Stress degradation studies and stability-indicating TLC-densitometric method of glycyrrhetic acid

Background Glycyrrhetic acid, a pentacyclic triterpenoid, possesses a broad range of pharmacological activities and serves as template to synthesize many bioactive drugs. This paper describes a simple, accurate, and sensitive stability-indicating TLC densitometric method for the determination of glycyrrhetic acid and its degradation product as per the ICH guidelines. Results Separation was carried out on TLC aluminium sheet pre-coated with silica gel 60F254 using chloroform, methanol and formic acid (9:0.9:0.1, v/v). Compact spot for glycyrrhetic acid was found at Rf value of 0.42 ± 0.03. Densitometric analysis was carried out in the absorbance mode at λmax 254 nm. Glycyrrhetic acid was found to be stable to the exposure of base, neutral, oxidation, dry heating treatment and wet heating treatment, but showed degradation under acidic and photochemical conditions. Moreover, fragmentation pattern of glycyrrhetic acid was developed by using a positive ion electrospray ionization quadrupole time-of-flight mass spectrometry (ESI-QqTOF-MS/MS) hybrid instrument. A photo-degraded product was characterized through comparison of mass spectrometric studies with glycyrrhetic acid. Conclusion The developed stability-indicating TLC-densitometric method can be applied for routine analysis of glycyrrhetic acid in the presence of its degradation products.


Background
Glycyrrhiza glabra Linn (Fabaceae) commonly known as Licorice, used worldwide as a natural sweetener and in certain cases, used as a flavor additive in the preparative of candies and foods. Moreover, powdered Licorice root is widely used in herbal drugs in the formulation of Ayurvedic and Chinese medicines. This herb has been reported with various biological activities including antitumour [1], anti-inflamatory [2], antiulcer [3], immunomodulatory [4], antimalarial [5], and anti-hypercholesterolmic [6]. The major constitute of Licorice is glycyrrhizin, which is potassium or calcium salt of glycyrrhizic acid. Glycyrrhizin is a pentacyclic triterpenoid glycoside, which is hydrolyzed to form aglycone glycyrrhetic acid [7]. Glycyrrhetic acid has shown antimicrobial [8] and anti-tumor activities [9]. Individual and simultaneously quantification of glycyrrhizin, 18α-glycyrrhetic acid, and 18β-glycyrrhetic acid in Licorice root and confectionary products have been developed with the help of various analytical tools including HPTLC [10][11][12], HPLC [13,14], microemulsion thin layer chromatography [15] and LC-ESI/MS/MS [16].
Majority of plants contain multiple compounds as active ingredients, which are frequently used in drugs. These active ingredients degraded and may alter their biological activities, therefore extensive study is required for estimation of their stability-indicating properties. The parent drug stability test guidelines (Q1AR) issued by International Conference on Harmonization (ICH) requires the stress testing to elucidate the inherent stability characteristics of the active substance. This guideline emphasizes the testing of those features which are susceptible to change during the storage under the influence of various environmental factors (temperature, light, humidity, oxidizing agent etc.). Quality, safety and efficacy must also be checked with validated stability-indicating testing methods [17,18].
A considerable attention is being paid to the development of stability-indicating TLC-densitometric method as it is fast, reliable and accurate technique. The major advantage of HPTLC over HPLC is that a number of samples can be processed at the same time by using a minute quantity of mobile phase, thus lowering the analysis time and cost per analysis. To the best of our knowledge, there is no report found for the stability-indicating method development of glycyrrhetic acid, while stability-indicating HPTLC method of glycyrrhizic acid has been reported [19]. In continuation of our work on the chromatographic method development of pharmaceutical important compounds [20][21][22][23][24], this paper describes a simple, accurate, and sensitive stability-indicating TLC densitometric method for the determination of glycyrrhetic acid (an active component of Glycyrhiza glabra) and its degradation product as per the ICH guidelines. Moerover, an extensive MS/MS study of glycyrrhetic acid and its photo-degraded products was also conducted using ESI-QqTOF-MS/MS and accurate mass measurements.

Materials
Glycyrrhetic acid was purchased from Tokyo chemical Industry (TCI, Japan). Roots (S-1) and commercial extract (S-2) of Glycyrrhiza glabra were purchased from the local market. Precoated silica gel aluminum sheets (60F254, 20 cm × 20 cm) were perchased from Merck (Germany). Sodium hydroxide was purchased from BioM Laboratories (Cerritos, USA) while hydrochloric acid (HCl) and hydrogen peroxide (H 2 O 2 , 35% v/v) were obtained from Fisher Scientific (UK). Deionized water was obtained from Millipore Milli Q Plus System (Bedford, USA). All solvents used were of HPLC grade and purchased from Merck, (Germany).

HPTLC analysis
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.

