Development and validation of analytical methods for estimation of Levosimendan in injectable dosage form
Patel A. K. 1*, Patel P. U. 2, Prajapati A.M.2, Patel M.M. 1
1Shankersinh Vaghela Bapu Institute of Pharmacy, Near Vasaniya Mahadev, Gandhinagar-Mansa Road, Vasan,
Dist. Gandhinagar- 382650 Gujarat
2Shree S. K. Patel College of Pharmaceutical Education and Research, Ganapat University, Kherva-382711, Gujarat, India
*Corresponding Author E-mail: akta_patel11@yahoo.com
ABSTRACT:
A simple, rapid, precise and accurate gradient reversed phase stability indicating HPLC method was developed and validated for the determination of levosimendan in injectable dosage form. The chromatographic separation was achieved on phenomenex Gemini C18 (250×4.6 mm, 5 μm) column using a mobile phase consisting of Acetonitrile, water and 0.1% TFA in the ratio of 40:10:50 v/v/v having pH 3.0 at a flow rate of 1.0 ml/min and UV detection at 375 nm. The linearity of the proposed method was investigated in the range of 0.1-15 µg/ml (r 2 =0.9967) for levosimendan. Degradation products produced as a result of stress studies did not interfere with the detection of levosimendan and the assay can thus be considered stability-indicating.
KEYWORDS: Levosimendan, RP-HPLC.
1.0 INTRODUCTION:
Levosimendan (Fig.-1) is a calcium sensitiser a novel class of positive inotropic agents that may possess potential advantages for the treatment of acute decompensated heart failure, compared with conventional cardiotonic drugs. It reduce peripheral vascular resistance, and enhance the contractility of the failing heart, without significantly increasing myocardial oxygen uptake it increases the sensitivity of the heart to calcium, The cardiovascular effects of calcium sensitizers are mediated mainly by two mechanisms of action: (i) calcium sensitization via binding to the Ca2+-saturated troponin C (cTnC) in cardiomyocytes; and (ii) opening of the ATP-sensitive potassium channels on the sarcolemma and mitochondria.
Figure 1: Chemical structures of levosimendan.
A survey of literature revealed that number of analytical methods has been reported for the estimation of Levosimendan. Levosimendan is not official in IP, BP and USP. There are no any official methods available for estimation of Levosimendan.
Levosimendan is one of the new calcium sensitizer drug available in the market. This drug is not official in any pharmacopoeia; hence no official method is available for the estimation of this drug in pharmaceutical dosage forms. So it was thought of interest to develop and validate the chromatographic methods for the estimation of this drug.
2.0 MATERIALS AND METHODS4,5:
2.1 Apparatus
A gradient high performance liquid chromatography from younglin HPLC system, equipped with a UV detector and Autochro-3000 software was used. A reversed phase Varian C18 (250 Χ 4.6 mm i.d, 5 μm particle size) analytical column was used for the present analysis. Shimadzu electronic balance, ultrasonic cleaner (225 Χ 125 Χ 60 mm s.s-304, 1.5 Liter capacity of tank) and pH meter LI127 (Elico Limited) were used during the study.
2.2 Reagents and materials
Pharmaceutical grade of levosimendan (N-{4-[(4R)-4-methyl-6-oxo-1,4,5,6-tetrahydropyridazin-3-yl]phenyl} cyanomethanecarbohydrazonoyl cyanide was kindly supplied as a gift sample from Torrent Research Centre, Gujarat (India). All chemicals and solvents were of analytical reagent grades. Acetonitrile was obtained from Finar Chemicals Ltd., Mumbai, India. The water for RP-HPLC was prepared by triple glass distillation and filtered through a nylon 0.45 µm – 47 mm membrane filter. All solvents and solutions were filtered through a membrane filter (ultipor N66 Nylon 6,6, 0.2μm pore size) and degassed using ultrasonic cleaner before use.
Optimization of wavelength maxima:
The standard solution of levosimendan was scanned over the range of 200 nm to 400 nm wavelength. Detection was carried out at different wavelength. Best response was achieved at 375 nm with PDA detector. So this 375 nm was selected as a detection wavelength for determination of levosimendan in its injectable dosage form (Fig.-2).
