Development and Validation of UV Spectrophotometric Method for Doxazosin Mesylate in Bulk and Tablets
Priyanka S. Yadav1, Ashok A. Hajare1*, Kiran S. Patil2
1Department of Pharmaceutics, Bharati Vidyapeeth College of Pharmacy,
Kolhapur - 416008, Maharashtra, India.
2Department of Pharmaceutical Quality Assurance, Tatyasaheb Kore College of Pharmacy,
Warnanagar, Kolhapur - 416113, Maharashtra, India.
*Corresponding Author E-mail: ashok.hajare@bharatividyapeeth.edu
ABSTRACT:
Doxazosin mesylate (DX) is an antihypertensive agent belonging to BCS class II. There is no reported literature available on development of spectrophotometric method for its analysis in bulk and pharmaceutical dosage forms using 0.1N HCl. Thus, attempt was made to develop and validate a new UV spectrophotometric method using 0.1N HCl as the solvent for its quantitative estimation in tablets that would be fast, simple, accurate, and sensitive. DX had a maximum absorbance wavelength of 245 nm and was linear with a correlation coefficient (R2) of 0.9981 across the concentration range of 2-14 μg/mL. The present method was utilized to determine the drug content of two commercial brands namely; brand I and brand II. The estimated amount of DX was 99.13% and 99.02% in these brands, respectively. The proposed method generated results that confirm the label claim and was accurate, precise, sensitive, and rugged during its validation study. The accuracy of the technique was tested using recovery research at three different levels, namely 80%, 100%, 120%, and the percent recovery rate was determined between 98% and 102% suggesting that the proposed approach is accurate. Precision and robustness were within the acceptable limits, complying with ICH standards. The proposed method could be used to quantify DX in API and dosage forms.
KEYWORDS: Doxazosin mesylate, UV spectrophotometry, Method development, Validation, Statistical analysis, ICH guideline.
INTRODUCTION:
Doxazosin mesylate is a white or almost white crystalline powder slightly soluble in methanol and water and a combination of 15 parts water and 35 parts tetrahydrofuran. It is insoluble in acetone.5, 6, 7
Spectrophotometry is a very useful technique for assessing a material's ability to reflect or transmit light as a function of wavelength. There are several advantages of using this technique that including its high potential for rapid development with great precision. UV spectrophotometry has been considerably used in the quantitative and qualitative assessment of pharmacological dosage formulations since the past few years.6 Instrumentation and methodology should be established early in the process of developing the analytical procedure, following objective and scope of the analytical technique. Precision, specificity, linearity, limits of detection (LOD), and limits of quantitation (LOQ) are a few of the characteristics that must be considered while developing a technique.7
Validation of analytical methods is the process of ensuring that an analytical procedure is suitable for its intended purpose. Scientific method development and optimization are performed to ascertain the method strategy and objective of operations analysis. The International Conference for Harmonization (ICH) Q2 (R1) provides the most authoritative advice on validation characteristics for analytical techniques.7
Prior to developing pharmaceutical formulations, the first and most critical step is to authenticate the raw ingredients. To accomplish this, analytical suitability must be established via the development of analytical techniques. Developing a new method for the quantitative determination of doxazosin mesylate is an essential step in the product and process development and hence attempt was made to develop a new method for the quantitative determination of Doxazosin mesylate using UV spectrophotometric analysis from the bulk and pharmaceutical dosage formulations employing 0.1 N HCl as the solvent.
The objective of current analytical method development was to compare the analytical properties of both the bulk doxazosin and its finished product to a pre-determined acceptable level.
Figure 1: Chemical structure of doxazosin mesylate
MATERIALS AND METHODS:
Materials:
Doxazosin mesylate was procured from Dr. Reddy's laboratory, Hyderabad. All chemicals and reagents used were of analytical grade purchased from Finar Chemicals Ltd., Ahmadabad, India. UV-Visible double beam spectrophotometer (UV-1800, Shimadzu, Japan) and two identical quartz cells of 1 cm path length were used for the spectral measurements. Digital Electronic Balance (Contech & Citizen, Mumbai, India) was used for weighing purposes.
