The fresh bark of the tree of Adenanthera pavonina was collected in October 2013 from LM College of Pharmacy, Ahmedabad, Gujarat. The plant was authenticated at the Department of Botany, Gujarat University, Ahmedabad, Gujarat. The bark of Adenanthera pavonine was shade dried, pulverized using a mixer grinder, and passed through a 200-mesh sieve to obtain a fine powder. The bark powder (500 g) was weighed, transferred to a round bottom flask (RBF), and dissolved in 500 mL of various solvents like petroleum ether, hexane, chloroform, ethanol, methanol, and ethyl acetate. Finally, petroleum ether was selected as an extraction solvent because it showed maximum extraction and it showed maximum spot on TLC plate. The bark powder was suspended in petroleum ether and was refluxed for 3 h at 85˚C. The slurry was filtered through Whatman filter paper no.42. It was further re-extracted (500 mL×4), following the same procedure. All the filtrates were combined, concentrated to 100 mL via distillation, and evaporated to dryness.

All the solvents,including methanol, toluene, chloroform, petroleum ether, diethyl ether, ethyl acetate, rectified spirit acetone, n-hexane, benzene, ortho-phosphoric acid, and the chemicals silica gel 60–120 #, sodium hydroxide, hydrochloric acid used were of analytical grade and obtained from Finar Chemicals Ltd., Ahmedabad, India.

The HPTLC system (CAMAG, Switzerland) included CAMAG Linomat IV semi-automatic spotting device, CAMAGTLC Scanner3, CAMAG Twin Trough Chamber (10× 10cm), and CAMAGCATS4 Software. TLC aluminum sheets pre-coated with silica gel 60 F₂₅₄ 20× 20 cm² with 0.2 mm layer thickness were purchased from E. Merck, Darmstadt, Germany.

The chromatographic method was developed on pre-coated silica gel 60 F_{254 -}aluminum sheets (20× 20 cm²) with 0.2 mm layer thickness as stationary phase using different mobile phase compositions. Before applying the sample, the TLC plates were pre-washed with methanol and activated in an oven at 60 °C for 5 minutes. The solutions were applied on a TLC plate in the form of bands of 6 mm width under a stream of nitrogen gas using a CAMAG Linomat IV semiautomatic spotting device with a constant application rate of 10 sec/µL. The space between the two spots was kept at 5 mm. The plates were developed in a twin trough chamber saturated with the mobile phase for 15 min at 25 ± 2 °C using ascending development technique to 80 mm in length. After development, the plates were air-dried and derivatized with vanillin –phosphoric acid solution. Then the plates were heated for 3 min at 110 °C in a hot air oven, and densitometric scanning was done using CAMAG TLC scanner 3 using CATS 4 software at 550 nm.

HPTLC preliminary TLC analysis was performed using n-Hexane: ethyl acetate (90:10v/v) as the mobile phase. The plate was sprayed with respective reagents like 5% conc. sulphuric acid, p-anisaldehyde, vanillin- phosphoric acid, and Liebermann-Burchard to detect steroids or triterpenoids. Several standards of triterpenoids and steroids available in the laboratory were used for comparison with the isolated compound I and II. The R_f value of compound I was found as 0.43 ±0.02, which was similar to LPL, while the R_f value of compound II was found as 0.55 ± 0.02, which was similar to STIGMA (Figure 1).

Calibration was carried out with the dilutions of standards, and triplicate measurement was performed. The linearity of each standard was achieved by plotting peak areas versus standard concentrations (Figure 2 and Figure 3). Peak identification was attained by comparing retention time (RT) and UV absorbance (λmax).

Validation was carried out according to International Conference on Harmonization (ICH) guidelines. 13

Linearity and range of the method were determined by spotting serial dilution of STIGMA and LPL in the concentration range of 100-500 ng/spot and 400-2000 ng/spot, respectively. There is no reported optimized range as these are not used in any dosage forms. Three sets of each solution were evaluated to obtain a calibration curve. Linearity was obtained by estimating the correlation coefficient, slope, and intercept of the curves.

