A new analytical method development and validation for the estimation of Midodrine HCl by UV and HPLC

http://dx.doi.org/10.21276/IJRDPL.22780238.2018.7(4).3060-3070 ABSTRACT:Two simple, accurate, sensitive and precise reverse phase-high performance liquid chromatographic and U.V. assay method for estimation of Midodrine hydrochloride (MD) was developed in tablet formulation. The chromatographic separation was performed on Thermo C18, (250 mm X 4.6 mm, 5 μm) column. The mobile phase consists of water: acetonitrile (30:70 v/v) pH adjust 3.5 with TEA was delivered at a flow rate of 1.0 ml/min and UV detection at 272 nm. The retention time of the drug was found to be 3.89 min. The developed method was found to be linear in a concentration range of 5-25μg/ml of the drug (r2= 0.999). The low value of % RSD indicates reproducibility of the methods. The low value of LOD and LOQ suggests the sensitivity of the method. Thus, this method can be used for routine analysis of Midodrine hydrochloride formulation and to check the stability of bulk samples.


INTRODUCTION
Every year many new drugs and newer drug combinations enter the pharmaceutical area. The analytical methods for these new and first timer drugs are mostly confined only to the manufacturing company. However, availability of multiple analytical methods for the same drug/drug combinations in their formulations is always advantageous.
Moreover, development of such methods helps in training the analysts for skillfully handling the sophisticated analytical instruments and the way for research approach. Reference literature and general survey reveals that similar work of development of UV and HPLC methods for new drugs and their combinations introduced in the market is continuously underway in many academic institutions. The present work is also planned on similar lines [1,2].
The non-availability of analytical methods till now for the concurrent analysis of single and multi-component formulations made it worthwhile to pursue the present research work. It was also planned to validate the developed methods as per ICH guidelines.
Hence, they offer wide area for research activity with relatively minimum chances of exactly repetitive work. The pharmaceutical dosage forms are widely present with multiple active components i.e. in combined dosage forms. This has opened new task for analyst for simultaneous estimation of Midodrine hydrochloride inmarketed formulation.
Therefore, in proposed project, to attempt to develop New, simple, accurate, cost effective and precise method for analysis of drugs in marketed formulation and validate them [2][3][4].
Melting point-M.P. of the drug was 200-203 o C found through Melting point apparatus.
Solubility-Solubility of Midodrine hydrochloride was established by I.P. method. For HPLC method:

Chemicals and solvents
The HPLC system consisted of a Waters isocratic System UV detector. Model no. 784. The software used was Data Ace, Separation was achieved on a reverse C 18 (250mm×4.6mm 5µm).

Determination of solubility
Solubility of Midodrine hydrochloride was performed in different solvents.

Determination of  max of Drug
Standard solution (10g/ml) of pure Midodrine hydrochloride was prepared. The pure drug solution was scanned on UV spectrophotometer, which showed maximum absorbance at 272.0 nm.

Experimental Procedure:
Preparation of Standard Stock Solution 10mg of Midodrine hydrochloride was weighed accurately and transferred to a 10ml volumetric flask, and the volume was adjusted to the mark with the diluent water, to give a stock solution of 1000ppm.

Preparation of Working Standard Solution
From stock solutions of Midodrine hydrochloride 1 ml was taken and diluted up to 10 ml. from this solution 0.5, 1.0, 1.5, 2.0 and 2.5 ml solutions were transferred to 10ml volumetric flasks and make up the volume up to 10 ml with water gives standard drug solution of 5, 10, 15, 20, 25µg/ ml concentration.

Preparation of the calibration curves of the drug
The standard drug solutions were taken absorbance 3 times and the mean area of drug was calculated and plotted against the concentration of the drug. The regression equation was found out by using this curve. A typical spectrum and the calibration curve were obtained.

Preparation of analysis of tablet formulation
20 tablets were weighed and ground to a fine powder. Tablet powder equivalent to 10 mg Midodrine hydrochloride was weighed and transferred to a 10 ml volumetric flask and volume was made up to 10 ml with diluents (water) to obtain concentration of 1000µg/ml. Resultant solution was filtered through Whattmann filter paper.
1 ml of filtrate was taken in 10 ml volumetric flask and volume was made up to 10 ml with diluent to obtain concentration of 100µg/ml. Further 1.0 ml of this solution was taken and diluted up to 10 ml obtain final concentration of 10 µg/ml. Absorbance of the sample solutions at 272.0 nm was measured and from the absorbance values, the concentration of drugs in the sample solution was determined by using calibration curve Y=mx+c.

Linearity
Linearity of analytical procedure is its ability (within a given range) to obtain test, which are directly proportional to absorbance of analyte in the sample. The calibration plot was constructed after analysis of five different (from 5 to 25µg/ ml) concentrations and area for each concentration were recorded three times, and mean absorbance was calculated.
The regression equation and correlation coefficient of curve and the standard curve of the drug is shown in figure.

Precision (A)Repeatability
Standard dilutions were prepared, and three replicates of each dilution were analyzed in same day for repeatability and results were subjected to statistical analysis. Standard dilutions were prepared, and three replicates of each dilution were analyzed in different days and by different analysts. Statistical analysis was carried out.

(B) Intermediate Precision (a) Day to Day (b)Analyst to Analyst
The intermediate precision expresses with in laboratories variation: different days, different analysts, different equipment etc. The standard dilution was prepared, and three replicates of each dilution were analyzed by different analysts for all the developed methods.

LOD (Limit of Detection):
The Limit of Detection (LOD) is the smallest concentration of the analyte that gives the measurable response. LOD was calculated using the following formula.