Methods:
Wavelength selection:
About 10 µg/ml of doxazosin mesylate was accurately prepared in spectroscopy grade 0.1 HCl. This solution was then scanned in the 200 - 400 nm region. The wavelength maxima (λmax) were observed at 245 nm and this wavelength was adopted for absorbance measurement as shown in Fig. 2.
Selection of the solvent:
The criterion for the selection of the solvent was the solubility of the drug. Based on the solubility of doxazosin mesylate, 0.1 N HCl was selected as a solvent.
Preparation of standard solution:
Accurately weighed 100 mg of doxazosin mesylate standard was transferred to a volumetric flask containing 0.1N HCl to obtain a standard stock solution of concentration 1000 μg/ml.
Preparation of working standard solution:
Doxazosin solution of concentration 100 μg/ml was prepared by diluting 10 ml standard stock solution to 100 ml using 0.1N HCl.
Validation:
Linearity:
A linear relationship was investigated to determine the range of an analytical technique. To determine linearity, a graph of absorbance vs. analyte concentration was plotted. The regression equation was generated as output data from the absorbance and analyte concentration data input. A graph is included showing the data, Fig. 4. Linearity analysis is often employed to ascertain the required range.8
Multiple aliquots of the working standard solution of concentrations of 2, 4, 6, 8, 10, 12, and 14 μg/ml were prepared by dilution with 0.1N HCl. These sample solutions were scanned between wavelengths ranging from 200 to 400 nm against a blank 0.1N HCl. The absorbance of these sample solutions was determined, and the data were utilized to construct a linear calibration curve.
Accuracy:
The precision of an analytical process is the degree to which the experimental result is near to the true value. At least nine determinations spanning three concentration levels with three replicates throughout the prescribed range should be assessed for accuracy. Accuracy assessment in pre-analyzed sample solutions should be seen as a recovery study using a known quantity of analyte. The recovery rate should be expressed in percentage.8, 9, 10,11
A standard stock solution of known concentration was added at three different concentrations to the previously examined sample solutions, namely 80%, 100%, and 120%. These alternatives were re-examined. At each level, solutions were analyzed thrice and the accuracy was calculated and reported as a percentage of the recovery.
Precision:
Precision was determined by examining intra- and inter-day variance. The accuracy was determined by measuring the absorbance of three duplicates of fixed concentration solutions of doxazosin mesylate at three distinct time points during the day and three additional days. The precision investigation findings were reported as a percent percentage of relative standard (RSD) deviation.12, 20
Sensitivity:
The sensitivity of the technique under investigation was evaluated by examining characteristics such as the limit of detection (LOD) and the limit of quantitation (LOQ). The LOD and LOQ values for the test were determined using the following formula.20, 21
Standard deviation of the y-intercept of the regression line
LOD = 3.3 × ------------------------------------------------------------ =. (1)
Slope of the calibration curve
Standard deviation of the y-intercept of the regression line
LOQ = 10 × -------------------------------------------------------------- =. (2)
Slope of the calibration curve
Assay:
The current method was used to determine the quantity of doxazosin mesylate contained in two distinct brands of a commercial tablet formulation and bulk formulation. To produce powder, twenty pills were triturated. To achieve a concentration of 100µg/ml, 2 mg of tablet powder was weighed and added to 100 ml 0.1N HCl, which was shaken until dissolved. This solution was filtered using Whatman filter paper #41 to remove any undissolved traces. Additional dilution with 0.1N HCl was performed to produce a solution with a concentration of 10 µg/ml. The absorbance of the solution was measured, and the drug concentration was calculated using the calibration curve. 9, 11
Short-term stability study:
Doxazosin mesylate solutions prepared for the stability study were stored for 24 h at ambient temperature, and the absorbance of solutions was determined on a subsequent day.13
Ruggedness:
The ruggedness of the developed method was analyzed by using a standard stock solution. A sample solution of concentration 10 μg/ml was prepared and analyzed by two distinct analysts at the same environmental conditions, Table 6.
