Analytical method development and validation of doxazosin mesylate uncoated tablets by RP-HPLC

A Simple, Precise, Stability indicating reverse phase HPLC method has been developed and validated in order to determine Doxazosin mesylate uncoated tablet dosage form. A Chromolith RP-18 column made up of Stainless Steel has been used with dimensions 100mm x 4.6mm x 10m. An Isocratic method with Mobile phase consisting of phosphate buffer: Methanol (40:60) ratio. A UV-Detector is used and the wavelength was set at 251nm.At the flow rate of 1.0ml/min the mobile phase was run for 10 minutes. The method was validated for Linearity, Precision, Accuracy, Robustness, and Stability of analytical solution. The Linearity range for the standard and the placebo was found to be between 50% and 150%. The method was validated for precision in method, system and intermediate. All the validation parameters have come under the limits.


Column
Chromolith RP -C18, 100mm x 4.6mm x 10µ Buffer Weigh accurately and transfer 6.8 g of Potassium dihydrogen orthophosphate to a 1000 ml volumetric flask. Add about 980 ml of water, dissolve and dilute to volume with water.
Mobile Phase For isocratic system, prepare a mixture of Methanol, and buffer in the proportion 60 : 40 respectively. Mix well, adjust the pH to 5.0 ± 0.05 with sodium hydroxide pellets. Filter through 0.2 µ Nylon membrane filter paper and degas prior to use.

Results and discussions
3.1 Specificity / Selectivity: Selectivity was performed by injecting the diluent blank solution, excipient blend, system suitability solution, test solution 3.1.1 Acceptance criteria: The Doxazosin mesylate peak should be well resolved from any other peak and from each other. The diluent blank solution, excipient blend solution should not show any peak at the retention time of the Doxazosin mesylate.

Results:
The system suitability criteria were found to meet the pre-established acceptance criteria as per the analytical method. All the injections were processed at the wavelength provided in the method. There was no interference observed from diluent blank solution, excipient blend solution with Doxazosin mesylate peak.

Linearity and Range for standard:
For the linearity study five standard solutions of Doxazosin mesylate were prepared from the range starting from 50% to 150% of the theoretical concentration of assay preparation.
The system suitability solution and the linearity solutions were injected as per the protocol. The linearity graph of concentration against peak response was plotted and the correlation coefficient was determined.

Acceptance criteria:
Correlation coefficient should be greater than or equal to 0.999.

Results:
The system suitability criteria were found to meet the pre-established acceptance criteria as per the analytical method. (Refer to Table-2 for system suitability results). (%) Relative Standard Deviation 0.42 The average peak area of Doxazosin mesylate peak at each concentration level was determined and the linearity graph was plotted against the sample concentration in percentage. The results of linearity study are as given in Table 3.  IJBR (2013) 04 (09) www.ssjournals.com

Linearity level Sample Concentration (%) Sample Concentration (in ppm) Peak Area Correlation coefficient
The system suitability solution and the linearity solutions were injected as per the protocol. The linearity graph of concentration against peak response was plotted and the correlation coefficient was determined.

Acceptance criteria:
Correlation coefficient should be greater than or equal to 0.999.

Results:
The system suitability criteria were found to meet the pre-established acceptance criteria as per the analytical method. (Refer to Table 4 for system suitability results). (%) Relative Standard Deviation 0.42 The average peak area of Doxazosin mesylate peak at each concentration level was determined and the linearity graph was plotted against the sample concentration in percentage. The results of linearity study are as given in Table -5. The system precision was performed by injecting 10 replicate injections of system suitability solution and the chromatograms are reviewed for the system suitability criteria. 3.4.1.2 Acceptance criteria: % RSD of peak areas of ten replicate injections of system suitability solution should not be more than 2.0% and system suitability criteria should pass as per analytical method.

Results:
The system suitability criteria were found to meet the pre-established acceptance criteria as per the analytical method.

Results:
The system suitability criterion was found to meet the pre-established acceptance criteria as per the analytical method. The results of assay obtained from six test solutions preparations are presented in Table -8     These test solutions were prepared by adding a quantity of Doxazosin mesylate API to excipient blend to produce three different concentration solutions equivalent to 50%, 75%, 100%, 125% and 150% of test concentration.
3.5.2 Acceptance criteria: Mean recovery at each concentration level should be between 98.0% and 102.0%.

Results:
The system suitability criteria were found to meet the pre-established acceptance criteria as per the analytical method. (Refer to Table -12 for system suitability result). The results of accuracy study obtained are presented in Table-13.  The system suitability criteria were found to meet the pre-established acceptance criteria as per the analytical method. (Refer to Table -14 for system suitability results).

IJBR (2013) 04 (09)
www.ssjournals.com (%)Relative Standard Deviation 0.14 0.12 The assay results obtained with different flow rate conditions are as given in Table -15. The system suitability criteria were found to meet the pre-established acceptance criteria as per the analytical method. (Refer to Table -16 for system suitability results). The system suitability criteria were found to meet the pre-established acceptance criteria as per the analytical method. (Refer to Table -18 for system suitability results).
IJBR (2013) 04 (09) www.ssjournals.com  The system suitability criteria were found to meet the pre-established acceptance criteria as per the analytical method (Refer to Table -20 for system suitability results).  System suitability solution and test solution of DOXACARD-2 were prepared on 0 th ,12 th , 24 th , 36 th and 48 th hour of experiment and stored these solutions at room temperature for every time interval up to 48 hrs and analyzed these solutions on 48 hrs with freshly prepared test solution. The system suitability solution was prepared freshly at the time of analysis. The assay of DOXACARD-2 in the sample was calculated.

Acceptance criteria:
The analyte is considered stable if there is no significant change in % assay.

Results:
The assay results obtained during solution stability experiment are as given in Table-