Z. rhetsa plants were identified and collected from Medicinal plants garden of Kerala Forest Research Institute (KFRI) of Peechi, Kerala, India and it was authenticated by Prof. T. Christopher, Taxonomist, department of Botany, Kakatiya University, Warangal, Telangana, India. Voucher specimen of the plant was deposited in the author laboratories.

All solvents and reagents used were purchased from Merck, Mumbai, India. Standard drugs nitidine (≥97%), chelerythrine (≥95%) and sanguinarine (≥98%) were purchased from Sigma, Mumbai, India.

1 mg/ml stock solutions of nitdine, chelerythrine and sanguinarine were prepared by dissolving an accurately weighed 10 mg of each standard in 10 ml of 70% methanol in volumetric flask. Further dilutions were made from this stock.

5 gms of dried leafy callus of Z. rhetsa, was taken and extracted with 10 ml of 70% v/v methanol by refluxing for 30 minutes, and then concentrated to dryness by vacuum. Dried extract was re-dissolved in 70% v/v methanol to get sample stock solution.

The method was developed on Camag HPTLC system, consisting of Linomat V 10 AT semi automatic applicator (Muttenz, Switzerland), Camag twin trough chamber (20 cm × 20 cm) for TLC plate development and Camag TLC scanner 3 20AT, equipped with software (version 1.4.3) win CATS and 100 µl capacity Camag Syringe. HPTLC analysis was performed by application of 10 µl of each standard drug on 10 cm × 10 cm, 0.2 mm layer thickness silica gel 60F₂₅₄ (Merck, Germany) pre-coated aluminum plates as 8 mm band width with the help of semi automatic applicator under pressure of nitrogen gas. The space between each band is 6 mm, 15 mm from side and 8 mm from bottom. Development was done through twin trough chamber by linear ascending mechanism. The chamber is pre saturated with mobile phase i.e., ethyl acetate: methanol: water: di ethyl amine (30:5:2:0.5 v/v) for 20 minutes at room temperature in prior to insertion of plate into solvent system. The development distance was 80 mm. After this process the plates were dried. Densitometric scanning at 280 nm was selected the maximum absorption of band, performed with Camag TLC scanner in reflection absorbance made by using a slit width 6 mm × 0.3 mm, data resolution 100 mm∙sec⁻¹, 20 mm∙sec⁻¹ scanning speed. For continuous radiation purpose deuterium lamp was used for UV-Visible region 190 - 800 nm.

5, 10, 20, 40, 80, 160 µg/spot concentrations of standards were loaded on to TLC plate by using semi automatic applicator, which were prepared from standard solutions. Each different concentration was loaded for 3 times on the plate. The plate was developed by using mobile phase and plotted the peak areas of each spot against concentration to obtain the calibration curve.

Slope and standard deviation of the calibration curve were used for calculation of LOD and LOQ.

LOD = 3.3 σ/S

where σ is the standard deviation of the response and S is the slope of the calibration curve.

LOQ = 10 σ/S

Specificity of the method was analyzed by comparing the callus extracts and standards. The spot for nitidine, chelerythrine and sanguinarine was confirmed by comparing their R_f values with standard compounds.

Accuracy of the method was established by performing recovery experiments using the standard addition method. To the pre analyzed samples of callus extract, standard nitidine, chelerythrine and sanguinarine solution was added by spiking at 100 µg level and the mixture was analyzed by the proposed HPTLC method.

Random errors were identified by precision. Results were expressed in relative standard deviation (% RSD). Standard solution of nitidine, chelerythrine and sanguinarine (5, 20, 80 µg/band) were applied. Inter day precision was evaluated by applying each concentration for 3 times on three different days with an interval of 24 hrs. Intraday precision was evaluated by applying each concentration three times within the day.

To test the robustness of the method, deliberately small changes were made in the chromatographic parameters that may affect the performance of the method, i.e., mobile phase composition, mobile phase value. The RSD of the peak areas was calculated for each parameter.

System suitability was carried out to check the reproducibility and resolution of the method. After development, the plates were scanned and peak area of each spot and their R_f values were calculated.

The present study deals with simultaneous quantification of three benzophenanthridine alkaloids namely nitidine, chelerythrine and sanguinarine by using densitometric HPTLC method.

The Present paper aimed to establish optimum mobile phase for TLC analysis, which would shows clear separation of nitidine, chelerythrine and sanguinarine. A number of TLC analysis as preliminary tests to separate above said alkaloids were performed by using different combinations of solvents and modifications of mobile phases. Different methods which were proposed by earlier authors for HPTLC individual quantification of nitidine (Praveena and Veeresham in 2014 and 2015 [19] [20] , Baerhein et al., in 1983) [25] , chelerythrine (Petruczynik et al., in 2008) [26] , sanguinarine (Ghosh et al., in 2005, Garcia et al.,) ( [27] [28] ) for various mobile phases chloroform:methanol (7:1 v/v), n-butanol:pyridine: water (6:4:3 v/v), acetone:diisopropyl ether:diethyl amine (1:1:0.1 v/v), hexane: acetone:methanol (80:15:5 v/v), hexane:ethyl acetate:ammonia (25%) (6:4:0.1 v/v) respectively, similarly simultaneous estimation of sanguinarine and chelerythrine (Bogucka-Kocka and Zalewski in 2017 [21] and Baerhein et al., in 1983 [25] ) for various mobile phases “toluene:ethyl acetate:methanol (83:15:2) and benzene:methanol (6:1), chloroform:ethyl acetate:methanol (2:2:1), were tried with different modifications. Because there is no report on simultaneous estimation of proposed three benzophenanthridine alkaloids, the present study was carried out with a mobile phase of ethyl acetate:methanol:water:diethylamine (30:5:2:0.5), which gave good resolution for nitidine, chelerythrine and sanguinarine with a sharp and well defined peaks at R_f = 0.28, 0.49 and 0.73 and when the chamber was saturated with mobile phase for 20 min at room temperature (25˚C ± 2˚C) during HPTLC determination of nitidine, chelrythrine and sanguinarine from plant tissue culture extracts. The plate was visualized under UV light at 280 nm without any derivatization. Identity of nitidine, chelerythrine and sanguinarine bands in sample chromatograms was confirmed by the comparison of chromatograms obtained from the sample with that obtained from the standard chromatograms (Figure 1 and Figure 2) and also by comparing retention factor (Rf―0.28, 0.49 and 0.73). The peaks corresponding to nitidine, chelerythrine and sanguinarine from the sample solutions had the same retention factor as that of three standard drugs. Praveena and Veeresham in 2014 and 2015 [19] [20] reported that the nitidine has R_f value 0.28 from the roots and tissue culture extracts of Toddalia asiatica. Similarly Bogucka and Zalewski in 2017 [21] reported that the R_f values of chelerythine (0.35) and sanguinarine (0.45) from roots and herbs of Chelidonium majus. The present results are also in line with the reports of Praveena and Veeresham [20] and Bogucka and Zalewski [21] .