INTRODUCTION:

Ursolic acid is a carboxylic acid present in a wide variety of plants in the form of a free acid or an aglycone of triterpene saponins¹. It is pentacyclic triterpenoid compound which naturally occurs in a large number of vegetarian foods, medicinal herbs, and plants. Ursolic acid is a white to light yellow crystal powder. Chemically, it is (3b)-3-hydroxy-urs-12-en-28-oic acid. Prominent pharmacological actions of Ursolic acid are anti-inflammatory, anti-tumor, anti-ulcer, anti-viral, etc. Additionally, ursolic acid has also been shown to retard the growth of cancer cells². Literature survey revealed that various analytical methods were reported for determination of Ursolic acid like HPTLC^3-7, RP-HPLC^8-16, UPLC-MS¹⁷, LC-MS^18-19, LC-MS-MS²⁰, tandem MS²¹, GC²² and GC-MS²³. No stability indicating method has been reported for estimation of Ursolic acid. The aim of the present work was to develop and validate a stability indicating HPTLC method for estimation of ursolic acid.

MATERIALS AND METHOD:

Materials

Analytical pure samples of ursolic acid procured from Yucca Enterprises, Mumbai was used in the study. All reagents and chemicals used in the study were of analytical grade (LOBA, India).

Instrument

The HPTLC system (Camag, Switzerland) consisted of a Linomat V semi-automatic sprayer fitted with 100 μL syringe and connected to a nitrogen cylinder, a twin trough chamber (20×10 cm), dip tank (20×10 cm with lid), a TLC scanner IV with win CATS software (version 1.4.7).

Preparation of standard solution

A standard solution of pure ursolic acid was prepared by dissolving the required amount of drug in methanol to obtain final concentration of 1000 ng/μl from which working standard solution (100 ng/μl) was prepared by suitable dilution.

Optimized Chromatographic conditions

· Stationary phase: TLC silica gel 60 F₂₅₄ precoated on aluminium sheets

· Mobile phase: Toluene: Ethyl acetate: Formic acid (7.0: 1.0: 0.1, v/v/v)

· Chamber saturation time: 10 minutes

· Slit dimension: 4 × 0.3 mm

· Syringe capacity: 100 µl

· Band width: 6 mm

· Derivatization: Anisaldehyde-sulphuric acid and heating at 110⁰C for 10 minutes

· Scanning wavelength: 530 nm

Method Validation²⁴

The method was validated in compliance with ICH Q2(R1)guidelines. The following parameters were used for validation of the developed method.

Linearity and range

Different volumes (2, 3, 4, 5 and 6 μl) of working standard solution were spotted on TLC plate in six replicates to obtain concentration ranging from 200 to 600 ng/spot of ursolic acid. The peak areas obtained were plotted against the corresponding concentrations to construct the calibration curve.

Precision

Precision was determined by performing Intra-day and Inter-day studies. The Intra-day Precision was determined by analyzing 3 different concentrations of standard solution (200, 400, and 600 ng/spot) of ursolic acid for six times on the same day. The Inter-day Precision was determined by analyzing 3 different concentrations (200, 400, and 600 ng/spot) for six different days. % RSD for peak areas was calculated.

Specificity

Peak purity was assessed to evaluate the specificity of the method. The standard bands were scanned at three different levels, i.e., peak start (s), peak middle (m), and peak end (e) positions. The specificity of the method was also determined by performing forced degradation studies.

Limit of Detection (LOD) and Limit of Quantitation (LOQ)

The LOD and LOQ were determined based on the calibration curve. The standard deviation of the y-intercepts and slope of the regression lines were used.

Robustness

The effects of deliberate variations in method parameters like the composition of the mobile phase, saturation time, detection wavelength and run distance were evaluated in this study. The effect of these changes on both the R_f values and peak areas was evaluated by calculating the relative standard deviations (%RSD) for each parameter.

Stability studies²⁵

To evaluate the stability indicating properties of the developed HPTLC method, forced degradation studies were carried out. The standard drug was subjected to acid, base, oxidation, heat, humidity and photo-degradation studies.