Figure 2: Absorbance spectra of standard solution of levosimendan at 375 nm
2.3 Optimized chromatographic conditions
The samples were chromatographed on a reversed phase C18 (250 Χ 4.6 mm i.d, 5 μm particle size) column with a flow rate of 1.0 ml/min. All analyses were carried out at isocratic conditions. The mobile phase consisted of a mixture of Acetonitrile: Water: 0.1 % TFA Buffer having pH 3 (40:10:50, v/v/v) The mobile phase was filtered through a Nylon 0.2µm membrane filter and degassed before use. The volume of injection was 20µl and the detection was made at 375nm.
2.4 Preparation of solutions
Trifluroacetic acid Buffer
A solution of 0.1% tri fluoro acetic acid having pH 3 was prepared by accurately taken 1 ml of tri fluoro acetic acid and was transferred to a 1000 mL volumetric flask, diluted up to mark with water.
Mobile phase preparation
HPLC grade solvents were used in separate bottles of gradient pump as mobile phase. Acetonitrile, water and 0.1% TFA in the ratio of 40:10:50 v/v/v were adjusted by gradient pump operated by LC solution software. Mixed solvents were degassed by the instrument and use as a mobile phase.
Standard preparation
Stock solution of levosimendan was prepared in methanol at concentrations of 100 µg/ml. The solution was stored at room temperature (22±1°) until analysis. Series of standards were prepared by progressive dilution of the stock solution for calibration study.
Sample preparation
Marketed injection having a dose 2.5 mg/ml was taken and from that 5 ml was transferred to a 50 ml volumetric flask. The solution was diluted to 50 ml with methanol. The resulting solution (4 ml) was diluted to 100 ml with methanol (10µg/ml) in 100 ml volumetric flask.
Preparation of 1 N HCl
A solution of 1 N HCL was prepared by accurately taken 8.6 ml of HCL in 100 ml volumetric flask and adjusted volume up to the mark with methanol.
Preparation of 0.5 N NaOH
A solution of 0.5 N NaOH was prepared by accurately weighed 2.0 gm of NaOH in 100 ml volumetric flask and adjusted volume up to the mark with methanol.
Preparation of 30% H2O2
A solution of 30% H2O2 was directly used.
Forced degradation study
In order to establish whether the analytical method for the assay was stability-indicating, pure active pharmaceutical ingredient (API) of levosimendan was subjected to various stress condition to conduct force degradation studies. Stress studies were carried out under the conditions of acid, base hydrolysis and oxidation in ICH Q1A (R2). Photo and thermal degradation of drug substances and drug product was performed in the solid state. All solutions for force degradation studies were prapared to yield starting condition 100 µg/ml of levosimendan.
In Acid degradation drug was heated under reflux condition with 1N HCL in methanol at 70˚C for 2 h to facilitate acid degradation of levosimendan.
In Alkali degradation drug was heated under reflux condition with 0.5 N NaOH in methanol at 70˚C for 4 h to facilitate base degradation of levosimendan
In Oxidative degradation drug was heated under reflux condition with 30% H2O2 in methanol at 70˚C for 2 h to facilitate oxidative degradation of levosimendan
Photo degradation was performed by exposing solid drug to sun light at 1000 C for 48 h and
Thermal degradation was performed by exposing solid drug to dry heat in convection oven at 1000 C for 48 h.
2.5 Method validation
2.5.1 Specificity
The specificity of the method was evaluated by assessing interference from blank without addition of drug. The specificity of the method for the drug was also established by checking for interference with drug quantification from degradation products formed during the forced degradation study.
2.5.2 Linearity
Test samples for assessment of linearity of method were prepared at seven concentrations from 0.1-15 µg/ml (i.e, 0.1, 0.5, 1.0, 3.0, 5.0, 10.0, 15.0 µg/ml). Peak area and concentration data were evaluated by regression analysis from regression equation.
