Development of validated stability-indicating HPTLC method for the estimation of ulipristal acetate in bulk and dosage form

Materials

UPA was obtained as a generous gift from Mylan Laboratories Ltd, Telangana, India. Fibristal^TM (5 mg label claim by Akumentis Healthcare Ltd) was purchased from local retail pharmacy. Analytical grade solvents, viz. methanol and dichloromethane, were purchased from Merck Millipore (Mumbai, India).

Standard solution preparation

Stock solution was prepared by accurately weighing UPA (10 mg) and dissolving it in 10 ml of methanol (concentration 1,000 μg ml⁻¹). It was further diluted 10 times with methanol to get the concentration of 100 μg ml⁻¹ and further a 20–150 ng/spot concentration range was applied on thin layer chromatography (TLC) plate with Linomat V autosampler.

Sample solution preparation

The average weight of 20 tablets of Fibristal^TM was calculated and finely powdered. The quantity equivalent to 10 mg of UPA was taken and dissolved in 10 ml methanol; the content was sonicated for 15 minutes before being centrifuged at 2,000 rpm for 5 minutes. The supernatant was then analyzed on TLC plate for drug content of 90 ng/spot.

Chromatographic conditions

The prepared samples of UPA and its marketed preparation were spotted as 8 mm bandwidth on precoated silica gel 60 F_254, aluminum plates (20 × 10 cm with 250 μm thickness; E. Merck, Germany) using Camag Linomat V autosampler consisting of Camag microliter syringe (Switzerland). Methanol was employed for prewashing the plates, followed by activation for 5 minutes before chromatographic application. Twin trough glass chambers (Camag) were saturated using saturation pads for 20 minutes, using mobile phase dichloromethane: methanol (9.5:0.5, v/v). Migration time was 15 minutes covering migration distance of 7 cm. The densitogram scanning was carried out at 312 nm on Camag TLC scanner III, vision CATS version 2.5.18262.1 software. The deuterium lamp was utilized as source of UV radiation between 200 and 400 nm.

Analytical method validation

Linearity and range

Standard solution of the drug in the range of 30–150 ng/spot was applied on plate for the development and then analyzed to evaluate linearity (as per guidelines of ICH 5 spots were spotted). The calibration curve was plotted for peak area vs. drug concentration with the help of vision CATS software and the analysis of linear regression was carried out.

Sensitivities [limit of detection (LOD) and limit of quantification (LOQ)]

Method sensitivities were determined in terms of LOD and LOQ. LOD indicates the minimal amount of an analyte that can be identified but not normally quantified, whereas LOQ is the minimal quantity of the drug, which is estimated with proper precision and accuracy. The LOD and LOQ were calculated by using the following formula as per ICH guidelines:

$\mathrm{LOD}\mathrm{=}\frac{3.3\times \sigma }{\mathit{S}}$

$\mathrm{LOQ}\mathrm{=}\frac{10\times \sigma }{\mathit{S}}$

where ‘σ’ is the standard deviation of linear responses based on the calibration curve and ‘S’ is the slope of the calibration curve. Standard deviation is calculated through the residual value between a set of observed and predicted values shown by points in a regression analysis.

Accuracy

The accuracy of the analysis is presented as the percentage recovery and was carried out by spiking 80%, 100%, and 120% of the standard drug to the formulation by standard addition method in triplicates.

Precision

Three different concentrations of UPA (70, 90, and 110 ng/spot) were applied and analyzed for intraday and interday precision.

Specificity

Specificity is the capability of a method to identify the analyte when other components are expected to present. It was determined by checking the interference of the drug with the diluent and mobile phase. For this standard solution and sample solution of ulipristal (each having 90 ng/spot), mobile phase and diluent was applied on the plate and chromatographic run was assessed.

Degradation study

Forced degradation studies provide the approach to analyze the stability of drug samples. The ICH guidelines that are applicable to forced degradation studies are ICH Q1A (stability testing of new drug substances and products) and ICH Q1B (photostability testing of new drug substances and products).

Acid and alkaline degradation

Acid degradation study was carried out by dissolving the drug in 0.1 N methanolic hydrochloric acid solution to get the stock solution of 1 mg ml⁻¹ concentration and refluxing it at 60°C in dark for 1 hour to avoid any possible side effects of light. However, alkaline degradation of UPA was carried out for the concentration of 1 mg ml⁻¹ using 0.01 N methanolic sodium hydroxide solution for 15 minutes in the dark. Solution was applied on a precoated plate of TLC after dilution to achieve concentration of 100 ng/spot and then chromatogram was run.

Oxidative degradation

Drug solution of concentration 1 mg ml⁻¹ was prepared with methanol and solution was exposed to 10 ml of 6% v/v H₂O₂ at room temperature for 1 hour inthe dark, after dilution it was applied on the precoated TLC plate to get 100 ng/spot.

