Journal of Advanced Pharmaceutical Technology & Research

: 2012  |  Volume : 3  |  Issue : 4  |  Page : 210--215

Quantitative analysis of Glycyrrhizic acid from a polyherbal preparation using liquid chromatographic technique

Amit K De, Sriparna Datta, Arup Mukherjee 
 Department of Chemical Technology, University College of Science and Technology, University of Calcutta, Kolkata, West Bengal, India

Correspondence Address:
Amit K De
92, A.P.C. Road, Kolkata, West Bengal


Glycyrrhizic acid has been used in Indian traditional medicine for ages. It is obtained from the root extract of Glycyrrhizaglabra. There is seasonal variation of Glycyrrhizic acid content in the roots of the plant. So a proper method for quantification of the same is necessary from the polyherbal preparation available in the market. A simple, rapid, sensitive and specific reverse phase high performance liquid chromatographic method have been developed for the quantitative estimation of glycyrrhizic acid from polyherbal preparation containing aqueous root extract of Glycyrrhizaglabra using a photodiode array detector. The identity confirmation was carried out using mass spectrometry. Baseline resolution of the glycyrrhizic acid peak was achieved on a reverse phase C18 column (125 mm × 4.0 mm, 5 μ) using an isocratic mobile phase consisting of 5.3 mM phosphate buffer and acetonitrile in the ratio 65:35 v/v. Chromatograms were monitored at 252 nm.5.3 mM phosphate buffer was replaced with 0.5mM ammonium acetate buffer in the mobile phase when MS detector was used. The method was found to be linear in the concentration range of 12.4 to124 μg/ml with a correlation co-efficient of 0.999. The limit of detection and the limit of quantitation were 3.08 μg/ml and 10.27 μg/ml respectively. The average recovery from three spike levels was 99.93 ± 0.26%. Identity confirmation of the chromatographic peak was achieved by electrospray ionization mass spectrometry and similar molecular ion peak was obtained for both sample and standard. The developed method is suitable for the routine analysis, stability testing and assay of glycyrrhizic acid from polyherbal preparations containing aqueous extracts of Glycyrrhizaglabra.

How to cite this article:
De AK, Datta S, Mukherjee A. Quantitative analysis of Glycyrrhizic acid from a polyherbal preparation using liquid chromatographic technique.J Adv Pharm Technol Res 2012;3:210-215

How to cite this URL:
De AK, Datta S, Mukherjee A. Quantitative analysis of Glycyrrhizic acid from a polyherbal preparation using liquid chromatographic technique. J Adv Pharm Technol Res [serial online] 2012 [cited 2022 Jan 18 ];3:210-215
Available from:

Full Text


Yastimadhu (Glycyrrhizaglabra) is being used as expectorant, emollient, anti-inflammatory, anti-hepatotoxic and anti-bacterial from ages in Indian Ayurveda, [1],[2] Chinese and Japanese system of medicine. It is used in ayurvedic preparations like churnas, avaleh, asav and arista. [3] It is also used as cosmetic ingredient for its skin whitening properties. The bioactive components reported from G. glabra include liquiritin, isoliquiritin, liquiritinapioside, glabridin, glycyrrhetic acid and glycyrrhizic acid (GA). [4],[5],[6],[7]

GA is a major triterpene saponin found mainly from the roots of Glycyrrhizaglabra [Figure 1]. It has wide spectrum of medicinal applications which includes hepato-protective, anti-inflammatory, antidotal, anti-allergic, immunomodulatory and antiviral and all these have been substantiated by modern pharmacological analysis. [8],[9],[10],[11],[12],[13] It has been found to be effective in the amelioration of peptic ulcer diseases, inflammatory bowel disease [14],[15],[16] in human immunodeficiency virus infection [17],[18],[19] and anti-angiogenic properties in tumor progression. [20] GA is commonly used as sweetening and flavoring agent in food, tobacco and confectionary products and is about 170 times sweeter than sucrose. [21]{Figure 1}

