Analytical Method Development and Validation of Drospirenone by UV-Vis Spectroscopy in Accordance with ICH Guidelines

UV-Visible (UV-VIS) spectroscopy is an analytical technique that studies the interaction of electromagnetic radiation with matter, specifically focusing on the absorption and emission of UV and visible light by atoms, ions, or molecules as they transition between energy states.(1) It is widely used in the analysis of inorganic, organic, and biochemical substances in research, industry, clinical laboratories, and environmental monitoring(2). The technique is based on the principle that molecules absorb light energy, causing electrons to move from a ground state to an excited state, with the energy absorbed corresponding to the difference between these states(3). This is described by Beer-Lambert’s Law, which establishes a linear relationship between the absorbance (A) and the product of molar absorptivity (a), path length (b), and concentration (c) of the solution (A = abc). However, this law has limitations at high concentrations due to deviations in absorptivity, light scattering, sample fluorescence, changes in refractive index, and chemical equilibria(4). The instrumentation of UV-VIS spectroscopy includes components such as light sources (hydrogen, deuterium, tungsten, xenon, mercury lamps), wavelength selectors (filters and monochromators), sample holders or cuvettes made of quartz or glass, and detectors like photovoltaic cells, phototubes, and photomultiplier tubes that convert light energy into electrical signals(5). Ideal detectors are characterized by high sensitivity, low noise, and quick response. Spectrophotometers can be of single-beam design, where light passes through a sample and reference sequentially, or double-beam design, either in-space or in-time, where light is split and directed through both paths simultaneously or alternately(6). Double-beam-in-time spectrophotometers are preferred due to better accuracy and fewer matching issues with detectors(7). Drospirenone is a synthetic spironolactone analogue and progestin, exhibiting both pregestational and anti-mineralocorticoid activity.(8,9) Its IUPAC name is (6R,7R,8R,9,10R,13S,14S,15S,16S,17S)-1,3',4',6,6a,7,8,9,10,11,12,13,14,15,15a,16-hexadecahydro-10,13-dimethylspiro-[17H-dicyclopropa[6,7:15,16]cyclopenta[a] phenanthren - 17,2'(5'H)-furan] -3,5'(2H)-dione.(10) It has the molecular formula C??H??O? and a molecular weight of 366.493 g/mol. Drospirenone has a melting point of approximately 204°C, indicating its thermal stability. In terms of solubility, it is soluble in dimethyl formamide (DMF) at about 2 mg/mL, though DMF must be purged with an inert gas prior to use. However, drospirenone is only sparingly soluble in aqueous buffers, which may influence its formulation and delivery strategies.(11–13) Chemically, it is classified as a steroid lactone and a 3-oxo-Δ? steroid. It functions pharmacologically as a contraceptive agent, an aldosterone antagonist, and a progestin. The compound exhibits a pKa of 5, which provides insight into its ionization characteristics and behaviour under physiological pH conditions.(14,15) The International Council for Harmonisation (ICH) aims to achieve global harmonization in the development, registration, and maintenance of safe, effective, and high-quality medicines in the most efficient manner while adhering to high standards.(16) ICH has established over 60 guidelines categorized into four areas: Safety (14 guidelines), Quality (23 guidelines), Efficacy (21 guidelines), and Multidisciplinary (6 guidelines). Among these, ICH Q2(R1) specifically addresses analytical method development and validation.(17) Key parameters include accuracy, precision, specificity, detection limit, quantitation limit, linearity, range, and validation. Accuracy is the closeness of test results to the true value and can be established using highly pure analytes, independent procedures, spiking techniques, or validation parameters like precision and linearity(18). For impurity methods, accuracy involves spiking known impurities or degradants. Precision, the agreement among repeated measurements, includes repeatability (same conditions), intermediate precision (variations within the lab), and reproducibility (different labs)(19). Specificity is the method's ability to measure the analyte in the presence of other components and may require complementary techniques like titrimetric assays and HPLC, especially for complex biologicals such as peptides or oligonucleotides(20). Detection limit is the smallest amount of analyte detectable but not quantifiable, while the quantitation limit is the lowest amount that can be quantified accurately and precisely, often calculated using signal-to-noise ratios or standard deviation methods.(21) Linearity refers to the method’s ability to provide results directly proportional to analyte concentration, typically evaluated using regression analysis with at least five concentrations(22). The range is the interval between the lowest and highest analyte concentrations showing acceptable accuracy, precision, and linearity—commonly 80–120% for assays, 70–130% for content uniformity, and ±20% for dissolution testing.(17,23) Validation confirms that procedures consistently yield expected results and must be documented in a protocol including acceptance criteria, methodological details, and justification for non-pharmacopeial methods when used. (23,24) Analytical method validation protocols should ensure repeatable, accurate, and specific measurements, and include complete method descriptions, system suitability tests, and calculations necessary for consistent and reliable analysis.(25)

MATERIAL AND METHODS:

Instrumentation and Materials:

U.V. Visible double beam spectrophotometer Jasco international co. V-730, equipped with spectra manager software, utilized path length 1cm u.V. matched quartz cells. Drospirenone sample and standard gifted from Mylan, Hyderabad. All the chemicals, solvents, and reagents (ethanol, water, and methanol) used were of analytical grade.