Acid induced degradation study

25 mg of ursolic acid was dissolved in 3ml of methanol. To the above solution, 10 ml of 0.1 N HCl was added and refluxed for 1 hour at 40⁰C. The acid degraded sample was neutralized, and diluted up to 25 ml with methanol to give expected concentration of 1000 ng/μl of ursolic acid. The above solution was further diluted with methanol to give expected concentration of 100 ng/μl. The resultant solution (4 μl) was applied on TLC plate. The chromatogram was run as described earlier.

Base induced degradation study

25 mg of ursolic acid was dissolved in 3ml of methanol. To the above solution, 10 ml of 0.1 N NaOH was added and refluxed for 1 hour at 40⁰C. The acid degraded sample was neutralized, and diluted up to 25 ml with methanol to give expected concentration of 1000 ng/μl of ursolic acid. The above solution was further diluted with methanol to give expected concentration of 100 ng/μl. The resultant solution (4 μl) was applied on TLC plate. The chromatogram was run as described earlier.

Oxidation induced degradation study

10 mg of ursolic acid was dissolved in 3ml of methanol. To the above solution, 10 ml of 3.0 % v/v H₂O₂ solution was added to give expected concentration of 1000 ng/μl of ursolic acid and allow to stand for 1 hour at room temperature. The above solution was further diluted with methanol to give expected concentration of 100 ng/μl of ursolic acid. The resultant solution was applied on TLC plate, (4 μl). The chromatogram was run as described earlier.

Heat degradation study

10 mg of ursolic acid was stored at 100°C for 7 hours in oven. It was transferred to 10 ml volumetric flask and volume was made up to the mark with methanol to give expected concentration of 1000 ng/μl of ursolic acid. The above solution was further diluted with methanol to give expected concentration of 100 ng/μl of ursolic acid.. The resultant solution was applied on TLC plate, (4μl). The chromatogram was run as described earlier.

Photo-degradation study

10 mg of ursolic acid was spread in 1 mm thickness on petri dish and was exposed to direct sunlight for 21 hours. It was transferred to 10 ml volumetric flask and volume was made up to the mark with methanol to give expected concentration of 1000 ng/μl of ursolic acid. The above solution was further diluted with methanol to give expected concentration of 100 ng/μl of ursolic acid. The resultant solution was applied on TLC plate, (4 μl). The chromatogram was run as described earlier.

Humidity induced degradation study

10 mg of ursolic acid was spread in 1 mm thickness on petri dish and was kept in humidity chamber (40 ⁰C and 75 % RH) for 48 hours. It was transferred to 10 ml volumetric flask and volume was made up to the mark with methanol to give expected concentration of 1000 ng/μl of ursolic acid. The above solution was further diluted with methanol to give expected concentration of 100 ng/μl of ursolic acid. The resultant solution was applied on TLC plate, (4 μl). The chromatogram was run as described earlier.

RESULTS AND DISCUSSION:

Optimization of Method

TLC procedure was optimized to develop a stability-indicating assay. The mobile phase toluene: ethyl acetate: formic acid (7.0: 3.0: 0.1, v/v/v) give compact band of ursolic acid at R_Fvalue of 0.45 ± 0.02 (Figure 1).

Figure 1: HPTLC chromatogram of standard ursolic acid

Method Validation

Linearity (n=6)

The linear regression data for the calibration curve as shown in Table 1 showed a good linear relationship over the concentration range 200–600 ng/spot with respect to peak area.

Table 1: Linear regression data for Calibration curve

Parameters	Ursolic acid
Linearity range (ng/spot)	200-600
Correlation coefficient	0.9986
Slope	8.5247
Intercept	200.17
Linearity Equation	y = 8.5247x - 200.17

Precision (n=6)

Intra-day precision and inter-day Precision of the developed method was evaluated at three different concentrations. % RSD of peak area of spots was used to evaluate method precision and the results obtained are shown in Table 2.

Specificity

The result of peak purity of ursolic acid obtained is shown in Figure 2. From the spectra, purity of the peak for Ursolic acid r_(s,m)=0.9997, r_(m,e)=0.9998 was obtained. So, it was concluded that no other compounds were present in the standard.

Figure 2: Spectra of Ursolic acid

LOD and LOQ

The limits of detection and quantitation were found to be 10.02 and 30.36 ng/spot, respectively which indicates the sensitivity of the developed method.

Robustness (n=3)

The low values of % RSD obtained after introducing small changes in mobile phase composition and volume were indicative of the robustness of the method. The results of robustness are shown in Table 3.