Extensive literature survey revealed only one analytical method for the estimation of GA from extract that has been properly substantiated by a mass spectroscopic analysis. [22] However, several chromatographic method of estimation has been reported where an UV detector has been used and the eluent is monitored at 254 nm following an isocratic elution. A C18 column is used with a mobile phase consisting of either methanol or acetonitrile as organic component along with an aqueous phase containing either an acid modifier like acetic acid or phosphoric acid. [23],[24],[25],[26],[27],[28],[29],[30],[31],[32],[33],[34],[35],[36] GA being a triterpinesaponin, its content in the plant tissue varies significantly at different time periods of the year. Thus a proper quantification of the same in the marketed polyherbal preparation is essential. In our study we have developed a simple, rapid and sensitive method for the estimation of GA from a polyherbal preparation containing extract of Glycyrrhizaglabra. We have used LC- DAD method for quantification and LC MS-MS system with gradient elution for identity confirmation. The earlier methods [37],[38],[39] are much more tedious and time consuming compared to the newly developed method which is rapid and reproducible and has been validated as per ICH guidelines. [40]

 Materials and Methods


The solvents used were of analytical reagent grade and HPLC grade (for chromatography) and were purchased from Spectrochem India limited. Phosphoric acid of HPLC grade (Spectrochem, India) was used for pH adjustment of the eluent. Glycyrrhizic acid mono-ammonium salt (purity = 98.1%) was purchased from Sigma Aldrich India Ltd. The polyherbal formulation (cough syrup) containing Glycyrrhizaglabra extract as one of the principle component was purchased from market with batch number HD514, manufacturing date January 2012 and expiry date December 2014.


Instrumentation and chromatographic conditions for LC- DAD and LC-MS/MS analysis (API mode)

The chromatographic determinations were carried out on a Waters (Waters, USA) binary gradient system equipped with Waters 515 pump (two numbers), a manual Rheodyne injector port attached with a 20 μl loop and Waters 2996 PDA detector. The system control and data acquisition was carried out using Empower 2 software (Waters, USA). The separation was carried out in reverse phase Kromasil C18 column (125 mm × 4.0 mm, 5 μ; Akzonobel, USA). The mobile phase was a mixture of 65%, 5.3 mM phosphate buffer (pH = 3.0) and 35% acetonitrile which was filtered through 0.45 μm Millipore filter paper. The flow rate was 1.0 ml/minute and the column was maintained at ambient temperature. The column effluent was monitored at 252 nm with PDA detector. Prior to chromatographic separation both the standard and the sample were filtered through 0.2 μm membrane filter (Pall Life science, India).

For identity confirmation the analysis was carried out on a LC-MS/MS equipped with Shimadzu SILHTC autosampler-pump module (Shimadzu, Japan) and Mass detector with Atmospheric Pressure Ionization -ESI (API2000, Applied Biosystems, California). For LC-MS/MS analysis a different chromatographic condition was maintained. Waters X-bridge column (Waters X-Bridge, C18, 4.6 mm × 50 mm) was used. The 5.3 mM phosphate buffer (pH = 3.0) was replaced with 0.5 mM ammonium acetate buffer and a linear gradient elution at a flow rate 1.2 ml/minutes was used. The LC MS/MS system was operated at source gas pressure 100 psi, exhaust gas pressure 50 psi and curtain gas pressure 30 psi. The ESI was operated at Ion spray voltage 5500 V. The source temperature was 200°C. The cone voltage was 50 V, focusing potential 200 V and entrance potential 10 V.

