Steviol glycosides are natural sweetener constituents found in the leaves of Stevia rebaudiana Bertoni (Asteraceae). The specifications for steviol glycosides were established by the Joint FAO/WHO Expert Committee on Food Additives (JECFA) in 2008, although there was a call in the following year for the modification of this assay method to enable the determination of nine steviol glycosides rather than just seven. In response, based on a proposed method by the Japan Stevia Association, we developed an improved method by changing the HPLC conditions and including the use of an octadecylsilyl column instead of an amino-bonded column to enable the rapid and reliable determination of the nine steviol glycosides by an isocratic HPLC-UV method. With the developed method, the nine steviol glycosides can be separately determined, and identified using individual reference chemicals as standards, unlike the previous identification method, which was based on the relative retention times. In addition, the single stevioside quantification standard was replaced with both stevioside and rebaudioside A quantification standards. Importantly, the validation of the developed method was successful. The limits of quantification for the nine steviol glycosides were between 0.2% and 0.6%. The developed assay method for the nine steviol glycosides was proposed to JECFA and adopted as the revised assay method for the steviol glycosides specifications at its 73rd meeting in 2010.
Steviol glycosides are natural sweeteners found in the leaves of Stevia rebaudiana Bertoni (Asteraceae), and their principal components are stevioside and/or rebaudioside A. Stevia extract was accepted as an existing food additive by the Ministry of Health, Labor and Welfare in Japan (1996), and it has been used in many types of Japanese foods. The specifications for “steviol glycosides” were established by the FAO/WHO Joint Expert Committee on Food Additives (JECFA) in 2008 [
In the specifications established by JECFA in 2008 [
In 2009, JECFA required that additional information for the detection of the steviol glycosides rebaudioside D and rebaudioside F (
and the quantification was achieved using both stevioside and rebaudioside A standards. Thus, this analysis provided higher accuracy and reliability. Subsequently, this method was adopted as the revised assay method for steviol glycosides at the 73rd JECFA meeting in 2010 [
The standards for stevioside (99.0+% (HPLC, dried), Code No. 193-15351) and rebaudioside A (99.0+% (HPLC, dried), Code No. 183-02361) used for the quantification were purchased from Wako Pure Chemical Industries Co., Ltd. (Osaka, Japan). The other seven steviol glycosides (rebaudioside B, rebaudioside C, rebaudioside D, rebaudioside F, dulcoside A, rubusoside, and steviolbioside) were prepared from stevia extracts by the Laboratory of Creative Science Co., Ltd. (Osaka, Japan). The purity of rebaudioside F was approximately 20%, while that of the other compounds was greater than 90%. A reference standard mixture containing the nine steviol glycosides for peak identification was prepared by dissolving nine standards in a 30:70 (v/v) mixture of acetonitrile and water, which are currently marketed by Wako Pure Chemical Industries. Two samples of commercial stevia extracts used as food additives in Japan, RA95 and RA60, were obtained from Morita Kagaku Kogyo Co., Ltd., Japan.
The analysis was performed using an Alliance 2695 HPLC system (Waters Co., MA, USA) with an SPD- 10AV UV-Vis detector (Shimadzu Corporation, Kyoto, Japan). Separation was carried out on 4.6 × 250 mm ODS columns with a particle size of 5 µm: Capcell Pak C18 MGII (Shiseido Co., Ltd., Tokyo, Japan), Shim-pack CLC-ODS (Shimadzu Corporation), and Luna C18 (Phenomenex Co., Ltd., CA, USA). For comparison, a SunFire C18 column (Waters Co., Ltd.) and other ODS columns were also used. The column temperature was maintained at 40˚C. Elution was achieved using a 32:68 (v/v) mixture of acetonitrile and 10 mmol/L sodium phosphate buffer (pH 2.6) as the mobile phase for 30 min. The flow rate was maintained at 1.0 mL/min, and the chromatographic profile was monitored at 210 nm.
For comparison, the existing JECFA method was performed using a Supelcosil LC-NH2 amino-bonded column (4.6 mm i.d. × 250 mm, 5 μm, Sigma-Aldrich Co., MO, USA). The column temperature was maintained at 40˚C. Elution was achieved at a flow rate of 1.0 mL/min using an 80:20 (v/v) mixture of acetonitrile and water adjusted to a pH of 3.0 with phosphoric acid as the mobile phase, and the chromatographic profile was monitored at 210 nm.
For the standard solutions, each of the stevioside and rebaudioside A standards (50 mg each after drying at 105˚C for 2 h) was accurately weighed into separate 50 mL volumetric flasks, and the volume was made up with a 30:70 (v/v) mixture of acetonitrile and water. They were diluted as appropriate with the same solvent mixture. For the sample solutions, RA95 and RA60 (50 mg each after drying at 105˚C for 2 h) were accurately weighed into separate 50 mL volumetric flasks, and the volume was made up with a 30:70 (v/v) mixture of acetonitrile and water. The standard and sample solutions (5 µL) were injected into the HPLC system. Steviol glycosides were identified on the basis of their correspondence of retention times using the reference standard mixture, and the peak areas were measured. Each solution was injected in triplicate, and the mean value was used for quantitation.
The concentration of eight (not rebaudioside A) steviol glycosides (x) in the sample was calculated by the following formula:
Here Cx (%) is the concentration of a steviol glycoside, Wstv-stn is the weight of the stevioside standard (dried basis), Wsmp is the weight of the sample (dried basis), Ax-smp is the peak area of the steviol glycoside (x) in the sample solution, Astv-stn is the peak area of the stevioside in the standard solution, and fx is the coefficient value for each steviol glycoside (1.00 (rebaudioside B), 1.18 (rebaudioside C), 1.40 (rebaudioside D), 1.16 (rebaudioside F), 0.98 (dulcoside A), 0.80 (rubusoside), or 0.80 (steviolbioside)).
