JOURNAL OF FOOD COMPOSITION AND ANALYSIS (2001) 14, 383}388doi:10.006/jfca.2000.0974Available online at http://www.idealibrary.com on

ORIGINAL ARTICLE

Determination of Stevioside in Plant Material and Fruit Teas

TomaH s\ Vane\ k*,1, Ales\ NepovıHm*, and Pavel ValıH c\ ek-

*Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic, Flemingovo naH m.2, 166 10 Praha 6, Czech Republic; and -Institute of Tropical and Subtropical Agriculture, Czech Agriculture

University, KamyHckaH 129, 165 21 Praha 6, Czech Republic

Received February 11, 2000, and in revised form October 27, 2000

Stevioside, a diterpene glucoside from Stevia rebaudiana, belongs to the strongest naturalsweeteners. Its potential widespread use requires an easy and accurate analytical method.A simple method for stevioside analysis based on the water extraction, hydrophobic chromatog-raphy (Sep-Pak C

18cartridges) and HPLC using linear gradient of acetonitrile in water is

described. The feasibility of this procedure was tested analyzing the stevioside level in Stevialeaves as well as in tea &&Fruit tea with Stevia''. Stevioside content did not show statisticallysigni"cant di!erence when a set of 20 tea bags randomly chosen from "ve boxes was determined(P(0.01). The mean value of stevioside content in fruit tea with Stevia was 8.14$2.71 mg/mL.The described method proved to be fast and friendly to the environment by minimization oforganic solvent consumption. ( 2001 Academic Press

Key=ords: Stevia rebaudiana; stevioside; HPLC; fruit tea.

Stevioside (Fig. 1), a diterpene glucoside isolated from Stevia rebaudiana Bertoni(Bridel and Lavieille, 1931), a perennial herb from Paraguay, is reported to be c. 300times sweeter than sucrose (Soejarto et al., 1983) and has recently gained importanceas a natural sweetener. This herb is more frequently used in the food industry of Japanand Korea than of Brazil. The establishment of rules for the use of stevioside andStevia leaves in U.S.A. is now in progress (U.S.A. FDA import alert; http://www.fda.gov/ora/"ars/ora}import}ia4506.html).

S. rebaudiana can be cultivated in the climatic conditions of the Czech Republic(CZ), too (with the stevioside content from 6 to 11% of dry weight) (NepovmHm et al.,1998), and it is now being used as a food additive here (Jemc\ a Comp., Jemnice, CZ).For such purposes it has been necessary to develop a suitable analytical method aswell as a simple method for sample preparation.

Methods described for estimation of stevioside content in Stevia leaves includeenzymatic determination (Mizukami et al., 1983), which is time consuming, near-infrared re#ectance spectroscopy (NIRS) (Nishiyama et al., 1992) requiring expensiveinstrumentation or most frequently HPLC (Ahmed et al., 1980; Makapugay et al.,1984; Nikolova-Damyanova et al., 1994; Liu and Li, 1995). Recently, stevioside

1To whom correspondence and reprint requests should be addressed: Tel.: #420/2/20183342. Fax:#420/2/20183574. E-mail: vanek@uochb.cas.cz

0889}1575/01/040383#06 $35.00/0 ( 2001 Academic Press

FIGURE 1. The structure of stevioside.

384 VANE[ K, NEPOVIDM, AND VALIDC[ EK

detection by ELSD detector after HPLC separation using a normal phase waspublished (Tateo et al., 1999). For stevioside extraction, methanol and water(Fujinuma et al., 1986) have been usually used.

This contribution describes a simple HPLC method using inexpensive C18 columnfor determination of stevioside in Stevia leaves and fruit tea. For the sample prep-aration, the extraction of analyzed material by boiling water was used, which dimin-ished the use of hazardous organic solvents.

MATERIAL AND METHODS

Material

Stevioside standard was obtained from Sigma Ltd., U.S.A. (95% purity). Acetonitrilewas of HPLC grade from Merck, Germany.