Preparation of standard solution and sample solution

The GA stock solution (620 μg/ml) was prepared by dissolving 15.5 mg of glycerrhyzic acid mono-ammonium salt in 25 ml of hot water. The stock solution was diluted to the range 124 μg/ml to 12.4 μg/ml of GA for analysis. A six point calibration curve [Figure 2] was drawn for linearity study and for quantification purpose. Each dilution of the stock was injected in triplicates. The least square method was used. The sample solution was prepared by diluting the sample cough syrup to a dilution range 100 μg/ml.{Figure 2}

Validation of the Developed Method

The analytical method was validated as per USP and ICH guidelines. The studied parameters were accuracy, precision, linearity, range, ruggedness and robustness. To ensure reliability and accuracy of the proposed method, recovery studies were carried out by mixing a known quantity of the standard drug with the sample at three different concentration levels (10, 20 and 30% above assay value labeled as A, B and C). Six injections of the standard solutions were done to study the precision of the method. The linearity of the method was established by triplicate injections of standard solution in the concentration range of 12.4 to124 μg/ml. The intra-day precision was calculated using six injections at the higher concentration range (124 μg/ml) on the same day. These studies were repeated with solutions of different concentration on different days to obtain the inter-day precision. The specificity of the method was studied from purity plot of PDA detector. The limit of detection (LOD) and the limit of quantitation (LOQ) were determined by injecting progressively low concentrations of standard solution under optimized chromatographic conditions. [41] Ruggedness of the method was studied by carrying out experiment on instruments of different make. The robustness of the method was determined by making slight changes in chromatographic conditions like composition (±5%) and pH (±0.1%) of mobile phase.

Statistical Analysis

The statistical analysis was carried out on Sigma plot software (Version 8.02 SPSS Inc., USA) and MS Excel 2007. The results were represented as mean ± S.D. values for three to six replicate injections.

 Results and Discussions

LC-DAD and LC MS/MS identity confirmation

The newly developed simple method was more specific and less time consuming compared to the previously reported methods. [37],[38],[39],[42],[43] The maxima in case of LC-DAD analysis was observed at 252 nm. The chromatogram was subjected to peak purity analysis in order to study any co-elution. The LC-DAD analysis revealed the peak to be pure and spectrally homogenous with peak purity angle 0.357. This was less than the peak purity threshold 0.438. A reasonable resolution between the GA peak and the closely eluting peaks was observed in the sample [Figure 3].{Figure 3}

The average retention time of GA peak was 8.5 ± 0.09 minutes (± S.D.; n=3). The representative chromatograms presented in [Figure 3] showed the analyte peaks to be symmetrical and well resolved from the closely eluting peaks. In case LC-MS/MS analysis the mass spectrum represented a high degree of identity confirmation. The presence of molecular ion peak at m/z = 839 for both the sample and the standard spectrum was taken as confirmation that the peak observed was of GA in both sample and standard solutions [Figure 4].{Figure 4}

Validation of the Developed Method

The liquid chromatographic method was validated as per USP and ICH guidelines and the various parameters were evaluated which are presented as follows.


A HPLC method was considered to be specific if it ensures that the measured peak was only due to the substance being analyzed and it was free from potential impurities or co-eluting components. The specificity with regard to other co-eluting components of GA was investigated. Sufficient resolution between GA peak and the closely eluting peaks was observed under optimized chromatographic conditions [Figure 3]d. In all cases the purity angle was less than the peak purity threshold indicating the absence of any co-eluting peaks. The resolution factor between the GA peak and closely eluting peak in the chromatogram of polyherbal preparation of G. glabra was 1.9.

Linearity and Sensitivity (LOD and LOQ)

LOD denotes the lowest amount of analyte in a sample could be detected but not necessarily quantified and LOQ denotes the lowest amount of analyte that could be quantified with precision and accuracy. [44] Reference standard solutions of six different concentrations ranging between 12.4 μg/ml to 124 μg/ml were injected into the chromatographic system and the area of the major peak was recorded separately (six replicates each). An area verses concentration curve was drawn with the concentration on X-axis and the peak area on the Y-axis. The linear regression parameters were slope 21823 ± 33.4103 and Y- intercept 1855.3 ± 8168.0187 (P < 0.0001). The correlation coefficient for GA was r 2 = 0.9999. The residual plot analysis demonstrated that the residual values were randomly distributed around zero. This confirms the choice of linear model. The sensitivity of the method was evaluated on the basis of LOD and LOQ values which were 3.08 μg/ml and 10.27 μg/ml respectively.