2.5.3 Accuracy
The accuracy of the method was determined by calculating recovery of levosimendan by the standard addition method. Accuracy was studied by adding three different amounts (corresponding to 50, 100 and 150% of the test preparation concentrations) of levosimendan to the prequantified separate sample solutions of levosimendan 5 µg/ml and comparing the actual and measured concentrations. For each level, three solutions were prepared and each was injected in duplicate.
2.5.4 Precision
The precision of the method, as repeatability was evaluated by repeatedly injecting (n = 6) standard solutions of levosimendan (3 μg/ml) under the same chromatographic condition. Intermediate precision (Reproducibility), as intraday and interday precision of the proposed method was determined by analyzing the corresponding responses 3 times on the same day and on 3 different days over a period of 1 week for 3 different concentrations of standard solutions of levosimendan (1, 5, and 10 μg/ml).
2.5.5 Limit of detection and quantification
LOD and the LOQ of the drug were calculated using the following equations as per International Conference on Harmonization (ICH) guidelines.
LOD = 3.3 × δ /S
LOQ = 10 × δ/S
Where δ = the standard deviation of the response
S = Slope of calibration curve
2.5.6 Robustness
The robustness was studied by evaluating the effect small but deliberate variations in the chromatographic conditions like change in the mobile phase composition, wavelength and flow rate. For the same, mobile phases having different compositions, like acetonitrile : water : buffer (39.5: 10.5: 50 , v/v/v), (39.5: 10: 50.5 , v/v/v), (40: 10.5: 50.5 , v/v/v), (40.5: 9.5: 50 , v/v/v), were tried and chromatograms were run. The changes made in wavelength and flow rate were ± 1 nm and ± 0.02 ml/min, respectively. Robustness of the method was evaluated by calculating the % RSD values.
2.5.7 Solution Stability
Solution stability is the stability of sample and standard in solution form for specified period of time. Standard and sample preparations were prepared. The standard and sample preparations were analyzed by HPLC system at regular intervals for 48 hr. The areas of the analyte peak for both standard and sample solutions were monitored. The % difference of analyte peak area from initial for both standard and sample solutions were calculated and recorded.
2.5.8 System suitability
|
System suitability was performed and calculated at the start of study of each validation parameter. Standard solution of the levosimendan 100 µg/ml was injected and system suitability parameters like retention time, peak asymmetry, theoretical paltes, resolution were measured from the chromatogram.
3.0 ESTIMATION OF LEVOSIMENDAN IN PHARMACEUTICAL FORMULATION:
The response of the sample solution was measured at 375 nm under the chromatographic condition mentioned earlier for the quantitation of levosimendan. The amount of levosimendan present in sample solution was determined by applying values of the peak area to the regression equations of the calibration graph.
4.0 RESULT AND DISCUSSION:
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In this work HPLC method with PDA detection for analysis of Levosimendan in a injectable dosage form was developed and validated. The analytical conditions were selected after testing the different condition effecting HPLC analysis, for example buffer composition, buffer concentration, organic solvent in the mobile phase, buffer to organic solvent ratio and other chromatographic conditions. Preliminary trials with mobile phases comprising mixtures of water with methanol or Acetonitrile did not give good peak shape. The best peak shape was obtained by use of Acetonitrile, water and 0.1% TFA in the ratio of 40:10:50 v/v/v having pH 3.0 at a flow rate of 1.0 ml/min and UV detection at 375 nm which gave sharp, well-resolved peak with minimum tailing factor (Fig.-3).
|
Figure 3: Chromatogram of standard solution of levosimendan at 375 nm
The specificity of the method (Fig.-4) was determined by checking for interference with the analytes from blank and forced degradation products by measuring peak purity of the levosimendan. Peak purity was satisfactory under different condition as shown in table 1. There was no interference of any degradation products peak with the main drug peak. Maximum degradation of levosimendan was achieved in alkaline condition. Acid, alkaline, oxidative. thermal and photo degradation of levosimendan were shown in table-2 and figure 5-9, respectively. There were no any degradation product found during thermal and photo degradation studies.
