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Anal Bioanal Chem (2007) 388:18471857 DOI 10.

1007/s00216-007-1404-y

ORIGINAL PAPER

Comparison of two different solvents employed for pressurised fluid extraction of stevioside from Stevia rebaudiana: methanol versus water
ov Ostr & Pavel Karsek & Jaroslav Pl & Elena Varad Michal Roth & Karolnka Beneov & Pavla Kotlakov & Josef slavsk

Received: 15 October 2006 / Revised: 23 May 2007 / Accepted: 25 May 2007 / Published online: 27 June 2007 # Springer-Verlag 2007

Abstract Pressurised fluid extraction using water or methanol was employed for the extraction of stevioside from Stevia rebaudiana Bertoni. The extraction method was optimised in terms of temperature and duration of the static or the dynamic step. Extracts were analysed by liquid chromatography followed by UV and mass-spectrometric (MS) detections. Thermal degradation of stevioside was the same in both
ov Ostr : P. Karsek : M. Roth : K. Beneov : J. Pl : E. Varad P. Kotlakov : J. slavsk Institute of Analytical Chemistry, Academy of Sciences of the Czech Republic, Veve 97, 611 42 Brno, Czech Republic e-mail: pol@iach.cz Present address: J. Pl (*) Division of Pharmaceutical Chemistry, Faculty of Pharmacy, University of Helsinki, P.O. Box 56, 00014 Helsinki, Finland e-mail: jaroslav.pol@helsinki.fi Present address: K. Beneov State Phytosanitary Administration, Zemdlsk 1a, 613 00 Brno, Czech Republic Present address: P. Kotlakov Pliva-Lachema, Karsek 1, 621 33 Brno, Czech Republic Present address: J. slavsk Faculty of Chemistry, Brno University of Technology, Purkyova 118, 612 00 Brno, Czech Republic

solvents within the range 70160 C. Methanol showed better extraction ability for isolation of stevioside from Stevia rebaudiana leaves than water within the range 110160 C. However, water represents the green alternative to methanol. The limit of detection of stevioside in the extract analysed was 30 ng for UV detection and 2 ng for MS detection. Keywords Pressurised fluid extraction . Liquid chromatography . Mass spectrometry . Stevia rebaudiana . Stevioside

Introduction Recently, an increasing demand has been noted for new natural substitute sweeteners for sucrose and synthetic sweeteners such as saccharine and aspartame. Stevioside is a sweetener of plant origin possessing a 200350 times higher sweetening property than sucrose. Stevioside comes from the South American perennial shrub Stevia rebaudiana Bertoni, and it is the most abundant among the nine known sweet glycosides of the plant [1]. Application of stevioside varies among countries according to their laws and traditions; however, in the USA and Japan it has been a popular sweetener for years because of its low caloric intake and no side effects. The reason for not permitting the use of stevioside in some countries (e.g. EU countries) is steviol, a possible harmful aglycone of stevioside. Steviol has been proved to be a metabolite of stevioside produced by rat [2] and human [3, 4] digestion. On the other hand, it has been demonstrated that stevioside is not considerably metabolically converted to steviol by chicken [5] and, moreover, causes no harm to chicken embryos or to the individuals development [6]. Konoshima and Takasaki [7]