The precision was measured on the basis of repeatability and intermediate precision. The repeatability was measured on the basis of six replicate injection of the 124 μg/ml GA solution on the same day. The intermediate precision was obtained by triplicate injection of the sample solution on three different days. The intra-day and inter-day precision values were calculated from the linearity curve and the observed % RSD value was found to be < 2.0 [Table 1].{Table 1}


The accuracy of a method was defined as a percentage of systematic error and was calculated as the deviation agreement between the measured value and the true value. The acceptable value for deviation was 15% of the actual value. [45] The sample solution was spiked at three levels and the recovery was 99.99%, 99.84% and 99.97% respectively [Table 2]. The average recovery was 99.93 ± 0.26%.{Table 2}


The robustness was tested in order to evaluate the variation of analytical result due to deliberate changes in analytical conditions. [46] The changes in operational parameters (change in person) did not lead to significant changes in the performance of chromatographic analysis. The observed variation was only 0.98%. Thus the robustness of the study could be established.

System Suitability

System suitability was evaluated to verify if the chromatographic system was adequate for performing the analysis. The approximate results were theoretical plates (N = 11791), capacity factor (k = 2.30), peak asymmetry, or tailing factor (t = 0.10). The values for these parameters were satisfactory in accordance with the literature [47] [Table 1].

Solution Stability

The prepared solution was found to be stable for seven days for evaluation purpose only when kept at 4°C.


The developed RP-HPLC method was validated in terms of linearity and precision in the studied concentration range. The retention time of GA was only 8.5 minutes. Thus this rapid, simple, precise and accurate method could be used for routine analysis, stability testing and estimation of GA from herbal formulations available in the market.


The authors are thankful to Dr. Swagata Kumar Biswas for his kind help in carrying out the LC MS/MS analysis.


