Analytical Method Development and Validation for Estimation of Mifepristone in Pure and Pharmaceutical Dosage Form

Mifepristone structurally belongs to the class of anti-progesterone steroids, which are used as an oral contraceptive. The reverse phase HPLC method was designed in a simplified and rapid way for the estimation of Mifepristone in bulk as well as tablets. The method was established using a Kromasil C18 column of dimensions of 250mm×4.6mm and a particle size of 5m.The used mobile phase was Acetonitrile: Water (70:30, v/v). The pump was pumped at 1 ml/min at a temperature of about 30 ± 2 °C and the eluted analyte was identified at 305 nm. Mifepristone eluted with a mean retention time of 6.27 minutes. The intended method was validated as per ICH (International Council for Harmonisation) guidelines, indicating a high degree of specificity, system suitability, accuracy, precision, and robustness. The LOD (Limit of detection) was found to be 0.7238 ppm and the limit of measurement was 0.9562 ppm. The method linearity was found to be between 16μg/ml, with an R 2 of 0.9923. In accuracy studies, the percent recovery was found to be between 99.39% 100.50%. The method was discovered to be precise as the values of the percent RSD Original Research Article Bastia et al.; JPRI, 33(60B): 120-130, 2021; Article no.JPRI.79719 121 were found to be less than 2.0% for both intraday and interday. The method was discovered to be reliable and robust. Mifepristone in marketed pharmaceutical tablet dosage form was effectively quantified using the established Reverse Phase HPLC method.

were found to be less than 2.0% for both intraday and interday. The method was discovered to be reliable and robust. Mifepristone in marketed pharmaceutical tablet dosage form was effectively quantified using the established Reverse Phase HPLC method.

INTRODUCTION
Unintended pregnancies are those that are unplanned or unwanted during conception. It is one of the most troubling public health complications and a severe reproductive health issue, which includes accidental pregnancy [1]. The World Health Organization (WHO) states that approximately 70,000 maternal deaths are due to the complications of unintended pregnancy, which further results in legal unsafe abortion, and that about 585,000 women die each year due to the complications of unintended pregnancy that result in child birth [2]. Mifepristone is a 19-nor steroid with an effective and competitive antagonist of glucocorticoids and progesterone to give anti-progesterone activity [3]. It has major special effects on ovulation [4]. If given precisely in between the follicular phases, it leads to delayed follicular maturation and ovulation as compared to normal. If this drug is given continuously, then ovulation is prevented [5]. It inhibits the growth of secretory endometrium, which leads to the production of menstruation [6].Mifepristone has antiglucocorticoid and anti-androgenic properties when combined with glucocorticoid and androgen receptors [7][8][9].The use of mifepristone in the first trimester of pregnancy further leads to abortion. It is also used as a post-coital contraceptive [8][9][10][11]. The mechanism of action of Mifepristone is presented in: An exhaustive search reveals that no analytical techniques like UV, HPLC, and stability indicating methods for the determination of Mifepristone as an individual have been reported [12][13][14]. There was no method reported to quantify Mifepristone by HPLC. However, a fresh method has been created for estimating Mifepristone that is accurate, specific, precise, and repeatable [14][15][16].

Chromatographic Conditions and HPLC Instrumentation
HPLC studies were carried out using the Jasco HPLC system 4000 series, consisting of a quaternary pump (PU-4180-LPG), a degasser, an auto-sampler (AS-4050) and a UV detector. All the chromatograms obtained were evaluated using Chrom-NAV version 2.0 software. The used mobile phase was Acetonitrile: Water (70:30, v/v). Flow rate of 1 ml/min, which was fine-tuned by trial and error. At a distance of 305 mm, detection was made.

Standard stock solution preparation.
The Mifepristone standard stock solution was prepared by properly weighing 5 mg of medication and transferring it to a 50-ml flask with a modest amount of mobile phase. i.e., Acetonitrile: Water(70:30,v/v) to dissolve the drug was then sonicated for 10 minutes, and the remaining mobile phase was added to make the final volume up to 50 ml to obtain a stock solution containing 100 ppm of Mifepristone.

Sample solution preparation
To prepare the sample solution, weigh 5mg of equivalent Mifepristone tablet powder into a 50ml flask and add a tiny amount of mobile phase into the volumetric flask. (Acetonitrile: Water (70:30), v/v), it was then sonicated for about 10 minutes and the remaining mobile phase was added to make the final volume up to 50 ml to obtain a stock solution containing 100 ppm of Mifepristone.

Method Validation
Specificity, method suitability, precision, LOD, LOQ, linearity, accuracy, and robustness were used to validate the optimised method according to ICH guidelines.

Specificity
Specificity was evaluated by analysing the standard (100ppm) and test (100ppm) and comparing the spectra, and the presence of interference was checked.

Limit of Detection (LOD) & Limit of Quantification
The limit of detection and the limit of quantification Mifepristone for the proposed method were estimated using the standard deviation method. A calibration curve was prepared in the detection and quantitation range (1-6 ppm).
LOQ =10×σ/S S = Slope of the Calibration curve. = Std deviation of y-intercepts of calibration line.