RESULTS AND DISCUSSION:
The developed method was validated as per ICH guidelines.8, 15, 16
Method Validation:
Linearity Study:
The λmax for doxazosin mesylate was measured at 245 nm in 0.1N HCl, Fig. 2. The drug exhibits a linear relationship across the concentration range 02-14 μg/ml with R2 = 0.9981, Fig. 3 and Fig. 4. The result of regression analysis is presented in Table 1. The sensitivity of the present method was determined by Sandell’s sensitivity (μg/cm2/0.001 Abs unit). Sandell’s sensitivity was calculated using formula:
0.001
Sandell ‘s sensitivity (π) = Conc. (µ g/100ml) × ---------- .. (3)
Dl value
Figure 2: λmax for doxazosin mesylate in 0.1 N HCl
Figure 3: Doxazosin mesylate overlay of linearity absorbance spectrum
Figure 4: Linearity curve of doxazosin mesylate at 245 nm
Table 1: Result of doxazosin mesylate regression analysis
|
Observation |
Value |
|
λ max |
245 nm |
|
Beers law limit |
2-14 μg/ml |
|
Regression equation (y= mx ± c) |
y = 0.0982x + 0.0203 |
|
Correlation coefficient |
0.9981 |
|
Slop |
0.982 |
|
Intercept |
0.0203 |
|
Molar absorptivity |
0.1034×104 Lmol-1cm-1 |
|
Sandell’s sensitivity |
0.0814 μg cm-2 |
Accuracy:
The results of recovery studies are reported in Table 2, which reveals that the percentage recovery of the drug was between 98% and 102%. Average recoveries of doxazosin mesylate were 101.93%, 99.80%, 98.74%, at 80%, 100%, and 120%, respectively.11, 12 Hence, the method under investigation was accurate.
Table 2: Results of statistical analysis of accuracy
|
Concentration level |
Pre-analyzed sample solution (μg/ml) |
Amount of drug added (μg/ml) |
Amount of drug recovered (μg/ml) |
% Recovery Mean ± SD |
% RSD |
|
80% |
10 |
8 |
18.15 |
101.93±0.1102 |
0.1123 |
|
100% |
10 |
10 |
19.98 |
99.80±0.1545 |
0.1541 |
|
120% |
10 |
12 |
21.84 |
98.74±0.4126 |
0.4074 |
Values are mean ± SD, n = 3
Table 3: Results of statistical analysis of intra-day assay and inter-day assay
|
Concentration (μg/ml) |
Intra-day precision |
Inter-day precision |
||
|
Concentration found (μg/ml) Mean ± SD |
%RSD |
Concentration found (μg/ml) Mean ± SD |
%RSD |
|
|
2 |
2.0295±0.1915 |
0.1943 |
2.0291±0.1693 |
0.1717 |
|
4 |
3.9629±0.0941 |
0.0932 |
3.9608±0.0294 |
0.0291 |
|
6 |
5.9585±0.0555 |
0.0551 |
5.9602±0.0385 |
0.0382 |
Values are mean ± SD, n = 3
Precision:
The precision of an analytical procedure refers to the similarity of findings acquired from several samplings of the same homogenous material under specified circumstances. Precision was evaluated using homogenous and genuine.9, 12, 13
The method's precision under study was mentioned in terms of % relative standard deviation. This result reveals the reproducibility of the assay procedure. As %RSD values for intra-day and the inter-day precision study found less than 2 indicates that the proposed method is precise for the quantitative determination of doxazosin mesylate in the formulation.12,13
LOD and LOQ:
The detection limit of a test technique is its capacity to detect the smallest quantity of a chemical entity in a sample without requiring quantitative determination. By contrast, the quantitation limit refers to a testing method's capacity to identify the lowest concentration of a chemical in a sample, quantitatively. This quantitation limit parameter is most often used to determine contaminants and degradation products in sample matrices, as well as quantitative tests of low analyte levels.9-13
The LOD and LOQ for UV and HPLC were calculated using the slope and SD of response (intercept). The linearity equation was found Y = 0.0982x + 0.0203. The limit of detection (LOD) and limit of quantification (LOQ) were found at 0.0229 μg/ml and 0.0694 μg/ml, indicating desired sensitivity of the proposed UV method.12, 13
Assay:
The assay method must provide an accurate result experimentally to supports the claim about the analyte's concentration or potency in samples.9, 10 The results reported in Table 4 show the 99.00 to 99.13 %purity.