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have shown that stevioside has cancer-chemopreventive effects in mice. As reviewed by Geuns [8], stevioside is safe when used as a sweetener and there are no side effects like mutagenicity, carcinogenicity or teratogenicity. Stevioside is usually determined in Stevia rebaudiana by hot water leaching or supercritical fluid extraction (SFE) followed by liquid-chromatographic analysis of the extract. Water leaching has been performed by multiple mixing of leaves with boiling water [9, 10], followed by filtration and cleaning by passing the aqueous extract through an SPE cartridge prior to liquid-chromatographic analysis. The low cost of the instruments required has been countered by the time and labour consumption. When water is replaced with ethanol [11], the extraction step can be done more rapidly while keeping the same extraction recovery. SFE employing CO2 as a medium for extraction is faster than the previous method. It benefits from the physical-chemical properties of supercritical CO2, which possesses a higher diffusivity and lower viscosity than conventional liquid solvents. However, pure CO2 does not have sufficient solvation power for polar stevioside and therefore a polar cosolvent has to be added. Investigated cosolvents have included methanol [12], water and/or ethanol [13], a mixture of methanol and water [14], and water [15]. In most of the published papers, the highest extraction recoveries reported were obtained at a pressure of 200 bar and a subcritical temperature of 30 C. The procedures for isolation of stevioside from Stevia rebaudiana leaves on a pilot scale have been summarised by Pasquel et al. [13]. The methods include liquid extraction with different solventssupercritical CO2 and cosolvent, hot water or hot alcohols both employed below their respective boiling points at ambient pressure. The extracts have been purified by an additional adsorption, or precipitation with a salt or alkali. Stevioside is made up of the terpenic steviol with several glycosidic substitutents attached. From this point of view, the chromatographic separation of steviol glycosides resembles that of sugars. A wide range of various chromatographic systems have been employed for the purpose. The two systems used most frequently [16] are described below. Both of them are reversed-phase systems using an acetonitrilewater mixture as a mobile phase. The first one is based on the amino-substituted stationary phase [11, 17], whilst the second one uses an octadecyl silica stationary phase [9, 18]. UV photometry has been used quite often for the detection [11, 18]. UV-spectra of stevioside are very simple, with a maximum close to 200 nm. Unfortunately, the detection close to 200 nm causes problems during gradient elution in acetonitrilewater mixtures. Pulsed amperometric detection [17] offers much better sensitivity, but there is the necessity of postcolumn addition of 0.1 M NaOH solution to the acetonitrilewater eluent. Mass-

spectrometric detection offers better sensitivity and unbeaten selectivity compared with UV detection. Electrospray ionisation and negative ion detection have mostly been applied [3, 14, 19]. Recently, two-dimensional liquid chromatography with time-of-flight mass spectrometry (MS) has been employed for analysis of aqueous Stevia extract; it has been used to separate stevioside and other sweet glycosides from the plant matrix within a single chromatographic run [20]. Pressurised fluid extraction (PFE) has become a popular alternative to Soxhlet extraction or sonication because of its multiple rate and lower solvent consumption. PFE operates at temperatures above the boiling point of the extraction solvent and therefore elevated pressure is used to keep the solvent in the liquid state. Since high temperatures are used, the method is limited only to thermally stable analytes. The principal benefits of the solvent properties in PFE include enhanced mass transfer and increased solvation power. PFE has been employed for the extraction of different target analytes from various matrices. Applications of PFE to environmental samples were reviewed in [21]. If the organic solvent in PFE is replaced with water, the method is often termed pressurised hot water extraction (PHWE) or subcritical water extraction when the extraction temperature employed is below the critical temperature of water. The relative permittivity of water in the liquid state decreases noticeably as the temperature increases, which makes it possible to employ water as an extraction solvent for nonpolar compounds [22]. Last but not least, water is also an environmentally friendly and economically beneficial alternative to harmful organic solvents. Many papers have reported hot water extraction of both environmental and food samples and they were summarised in a recent review [23]. This paper presents a comparative study of two extraction methods, PFE employing methanol and PHWE, for Stevia rebaudiana leaves. Extraction efficiency and degradation of solutes were studied. Extracts were analysed by liquid chromatography (LC) with both UV and massspectrometric detection.

Experimental Chemicals Dried leaves of Stevia rebaudiana were obtained from Sigma (Prague, Czech Republic). Stevioside (purity 97 mol%) was purchased from Latoxan (Valence, France). Deionised water used as an extraction medium for PHWE and as a mobilephase component for LC was prepared in our laboratory using an Ultra Clear UV device (SGWasseraufbereitung und Regenerierstation, Hamburg, Germany). Acetonitrile (SupraGradient Grade) used in the LC mobile phase and

