Chromatography was performed by spotting the samples in the form of bands of width 6 mm with a CAMAG 100 μL syringe on pre-coated silica gel TLC aluminum sheets using Linomat V (CAMAG Muntenz, Switzerland) autosampler. A constant sample application rate of 0.1 μL/s was employed and the space between the two bands was 9.1 mm. 10 mL of mobile phase (chloroform: methanol: formic acid, 9:1:0.1, v/v) was used for linear ascending development and chromatogram was allowed to move to a distance of 80 mm, in twin trough glass chamber (CAMAG). The chamber saturation time for mobile phase was 10 minutes at 25 ± 2°C with relative humidity 42 ± 5%. The developed TLC plate was dried with the help of air dryer for 4 min. Video densitometry was carried out with CAMAG Reprostar III and scanning was performed on CAMAG TLC Scanner III at λ
max 254 nm which operates in reflection absorbance mode by winCATS software. Deuterium lamp with range between 190 nm and 400 nm was used as source. Evaluation was carried out via peak areas using linear regression.
UPLC analysis were performed on Agilent 1200 Series, Rapid Resolution LC (RRLC) system, comprised of an Agilent 1260 binary pump with degasser, a high performance 1260 ALS autosampler with 1290 thermostat, a thermostated column compartment (1290 TCC), and a diode-array detector VL (1260 DAD VL). Data acquisition and integration was controlled by Agilent Technologies Chem-Station software. An Agilent Zorbax XDB-C18 column (50 × 3 mm i.d., 1.8 μm) was used. The mobile phase was a binary gradient system prepared from water (eluent A) and acetonitrile (eluent B), properly filtered and degassed for 15 min in ultra sonic bath before to use. The gradient program was: 50–60% B from 0–0.5 min, 60–65% B from 0.5–1 min, 65–70% B from 1–2 min, 70–75% B from 2–2.5 min, 75–100% B from 2.5–3 min, and 100–50% B from 3–4 min. The injection volume and flow rate were 5 μL and 1 mL min-1, respectively. The assay was performed at 40°C. The detection wavelength of DAD was 254 nm.
The standard and its photo-degraded products were dissolved in methanol and working dilution was prepared in 50:50 acetonitrile–water containing 0.1% tetrafluoro acetic acid (TFA). Analysis was performed by electrospray ionization (ESI) and collision-induced dissociation (CID), positive ion mode, on Qq-TOF-MS/MS instrument (QSTAR XL mass spectrometer Applied Biosystem/MDS Sciex, Darmstadt, Germany) coupled with 1100 HPLC system (Agilent). High-purity nitrogen gas was used as the curtain gas and collision gas delivered from Peak Scientific nitrogen generator. The ESI interface conditions were as follows: ion spray capillary voltage of 5500 V, curtain gas flow rate 20 L min−1, nebulizer gas flow rate 30 L min−1, DP1 60 V, DP2 10 V, and focusing potential of 265 V. The collision energy was swept from 20 to 45 eV for MS/MS analysis. Sample was introduced into the mass spectrometer using a Harvard syringe pump (Holliston, MA) at a flow rate of 5 μL min−1.
Calibration curve of glycyrrhetic acid
Stock solution of glycyrrhetic acid (1 mg mL-1) was prepared in methanol. The stock solution was further diluted to obtain working standard of various concentrations including 200, 400, 600, 800, 1000, and 1200 ng spot-1 for calibration curve. Each working standard was spotted three times on TLC plate. The spotted plate was developed as mentioned in previous section. This practice was repeated six times to get an average standard calibration curve. The data of peak areas plotted against the corresponding concentrations were treated by linear least-square regression analysis.