Table 1: Data indicating peak purity index of levosimendan
Sr no. |
Preparation |
Peak purity index |
1 |
Standard preparation |
0.999 |
2 |
Sample preparation |
0.999 |
(a)
9.101 min
(b)
|
(c)
Figure 4: Chromatograms of (a) diluent, (b) standard and (c) sample preparation obtained using optimized protocol indicating specificity of proposed method
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Figure 5: Chromatogram of acid degraded levosimendan in standard solution
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Figure 6: Chromatogram of base degraded levosimendan in standard solution
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||||
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Figure 7: Chromatogram of oxidative degraded levosimendan in standard solution
Table 2: Results of force degradation studies of API and sample solution of levosimendan indicating specificity of the developed method
Sr No. |
Stress condition/ duration/ state |
Retention time of API and impurity formed
|
% Degradation of API |
% Degradation of sample solution |
|
1 |
Acidic/1N HCl/70°C/4hr/ solution |
Impurity A |
7.00 ±0.20 |
------- |
---- |
Levosimendan |
9.15 ± 0.10 |
25.66 |
23.87 |
||
2 |
Alkaline/ 0.5 N NaOH / 70°C /2 hr/solution |
Impurity B |
5.05 ± 0.30 |
---- |
----- |
Levosimendan |
8.10 ± 0.25 |
33.81 |
32.96 |
||
3 |
Oxidative/30% H2O2 / 70°C / 2 hr/solution |
Impurity A |
7.13 ± 0.23 |
----- |
---- |
Levosimendan |
9.18 ± 0.17 |
17.47 |
15.56 |
||
4 |
Thermal/100°C/ 48 hr/solid |
--- |
--- |
------- |
------- |
Levosimendan |
9.12 ± 0.33 |
2.9 |
2.5 |
||
5 |
Sun light/48hr/ liquid |
--- |
--- |
------ |
------ |
Levosimendan |
9.13 ±0.15 |
2.1 |
1.9 |
Table 3: Recovery data for the levosimendan by stability indicating RP-HPLC method
Drug |
Level |
Amount of sample taken (µg/ml) |
Amount of standard spiked (%) |
Mean % Recovery ± SD* |
Levosimendan |
I |
5 |
50 % |
100.13 ± 1.02 |
II |
5 |
100 % |
100.26 ± 0.75 |
|
III |
5 |
150 % |
99.31 ± 1.10 |
* Mean % Recovery ± SD where n=3.
Figure 8: Chromatogram of thermal degraded levosimendan in standard solution
Figure 9: Chromatogram of photo degraded levosimendan in standard solution
Levosimendan showed linearity in the concentration range of 0.1-15 µg/ml. The calibration curves of levosimendan at 375 nm are shown in Figure 10.
Figure 10: Calibration curve of levosimendan at 375 nm
Accuracy was performed by the standard addition method. The recoveries obtained were 99.9 ± 0.52 % for levosimendan (Table 3).
The RSD values for levosimendan were found to 0.21 %, (Table 4). The RSD values were found to be < 2 %, which indicates that the proposed method is repeatable. The low RSD values of interday 0.35-0.87 % and intraday 0.27-0.75 % for levosimendan reveal that the proposed method is precise (Table 7).
Table 4: Precision data for levosimendan by stability indicating RP-HPLC method
Sr. No. |
Retention time |
Peak area |
Tailing factor |
1 |
9.140 |
224765 |
1.21 |
2 |
9.127 |
225650 |
1.193 |
3 |
9.135 |
225752 |
1.191 |
4 |
9.135 |
224769 |
1.179 |
5 |
9.145 |
225045 |
1.165 |
6 |
9.118 |
224756 |
1.169 |
Mean |
9.13 |
225122.8 |
1.18 |
SD |
0.009 |
462.0872 |
0.016 |
% CV |
0.10 |
0.21 |
1.42 |
In all deliberately varied conditions, the RSD of the peak was found to be less than 2 %. The low value of % RSD indicated robustness of the proposed method.
