INTRODUCTION:

Stevia rebaudiana Bertoni is a plant that has a sweet taste because it contains steviol glycosides. The main steviol glycoside in stevia is stevioside, which has a sweetness level of 100-300 times sucrose so that it can be used as a substitute for sucrose. In addition to the higher level of sweetness, stevioside also provides another benefit, which is low in calories so it does not cause an increase in blood sugar levels¹.

Some countries use both stevia leaves and extracts for various purposes, including natural sweeteners, drugs (ethnobotany), and supplements².

Thus, stevioside is a potential material for use in various food and drug industries. Therefore, the use of steviol glycosides is increasingly widespread, leading to the need for quantitative analysis of stevioside.

The stevioside content in stevia leaves varies between 4-13%³. Various methods for quantification of stevioside have been reported. In this paper, the quantification method of stevia will be presented in the last decade so that an effective method for the quantification of stevioside can be compared.

Stevia rebaudiana Bertoni:

Moisés Santiago Bertoni classified Stevia rebaudiana Bertoni botanically in 1899. Initially, this plant was called rebaudianum Eupatorium, then in 1905 its name was changed to Stevia rebaudiana (Bertoni) Bertoni⁴.

Stevia is a shrub of Asteraceae family. Its height reaches 1 m. Stevia has broad, slightly branched roots and brittle stems, elliptical leaves measuring 2-3cm with serrated edges. The stem is woody and weak at the bottom⁵. The flowers are white with a pale purple throat and are small in size and the fruit is in the shape of a five ribbed spindle⁶.

Stevia leaves contain sweet components of steviol glycosides, with the main constituents of stevioside (4-13%). In addition, there are also other steviol glycosides which are rebaudioside A (2-4%), rebaudioside c (1-2%) and dulcoside A (0.4-0.7%)³. The difference in the content of each steviol glycoside from and stevia is influenced by the time of harvest, the age of the plant, and the place of growth⁷. Stevia is a semi-humid subtropical plant that can be planted in areas with temperatures ranging from -6-43.8ºC and rainfall ranges from 1500 to 1800 mm per year⁸.

This plant grows easily in the soil with a pH of  6.5-7.5. Its habitat stretches from the southwestern United States to the Brazilian highlands¹.

Stevioside:

The main steviol glycoside from stevia leaves with the largest quantity is stevioside. Stevioside is described as a glycoside consisting of three glucose molecules bound to aglycone, the steviol part. This compound was first purified in 1931, then its chemical structure was known in 1952 as diterpenic glycosides⁴.

Figure 1. Structure of stevioside⁹

Stevioside or 13-[(2-O-β-D-glucopyranosyl-β-D-glucopyranosyl) oxy] kaur-16-en-18-oic acid, β-D-glucopyranosyl ester is a compound with molecular weight 804.88 g/moles have the structural formula C₃₈H₆₀O₁₈¹⁰. It is a white powder to light yellow, odorless or has a slight characteristic odor with around 200-300 times sweeter than sucrose, and it is easily soluble in water⁹.

Stevioside can be used as a sweetener¹⁰ in food and various products because it has a high sweetness level but is low in calories. Besides, stevioside also has various pharmacological activities including lowering blood sugar levels, lowering blood pressure by inducing vascular vasodilation and diuresis, anti-inflammatory, immunomodulatory and inhibiting TPA-induced tumors (12-O-tetradecanoylphorbol-13-acetate)¹¹.

QUANTIFICATION OF STEVIOSIDE:

The challenge in developing a method for quantification of stevioside from Stevia rebaudiana is its selectivity. Because stevioside has a compound structurally similar to but not identical to other steviol glycosides contained in Stevia rebaudiana, especially rebaudioside A. With a similar structure, the two components of this compound have similar physicochemical properties so that the results of analysis often overlap¹². So that the analysis requires a method that is able to separate and determinate the types of steviol glycosides from one another so that quantification can be carried out correctly. The ability of an analytical method to test analytes can be assessed by validating. Includes an assessment of accuracy, precision, linearity, specificity, limit of detection (LOD), limit of quantitation (LOQ), range, and ruggedness¹³.