ESI-QqTOF-MS/MS analysis
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.

Method validation
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 intraday 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. [15]. 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 H 2 O 2 (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 H 2 O 2 (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.

Method optimization
The TLC procedure was optimized with a view to develop stability-indicating assay method. Both the standard and degraded products were spotted on the TLC plates and developed in different solvent systems. Different mobile phases were tried to resolve glycyrrhetic acid from its degraded product. Different compositions of mobile phase and resulting R f values of standards are summarized in (see Additional file 1: Table S1). Suitable separation with best resolution was achieved with chloroform: methanol: formic acid in the ratio of (9:0.9:0.1, v/v) which showed sharp and symmetrical peaks with R f value of glycyrrhetic acid at 0.42 ± 0.03 (Figure 1). Well defined spots were  obtained when the chamber was saturated with the mobile phase for 10 min at room temperature. The linear regression data for the calibration curve (n = 6) showed a good linear relationship with r 2 ± SD = 0.998 ± 0.0019 over concentration range of 200-1200 ng spot -1 with respect to the peak area. Calibration curve for glycyrrhetic acid is shown in (Additional file 1: Figure S1).

Method validation
Repeatability and reproducibility of the method was determined using intra and inter-day analysis. The % relative standard deviation (R.S.D. %) for glycyrrhetic acid were found to be less than 1% which shows good precision of proposed method ( Table 1). Limit of detection at 3:1 signal to noise ratio was found to be 1.56 ng spot -1 while limit of quantification at 10:1 signal to noise ratio was found to be 4.74 ng spot -1 , which indicate the adequate sensitivity of the method. For robustness analysis, the standard deviation of standard levels (300, 500 and 700 ng) was estimated for each parameter. Mean % R.S.D. was 0.94 for varying in mobile phase composition, 0.79 for varying chamber saturation time, 1.73 for the use of different TLC plates, 2.41 for varying time from spotting to chromatography, 1.25 for varying time from  chromatography to scanning ( Table 2). Spiking studies showed recovery of glycyrrhetic acid (96.7-101.5%) in Licorice root extract ([see Additional file 1: Table S2]). The linear regression data and the method validation results are summarized in Table 3.

Stability-indicating property
The chromatogram of glycyrrhetic acid treated with acid and sunlight showed well-separated peak of glycyrrhetic acid as well as some additional peaks (Figure 2, 3A). No degradation was observed after exposing the glycyrrhetic acid to stressed conditions of base, neutral, hydrogen peroxide, wet heating, and dry heating. Results of the stress degradation studies are summerized in Table 4 Figure S2). Moreover, screening of Licorice root extract (S-1) and its commercially available extract (S-2) showed the presences of its phtoto-degraded product at R f = 0.49 ( Figure 4). MS/MS analysis of glycyrrhetic acid and its photo-degradation products UPLC analysis of photo-degradation reaction mixture of glycyrrhetic acid showed the elution of degraded products within 3 min ( Figure 3B). The retention times (R t ) and proposed formula of all the peaks are shown in Table 5. ESI-QqTOF-MS (positive mode) scan of glycyrrhetic acid and its degradation products showed peak   Table 5. These compounds were not isolated due to further degradation during chromatographic processing, and their structures were not characterized due to miss matched fragmentation pattern with the available structure according to the molecular formula in Dictionary of Natural Products.
The   Figure 5b and Scheme 2, respectively. Degradant (peak 4) showed only 14 amu difference in comparison to glycyrrhetic acid, while MS/MS spectra showed similar fragmentation pattern as observed in glycyrrhetic acid. The structure of peak 4 was proposed as 11-deoxy-glycyrrhetic acid. Isolation of this degraded product was carried out and the structure was also confirmed by 1 H and 13 C NMR techniques.
All proposed fragmentation pathways have been confirmed by accurate mass measurements, which are summarized in (Additional file 1: Table S3). Degraded product was identified through comparative MS/MS studies with glycyrrhetic acid with the help of data base (Dictionary of Natural products). Degradation product was searched in the update Dictionary of Natural Products (DNP, version 20.2) on the basis of deprotonated molecular mass and respective formulae for the identification of compound. In the case of more than one match, the search was narrowed down to the plant species (Glycyrrhiza glabra) and to the other species of Glycyrrhiza.

Conclusion
The developed and validated TLC-densitometric method is precise, accurate, and stability-indicating for the quantification of glycyrrhetic acid in the presences of its degradation products. Glycyrrhetic acid showed extensive degradation in acidic and photochemical stress conditions, while stable to alkaline, neutral, oxidative, dry heating and wet heating stress conditions. A photo-degradation product was also characterized with the help of the ESI-QqTOF-MS/MS experiments combined with accurate mass measurements of precursor and fragment ions. The results showed the importance of appropriate light protection during the drug development process, storage and handling.