Instrumentation

Analytical instrumentation consisted of the CostaMetric I pump (LDC, U.S.A.),GM-3 gradient master (LP Praha, CZ), Rheodyne 7125 injection valve (Rheodyne,U.S.A.) and DAD detector PDA991 (Waters, U.S.A.) Analyses were performed ona stainless-steel column (250]4 mm i.d.) packed with SiC18 reverse phase (7 lm,Biospher, Labio Ltd., CZ).

Sample Preparation

&&Fruit tea with Stevia''. A tea was prepared according to the instruction for use:A tea bag of fruit tea (under the trade name &&Fruit tea with Stevia'', Jemc\ a Comp.,Jemnice, Czech Republic) weighing 2 g was added to 100 mL of boiled distilled water.After 5 min extraction the bag was removed and tea was let to cool to 253C. Thevolume was adjusted to 100 mL. The concentration of stevioside was determined ina set of "ve boxes. Four tea bags (20 pieces per box) were randomly chosen from eachbox and analyzed for stevioside content. The content of stevioside in tea bags wasstatistically evaluated by one-way analysis of variance (ANOVA).

Stevia leaves. Stevia leaves (Lednice, CZ) were dried at 503C for 24 h in dark andpulverized to uniform size (c. 18 mesh). This material (2 g) was extracted by boilingwater (2]50 mL) and further boiled for 30 s. Cooled extract was "ltered through the"lter paper and micro"lter (0.45 lm) to be ready for analysis.

DETERMINATION OF STEVIOSIDE IN PLANT MATERIAL 385

Processing of tea extract. 50 mL of water extract was "ltered using 5 lm "lter(Millipore,) and passed through the Sep-Pak C18 cartridge (Waters, U.S.A.). Thecartridge was washed with water (30 mL) and 30% methanol}water mixture (10 mL)and the stevioside was eluted from the cartridge with 60% methanol}water mixture(10 mL). The extract was evaporated in vacuo, redissolved in methanol (1 mL) andanalyzed by HPLC. The potential residue of stevioside on Sep-Pak cartridge waschecked in the methanol eluate.

Recovery test. A tea bag without Stevia leaves (&&Fruit tea'') was extracted withboiled water (50 mL) for 5 min and allowed to cool to laboratory temperature (253C).Stevioside was added in a concentration of 1 mg/mL.

HP¸C Analyses

Analyses were performed on a C18 reverse phase (Biospher 7 lm, Labio, CzechRepublic) packed stainless-steel column (4]250 mm, i.d.) at 253C. The linear gradientelution pro"le started with water}acetonitrile (A; 75 :15) and ended with water}acetonitrile (B; 50 :50) within 30 min. The initial conditions were re-established by5 min wash in both solvents. The #ow rate was 1 mL/min and all chromatogramswere plotted at 205 nm, using a high performance liquid chromatography systemequipped with a photodiode array UV detector (Waters, U.S.A.). Throughout theexperiment, all injection volumes were 20 lL. All solvents and samples were "lteredthrough a 0.45 lm "lter prior to use in HPLC analyses. Identi"cation of stevioside insamples was done by means of retention time, UV spectra and mixed inject withstandard.

Calibration curve was constructed from results obtained from analysis of samples(20 lL each) containing stevioside standard in the concentration range from 0.05 to2 mg/mL. Each analysis was repeated three times and mean value was used.

RESULTS AND DISCUSSION

Calibration Curve

Calibration curve was linear in the range from 0.2 to 2 mg/mL. The detection limit(2]noise level) was 1 lg per injection (0.05 mg/mL). Standard deviations are shown inFigure 2. The concentration was calculated according to formula: concentration(mg/mL)"2.24]10~8]area (n"8; r2 "0.997).

Stevioside Detection

Individual tea boxes as well as tea bags within a set of 20 pieces of fruit tea did notdi!er signi"cantly in the stevioside level. The concentration of extractable steviosideinto the boiled water was 8.14$2.71 mg/mL. The variability of the stevioside contentin tea bags could be caused by di!erences in natural plant material (Tateo et al., 1998;Truong et al., 1999) and/or by the impact of the individual ingredients in the teamixture.