The present method was validated for intraday and interlay precision. Intraday precision was determined in triplicate with the same method on the same day for three different concentrations of STIGMA (100, 300 and 500 ng/spot) and LPL (400, 1200 and 2000 ng/spot). The interlay precision of the method was verified by performing a similar method on different days under the same set of experimental situations. The repeatability of the sample application and calculation of the peak area for the analyte were articulated in terms of the % RSD.

The repeatability of the peak area was assessed by applying 10 µL of working mixed standard solution of STIGMA (200 ng/spot) and LPL (800 ng/spot) on the pre-coated plate. The plate was developed using selected chromatographic conditions. The developed spot was scanned six times, peak areas were recorded, and % RSD was calculated.

The repeatability of the sample application was assessed by applying six spots of 10 µL of working mixed standard solution of STIGMA (200 ng/spot) and LPL (800 ng/spot) on the pre-coated plate. The plate was developed using selected chromatographic conditions. The developed spots were scanned, peak areas were recorded, and % RSD was calculated.

The accuracy of an analytical method was determined by calculating the recovery of STIGMA and LPL at three different concentration levels (50%, 100%, and 150%) following the standard addition method. The amount of the drug was calculated by employing corresponding linear equations. The average recovery obtained at all three levels was reported as % recovery.

LOD is the lowest concentration of an analyte in a sample that can be detected but not necessarily quantified under the stated analytical conditions. In contrast, LOQ is the ability of the analytical method to detect analytes quantitatively in the presence of other compounds. It was calculated as LOD= (3.3 x σ)/S and LOQ= (10 x σ)/S, where ‘σ’ is the standard deviation of the Y-intercept and ‘S’ is the mean slope of the calibration curve line.

The first isolated compound was a white crystalline solid with a melting point in the range of 204–208 °C, which was near the melting point of LPL. 13 The second isolated compound was a white solid with a melting point in the range of 164–167 °C, which was similar to the melting point of stigmasterol reported in the literature. 14 When checked qualitatively using HPTLC with various spraying reagents, vanillin-phosphoric acid spray confirmed the presence of the compounds. Several standard protocols for the steroids and triterpenoids available in the laboratory were used for comparison purposes. The R_f value for the isolated compound I (0.43)was similar to LPL, and that of compound II (0.55) matched with the STIGMA.

On the silica gel 60 F₂₅₄ pre-coated plate, mixed standard solutions were applied as a 6 mm band in the concentration range of 100–500 ng/spot of STIGMA and 400–2000 ng/spot for LPL. The plate was developed using selected chromatographic conditions, and the respective peak areas were recorded (Figure 1). A graph was plotted using peak area vs. concentration of STIGMA and LPL, respectively. Both graphs were linear with R² values of 0.9914 and 0.9871 for STIGMA and LPL, respectively

Figure 2

Figure 3

The sample analysis of the dried extract revealed that STIGMA and LPL were present in the bark in the concentration of 0.0743 % w/w and 0.146 % w/w, respectively.

Validation of the developed HPTLC method revealed linearity in the range of 100–500 ng/spot for STIGMA with an R² value of 0.99 and 400–2000 ng/spot for LPL with an R² value of 0.9871. The method was precise as % RSD for all the studies was less than 2%. The results are presented in the Table 3 and Table 4.

The accuracy of the method was further checked using the standard addition method at 50 %, 100 %, and 150 %. Recovery was observed in the range of93.8 to 97.2 % for STIGMA and 94.9 to 95.6 % for LPL. Recovery study data is shown in Table 5 and Table 6. The LOD and LOQ were calculated as11.74 ng/spot and 35.58 ng/spot, respectively, for STIGMA and 90.44 ng/spot and 274.06 ng/spot for LOQ.