HPLC (High Performance Liquid Chromatography):

The quantitative method of stevioside from Stevia rebaudiana Bertoni extract using HPLC has been developed. JECFA (The Joint FAO/WHO Expert Committee on Food Additives) has explained the quantification of steviol glycosides by HPLC-UV. JECFA states that the quantification of steviol glycosides can be done using Capcell non-polar pack C18 MG II (Shiseido Co.Ltd), Luna 5μm C18 (2) 100A (Phenomenex), or other similar columns (250mm x 4.6 mm, particle size : 5μm). The mobile phase used was acetonitrile and 10mmol/L: sodium phosphate buffer (pH 2.6) with a ratio of 32:68. A flow rate of 1.0ml/min at 40⁰C with a UV detector at 210nm. The stevioside retention time for this method is 8.08 minutes (using capcell column pack c18 MG II)¹⁰.

The RP-HPLC method is confirmed by Chaturvedula et al., by optimizing column temperatures at 20, 40, 60, and 79⁰C. Different temperatures produce different retention times of 8.015 minutes for 20⁰C, 9.246 minutes at 40⁰C, 9.012 minutes at 60⁰C, and 9.069 at 79⁰C. The optimum temperature for separating steviol glycosides is 40 and 60⁰C. Lower temperatures cause incomplete separation while higher temperatures cause some components to degrade. The relative retention times obtained for stevioside against rebaudioside were 1.057 and 1.054 at 40 and 60ºC respectively. This study also states that the quantization limit generated from this method is 0.5ppm¹⁴.

Quantification of stevioside, in the extract of Stevia rebaudiana leaves from India using the RP-HPLC method with UV array detection at 219nm was established. The stevia leaves were extracted by deionization water at 55⁰C for 2 hours. The mobile phase consisting of methanol and water with 0.1% orthophosphoric acid (v/v) (70:30) at a flow rate of 1.0 ml/min. The calibration curve was obtained in the range of 5.0-75μg/ml with a regression coefficient of 0.9999. The results of the validation method show that intraday and interday precision studies have a relative standard deviation less than 2.5% and recovery was 97-99%. The LOD was 0.02μg/ml and LOQ 0.05μg/ml. Stevioside content ranges 7.5-7.87%¹⁵.

Stevioside analysis with RP-HPLC used Acclaim Mixed-Mode WAX-1 column (150×3.0mm, 3μm particle size; Dionex, Bannockburn, IL). was developed by Jaworska. Stevia leaves from China and Brazil were extracted by acetonitrile: water (3:7) for 1 hour. Before the elution was carried out the extract was passed on Acclaim Mixed Mode WAX-1 guard column (10×4.3mm, 5μm particle size). The solvent system consisted of deionized water with phosphoric acid (pH 3) (solvent A) and acetonitrile (solvent B). Elution was carried out at 32ºC, flow rate 1mL/min, with a photodiode array detector at 210nm. The elution ran in gradient mode by 90% B maintained for 2 min, from 90 to 70% B in 37.5 min, followed by 60% B for 10 min. The retention time of stevioside is 26.2 min. This method produces LOD 0.0060mg/mL and LOQ 0.020 lower than JECFA method. The linearity testing resulted in correlation coefficient 0.9990. Intraday precision 0.070% and interday 0.40%¹⁶.

The RP-HPLC method with UV detector was also developed by Martono et al. in their study, quantification of stevioside from ethanol 40% stevia leaves was carried out using the Eurosphere C-18 stationary phase (250×4.6 mm, 5μm) with the mobile phase of the mixture of methanol 10% pH = 3.0 with phosphoric acid), acetonitrile and TFA (5: 35: 0.01). The analysis was carried out at 30ºC with a flow rate of 0.6 mL/minute with a 210nm UV detector. Linearity made with a standard solution of 20-120mg/mL produces a regression coefficient of 0.9994. This method produces a resolution> 2.0 which indicates good separation. The accuracy obtained was 100.82%, intraday precision was 0.31-1.81% while interday was 0.37%. The lowest concentration that can be detected is 1µg/mL while LOQ is 3µg/mL. The results of quantification indicate that 40% ethanol extract of stevia leaves originating from central Java contains stevioside which ranges from 7.66-8.28%¹².