Thermal and photochemical degradation

For thermal degradation, UPA (10 mg) was kept in oven at 60°C for 4 hours, and its 1 mg ml⁻¹ solution was prepared in methanol and analyzed on a precoated plate of TLC to get a concentration of 100 ng/spot. While for photochemical degradation study, the drug was exposed in direct sunlight for 48 hours, after subsequent dilutions with methanol it was applied on plate to get concentration of 100 ng/spot.

Method development and validation

The HPTLC method was optimized for developing stability indicating method, using dichloromethane: methanol (9.5:0.5, v/v) as mobile phase, a sharp and symmetrical peak of UPA was observed with retention factor (R_f) of 0.60 ± 0.02 (Fig. 2). The current guidelines of the ICH Q2 (R1) were adopted for method validation.

Linearity

Calibration curve of UPA (Fig. 3 and Fig. 4) exhibited linear correlation between peak area and concentration in the range of 30–150 ng/spot (five data points). The regression data of graph was found to be linear with best correlation r² ≥ 0.998 (Table 1).

LOD and LOQ

The findings of LOD and LOQ regarding UPA were 9.577 and 29.022 ng/spot, respectively (Table 1).

Accuracy

Accuracy was calculated in terms of % recovery at each addition level with % relative standard deviation (RSD) (Table 2). The mean percentage recovery was determined as 98.56%.

Precision

Three concentrations (70, 90, and 110 ng/spot) of the drug were analyzed in triplicates for performing repeatability and interday precision. The consequence of the repeatability indicates no significant variation in intraday (%RSD = 1.14) and interday (%RSD = 1.68). The values were within the acceptable range (Table 1).

Analysis of marketed formulation

Densitogram of marketed tablet of UPA (Fibristal^TM) revealed only one spot at R_f 0.60 showing no interference from excipients of the tablet. The experimental finding regarding amount of ulipristal in tablets was estimated to be 99.6% which showed good conformity with label claim of tablet (5 mg per tablet), thereby re-emphasizing the fact of no interference with any excipients and indicating the method suitability for routine analysis of ulipristal and its formulation.

Figure 2. Densitogram of UPA (Rf = 0.60 ± 0.02) at 312 nm. [Click here to view]

Table 1. Summary of the HPTLC method validation parameters. [Click here to view]

Table 2. Accuracy studies of UPA. [Click here to view]

Figure 3. Linearity graph of UPA. [Click here to view]

Degradation studies

The UPA exhibited varied degradation pattern under different stress conditions.

Acidic degradation

In acidic degradation, two degradants were observed at R_f 0.49 and 0.83 along with peak of UPA (Fig. 5A).

Alkaline degradation

In alkaline degradation, seven degradation products were resolved having R_f value of 0.01, 0.03,0.30, 0.39, 0.45, 0.50, and 0.86, as shown in Fig. 5B, along with drug peak with 20.20% degradation.

Figure 4. 3D view densitogram of UPA (standard: 30, 60, 90, 120, 150 ng/ spot; track 2–6) (sample: two spots of 90 ng/spot; track 7 and 8). [Click here to view]

Figure 5. (A) Densitogram of acid degradation: peak 1 (ulipristal Rf: 0.58), peak 2 (degradant Rf: 0.49), and peak 3 (degradant Rf: 0.83); (B) densitogram of base degradation: peak 1 (ulipristal), peak 4 (degradant Rf: 0.01), peak 5 (degradant Rf: 0.03), peak 6 (degradant Rf: 0.30), peak 7 (degradant Rf: 0.39), peak 8 (degradant Rf: 0.45), peak 9 (degradant Rf: 0.50), and peak 10 (degradant Rf: 0.86); (C) densitogram of peroxide degradation: peak 1 (ulipristal Rf: 0.58), peak 11 (degradant Rf: 0.04), and peak 10 (degradant Rf: 0.86); (D) densitogram of thermal degradation: peak 1 (ulipristal Rf: 0.61) and peak 10 (degradant Rf: 0.86); (E) densitogram of photochemical degradation: peak 1 (ulipristal Rf: 0.59), peak 4 (degradant Rf: 0.01), peak 12 (degradant Rf: 0.75), and peak 10 (degradant Rf: 0.86). [Click here to view]

Oxidative degradation

UPA was susceptible to oxidative degradation amounting to 17.25% of the degradation and the degradants were identified at R_f 0.04 and 0.86 (Fig. 5C).

Thermal degradation

Thermal degradation at 60°C up to 4 hours resulted in 10.02% degradation, the degradant was resolved at R_f 0.86 (Fig. 5D), which was same as obtained in alkaline and peroxide induced degradation study.

Photochemical degradation

Photochemical degradation led to 19.29% degradation, with three peaks at R_f 0.01, 0.75, and 0.86, as shown in Fig. 5E. One degradant at R_f 0.86 was common in all degradation studies except in acidic degradation. The outcome of the degradation studies is summarized in Table 3.

Table 3. Degradation study of UPA. [Click here to view]