Stability Indicating Property

Acid Induced degradation study

The chromatogram of the acid degraded sample of ursolic acid showed two degradation peaks D1 and D2 at R_f0.24 and 0.69 respectively with 25.27 % degradation of standard (Figure 3).

Base Induced degradation study

The chromatogram of the base degraded sample of ursolic acid showed two degradation peaks D1 and D2 at R_f0.17 and 0.23 respectively with 22.82 % degradation of standard (Figure 4).

Table 3: Robustness study of Ursolic acid

Parameters	Mean area ± SD	% RSD
Mobile Phase Composition (±0.2 ml)	3244.62 ± 33.04	1.02
Saturation time (± 1 min)	3262.38 ± 34.57	1.06
Run distance (±5 mm)	3260.88 ± 42.51	1.30
Detection wavelength (± 2 nm)	3258.81 ± 32.90	1.01

Oxidation Induced degradation study

The chromatogram of the oxidative degraded sample of ursolic acid showed two degradation peaks D1 and D2 at R_f0.33 and 0.56 respectively with 28.52 % degradation of standard (Figure 5).

Heat degradation study

The chromatogram of the heat degraded sample of ursolic acid showed two degradation peaks D1 and D2 at R_f0.20 and 0.33 respectively with 13.70 % degradation of standard (Figure 6).

Photo-degradation study

The chromatogram of the photo-degraded sample of ursolic acid showed one degradation peak D1 at R_f0.28 with 11.69 % degradation of standard (Figure 7).

Humidity Induced degradation study

The chromatogram of the humidity degraded sample of ursolic acid showed one degradation peak D1 at R_f0.20 with 9.76 % degradation of standard (Figure 8).

Degradation peaks were well resolved from standard peak of ursolic acid. The peak of ursolic acid was not significantly shifted in the presence of the degradation peaks, which indicated the stability-indicating nature of the method. Ursolic acid in acid, base, hydrogen peroxide, heat, photo and humidity degraded standard drug solutions were scanned to access the peak purity. (Figure 9, 10)

Figure 3: Chromatogram of acid treated ursolic acid

Figure 4: Chromatogram of base treated ursolic acid

Figure 5: Chromatogram of H₂O₂ treated ursolic acid

Figure 6: Chromatogram of heat treated ursolic acid

Figure 7: Chromatogram photo-degraded ursolic acid

Figure 8: Chromatogram of humidity induced ursolic acid

Figure 9: Overlay spectra of Ursolic acid under acid, base and oxidative degradation condition

Figure 10: Overlay spectra of Ursolic acid under themal, sunlight and humidity degradation condition

Table 4: Result of force degradation study

Degradation condition	Time (hours)	% Drug degradation	Peak purity data r(_s,_m), r(_m,_e)
Acid (0.1 N HCl at 40⁰C)	1	25.27	0.9997, 0.9997
Base (0.1 N NaOH at 40⁰C)	1	22.82	0.9996, 0.9998
Oxidative (3.0 % v/v H₂O₂at room temperature)	1	28.52	0.9997, 0.9998
Heat (100⁰ C)	7	13.70	0.9996, 0.9999
Photo (Direct sunlight)	21	11.69	0.9997, 0.9998
Humidity (37⁰ C, 75 % RH)	48	9.76	0.9998, 0.9996

CONCLUSION:

In the present study, the drug was subjected to acid hydrolysis, base hydrolysis, oxidation, photolysis, dry heat and humidity to apply stress conditions. The results of the stress studies indicated the specificity of the method and it can separate the drug from its degradation products. Thus, it can be used as a stability-indicating method. The method was validated as per ICH Q2(R1) guideline. Developed stability indicating HPTLC method is considered to be valid, simple, precise, sensitive, specific and robust.

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Received on 28.04.2013 Modified on 15.05.2013

Asian J. Research Chem. 6(6): June 2013; Page 546-551

Concentration (ng/spot)	Intraday Precision		Interday Precision
Concentration (ng/spot)	Mean Area ± SD	% RSD	Mean Area ± SD	% RSD
200	1440.49 ± 15.77	1.09	1438.77 ± 17.62	1.22
400	3249.72 ± 40.65	1.25	3242.39 ± 42.53	1.31
600	4875.26 ± 52.70	1.08	4857.44 ± 56.03	1.15