HPLC-MS (High Performance Liquid Chromatography-Mass Spectrometry):

Woelwer et al., 2010 quantified steviol glycosides, stevioside from Stevia rebaudiana using normal phase HPLC. In his research, the preparation of leaf samples was extracted using water at 102⁰C followed by reverse phase SPE using a C18 cartridge. Recovery from SPE for stevioside is 99%. The analysis was carried out with the HILIC luna column with the mobile phase of acetonitrile: water (85:15 v/v) and the Luna NH₂ 100 A column using the mobile phase of acetonitrile: water (75:25 v/v) with a diode array detector at 210nm wavelength. Elution is done isocratically. The results showed that the Luna HILIC column produced a good separation between two similar components of steviol glycoside, stevioside with rebaudioside A. This was indicated by the stevioside retention time appearing at 7 minutes while rebaudioside at 9.7 minutes. Analysis with the Luna HILIC column resulted in repeatability of 0.43–0.59% and an interday precision of 0.52% for stevioside. The timing of equilibration generated by the Luna HILIC column is also shorter and provides robust analysis results. This column is also suitable for further analysis using MS. In this study, stevioside was reconfirmed using an MS detector with ESI (electrospray Ionisation) using negative ion mode. The stevioside has a molecular ion with m/z 803. The results of the sample show that stevia which grows in Germany contained stevioside ranges from 7.78 to 7.90%¹⁷.

Moronto et al. Performed quantification and determination of six steviol glycosides using LC-ESI MS/MS. Elution was carried out with Waters Atlantis column RP DC18 (150x2.1mm, 5μm) with mobile phase water and acetonitrile which both were acidified with 0.05% trifluoroacetic acid (TFA) in gradient mode with acetonitrile composition from 35% to 80% for 25 minutes. Positive ion mode is selected in this analysis with multiple reaction monitoring (MRM). The test conditions are regulated by 300⁰C capillary temperature, ion spray voltage of 5000, potential de-clustering, focusing, entrance 100 eV, 200 eV, 10 eV respectively, while the energy collision and collision exit cells are 80 eV and 15 eV consecutive. The results of ESI/MS showed pseudo-molecular ion peak at m/z 827 and MS/MS fragmentation showed m/z 665. Validation results on stevioside components showed a correlation coefficient of 0.996; LOD 1.8±0.4ng/mL; LOQ 7.8± 0.8ng/mL. Stevioside concentrations from various regions with seeds from Brazil and Paraguay range from 2.12-15.73%¹⁸.

The stevioside analysis was also carried out by Lorenzo et al., Who compared the analysis conditions using the Devosil ODS-HG column (250x4.6mm i.d., 5μm) and Luna HILIC (150x4.6mm i.d., 5μm Phenomenex). Elution is carried out in two conditions, using the mobile phase of water (A) and acetonitrile (B), isocratic (acn: water/80:20) and gradient (80% B 10 min; 80–50% B 2 min; 50–0% B, 2min; 0–80% B 2min; 80% B 2 min). The analysis was carried out at 36⁰C. DAD detectors were used with wavelengths 210, 256, 330, 360 and 450 nm. The chemical structure of each detected component was confirmed by MMESI/APCI-MS (multimode electrospray and atmospheric pressure chemical ionization) with negative ionization mode. The results showed the best separation was obtained using a Luna HILIC column with gradient elution. The retention time of stevioside obtained is 5.15 minutes. The standard solution linearity is 0.99; LOD 1.07mg/L; and LOQ 3.55mg/L. Sample preparation was carried out by extraction using 60⁰C water followed by centrifugation, microfiltration and ultrafiltration. The results showed that ultrafiltration of the impurity sample was lost but also followed by a decrease in the levels of the analyte being measured. The measurement of stevioside obtained by 10.86%¹⁹.

