Stress degradation studies and stability-indicating TLC-densitometric method of glycyrrhetic acid
© Musharraf et al.; licensee Chemistry Central Ltd. 2013
Received: 24 October 2012
Accepted: 14 January 2013
Published: 17 January 2013
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.
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.
The developed stability-indicating TLC-densitometric method can be applied for routine analysis of glycyrrhetic acid in the presence of its degradation products.
KeywordsGlycyrrhetic acid Stress degradation Electrospray ionization quadrupole time-of-flight mass spectrometry
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 , anti-inflamatory , antiulcer , immunomodulatory , antimalarial , and anti-hypercholesterolmic . 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 . Glycyrrhetic acid has shown antimicrobial  and anti-tumor activities . 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 [10–12], HPLC [13, 14], microemulsion thin layer chromatography  and LC-ESI/MS/MS .
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 . In continuation of our work on the chromatographic method development of pharmaceutical important compounds [20–24], 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.
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 (H2O2, 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).
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 λmax 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.
Results and discussion
Precision and accuracy for quality control standard of glycyrrhetic acid
Robustness Study (n = 3)
Mobile phase composition
Nature of TLC
Time from spotting to chromatography
Time from chromatography to scanning
Amount detected (ng ± S.D.)
Amount detected (ng ± S.D.)
Amount detected (ng ± S.D.)
Amount detected (ng ± S.D.)
Amount detected (ng ± S.D.)
306.16 ± 3.78
304.45 ± 3.66
`302.81 ± 2.72
303.39 ± 2.86
301.7 ± 5.67
512.97 ± 4.15
514.78 ± 3.32
515.1 ± 5.06
511.88 ± 4.01
517.9 ± 5.46
716.63 ± 5.62
711.2 ± 3.82
717.35 ± 10.24
720.48 ± 4.99
715.58 ± 5.76
Summary of validation parameters
Data of standard Glycyrrhetic acid (at λmax254nm)
200-1200 ng spot-1
Correlation coefficient r2 ± SD
0.998 ± 0.0019
Limit of detection (LOD)
1.56 ng spot-1
Limit of quantitation (LOQ)
4.74 ng spot-1
Y = mx + c
5.736x + 664.3
Slope ± SD
5.76 ± 0.58
Intercept ± SD
664.3 ± 25.54
Intra-day analysis (n = 3), % RSD
Inter-day analysis (n = 3), % RSD
Summary of stress degradation studies of glycyrrhetic acid
Rfof degraded products
Compound remained (ng /1000ng±S.D., n=3)
Acidic hydrolysis a
0.08,0.1, 0.12, 0.64
Basic hydrolysis a
Oxidation at room temp
Neutral hydrolysis a
Wet heating a
MS/MS analysis of glycyrrhetic acid and its photo-degradation products
LC-ESI-MS/MS analysis of photo-degradation mixture of glycyrrhetic acid
Characteristic MS/MS fragment ions
467, 449, 431, 421,403, 385, 259, 233, 187
451,423,405,387, 317, 271,235,189,175
Glycyrrhetic acid ( Peak 3)
453, 425, 407, 389, 317, 271, 263, 235, 217, 189, 149
439, 411, 393, 343, 249, 203, 191
The ESI-MS/MS spectrum of [M+H]+ ions of m/z 485 (Peak 1) and m/z 487 (Peak 2) , showed only 14 and 16 amu increase from glycyrrhetic acid, respectively. Characteristic MS/MS fragments are summarized in 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.
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.
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.
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