Table 4: Result of tablet assay
|
For mulation |
Label claim (mg/ tab) |
Amount of drug estimated (mg/tab) Mean ± SD |
%Amount found Mean ± SD |
% RSD |
|
Brand I |
2 |
1.9828±0.0008 |
99.13± 0.04189 |
0.04152 |
|
Brand II |
2 |
1.9804±0.0006 |
99.02± 0.02943 |
0.029141 |
|
Bulk formulation |
2 |
1.9801±0.0004 |
99.00± 0.02054 |
0.020334 |
Values are mean ± SD, n = 3
Short-term stability study:
The %RSD of doxazosin mesylate for the duration of solution stability, no notable changes were seen in the content of the drug. The solution steadiness and eluent steadiness experimentation statistics recommend that the sample solution used throughout the method was unwavering for 24 h.13
Table 5: Results of short-term stability study
|
Concentration (μg/m) |
The concentration found at 24 hrs Mean ± SD, (μg/ml) |
%RSD |
% of drug content |
% RSD |
|
14 |
13.9666±0.0033 |
0.0004 |
99.78± 0.0244 |
0.0244 |
Values are mean ± SD, n = 3
Ruggedness:
The ruggedness of the analytical procedure is considered during the method development stage, so that method should provide reliable results on minor changes in method parameters.9, 14 The results indicate that the %RSD values for different analysts and different instruments were found to be in the range of 0.11 and 0.13. A method is said to be robust if the % RSD value is <2%. The results are reported in Table 6.
Table 6: Results of statistical analysis of ruggedness
|
Concentration (μg/ml) |
% Amount found |
|
|
Analyst I (Mean ± SD) |
Analyst II(Mean ± SD) |
|
|
10 |
99.88±0.1143 |
99.85±0.1307 |
|
%RSD |
0.11 |
0.13 |
Values are mean ± SD, n = 3
CONCLUSION:
For the quantitative determination of doxazosin mesylate from bulk and tablet formulations, the UV approach was devised in this work. In compliance with ICH Q2 requirements, the suggested method has been confirmed for the determination of doxazosin mesylate. The results of tablet formulation analysis were within the acceptable limits, and recovery tests revealed that formulation excipients had no impact on the developed method. As a result, the proposed approach was demonstrated to be simple, linear, accurate, and cost-effective, making it ideal for routine doxazosin mesylate analysis from a pharmaceutical dosage formulation.
ABBREVIATIONS:
Ultraviolet spectrophotometer, (UV); International Conference for Harmonization, (ICH); Active pharmaceutical ingredient, (API); Hydrochloric acid, (HCl); Normality, (N); Relative standard deviation, (RSD); Standard deviation, (SD); Limits of detection, (LOD), Limits of quantitation, (LOQ), Correlation coefficient, (R2).
ACKNOLEDGEMENT
The authors appreciate Bharati Vidyapeeth College of Pharmacy, Kolhapur for providing the necessary facilities for conducting research.
CONFLICT OF INTEREST
The authors declare that they have no conflict of interest.
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Received on 20.04.2022 Modified on 27.05.2022
Accepted on 21.06.2022 © RJPT All right reserved
Research J. Pharm. and Tech. 2022; 15(6):2675-2680.
DOI: 10.52711/0974-360X.2022.00447