Analysis of stevioside was also carried out with LC-ESI-QqQ-MS/MS. The chromatographic system is run using Waters Atlantis T3 (150 x 2.1mm, 5μm) column. The solvent system was generated by a gradient elution program of water with trifluoroacetic acid 0.05% (A) and acetonitrile with trifluoroacetic acid 0.05% (B). Gradient elution started at 10% - 80% B in 25 min, with a flow rate of 0.200mL/min. The analysis is done with positive ion mode. Where the de-clustering potential of 155 eV, the focusing potential of 260 eV, the entrance potential of 12 eV, and other modes is the same as the research conducted by Moronto et al., The linearity obtained from testing a standard solution of stevioside 1-10μg/mL is 0.999. The results of the method validation show that LOD is 1.1ng/mL and LOQ is 2.1ng/mL. Tests on 50% stevia ethanol extract from Tunisia with various treatments produce stevioside levels ranging from 2.35 to 6.17% ²⁰.

UHPLC (Ultra High-Performance Liquid Chromatography):

In 2010, Gardana et al. developed the UHPLC-MS method for the analysis of steviol and steviol glycosides from southern Italy's Stevia rebaudiana. The sample was extracted with methanol, then SPE was carried out. The analyte was separated by UHPLC HSS C18 column (150×2.1 mm i.d., 1.8μm). The mobile phase used is 2 mmol/L ammonium acetate pH 6.5 and 0.1% CH₂Cl₂ in acetonitrile in gradient mode. The analysis is done by a single ion reaction (SIR) monitoring mode CH₂Cl₂ in the mobile phase, serves as a source of Cl^- which will increase measurement sensitivity. This UHPLC method produces a fast separation time of 3 minutes at a flow rate of 0.5ml/minute. But the retention time between stevioside and rebaudioside coincides, so it can interfere with the determination of the quantity of each component. Stevioside contained 5.8±1.3% in southern Italy's Stevia rebaudiana. Linearity obtained from standard testing with concentrations of 0.05-10μg/mL is 0.999 and LLOD for stevioside 15ng/ml. The results of the validation method for quantification of stevioside show an accuracy of 98-101%, intraday precision ranging from 1.8-3.5% (<4.3%), precision interday 2.2-3.8% (<5.7%) ⁷.

UHPLC coupled to Orbitrap mass spectrometer was developed for the analysis of stevioside from Stevia rebaudiana leaves. The analysis was optimized for various types of columns, where the amide (150 × 2.1 mm, 1.7μm) Waters, Milford, MA, USA resulted in an optimum separation. Optimization was also carried out in ionization mode selection, the results showed that the negative ionization mode with 0.05% formic acid was the best mode for this analysis. Elution was carried out by means of water (A) and acetonitrile (B) which were acidified with 0.05% formic acid. The flow rate 0.3 mL/min at 30⁰C, with gradient elution 13–17% solvent A in 5 min, 17–20% A in 5 min, 20–35% A in 5 min and then, 35–50% B in 10 min. The % RSD for intra-day precision ranges from 2.1–4.2% and inter-day precision 3.0–5.1%. The accuracy obtained is 95-102%. Before the analysis was carried out, samples of stevia leaves were extracted using water: acetonitrile (80:20). The resulting stevioside ranges from 0.02-48.1%. This method is a robust method because a slight change in the temperature of the analysis and flow rate and changes in the parameters of MS does not affect the results of the analysis²¹.

Two Dimentional HPLC:

Analysis of steviol glycosides with two-dimensional systems has been developed in 2011. The combination of the normal phase and reverse phase HPLC systems (LC x LC) will provide increased separation capability compared to one-dimensional HPLC. In this study, for a normal phase system, a micro-polyamine HPLC column (250x1.0mm, 5μm) was used as D1 which was then connected with a reverse phase UHPLC system with a C18 sub-2μm column (230x1mm, 1.8μm, 1200 bar) as D2. Separation on D1 was carried out by gradient elution using the mobile phase of acetonitrile: water which was set at pH 3.0 with H₃PO₄ in the mode, from 5 to 25% water 50 min; 25-70% water 15 min; 70% water 15min; from 70 to 5% water 1 minute. The flow rate was 20 mL/min. Separation continued at D2 using the same mobile phase and carried out gradient with 5% acetonitrile 0.01min; 5-70% acetonitrile 0.26 min; from 70 to 5% acetonitrile 0.1min; 5% acetonitrile 0.5min with a flow rate of 3.4 mL/min, at a temperature of 70⁰C. the analysis was carried out with PDA detectors at 210nm. The sample to be analyzed is extracted with water: acetonitrile (80:20). The result is that this method produces a good separation for each component of steviol glycosides, but the analysis time is longer > 60 minutes ³.