1Ray P, Gupta H, Roy M. Su?rutaSamhitá (A Scientific Synopsis). Indian National Science Academy, India;1980. p. 226.
2Sharma PV. A Treatise on Principles and Practices of Ayurvedic Medicine. Vol. 3. Chaukhambha Publishers, India;2002. p. 240-56.
3Ayurvedic Formulary of India.Vol. 1. Govt of India, India;1986; p. 127.
4Cinatl J, Morgenstern B, Baur G, Chandra P, Rabenau H, Doerr HW. Glycyrrhizin, an active component of liquorice roots, and replication of SARS-associated coronavirus. Lancet 2003;361:2045- 6.
5Hoever G, Baltina L, Michaelis M, Kondratenko R, Baltina L, Tolstikov GA, et al. Antiviral activity of glycyrrhizic acid derivatives against SARS-coronavirus. J MedChem2005;48:1256-9.
6Yin CY, So HT, Kadir KA. Effects of Glycyrrhizic Acid on Peroxisome Proliferator-Activated Receptor Gamma (PPARã), Lipoprotein Lipase (LPL), SerumLipid and HOMA-IR in Rats.Hindawi Publishing Corporation PPAR Research;2010.
7Kamei J, Nakamura R, Ichiki H, Kubo M. Antitussive principles of Glycyrrhizae radix, a main component of the Kampo preparations Bakumondo-to (Mai-men-dong-tang). Eur J Pharmacol. 2003;469:159-63.
8Armanini D, Karbowiak I, Funder JW. Affinity of liquorice derivatives for mineral ocorticoid and glucocorticoid receptors.ClinEndocrinol (Oxf) 1983;19:609-12.
9Okimasu E, Moromizato Y, Watanabe S, Sasaki J, Shiraishi N, Morimoto YM, et al. Inhibition of phospholipase A2 and platelet aggregation by glycyrrhizin, an anti-inflammation drug. Acta Med Okayama 1983;37:385-91.
10Ohuchi K, Kamada Y, Levine L, T surufuji S. Glycyrrhizin inhibits prostaglandin E2 production by activated peritoneal macrophages from rats. Prostaglandins Med 1981;7:457-63.
11 Kao T, Shyu M, Yen G. Glycyrrhizic Acid and 18β-Glycyrrhetinic acid inhibit inflammation via PI3K/Akt/GSK3β signaling and glucocorticoid receptor activation. J Agric Food Chem 2010;58:8623-9.
12Raèková L, Janèinová V, Petríková M, Drábiková K, Nosá¾ R, Štefek M, et al . Mechanism of anti-inflammatory action of liquorice extract and glycyrrhizin. Nat Prod Res 2007;21:1234-41.
13Bhattacharjee S, Bhattacharjee A, Majumder S, Majumdar SB, Majumdar S. Glycyrrhizic acidsuppresses Cox-2-mediated anti-inflammatory responses during Leishmaniadonovani infection. J A ntimicrob Chemother 2012;67:1905-14.
14Yuan H, Ji WS, Wu KX, Jiao JX, Sun LH, Feng YT. Anti-inflammatory effect of DiammoniumGlycyrrhizinate in a rat model of ulcerative colitis. World J Gastroenterol 2006;12:4578-81.
15Sun Y, Cai TT, Shen Y, Zhou XB, Chen T, Xu Q. Si-Ni-San, a traditional Chinese prescription, and its active ingredient glycyrrhizin ameliorate experimental colitis through regulating cytokine balance. Int Immunopharmacol 2009;9:1437-43.
16Zapesochnaya GG, Zvonkova EN, Kurkin VA, Kazakova EV, Pervykh LN, Sheichenko BI, et al. Some properties of glycyrrhizic acid. Chem Nat Comp 1994;30:236-50.
17Pliasunova OA, Egoricheva IN, Fediuk NV, Pokrovskiĭ AG, Baltina LA, Murinov I, et al. The anti-HIV activity of beta-glycyrrhizic acid. Vopr Virusol 1992;37:235-8.
18Hattori T, Ikumatsu S, Koivo A, Matsushita S, Maeda Y, Hada M, et al. Preliminary evidence for inhibitory effect of glycyrrhizin on HIV replication in patients with AIDS. Antiviral Res 1989;11:255-61.
19Clerq ED. Current lead natural products for the chemotherapy of human immunodeficiency virus (HIV) infection. Med Res Rev 2000;20:323-49.
20Kim KJ, Choi JS, Kim KW, Jeong JW. The anti-angiogenicactivities of glycyrrhizic acid in tumor progression. Phytother Res 2012 Aug 16. doi: 10.1002/ptr.4800. [Epub ahead of print].
21Mizutani K, Kuramoto T, Tamura Y, Ohtake N, Doi S, Nakaura M, et al. Sweetness of glycyrrhetic acid 3-O-beta-D-monoglucuronide and the related glycosides. Biosci Biotechnol Biochem 1994;58:554-5.
22Hennell JR, Lee S, Khoo CS, Gray MJ, Bensoussan A. The determination of glycyrrhizic acid in Glycyrrhizauralensis Fisch. ex DC. (ZhiGan Cao) root and the dried aqueous extract by LC-DAD. J Pharm Biomed Anal 2008;47:494-500.
23Chauhan SK, Singh BP, Agrawal S. Estimation of glycyrrhizin from Glycyrrhizaglabra and its extract by high pressure liquid chromatography. Indian Drugs 1999;36:521-3.
