Simultaneous quantification of stevioside and rebaudioside A in different stevia samples collected from the Indian subcontinent.

BACKGROUND: A high performance thin layer chromatographic (HPTLC) method was developed for simultaneous estimation of stevioside and rebaudioside A in Stevia rebaudiana samples collected from different regions of Indian subcontinent.
MATERIALS AND METHODS: THE SEPARATION WAS ACHIEVED BY USING ACETONE: ethyl acetate: water (5:4:1, v/v/v) as the solvent system on precoated silica gel 60 F(254) TLC plates. The densitometric quantification of stevia glycosides was carried out at wavelength 360 nm in absorption mode after spraying with anisaldehyde sulphuric acid as detecting reagent.
RESULTS: The well resolved peaks for stevioside and rebaudioside A were observed at R(f) values 0.31± 0.02 and 0.21± 0.02 respectively. The calibration curves were found linear with a wide range of concentration 100 - 2000 ng spot(-1) with good correlation coefficient 0.996 and 0.991 for stevioside and rebaudioside A, respectively.
CONCLUSIONS: The proposed method was validated as per the ICH (International Conferences on Harmonization) guidelines and found simple, sensitive, economic, reproducible, robust and accurate for quantitative analysis of stevia glycosides, which can be applied for quality control of stevia as well as to check.

Stevia rebaudiana Bertoni (Family Compositae) is one of the most valuable tropical medicinal plants, native to South America.[1] It is among the 154 members of the genus Stevia, which produces sweet stevia glycosides.[23] Stevia leaves as well as their extracts and pure compounds are currently in wide use for the preparation of several medicines, food products, and nutraceuticals. The study on leaves proved it to be antioxidant,[4] anti-inflammatory,[5] antimicrobial,[6] and was found safe due to low mutagenicity and toxicity.[7-10] The principal chemical constituents of stevia leaves are stevioside, rebaudioside A–F, and isosteviol.

A wide range of analytical techniques have been employed to assess the distribution and level of sweet diterpenoid glycosides in S. rebaudiana. These include thin layer chromatography,[11-14] high-performance thin layer chromatography (HPTLC),[15] over-pressured layer chromatography,[16] droplet countercurrent chromatography,[17] capillary electrophoresis,[1819] and high-performance liquid chromatography (HPLC).[20-23] HPTLC method for quantification is cheap, less time consuming, and high throughput. Earlier report on stevia used HPTLC, which was less sensitive for determination of very little amount of stevia glycosides in plant samples, and also the method employed acetic anhydride as the detecting reagent, which is harmful, restricted in many countries, and a US DEA List II solvent.

Hence, it was thought worthy to develop a simple, safe, reliable, and sensitive HPTLC method for the determination of stevioside and rebaudioside A in different samples collected from the Indian subcontinent to investigate their variability when grown at different locations under different environmental conditions. The methodology can be used for selection of plants yielding high level of active constituents by screening large number of samples for commercial production [Figure 1].

Figure 1

Structure of stevioside and rebaudioside A

Experimental

Collection of samples and chemicals

The samples of S. rebaudiana were collected from different regions of the Indian subcontinent and the same were identified by Dr. Altaf Ahmad, Botanist, Department of Botany, Hamdard University. The samples were also authenticated in Bioactive Natural Product Laboratory, Hamdard University, by running TLC using marker compounds of the plant. The plant samples were dried in hot air oven below 60°C. Standard stevioside and rebaudioside A (98%, w/w) were obtained from Sami Labs Ltd., Bangalore (India) as gift samples, and other chemicals and reagents used were of analytical grade (AR) and procured from Merck Ltd. Mumbai (India).

Preparation of standard and sample solutions

The stock solutions of stevioside and rebaudioside A were prepared in methanol (1.0 mg/ml). The samples were prepared by weighing 500 mg of each of the powdered drug and macerating them separately in 25 ml of methanol for 4 h. The macerated extracts were sonicated for 30 min. The methanolic extract was then filtered through Whatman filter paper number 41 to remove any insoluble components and concentrated under vacuum. The residue was taken in a 5.0 ml volumetric flask and the volume was adjusted using methanol.