Method Development

a) To check the solubility of Drospirenone:

10 mg of Drospirenone was weighed and solubility of this sample was checked in double distilled water, methanol, ethanol, acetonitrile. The drug was found to be soluble in ethanol as well as methanol but ethanol being greener solvent, hence Ethanol was selected.

Table.1 Solubility table of Drospirenone

b) To identify the λ_max of Drospirenone:

100 mg of the pure drug was accurately weighed and dissolved in 75mL ethanol, and the volume was made up to 100mL with ethanol to give a standard stock solution of 1000 µg/mL. Aliquots of standard stock solution were pipetted out and suitable dilutions were made with water to get standard solutions of concentration: 10-50 µg/mL. These were scanned in UV range from 200-400nm, which could be utilized for analysis and spectrum was recorded.

c) Selection of solvent system for method development:

50 mg of Drospirenone was weighed and solubility of this sample was checked in ethanol and other solvents. The drug was found to be soluble in ethanol. Thus, the dilution was made in ethanol.

Fig. 1 Lambda max of Drospirenone

Linearity:

Estimation of Drospirenone was carried out spectrophotometrically at 274 nm in Ethanol. The absorbance obtained from 3 to 15µg/mL. The data were represented in Table 2. Based on the experimental data the standard calibration curve was plotted in Excel sheet. The regression analysis showed very good correlation. (r²=0.9999)

From the figure, regression coefficient slope and intercept were found to be 0.0638x. Table 2. shows the absorbance of Drospirenone standard solution containing 3-15 µg/mL of drug in water. Figure 2. shows a representative standard calibration curve with slope. regression coefficient, and intercepts of 0.203, 0.3916, and 0.5836 respectively. The curve was found to be linear in the range of 3-15 ug/ml at max 274 nm. The calculation of drug content is based on this calibration curve.

Table 2. Calibration Table of Drospirenone

Fig.2 Calibration Curve of Drospireneone

Fig 3. Linearity of Drospireneone

LOD & LOQ :

The detection limit and the limit of quantification (LOQ) for Drospireneone were determined by the standard deviation of the Y-supplies regression line combined with the slope of the calibration curve according to the instructions of the ICH.

The LOD can be determined by the following equation:

Limit of Detection=3.3σS

Similarly,

Limit of Quantification =10σS

the LOQ is determined by the following equation:

Where σ means a standard deviation of response and S means the inclination of the regression line.

Table 3. Sensitivity of Drospirenone

Precision:

Repeatability was performed by choosing 6 µg/mL concentration and multiple samplings of the same sample were prepared and analysed by UV at 274 nm wavelength. The experiment was performed as shown in table 4 and mean was calculated for further calculations. The data represented in Table 4 showed that the method was precise for detection of Drospirenone %RSD value was found to be less than 2.

Assay:

Twenty tablets were weighed accurately and crushed in mortar & pestle tablet powder equivalent to 10 mg (equivalent to 0.4 mg) of drospirenone was weighed and transferred to 25 mL volumetric flask, then 2 mL of ethanol was added to it and sonicate for 10-15min, volume was adjusted with ethanol to get 400 µg/mL solution. The solution was filtered via Whatman filter paper and 1 mL of this solution was diluted to 10 mL with distilled water to get the solution containing 40µg/mL. Further 0.3 mL of above solution was diluted to 10 mL with distilled water to get solution containing 3 µg/mL solution. The absorption spectra of the prepared solution were recorded, and the concentration of each absorbance was determined by applying the calibration curve equation in Table 5.

Table 5. Table of Assay of Drospireneone

Accuracy:

Precision experiments were performed by conventional augmentation technique. Different concentrations of pure drospirenone were incorporated into the drug sample, with levels ranging from 80% to 120% in each coated tablet containing 4 mg of drospirenone. The correct dosage of 10mg of drospirenone was precisely measured and added to a 25mL volumetric flask. This was dissolved in ethanol and the volume was adjusted to 25mL to create a standard stock solution of 1000µg/mL. From these 1.28, 1.6, and 1.92 µg/mL solutions, three separate preparations were made. To prepare a 1.28 ug/mL tablet solution, divide the required amount into three equal parts and measure each part using a volumetric flask. 1.6 micrograms per millilitre and 1.92 micrograms per millilitre of standard solutions were added respectively. The volume was adjusted to the desired level by adding distilled water, resulting in concentrations of 2.88µg/mL (80%), 3.2µg/mL (100%), and 3.5µg/mL (120%). These were examined using absorption maxima and area under the curve techniques. The procedure was repeated three times for 80%, 100%, and 120% of the level to assess recovery in Table 6.

Table 6. Table of accuracy of Drospireneone

Accuracy of analytical methods was evaluated by estimation of % recovery of known amount of standard. The attained percentage of recovery were within the range of 98-102%.

Robustness:

Robustness, the Uv method was achieved by using varying percentages of ethanol in a co-solvent system. The ethanol concentration in the co-solvent system was intentionally adjusted to 80 and 90% by adding water. Drospirenone (6μg/mL) was made using the above mentioned co-solvent system on its own, and the sample was analysed at a maximum wavelength of 273nm to determine the content of drospirenone. The result was determined in terms of %RSD in Table 7.

Table 7. Table of robustness of Drospireneone