
established in clinical trials, and the current version of the ACC/AHA guidelines on congestive heart failure
management does not give preference to carvedilol over other beta-blockers.The most common side effects include
dizziness, fatigue, hypotension, diarrhea, asthenia, bradycardia, and weight gain.[5]A case report of a patient with
panic disorder associated sleep disturbances and nightmares with the improper usage of carvedilol.[6]Carvedilol
has enantiomers with distinct pharmacodynamics.[7]The term "racemic carvedilol" is sometimes used to explicitly
denote that both enantiomers are applied.[8]
Carvedilol is indicated in the management of congestive heart failure (CHF), as an adjunct to conventional
treatments (ACE inhibitors and diuretics). The use of carvedilol has been shown to provide additional morbidity
and mortality benefits in CHF.[9] Carvedilol (Carvil) is available at the following doses 3.125 mg (smallest), followed
by 6.25 mg,12.5 mg, and 25 mg white tablets.On January 10, 2006 carvedilol supply became limited in the United
States, due to changes in documentation procedures at a plant. This was lifted on April 27, 2006 in a Dear
International Journal of Science Innovations and Discoveries, Volume 1, Issue 3, November-December 2011
434
Subhashini Edla et al., IJSID, 2011, 1 (3), 433-440
Pharmacist letter.[10]On October 20, 2006, the FDA approved a controlled release formulation of carvedilol; it is
marketed as Coreg CR.
EXPERIMENTAL
Chemicals and reagents
All HPLC solvents used like Acetonitrile, Methanol and Orthophosphoric Acid which are of HPLC grade
were purchased from E.Merck. Samples are purchased from local market. Pure form of CARVEDILOL from Bulk
drug industry JAIN PHARMA in Gulbarga
Instrumentation and analytical conditions
The analysis of the drug was carried out on Shimadzu HPLC model (VP series) containing LC-10AT (VP
series) pump, variable wave length programmable UV/visible detector SPD-10AVP and rheodyne injector (7725i)
with 20µl fixed loop. Chromatographic analysis was performed using Inertsil ODS C-18 column with 250 x 4.6mm
internal diameter and 5µm particle size. Shimadzu electronic balance (AX-200) was used for weighing. Isocratic
elution with Methanol, Acetonitrile, OPA 75:20:05 (v/v/v) was selected with a flow rate of 1.0 ml min-1.The
detection wavelength was set at 243nm with a runtime of 10 min. The mobile phase was prepared freshly and it
was degassed by sonicating for 5 min before use. The column was equilibrated for at least 30min with the mobile
phase flowing through the system. The column and the HPLC system were kept at ambient temperature.
Preparation of Stock, working standard solutions and Sample solutions
100mg of Carvedilol was weighed and transferred (working standard) into a 100ml volumetric flask. The
diluent methanol was added and sonicated to dissolve it completely and made up to the mark with the same
solvent. Further 1ml of the above stock solution was pipetted into a 10ml volumetric flask and diluted up to the
mark with diluent. The contents were mixed well and filtered through Ultipor N66 Nylon 66 membrane sample
filter paper. The calibration curve was plotted with the concentrations of the 1 to 0.2 mg/ml working standard
solutions. Calibration solutions were prepared and analyzed immediately after preparation.
The formulation tablets of Carvedilol were crushed to give finely powdered material. Powder equivalent to
25 mg of drug was taken in 10 ml of volumetric flask containing 5 ml of mobile phase and was shaken to dissolve
the drug and then filtered through Ultipor N66 Nylon 6,6 membrane sample filter paper. Volume of the filtrate was
adjusted to the mark with the same solvent to obtain concentration of 1mg/ml.