HPTLC instrumentation and procedure

The samples were spotted in the form of a band (3.0 mm) with a Camag microlitre syringe on TLC aluminum plate precoated with silica gel 60 F-254 (20 × 10 cm with 0.2 mm thickness; E. Merck, Darmstadt, Germany) using a Camag Linomat V sample applicator. A constant application rate of 120 nl/sec was employed and the space between two bands was 8.0 mm. The slit dimension was kept at 3.0 × 0.20 mm, and 20 mm/sec scanning speed was employed for scanning. The development was carried out in linear ascending manner in twin trough glass chamber (20 × 10 cm) saturated with mobile phase composed of acetone:ethyl acetate:water (5:4:1, v/v/v). The optimized chamber saturation time for mobile phase was 20 min at room temperature and the chromatogram was developed up to the length of 85 mm. Subsequent to the development, the TLC plate was air dried and sprayed with anisaldehyde–sulfuric acid reagent. The plate was kept in oven at 110°C for 5 min followed by air drying and further scanned at 360 nm using Camag TLC scanner III [Figures 2 and 3].

Figure 2

HPTLC chromatogram of samples of stevia extract in daylight after spraying with anisaldehyde–sulfuric acid solution (track no. corresponds to 1,2: Bangalore; 3,4: Dehradun (a); 5,6: Dehradun (b); 7,8: Delhi; 9,10: Haridwar; 11,12: Indore; 13,14: Jammu (a); 15,16: Jammu (b); 17,18: Kangra; 19,20: Mohali; 21,22: Surat)

Figure 3

HPTLC chromatogram of different stevia samples at wavelength 360 nm (A - Bangalore; B - Dehradun (a); C - Dehradun (b); D - Delhi; E - Haridwar; F - Indore; G - Jammu (a); H - Jammu (b); I - Kangra; J - Mohali; K - Surat)

Calibration curve

The calibration curve was prepared by applying 0.1, 0.2, 0.4, 0.6, 0.8, 1, 2, 2.5 μl of stock solution of stevioside and rebaudioside A in triplicate on TLC plate in order to obtain concentrations from 100 to 2500 ng/spot. The data of peak area versus drug concentration were treated by linear least-square regression by WINCATS software.

Validation

The method was validated for the specificity, accuracy, precision, robustness, limit of detection (LOD) and limit of quantitation (LOQ) parameters as per the International Conferences on Harmonization (ICH) guidelines. The statistical analysis was done using Excel 2007 (MS Office).

Specificity

The specificity of the method was established by analyzing standard drug and samples. The spot of stevioside and rebaudioside A in samples was confirmed by comparing the R f and the UV spectra of the peaks in samples with those of the standard. The peak purity of sample was examined by comparing the spectra at peak start, peak apex, and peak end position of the spot.

Accuracy

Accuracy was determined in terms of recovery. The recovery studies of stevioside and rebaudioside A were carried out by standard addition method. The pre-analyzed samples were spiked with extra 50%, 100%, and 150% of the standard stevioside and rebaudioside A, and the mixtures were re-analyzed by the proposed method. The experiment was conducted in triplicate.

Precision

The intra-day and inter-day variations for the determination of stevioside and rebaudioside A were determined at two concentration levels, 400 and 800 ng/spot. The relative standard deviation (% RSD) of peak area was calculated by repeating the experiment.

Robustness and ruggedness

The effects on the results by introducing small changes in the working conditions like temperature, relative humidity, and mobile phase composition were examined. Mobile phases having diverse composition of acetone:ethyl acetate:water (52:38:10 and 48:42:10, v/v/v) as well as small change in detection wavelength (357 and 363 nm) were used at two concentrations, 400 and 800 ng/spot. The % RSD of peak area was calculated.

Limit of detection and limit of quantification

LOD is defined as the lowest concentration of analyte in a sample that can be detected and LOQ is the lowest concentration of analyte in a sample that can be determined with acceptable precision and accuracy under the stated experimental conditions. The concentration of sample giving signal to noise ratio of 3 was fixed as the LOD. The concentration of the sample giving signal to noise ratio of 10 was fixed as the LOQ.