HPTLC (High Performance Thin Layer Chromatography):

The HPTLC method has been proposed for the quantification of stevioside. In this method, the test sample is bottled on TLC aluminum plates precoated with silica gel 60 F-254 (20×10cm x 0.2mm E. Merck, Darmstadt, Germany). The elution is performed with the mobile phase of acetone: ethyl acetate: water (5:4:1, v/v/v) until 85mm. The plate was then scanned with Camag TLC scanner III at a wavelength of 360nm with the appearance of anisaldehyde-sulfuric acid spots. The results show the content of stevioside in stevia rebaudiana which originates from various places in India ranging from 1,977-3,75%. The method validation shows that in the stevioside measurement the correlation coefficient obtained was 0.9965 and the accuracy was 97.75-103.54%. On observations of the precision, it was known that RSD for interday, intraday and inter-analysis were 0.91-1.73%; 0.93-1.76% and 0.99-2.23% respectively. LOD 35ng/spot and LOQ 100ng/spot. The robustness of the method is tested by changing the composition of the mobile phase and the wavelength of detection. The results show that the analysis method has good robustness with RSD 0.97-2.33%²².

HPTLC used a precoated silica gel 60 F254 plate also proposed by Saifi et al., by using chloroform: methanol: water (60:32:4, v/v/v) as eluent. Then Densitometric analysis using absorption-reflection mode at 400nm for stevioside was carried out. From the analysis, known that the stevioside Rf spot was 0.33±0.03 the validation of the method showed that the calibration plots around 200-1200ng/spot showed a good linear relationship with regression coefficient was found to be 0.99515. The accuracy of this HPTLC analysis of stevioside 98.97% indicating good reproducibility. Interday precision and intraday of the method was found to be 1.02 and 1.27% respectively. LOD from this proposed method was 100ng/spot and LOQ was 200ng/spot. Analysis of stevia leaves from India begins by extracted sample by ethanol 95% in 15min of reflux. The result showed that extract is contains 3,24-7,8% stevioside²³.

In 2013, Londe and Nanaware were developed the validated HPTLC for the quantification of stevioside in Stevia rebaudiana leaves from China. The precoated silica gel 60F254 HPTLC plates was used for separation using ethyl acetate: ethanol: acetone: water (15:3:6:6, v/v/v/v) as mobile phase. The quantification of stevioside was carried out by densitometric analysis at λ max 580nm with detecting reagent anisaldehyde–sulfuric acid. Rf value of stevioside was 0.34. The linearity in the range of 1000-7000ng/band was 0,9989. Percent RSD of intraday precision was <0.85% and interday precision was <0,97%. LOD and LOQ for this method were 127ng/band and 387ng/band respectively. This method is robust, specific and obtains 89.6–93.9% recovery. The ethanol extract of stevia leaves contains 6.94%²⁴.

Infrared Spectroscopy:

A rapid determination method of stevioside Stevia rebaudiana leaves using near-infrared trans-reflectance spectroscopy (NIRS) combined with multivariate analysis was developed. NIRS with multivariate analysis aimed to obtain a predictive model for the quantification of stevioside. NIR spectra of stevioside from 23 samples Stevia rebaudiana from Indonesia that diversity in ages of leaves, growing area and cultivar were recorded at 4000-10000 cm^-1 in 4cm^-1 data interval. This scanning carried out over 32 scans. The actual value of stevioside obtained from HPLC method and used as a reference. A prediction model was developed using partial least square (PLS) regression based on 2nd derivative spectra. The pre-processing spectra were used to improve the resolution of spectral. The spectral at wavenumber region of 4760-5016 cm^-1was used for stevioside analysis. PLS model performance was assessed in terms of R² (optimum determination coefficient) and minimum RMSEV (root mean square error of cross-validation). Cross-validation and leave one out calibration of the PLS component technique was selected for performing the PLS validation. Both techniques intended to find minimum RMSEV at the highest R². The result showed that PLS model for stevioside determination revealed R², RMSEC (root mean square error of calibration) and RMSEP (root mean square error of prediction) of 0.78, 0.97, and 1.63%, respectively. The validation showed that NIRS measurement with the PLS calibration model can predict stevioside content in Stevia rebaudiana dry leaves²⁵.