RESULT AND DISCUSSION
Optimization of the chromatographic conditions
The nature of the sample, its molecular weight and solubility decides the proper selection of the stationary
phase. The drug Carvedilol being non-polar is preferably analyzed by reverse phase columns and accordingly C18
column was selected. So the elution of the compound from the column was influenced by polar mobile phase. The
concentration of the methanol and Acetonitrile were optimized to give symmetric peak with short run time based
on asymmetric factor and peak area obtained. Different mobile phases were tried but satisfactory separation, well
International Journal of Science Innovations and Discoveries, Volume 1, Issue 3, November-December 2011
435
Subhashini Edla et al., IJSID, 2011, 1 (3), 433-440
resolved and good symmetrical peaks were obtained with the mobile phase Methanol,WATER,OPA
75:20:05
(V/V/V). The retention time of Carvedilol was found to be 3.0min, which indicates a good base line. The RSD values
for accuracy and precision studies obtained were less than 2% which revealed that developed method was
accurate and precise. The system suitability and validation parameters are given in Table 4. The high percentage of
recovery of Carvedilol was found to be 99.65 indicating that the proposed method is highly accurate. Proposed
liquid chromatographic method was applied for the determination of Carvedilol in tablet formulation. The result
for Carvedilol was comparable with a corresponding labelled amount (Table 6). The absence of additional peaks
indicates no interference of the excipients used in the tablets.
Figure 2: Typical chromatogram of Carvedilol Formulation
Method Validation procedure
The objective of the method validation is to demonstrate that the method is suitable for its intended
purpose as it is stated in ICH guidelines. The method was validated for linearity, precision, accuracy, specificity,
and limit of detection, limit of quantification, robustness and system suitability.
Linearity
The developed method has been validated as per ICH guidelines (Zucman D, 2007). Working standard
solutions of Carvedilol in the mass concentration range of 1mg/ml to 0.2 mg/ml was injected into the
chromatographic system. The chromatograms were developed and the peak area was determined for each
concentration of the drug solution. Calibration curve of Carvedilol was obtained by plotting the peak area ratio
versus the applied concentrations of Carvedilol. The linear correlation coefficient was found to be 0.999
International Journal of Science Innovations and Discoveries, Volume 1, Issue 3, November-December 2011
436
Subhashini Edla et al., IJSID, 2011, 1 (3), 433-440
Table 1: Linearity of Carvedilol
S.NO
CONC mg/ml
AREA
1
1.0
199182
2
0.8
163151
3
0.6
119227
4
0.4
81120
5
0.2
41003
Co-relation coefficient
0.9996
Figure 3: Calibration curve of Carvedilol
Table.2: Linear Regression Data for Calibration curve
Drug
Concentration range
Slope (m)
Intercept (b)
Correlation coefficient
% RSD
Carvedilol
1-0.2mg/ml
199194.5
1219.9
0.999
0.57
Precision
Repeatability of the method was checked by injecting replicate injections of 20 ppm of the solution for six
times on the same day as intraday precision study of Carvedilol and the RSD was found to be 0.43
Table 3: Precision parameters of Carvedilol
Injection
Concentration
Peak area
1
1mg/ml
199182
2
1mg/ml
199269
3
1mg/ml
199541
4
1mg/ml
199683
5
1mg/ml
199714
6
1mg/ml
199516
%RSD
0.108
Accuracy
The accuracy of the method was determined by calculating recovery of Carvedilol (0.2, 0.4, 0.6 mg/ml) by
the method of standard addition. Known amount of Carvedilol was added to a pre quantified sample solution and
International Journal of Science Innovations and Discoveries, Volume 1, Issue 3, November-December 2011
437
Subhashini Edla et al., IJSID, 2011, 1 (3), 433-440
the amount of Carvedilol was estimated by measuring the peak area ratios and by fitting these values to the
straight line equation of calibration curve. The recovery studies were carried out three times over the specified
concentration range and amount of Carvedilol was estimated by measuring the peak area ratios by fitting these
values to the straight line equation of calibration curve. From the above determination, percentage recovery and
standard deviation of percentage recovery were calculated.
Recovey
50%
100%
150 %
Table 4: Accuracy results of Carvedilol
Conc. of sample mg/ml
Recovery
0.5
0.498
1.0
0.996
1.5
1.504
% of recovery
99.6
99.6
100.2
Figure 4: Typical chromatogram of Carvedilol
Specificity
The specificity of the method was determined by comparing test results obtained from analysis of sample
solution containing excipients with that of test results those obtained from standard drug.
LOD and LOQ
Limit of detection (LOD) and limit of quantification (LOQ) were calculated as 50microgram/ml and
10microliter respectively as per ICH guide-lines.
Robustness
To determine the robustness of the method, two parameters from the optimized chromatographic
conditions were varied.