The recovery of extraction of stevioside from tea bags by boiling water was 98.2%.According to the published data, water and methanol appear to be the best solventsfor stevioside extraction with comparable e$ciency (Gagliardi et al., 1986; BovanovaHet al., 1998).

FIGURE 2. The calibration curve of stevioside. The detection limit 0.05 mg/mL. r, Stevioside;==, linearregression.

FIGURE 3. Chromatograms of raw tea extract (A) and the same extract after Sep-Pak C18

treatment (B).

386 VANE[ K, NEPOVIDM, AND VALIDC[ EK

TABLE 1

Quanti"cation of the sorption and the desorption processes during the treatment of the sample by cartridgeSep-Pak C

18

Eluted Sorbed Stevioside contentStep (mg) S.D. (mg) in eluate (mg) S.D.

Application of extract (50 mL)1 403.8 $7.7 56.2 nd2Application of water wash(30 mL) (87.8%) (12.2%)30% MeOH wash (10 mL) 39.6 $2.4 16.6 nd2

(8.6%)60% MeOH elution (10 mL) 8.5 $0.5 8.1 3.6 $0.2

(1.85%)MeOH wash (20 mL) 2.92 $0.5 5.2 nd2

(0.08%)Irreversible sorption * 5.2 *

(17.6%)

Note: 1Fifty milliliters of extract yields 460 mg of dry residue, which contains 3.75 mg of stevioside afterdirect evaporation.

2Not detected, detection limit (0.05 mg/mL).

DETERMINATION OF STEVIOSIDE IN PLANT MATERIAL 387

Solid Phase Extraction

The extract of fruit tea (100 mL) was divided into two parts, 50 mL was evaporated todetermine a total weight of extracted compounds. The other 50 mL were applied toSep-Pak. A total residue after evaporation of 50 mL of tea extract was 460 mg. Duringthe Sep-Pak C18 treatment only 12.2% of extracted compounds (but 100% ofstevioside) was adsorbed on the cartridge. Most polar compounds were not sorbed orwere washed out by 30% MeOH, only 8.5 mg (1.85%) was still adsorbed. Allstevioside (3.6 mg) was eluted from cartridge using 10 mL of 60% methanol}watermixture together with 4.5 mg of other compounds (together 17.6% of total residue).The rest of adsorbed compounds were eluted using 20 mL MeOH. Chromatograms ofthe raw tea extract (A) and extract treated by SPE on a cartridge Sep-Pak C18 demon-strated e$cacy of the method (Fig. 3) for sample preparation, where concentration ofstevioside is below the detection limit. The recovery rate of the SPE method was equalto 96%. The results concerning the processing of fruit tea extract are summarized inTable 1.

CONCLUSION

One-way analysis of variance used for comparison of stevioside content shows thatthere is no signi"cant di!erence among di!erent boxes and individual tea bags on thelevel of signi"cance P(0.01. The concentration of stevioside in fruit tea was 8.14$2.71 mg/mL.

The comparison of stevioside recovery using water extraction (98%) with the resultsof methanol extraction (Fujinuma et al., 1986) has shown similar e$ciency. The use ofwater is friendlier to the environment, especially in case of routine analyses to checkthe commercial product quality.

Sep-Pak cartridge (SiC18) has been found suitable for stevioside puri"cation fromthe &&Fruit tea with Stevia'' extract. By this way 98% of commonly extracted com-pounds were removed and stevioside was concentrated more than 60 times. Stevioside

388 VANE[ K, NEPOVIDM, AND VALIDC[ EK

quanti"cation by HPLC enabled to reach the detection limit of 1 lg per injection.This procedure can also be easily applied for routine analysis of stevioside in otherfood samples.

ACKNOWLEDGEMENT

This work was supported by grant GACR No. 503/95/0249 and NAZV project No. EP7227.

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