Other infrared spectroscopy method for quantitative of stevioside is FTIR (Fourier transform infrared) spectroscopy combined with multivariate analysis. The infrared spectrums used to build the prediction model were middle infrared spectrums that scanned at 4000–650 cm^-1wavenumbers region. The actual value of stevioside was determined by HPLC. Samples that analysis by that two methods was ethanol 40% extract Stevia rebaudiana leaves from Indonesia with 14 variations. The FTIR spectrums were pre-processing to select the optimal wavenumber region. Model by 2^nd derivatives transformation of SNV (standard normal variate) and MSC (multiplicative scatter correction) technique. The best PLS regretion model has resulted from the MSC technique. The spectra from selected wavenumber region than treated by siPLS (671-1450; 3278-3310 cm^-1), Second derivative, Baseline correction, First derivative, MSC, Normalization, Offset correction and SNV. The siPLS (synergy interval partial least square) produced best PLS model prediction for quantification of stevioside at 671-1450 and 3279-3301 cm^-1wavenumber region combination with RMSEC value of 0.84% an R² of 0.9952. That means this method could be used as an alternative method for quantitative analysis of stevioside²⁶.

Enzymatic:

The study of an enzymatic method using recombinant β-glucosidase BT_3567 (rBT_3567) for the quantification of stevioside was published in 2017. The enzyme was obtained from Bacteroides thetaiotaomicron HB-13. The quantification performed by reacted sample containing stevioside with rBT_3567 and glucose oxidase and peroxidase at pH 3 containing 3,3-diaminobenzidine as co-substrate. This process was incubated in 1h at 37⁰C. The colored product was produced, then measured by visible spectroscopy at 540nm. The validation of the method showed that this method obtained repeatability was 1.86-3.21% and the interday precision was 1.80-3.28%. The accuracy ranges were 95-105%. The stevioside content from steviol glycoside powder ranges were 21.88-33.30% and had 2.33-4.97% relative error compared with HPLC method. The enzymatic method using recombinant β-glucosidase BT_3567 is low cost, easy to perform, accurate, specific and reproducible ²⁷.

CONCLUSION:

Various analytical methods for quantification of stevioside from Stevia rebaudiana Bertoni have been developed, including HPLC, HPLC-MS, UHPLC, two-dimensional HPLC, HPTLC, Infrared Spectroscopy, and enzymatic methods with a colorimetric approach. The sensitivity of the best method was assessed from the lowest quantization limit obtained from the triple quadrupole LC-ESI-MS/MS method in SRM mode (selected reaction monitoring) with negative ionization with a value of 0.5ng/mL²⁸. The results of the method validation show that the best intraday precision is 0.07% obtained from the HPLC method with the mixed-mode RP column weak anion exchange chromatography using a PDA detector at 210nm¹⁶. However, the best interday precision was obtained from the RP-HPLC method with a mobile phase of 10% methanol (pH 3): acetonitrile: TFA (5:35:0.01) with an UV detector at 210nm which is 0.37%¹². Almost all methods that showing their accuracy has acceptable percent recovery values²⁹ except the HPTLC method using the mobile phase of ethyl acetate: ethanol: acetone: water (15:3:6:6, v/v/v/v) with densitometry at 580nm, resulting in an accuracy of 89.6-93.9%. Analysis of stevioside in stevia rebaudiana obtained from various regions using various methods produces levels of 2.12%¹⁸ and the largest content of 55% ²⁸. The prediction model obtained from the FTIR method is better than NIRS because it produces a smaller RMSEC which is 0.84% with R² greater than 0.9952 ²⁵^,²⁶.

CONFLICT OF INTEREST:

The authors declare no conflict of interest.

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Received on 26.06.2019 Modified on 19.08.2019

Research J. Pharm. and Tech. 2020; 13(11):5503-5509.

DOI: 10.5958/0974-360X.2020.00961.0