The concentration of rebaudioside A in the sample was calculated by the following formula:
Here CreA (%) is the concentration of rebaudioside A, WreA-stn is the weight of the rebaudioside A standard (dried basis), Wsmp is the weight of the sample (dried basis), AreA-smp is the peak area of rebaudioside A in the sample solution, and AreA-stn is the peak area of rebaudioside A in the standard solution.
The concentration of total steviol glycosides was then calculated from the sum of the nine steviol glycoside concentrations.
In the JECFA method established in 2008 [
As can be seen in
method, the peaks of rubusoside and steviolbioside were not fully separated, as shown in
To solve these problems, the use of an ODS column was attempted for the determination of the nine steviol glycosides. With respect to the organic solvent for the mobile phase, acetonitrile was found to give a better separation than methanol (data not shown). Next, to determine the appropriate pH of the mobile phase, six mobile phases based on 32:68 (v/v) mixtures of acetonitrile and acidic solutions, including 10 mmol/L sodium phosphate buffer (pH 2.6), 1.4 mmol/L phosphoric acid (pH 3.0), 5 mmol/L formic acid (pH 3.0), 5 mmol/L ammonium formate (pH 6.1), and 5 mmol/L ammonium acetate (pH 6.6), and H2O were used.
As shown in
Next, the ratio of acetonitrile and 10 mmol/L sodium phosphate buffer (pH 2.6) in the mobile phase was varied (35:65, 32:68, and 30:70). As can be seen in
columns (data not shown). Fortunately, with the 32:68 (v/v) mixture of acetonitrile and sodium phosphate buffer, the injection shock and rebaudioside D peaks were separated, and the rebaudioside B and steviolbioside peaks had a good shape and retention time. Based on these results, the 32:68 (v/v) mixture of acetonitrile and 10 mmol/L sodium phosphate buffer (pH 2.6) was selected as the mobile phase. Under the analytical conditions, the UV spectra of the steviol glycosides showed the strongest UV absorption near 200 - 210 nm (data not shown).
To confirm the equality of different ODS columns, the analysis was carried out using more than six commercial ODS columns, and the results were compared. As can be seen in
The calibration curves were constructed for a range of
nine concentrations of the stevioside and rebaudioside A standards (
each in the sample mixture. Good linearity was also achieved for the other seven steviol glycosides (data not shown) over the concentration range from approximately 0.001 to 0.5 mg/mL for rebaudioside D, rebaudioside C, dulcoside A, rubusoside, rebaudioside B, and steviolbioside, and 0.0001 - 0.1 mg/mL for rebaudioside F. The correlation coefficients for the seven steviol glycosides were more than 0.999. LOQ (S/N = 10) for the seven steviol glycosides—rebaudioside D, rebaudioside F, rebaudioside C, dulucoside A, rubusoside, rebaudioside B and steviolbioside—were 2, 4, 4, 4, 4, 6, and 5 μg/mL, respectively, in the standard solutions and 0.2%, 0.4%, 0.4%, 0.4%, 0.4%, 0.6%, and 0.5%, respectively, in the sample mixture. Therefore, the linearity and sensitivity of the method are acceptable.
As shown in
To ensure the applicability of the developed method, the quantities of the nine steviol glycosides in two types of commercial stevia extracts, RA 95 and RA 60, were determined. The reference standard mixture of the nine steviol glycosides was used for the identification of the peaks. As shown in
The quantification results are summarized in
standard was adopted for the determination of rebaudioside A, because high levels of rebaudioside A have recently been detected in many stevia extracts, and highpurity rebaudioside A standards have become commercially available. As shown in the table, the levels of rebaudioside A and total steviol glycosides in both RA95 and RA60 determined by the developed calculation method appear to be very slightly lower than those obtained by the previous calculation method. The levels obtained by the newly developed method are not significantly different from those obtained by the previous method, and both levels are within the range of the measurement error (0.73%). Scientifically, in an HPLC quantification method, the target compound should be quantified by a standard curve based on a standard of the identical compound. The levels of rebaudioside A determined using the rebaudiosideA standard are more precise and reliable than
aOn the dried basis. bRebaudioside A was quantified by rebaudioside A standard. cND: Not detected. Each value is a mean of three trials.
aOn the dried basis. bRebaudioside A was quantified by stevioside standard. cRebaudioside A was quantified by rebaudioside A standard.
those obtained using a standard that is a different compound and requires coefficient value, particularly for stevia extracts with high concentrations of rebaudioside A. Therefore the rebaudioside A standard was used for the quantification of rebaudioside A in the developed method.
We developed an isocratic HPLC-UV method by employing an ODS column using a reference standard mixture of nine steviol glycosides. On the ODS column, the steviol glycosides can be detected separately and sensitively within 30 min. In addition, the use of the reference standard mixture of nine steviol glycosides enables the correct identification of the steviol glycosides in different samples. The reference standard mixture of the nine steviol glycosides is now commercially available. The rapid and reliable determination of the nine steviol glycosides by an isocratic HPLC-UV method on an ODS column was first developed in this study and has not been previously reported [3-7]. Subsequently, this method was proposed as the revised assay method for steviol glycosides and adopted at the 73rd JECFA meeting in 2010 [
We are grateful to Wako Pure Chemical Industries Ltd., Maruzen Pharmaceuticals Ltd., Morita Kagaku Kogyo Ltd., the Japan Stevia Industry Association, and the Japan Food Additives Association for providing noncommercial steviol glycosides, stevia extract samples, and information on stevia extracts. This work was supported by a Grant-in-Aid for Scientific Research from the Ministry of Health, Welfare, and Labor of Japan.