Results And Discussion

Development of solvent system

The development of solvent system was carried out by hit and trial method using different solvents considering the results of earlier reports. The solvent system composed of acetone, ethyl acetate, and water gave some separation when tried in different ratios, but the mobile phase composed of acetone:ethyl acetate:water (5:4:1, v/v/v) produced good resolution with compact spots of active markers stevioside and rebaudioside A at Rf 0.31 and Rf 0.21, respectively [Figure 4].

Figure 4

HPTLC chromatogram of standard stevioside (800 ng) and rebaudioside A (800 ng) at a wavelength of 360 nm

The regression data obtained from calibration curves (n = 3) were found to be linear over a wide concentration range of 100–2500 ng/spot with respect to the peak area for both the compounds, which have better range as compared to the earlier reports [Table 1].[15]

Table 1

Linear regression data for calibration plot (n = 6)

Method validation was carried out to confirm that the analytical method employed for the analysis of stevioside and rebaudioside A is suitable for its intended use. Results from method validation can be used to check its quality, reliability, and consistency. The developed method was validated as per the ICH guidelines[24] as well as other chromatographic HPTLC methods reported by laboratory[25-29] for the quality control of herbal drugs.

The specificity of the method was ascertained by analyzing standard drug and sample. The spots for stevioside and rebaudioside A were confirmed by comparing the Rf and spectra of the peak with those of standard [Figure 5]. The peak purity of stevioside and rebaudioside A was assessed by comparing the spectra at three different levels, i.e. peak start, peak apex, and peak end positions.

Figure 5

Superimposed UV spectra of stevioside and rebaudioside A in standard and sample peaks showing λmax at 360 nm

Precision of the proposed method was obtained by repeatability and intermediate precision. Inter-day and intra-day precisions were analyzed by preparing and applying three different concentrations of standard (in triplicate) on the same day and on three different days, respectively. Inter-system precision was found by repeating the same procedure by using different system of the same make. The method precision and intermediate precisions were determined and reported in terms of % RSD [Table 2].

Table 2

Precision of the method (n = 6)

Accuracy as recovery

Accuracy may often be expressed as percent recovery by the assay of known, added amounts of analyte. The pre-analyzed samples were spiked with standard at four different concentration levels, i.e. 0, 50, 100, and 150%, and the mixtures were re-analyzed by the proposed method. The recovery experiment was conducted in triplicate, which demonstrated a good recovery in the range of 97.75-103.54% and 97.17-103.44% for stevioside and rebaudioside A, respectively [Table 3].

Table 3

Accuracy of the method (n = 6)

Robustness of the method

Robustness of the method was determined by introducing small changes in the composition of the mobile phase and detection wavelength, and the effect on the result was examined as % RSD. Mobile phase having different compositions were used, such as acetone:ethyl acetate:water (52:38:10, v/v/v), (48:42:10, v/v/v), and detection wavelengths 360 ± 3 nm were used and chromatograms were developed. The low value of % RSD indicated that the method withstood small changes and so the method was robust [Tables 4 and 5].

Table 4

Robustness of the method for stevioside (n = 3)

Table 5

Robustness of the method for rebaudioside A (n = 3)

Limit of quantification and limit of detection

The concentration of sample giving signal to noise ratio of 3 was fixed as the LOD. The concentration of the sample giving signal to noise ratio of 10 was fixed as the LOQ. It was found that 35 ng of stevioside and rebaudioside standard gave signal to noise ratio 3, and 100 ng of stevioside and rebaudioside A standard gave signal to noise ratio 10. Hence, 35 and 100 ng were fixed as the LOD and LOQ, respectively.

Estimation of stevioside and rebaudioside a in samples

The samples were applied in triplicate and the developed chromatograms were scanned according to the method. The average areas of the corresponding peaks of stevioside and rebaudioside A were used for quantification using regression equation [Table 6].

Table 6

Results of estimation of stevioside and rebaudioside A in samples of stevia collected from different regions of the Indian subcontinent

Conclusion

The validated sensitive, safe, and high throughput method developed for quantification of stevioside and rebaudioside A can be used for the quality control of S. rebaudiana as well as to check the content of stevia glycoside during different stages of crop for commercial production.