International Journal of Science Innovations and Discoveries, Volume 1, Issue 3, November-December 2011
438
Subhashini Edla et al., IJSID, 2011, 1 (3), 433-440
Ruggedness
Inter day variations were performed by using six replicate injections of standard and sample solutions of
concentrations which were prepared and analyzed by different analyst on three different days over a period of one
week. Ruggedness also expressed in terms of percentage relative standard deviation.
Table 5 : Ruggedness Results.
Parameter
Modification
Peak Area
M.phase
Methanlo: Water: 0.1% OPA 70:25:5
200179
pH
5.9
198756
Wavelength
248
202235
System Suitability Parameter:
% of change
0.5
0.214
1.5
System suitability tests were carried out on freshly prepared standard stock solutions of Carvedilol and it
was calculated by determining the standard deviation of Carvedilol standards by injecting standards in six
replicates at 6 minutes interval and the values were recorded.
Table 5: System suitability parameters of Carvedilol
Parameters
Values
λ max (nm)
243
Beer’s law limit (μg/ml)
1-0.2mg/ml
Correlation coefficient
0.999
Retention time
3.0 min
Theoretical plates
5538
Tailing factor
1.01
Limit of detection
50 microgram/ml
Limit of quantification
10microliter
Table 6: Tablet estimation of Carvedilol
Formulation
Carca Capsule
Tablet dosage
25mg
Sample concentration
0.6 mg/ml
CONCLUSION
Amount of drug estimated
99.04
A validated RP-HPLC method has been developed for the determination of Carvedilol in tablet dosage form.
The proposed method is simple, rapid, accurate, precise and specific. Its chromatographic run time of 6 min allows
the analysis of a large number of samples in short period of time. Therefore, it is suitable for the routine analysis of
Carvedilol in pharmaceutical dosage form.
REFERENCES
1. Press Release, FDA Approves First Generic Versions of Coreg, U.S. Food and Drug Administration, Sep. 5, 2007
2.
Stafylas PC, Sarafidis PA (2008). "Carvedilol in hypertension treatment". Vasc Health Risk Manag 4 (1): 23–30.
doi:10.2147/vhrm.2008.04.01.23. PMC 2464772. PMID 18629377.
3.
Othman AA, Tenero DM, Boyle DA, Eddington ND, Fossler MJ (2007). "Population pharmacokinetics of S(-)carvedilol in healthy volunteers after administration of the immediate-release (IR) and the new controlledrelease (CR) dosage forms of the racemate". AAPS J 9 (2): E208–18. doi:10.1208/aapsj0902023. PMC 2751410.
PMID 17614362.
International Journal of Science Innovations and Discoveries, Volume 1, Issue 3, November-December 2011
439
Subhashini Edla et al., IJSID, 2011, 1 (3), 433-440
4.
Vanderhoff BT, Ruppel HM, Amsterdam PB. Carvedilol: The new role of beta blockers in congestive heart
failure. Am Fam Physician 1998;58(7):1627-34. PMID 9824960
5.
Carvedilol Official FDA information, side effects and uses. Drugs.com, October 11, 2009.
6.
Maebara C, Ohtani H, Sugahara H, Mine K, Kubo C, Sawada Y (November 2002). "Nightmares and panic
disorder associated with carvedilol overdose". Ann Pharmacother 36 (11): 1736–40.
7.
Horiuchi I, Nozawa T, Fujii N, et al. (May 2008). "Pharmacokinetics of R- and S-carvedilol in routinely treated
Japanese patients with heart failure". Biol. Pharm. Bull. 31 (5): 976–80.
8.
Takekuma Y, Takenaka T, Yamazaki K, Ueno K, Sugawara M (November 2007). "Stereoselective metabolism of
racemic carvedilol by UGT1A1 and UGT2B7, and effects of mutation of these enzymes on glucuronidation
activity". Biol. Pharm. Bull. 30 (11): 2146–53.
9.
Packer M, Fowler MB, Roecker EB, et al. (October 2002). "Effect of carvedilol on the morbidity of patients with
severe chronic heart failure: results of the carvedilol prospective randomized cumulative survival
(COPERNICUS) study". Circulation 106 (17): 2194–9.
10. http://www.fda.gov/cder/drug/shortages/Coreg_Pharmacist_Letter_27Apr06.pdf PDF at FDA.govdead link
International Journal of Science Innovations and Discoveries, Volume 1, Issue 3, November-December 2011
440