Validation of the developed HPTLC method was carried out according to the ICH guidelines for sensitivity, precision, accuracy, robustness. The sensitivity of the method was determined with respect to LOD, LOQ, and correlation coefficient. Working solutions containing 200–1200 ng of glycyrrhetic acid were spotted on TLC aluminum sheet. In order to estimate limit of detection and limit of quantitation, calibration curve was used and were calculated by using following formula: LOD = 3.3 δ/S, LOQ = 10 δ /S where, δ = the residual standard deviation of regression line or the standard deviation of Y-intercept of regression line, S = the slope of the calibration curve. The LOD and LOQ were calculated as 3 and 10 times of the noise level, respectively. Furthermore, both were experimentally determined by diluting the known concentration of glycyrrhetic acid standard until the average responses were approximately three and ten times of the standard deviation of the responses for six replicate determinations. For method precision, the Intra- and inter-day variation for the determination of glycyrrhetic acid were carried out at three different concentration levels of 300, 500 and 700 ng spot-1. Repeated analyses were carried out in a same day for intra-day analysis while the same practice was repeated next day for inter-day analysis. Intra- and inter-day analyses were performed to check the repeatability and reproducibility of the method, respectively and results were statistically evaluated in terms of % R.S.D. In order to check the robustness, following parameters were intentionally changed within the range of ± 5% at three different concentration levels (300, 500 and 700 ng); mobile phase composition, time from spotting to chromatography, time from chromatography to scanning and chamber saturation time and using different type of TLC plates. Licorice root extract was prepared according to Cui Shufen et al. . All samples were spotted on TLC plate and developed as mentioned in previous section. The accuracy of the method was assessed by performing recovery study at three different levels of glycyrrhetic acid (50%, 100%, and 150%).
Preparation of forced degradation products
Stress degradation studies were performed using parallel synthesizer (Smart Start Synthesizer, Chem Speed Ltd., Switzerland) with sixteen reaction vessels. Stock solution containing 100 mg of glycyrrhetic acid in 100 mL of methanol was prepared. This stock solution (1 mg mL-1) was used for forced degradation studies in parallel synthesizer by refluxing the reaction mixtures for two hours at 80°C. After the reactions were completed, all the solutions were preserved at −20°C. Average peak areas of active components were analyzed after triplicate analysis.
For acidic hydrolysis, 3 mL of methanolic stock solution of glycyrrhetic acid (1 mg mL-1) were mixed with 3 mL of each 1N and 5N HCl separately and the resultant mixture solutions were refluxed for two hours at 80°C in the dark, in order to prohibit the possible degradative effects of light. 2 μL (1000 ng spot-1) of 1N and 5N HCl treated solutions of glycyrrhetic acid were applied on TLC sheet in triplicate and densitogram were developed. For alkaline hydrolysis, 3 mL of methanolic stock solution of glycyrrhetic acid (1 mg mL-1) were mixed with 3 mL of each 0.1N, 1N and 5N NaOH separately and the resultant mixture solutions were refluxed for two hours at 80°C in the dark. 2 μL (1000 ng spot-1) of 0.1N, 1N and 5N NaOH treated solutions of glycyrrhetic acid were applied on TLC sheet in triplicate and densitogram were developed. For neutral hydrolytic condition, 3 mL of methanolic stock solution of glycyrrhetic acid (1 mg mL-1) were mixed with 3 mL of milli Q water and the resultant solution was refluxed for two hours at 80°C in the dark. 2 μL (1000 ng spot-1) of treated solution of glycyrrhetic acid was applied on TLC sheet in triplicate and densitogram were developed. For wet heating, 3 mL of methanolic stock solution of glycyrhhetic acid was refluxed for two hours at 80°C in the dark. 1 μL (1000 ng spot-1) of resultant solutions were applied on TLC sheet in triplicate and densitogram were developed. Oxidative degradation was carried out by taken 3 mL stock solutions of glycyrrhetic acid mixed with 3 mL of H2O2 (35% v/v) and the resultant solutions were refluxed for two hours at 80°C in the dark and 2 μL (1000 ng spot-1) of resultant solutions were applied on TLC sheet in triplicate and densitogram were developed.
Photo-degradation studies were carried out by the exposure of stock solution of glycyrrhetic acid to direct sunlight for three days from 8 to 18 hrs at 30 ± 2°C. 1 μL (1000 ng spot-1) of resultant solutions were applied on TLC sheet in triplicate and densitogram were developed. Dry heating was performed by keeping standard glycyrrhetic acid in oven at 90°C for 4 hrs. 1 mg of treated standard was dissolved in 2 mL of methanol and 2 μL (1000 ng spot-1) of resultant solution of glycyrrhetic acid was applied on TLC plate in triplicate and densitogram were developed. For oxidation reaction at room temperature, 3 mL stock solution of glycyrrhetic acid was mixed with 3 mL of H2O2 (35% v/v) and the resultant solutions was kept for 24 hours at room temperature. 2 μL (1000 ng spot-1) of resultant solutions were applied on TLC sheet in triplicate and densitogram were developed.