Accuracy
The percentage recovery was used to assess the accuracy of the developed approach. Percentage Recovery was calculated at three levels (50%, 100%, and 150%) at three different concentrations of solutions (i.e.1.5 ppm, 3 ppm, and 4.5 ppm) in triplicates to evaluate the accuracy. 3 different concentrations (i.e. 1.5 ppm, 3 ppm, and 4.5 ppm) were prepared from 100 ppm of standard stock solution and one concentration (i.e. 1 ppm) from 100 ppm of sample solution. The resulting solutions were injected in triplicates to HPLC under optimised chromatographic conditions. A known amount of sample solution (1 ppm) was spiked into three different concentrations of standard solutions (i.e., 1.5 ppm, 3 ppm, and 4.5 ppm). These solutions were injected in triplicates into the HPLC for analysis. Percentage recovery at each level was calculated using a formula: % Recovery = x 100

Precision
The intraday and inter-day precision of the devised approach were investigated. Sample solutions of Mifepristone in three concentration ranges (i.e., 1.5 ppm, 3 ppm, and 4.5 ppm) were prepared and injected in triplicates into HPLC for analysis. At each concentration level, the peak area was measured and the percent RSD was calculated. Similarly, the intraday and inter-day precision studies were done.
Intraday precision: in intraday precision, analysis was conducted twice at various times on the same day.
Interday precision: In interday precision, analysis was conducted for two consecutive days.

Robustness
The method's robustness was assessed by fluctuating method parameters such as change wavelength (303nm, 307nm), flow rate change (0.9 ml/min, 1 ml/min), and mobile phase change composition to (Acetonitrile: Water) (69:31) and (71:29). The robustness was assessed by analysing standard solutions of 3ppm (n= 6) and sample solutions of 3ppm (n= 2) of Mifepristone, and the% RSD was determined.

ANALYSIS OF MIFEPRISTONE TABLETS
To analyse the tablet mixture, 5 mg of Mifepristone tablet powder was carefully weighed in a 50-ml volumetric flask, the mobile phase was added, and the flask was sonicated for 15 minutes. The mobile phase was used to dilute the solutions to a volume of 50 ml, shake the solution, and filter to obtain the stock solution containing 100 ppm of Mifepristone. The solution was runned in HPLC in triplicate. The% w/w of Mifepristone in each tablet was calculated using the formula:

Method Development
During method development, various mobile phases were tried at different flow rates and their effects on retention time, capacity factor, area, peak symmetry, and numbers of theoretical plates were evaluated in HPLC to get the optimum resolution. To assess the amount of Mifepristone, a simple, specific, selective, and accurate reverse phase HPLC technique was designed and validated according to ICH guidelines in this work. Acetonitrile and water in different quantities were tested, and a ratio of Acetonitrile: Water (70:30) was carefully chosen as a suitable combination which gave accepted resolution and system suitability. The chromatogram of the developed method of standard Mifepristone is shown in Fig. 3.

Specificity
It was found that there was no interference in tablets from the mobile phase and excipient. The chromatogram of the blank solution, sample solution, and standard solution is shown in Figs. 4,5,6.

System Suitability Test
The number of theoretical plates was found to be more than 100,000.The tailing factor was less than 2.0. Hence, the developed method is suitable for estimation of Mifepristone as shown in Tables (1 and 2).

LOD and LOQ
The suggested method's LOD and LOQ were found to be 0.7238 and 0.9562, respectively, at concentrations ranging from 1-6 ppm, as shown in Table 3. The developed method is sensitive enough to detect and quantify the drug at a ppm level.

Linearity
The method was linear over concentration, ranging between 1-6 ppm, as shown in Table 4. The R 2 was 0.9923 and the regression equation Y = 32780 x + 2571.8. The R 2 was greater than 0.99, so the method was stated to be linear and is shown in Fig. 7.

Accuracy
The result of the established method was discovered to be accurate, as the result of the percent recovery was within the range. The percentage recovery was found to be between 99.39% -100.50% as shown in Table 5 and 6.

Precision
The result was demonstrated by intraday and intraday precision at three concentration levels: 1.5 ppm, 3 ppm, and 4.5 ppm. The values of% RSD obtained at each level of both intraday and intraday precision were less than 2, as shown in Tables 7, 8, and 9,10. As a result, the proposed approach was discovered to be precise.

Robustness
The method was stated to be robust as the result of%RSD was less than 2. The% w/w was found between 98% and 102% for both unaltered conditions as shown in Table 11 and altered  conditions as shown in Tables 12, 13, 14. Mifepristone is currently used as a contraceptive. From a literature review, it was found that there is no reported Revere Phase HPLC method for estimation of Mifepristone. This research work describes the development and validation of an analytical method for the analysis of Mifepristone.
Kromasil C18 column of dimensions 250mm×4.6mm particle size 5 µm and Acetonitrile: Water(70:30, v/v) as the mobile phase at wavelength 305nm.Following the ICH guidelines, the devised approach was validated. With a concentration range of 1-6 ppm and a regression coefficient (R 2 ) of 0.9923, the desired approach was found to be linear. The method for determination of Mifepristone using HPLC met the acceptance criteria with respect to selectivity, system suitability, precision, accuracy, linearity, and robustness over a theoretical concentration range of 1-6 ppm. Mifepristone analysis could be performed using the designed and validated method.

CONCLUSIONS
In accordance with ICH guidelines, all validity parameters for the methods developed were studied. All limits have been shown to be exact, specific, targeted, reliable, and reproductive. The procedure should also be used in pure and pharmaceutical dosing for the regular study of mifepristone.

DISCLAIMER
The products used for this research are commonly and predominantly use products in our area of research and country. There is absolutely no conflict of interest between the authors and producers of the products because we do not intend to use these products as an avenue for any litigation but for the advancement of knowledge. Also, the research was not funded by the producing company rather it was funded by personal efforts of the authors.

CONSENT
As per international standard or university standard, patients' written consent has been collected and preserved by the author(s).

ETHICAL APPROVAL
It is not applicable.