24Hiraga Y, Endo H, Takahashi K, Shibata S. High-performance liquid chromatographic analysis of licorice extracts. J Chromatogr A 1984;292:451-3.
25Wang P, Li SF, Lee HK. Determination of glycyrrhizic acid and 18-b-glycyrrhetinic acid in biological fluids by micellarelectrokinetic chromatography. J Chromatogr A 1998;811:219-24.
26Tian M, Bi W, Row KH. Solid-phase extraction of liquiritin and glycyrrhizic acid from licorice using ionic liquid-based silica sorbent. J Sep Sci 2009;32:4033-9.
27Kondratenko RM, Baltina LA, Mikhailova LR, Danilov VT, Gabbasov TM, Murinov YI, et al. Obtaining glycyrrhizic acid and its practically useful salts from a commercial licorice root extract. Pharm Chem J 2005;39:84-8.
28Ong ES, Len SM. Pressurized hot water extraction of berberine, baicalein and glycyrrhizin in medicinal plants. Anal Chim Acta 2003;482:81-9.
29Pan X, Liu H, Jia G, Shu YY. Microwave-assisted extraction of glycyrrhizic acid from licorice root. Biochem Eng J 2000;5:173-7.
30Sagara K, Ito Y, Oshima T, Kawaura M, Misaki T. Application of ion-pair high-performance liquid chromatography to the analysis of Glycyrrhizin in Glycyrrhizae radix. Chem Pharm Bull 1985;33:5364-8.
31Tsai TH, Chen CF. Determination of three active principles in licorice extract by reversed-phase high-performance liquid chromatography. J Chromatogr A 1991;542:521-5.
32Okada K, Tanaka J, Miyashita A, Imoto K. Variation of chemical constituents in processed licorice roots: Quantitative determination of saponin and flavonoid constituents in bark removed and roasted licorice roots. Yakugaku Zasshi 1981;101:822-8.
33Kuwajima H, Taneda Y, Chen WZ, Kawanishi T, Hori K, Taniyama T, et al. Variation of chemical constituents in processed licorice roots: Quantitative determination of saponin and flavonoid constituents in bark removed and roasted licorice roots. Yakugaku Zasshi 1999;119:945-55.
34Kitagawa I, Chen WZ, Taniyama T, Harada E, Hori K, Kobayashi M, et al. Quantitative determination of constituents in various licorice roots by means of high performance liquid chromatography. Yakugaku Zasshi 1998;118:519-28.
35Liu S, Jiang X, Zheng Y, Xu P. Determination of glycyrrhizin in glycyrrhiza and its preparations by ion-pair HPLC. HuaXi YiKe DaXueXueBao 1993;24:111-4.
36Beasley TH, Ziegler HW, Bell AD. Separation of major components in licorice using high-performance liquid chromatography. J Chromatogr A 1979;175:350-5.
37Tian M, Yan H, Row KH. Simultaneous extraction and separation of liquiritin, glycyrrhizic acid, and glabridin from licorice root with analytical and preparative chromatography. Biot Biop Engin 2008;13:671-6.
38Shen S, Chang Z, Liu J, Sun X, Hu X, Liu H. Separation of glycyrrhizic acid and liquiritin from Glycyrrhizauralensis Fisch extract by three-liquid-phase extraction systems. Separation and purification technology 2006;53:216-23.
39Niu G, Xie Y, Lou J, Liu H. Isolation and purification of glycyrrhizic acid with solvent extraction. Separation and purification technology 2005;44:189-6.
40Validation of analytical procedures: Text and methodology Q2 (R1). ICH harmonized tripartite guideline. Geneva, Switzerland. 2005;1-13.
41Ahuja S, Scypinski S. Hand book of modern pharmaceutical analysis. Academic Press, U.S.A.;2005. p. 415-42.
42Krähenbühl S, Hasler F, Krapf R. Analysis and pharmacokinetics of glycyrrhizic acid and glycyrrhetinic acid in humans and experimental animals. Steroids 1994;59:121-6.
43Shen S, Chang Z, Liu J, Sun X, Hu X, Liu H. Simultaneous Determination of glycyrrhizic acid and liquiritin in Glycyrrhizauralensis extract by HPLC with ELSD detection. J Liq Chromatogr Relat Technol 2006;29:2387-97.
44Akhlaq M, Khan GM, Wahab A, Khan A, Hussain A, Nawaz A, et al. A simple high-performance liquid chromatographic practical approach for determination of flurbiprofen. J Adv Pharm Technol Res 2011;2:151-5.
45Singh G, Pai RS, Pandit V. Development and validation of HPLC method for the determination of trans-resveratrol in spiked human plasma. J Adv Pharm Technol Res 2012;3:130-5.
46United States Pharmacopeia. USP Convention, Rockville,MD, 2008. p. 1225.
47Snyder RL, Kirkland J,Glajch L. Practical HPLC Method Development. JonhWiley and Sons;U.S.A.1997.