LOQ (Limit of Quantitation):
The Limit of Quantification (LOQ) is the smallest concentration of the analyte, which gives a response that can be accurately quantified. LOQ was calculated using the following formula.

Mobile Phase Selection:
Initially to estimate Midodrine hydrochloride number of mobile phase in different ratio were tried. Taking into consideration the system suitability parameter like RT, Tailing factor, No. of theoretical plates and HETP, the mobile phase found to be most suitable for analysis was Water (pH adjust 3.5 with TEA): Acetonitrile in the ratio of (20:80v/v). The mobile phase was filtered through 0.45 filter paper to remove particulate matter and then degassed by sonication. Flow rate employed for analysis was 1.0 ml/min at the Λmax 272nm.

Selection of wavelength:
100 mg of Midodrine hydrochloride was weighed accurately and transferred to a 100 ml volumetric flask, and the volume was adjusted to the mark with the mobile Water (pH adjust 3.5 with TEA): Acetonitrile in the ratio of (20:80v/v). From above solutions of 0.1 ml was transferred to 10 ml volumetric flasks and make up the volume up to mark. Resulting solution was scanned over UV range (200-400nm), maximum absorbance was found at Lambda max 272nm.

Selection of Separation Variable:
Standard drug solution of Midodrine hydrochloride was prepared in different mobile phase and chromatograph was recorded by using different column (5 and 10 µm) at different chromatographic condition like different flow rate and temperature. Considering the theoretical facts and after several trials separation variables were selected which were constant during whole experiment.

System Suitability Parameters:
Separation variables were set, and mobile phase was allowed to saturate the column at 1.00 ml/min. After complete saturation of column, three replicates of working standard of Midodrine hydrochloride 10g/ml was injected separately. Peak report and column performance report were recorded for all chromatogram.

Preparation of Standard Stock Solution:
10mg of Midodrine hydrochloride was weighed accurately and transferred to separate 10ml volumetric flask, and the volume was adjusted to the mark with Acetonitrile to give a stock solution of 1000ppm.

Preparation of Working Standard Solution:
From stock solutions of Midodrine hydrochloride 1 ml was taken and diluted up to 10 ml. from this solution 0.5, 1.0, 1.5, 2.0, 2.5 ml solutions were transferred to 10ml volumetric flasks and make up the volume up to 100 ml with Acetonitrile, gives standard drug solution of 5, 10, 15, 20, 25 µg/ ml concentration.

Preparation of the Calibration Curves of the Drug:
Standard drug solutions were injected 3 times and the mean peak area of drug was calculated and plotted against the concentration of the drug. The regression equation was found out by using this curve.

Analysis of tablet formulation[15-17]:
Assay of Tablet formulation: 20 tablets were weighed and ground to a fine powder. Tablet powder equivalent to 10 mg Midodrine hydrochloride was weighed and transferred to a 10 ml volumetric flask and volume was made up to 10 ml with Acetonitrile to obtain concentration of 1000µg/ml. Resultant solution was filtered through Whattmann filter paper. 1 ml of filtrate was taken in 10 ml volumetric flask and volume was made up to 10 ml with diluents (Acetonitrile) to obtain concentration of 100µg/ml. Further 1.0 ml of this solution was taken and diluted up to 10 ml obtain final concentration of 10 µg/ml.
The amounts of Midodrine hydrochloride in Tablet formulation was calculated by extrapolating the value of area from the calibration curve. Analysis procedure was repeated six times with Tablet formulation.

Linearity:
Linearity of analytical procedure is its ability (within a given range) to obtain test, which are directly proportional to area of analyte in the sample. The calibration plot was contracted after analysis of five different (from 5 to 25 µg/ ml) concentrations and areas for each concentration were recorded three times, and mean area was calculated. The regression equation and correlation coefficient of curve are given, and the standard calibration curve of the drug is shown in fig. From the mean of AUC observed and respective concentration value, the response ratio (response factor) was found by dividing the AUC with respective concentration.

Accuracy:
Recovery studies were performed to validate the accuracy of developed method. To pre-analyzed sample solution, a definite concentration of standard drug (80%, 100%, and 120%) was added and then its recovery was analyzed.

Precision: (A)Repeatability
Standard dilutions were prepared, and three replicates of each dilution were analyzed in same day for repeatability and results were subjected to statistical analysis. Standard dilutions were prepared, and three replicates of each dilution were analyzed in different days and by different analysts. Statistical analysis was carried out.

(B) Intermediate Precision (a) Day to Day (b) Analyst to Analyst
The intermediate precision expresses with in laboratories variation: different days, different analysts, different equipment etc. The standard dilution was prepared, and three replicates of each dilution were analyzed by different analysts for all the developed methods. The statistical analysis method was carried out and the data is presented.

Robustness:
As per ICH norms, small, but deliberate variations, by altering the pH and / or concentration of the mobile phase were made to check the method capacity to remain unaffected. The effect of change in pH of mobile phase, flow rate, mobile phase ratio on the retention time, theoretical plates, area under curve and percentage content of Midodrine hydrochloride was studied.      Correlation Coefficient ( r 2 ) 0.996

Results of Validation Parameters
Results of Accuracy       Taking into consideration the system suitability parameter like RT, Tailing factor, No. of theoretical plates and HETP, the mobile phase found to be most suitable for analysis was Water (pH adjust 3.5 with TEA): Acetonitrile in the ratio of (20:80v/v) in the ratio of 20:80.

Results of LOD and LOQ
The mobile phase was filtered through 0.45 filter paper to remove particulate matter and then degassed by sonication. Flow rate employed for analysis was 1.0 ml/min