The results obtained from study of the stability of the test preparation. It was concluded that test preparation was stable upto 48 hr The difference in the initial value of % assay and the values obtained at 24 hours and 48 hours of % assay was not be more than 2.0%. (Table 5)
Table 5: Solution stability data for standard preparation
Time (hrs) |
Levosimendan |
||
Area |
% Assay |
% Difference |
|
Initial |
224765 |
100% |
- |
After 24 hrs |
223336 |
99.36 |
-0.64 |
After 48 hrs |
220929 |
98.29 |
-1.71 |
System suitability was (table-6) verified by measurement of peak asymmetry (A<2.0), resolution (Rs>2.0) and number of theoretical plates (N>2000) after chromatography of standard solution. The values of these properties were in accordance with in-house limits.
Table 6: Mean values of system suitability parameters (n=6)
Sr. No. |
Parameters |
Levosimendan |
1. |
Peak area ± % RSD |
225122.8 ± 0.21 |
2. |
No. of theoretical plates ± % RSD |
4408 ± 1.53 |
3. |
Retention time (min) ± % RSD |
9.13 ± 0.10 |
4. |
Tailing factor ± % RSD |
1.18 ± 1.42 |
Table: 7: Regression analysis data and summary of validation parameter for the stability indicating RP-HPLC method.
Parameters |
Levosimendan at 375 nm |
Concentration range (µg/ml) |
0.1 – 15 |
Slope |
56385 |
Intercept |
29198 |
Correlation coefficient |
0.9967 |
LOD (µg/ml) |
0.027 |
LOQ (µg/ml) |
0.081 |
Accuracy ± SD |
99.90±0.52 |
Repeatability (RSD, n = 6), % |
------ |
Precision (RSD), % |
0.21 |
Interday (n = 6), % Intraday (n = 6), % |
0.35-0.87 0.27-0.75 |
The proposed validated method was successfully applied to determine levosimendan in its injectable dosage form. The result obtained for levosimendan was comparable with the corresponding labeled amounts (Table 8). The RP-HPLC chromatogram for levosimendan in sample was recorded and is shown in Figure 11.
Figure 11: Chromatogram of sample solution of levosimendan (10 µg/ml)
Table 8: Analysis of marketed formulation of levosimendan by stability indicating RP-HPLC method (n = 6)
Sample No. |
Label claim (mg/ml) |
Amount found (mg/ml) |
% Label claim (mg/ml) |
1 |
2.5 |
2.49 |
99.6 |
2 |
2.5 |
2.48 |
99.2 |
3 |
2.5 |
2.50 |
100 |
4 |
2.5 |
2.51 |
100.4 |
5 |
2.5 |
2.50 |
100 |
6 |
2.5 |
2.49 |
99.6 |
Mean |
2.49 |
100.2 |
|
S.D. |
0.42 |
0.42 |
5.0 CONCLUSION:
In this proposed method the linearity is observed in the concentration range of 0.1-15 µg/ml with co-efficient of correlation, (r2) = 0.9967 for levosimendan at 375 nm. The result of the analysis of pharmaceutical formulation by the proposed method is highly reproducible and reliable and it is in good agreement with the label claim of the drug. The method can be used for the routine analysis of the levosimendan in injectable dosage form without interference from any degradation products.
6.0 REFERENCES:
1. Wikberg T, Korkolainen T, Karlsson M. Chirality. Enantiomeric bioanalysis of simendan and levosimendan by chiral HPLC. Chirality. 1996; 8(7): 511-517.
2. Li SR, Chen XY, Zhang YF, Li GX, Jiang CM, Zhong DF. Determination of levosimendan and its metabolites in human plasma with HPLC-MS/MS method. Acta Pharmaceutica Sinica. 2008 Oct; 43(10): 1053-9.
3. Niessen, W.M.A. “Liquid Chromatography-Mass Spectrometry” Marcell Decker Inc., New York, 2nd ed,. 1999.
4. ICH, Q2 (R1). “Validation of Analytical Procedures” Text and Methodology 2005.
5. ICH Q1A(R2). “Stability Testing of New Drug Substance and Product” 2003.
Received on 10.10.2012 Modified on 19.10.2012
Accepted on 24.10.2012 © RJPT All right reserved
Research J. Pharm. and Tech. 5(12): Dec. 2012; Page 1543-1548