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methanol used for PFE were purchased from Riedel-de Han (Prague, Czech Republic). Extraction apparatus One single laboratory-made apparatus was designed and constructed for both PFE and PHWE (Fig. 1). The solvent reservoir was equipped with a purging unit for removing residual oxygen by gently stripping with helium. The extraction solvent (water or methanol) was delivered to the extraction vessel using an LC1120 high-performance LC (HPLC) pump (GBC Scientific Equipment, Dandenong, Victoria, Australia) working in constant flow rate mode. The solvent was preheated prior to it entering the extraction vessel by an electronically controlled heater. Three types of extraction vessels were manufactured from a noncorrosive alloy (INCONEL 625, Special Metals Wiggin, Hereford, UK), and they could accommodate different volumes of samples: 11, 22, and 33 ml. They were equipped with a removable 10-m frit on their outlets to avoid clogging of the outlet capillaries. The extraction vessel was placed in a thermostatted block that allowed heating up to 350 C. The temperature of the thermostatted block was regulated with an accuracy of 0.1 C by a microprocessor unit with proportional-integrated-derivative (PID) regulation (3116 temperature and process controller, Eurotherm, Durrington, Worthing, UK). Further, the extract was led from the extraction vessel to a pair of two independently thermoFig. 1 High-pressure extraction apparatus for pressurised fluid extraction (PFE) and pressurised hot water extraction. 1 solvent reservoir, 2 high-performance liquid chromatography pump, 3 nitrogen tank, 4 preheating unit, 5 pressure sensor, 6 extraction vessel placed in a thermostatted block, 7, 8 thermostatted decompression units, 9 collection vial for extract, 10 fused-silica restrictor, 11 proportional-integrated-derivative control units, V2, V3 two-way valves, V1, V4 three-way SSI valves, T thermoinsulation covers

statted decompression units that either heated or cooled the extract. Each thermostatted decompression unit consisted of stainless steel tubing (0.03-in. inner diameter) tightly wound on a thermostatted aluminium block. The temperatures of the decompression units depended on the extraction temperature and the boiling point of the solvent at ambient pressure; details are discussed in Results and discussion. All connections were made via stainless steel capillaries (0.0625-in. outer diamter, 0.03-in. inner diameter). Valve V1 operated in three positions allowing (1) purging of the extraction vessel with nitrogen, (2) introduction of the extraction solvent into the extraction vessel, and (3) closing of both previously described inputs. Valve V2 was open during all experiments. Valve V3 had two positions and it controlled the duration of the static period of extraction by the closing time. The function of the twoposition valve V4 was to switch between the stainless steel capillary outlet and the silica capillary outlet, and it was used to control the dynamic mode of extraction. The extraction apparatus can operate in three modes: (1) static, (2) dynamic, and (3) semidynamic. In this work, only the modes 1 and 2 were employed and they are described in Experimental procedure. Liquid chromatographyUV detection The screening studies were performed using a laboratoryassembled HPLC setup that included an LC1150 HPLC

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quaternary pump (GBC Scientific Equipment, Dandenong, VIC, Australia), a C-model sampling valve fitted with a 20-l sampling loop (Ecom, Prague, Czech Republic), a Luna NH2 column (250 mm4.6-mm inner diameter, particle size 5 m, Phenomenex, Aschaffenburg, Germany) housed in an LCO 101 column oven (Ecom), and a SpectraFocus UV detector (Thermo Separation Products, Fremont, CA, USA) running in the scan mode. The column was kept at 40 C. The mobile phase flow rate was 1 ml/min with gradient elution by acetonitrile (solvent A) and water (solvent B): 0 15 min 10% solvent A, 1519 min 100% solvent A, and 19 24 min 100% solvent A. Liquid chromatographymass spectrometry LC with both UV and ion-trap MS detectors used for identification was performed using an Esquire-LC system (Bruker Daltonics, Bremen, Germany). The liquid chromatograph was an Agilent 1100 series instrument consisting of a vacuum degasser, a binary gradient pump, an autosampler, thermostatted a column compartment, and a diode-array UVvis detector. The mass spectrometer was equipped with an electrospray ion source and spherical ion trap analyser operated in negative ion detection mode. The column employed was a Discovery RP Amide C16, 15 cm2.1 mm, particles 5 m and precolumn 2 cm 2.1 mm (both Supelco), flow rate 0.25 ml/min, binary gradient acetonitrilewater: 0 min 30% acetonitrile , 10 min 60% acetonitrile, 13 min 60% acetonitrile, 15 min 30% acetonitrile, stabilisation period 5 min. The column temperature employed was 35 C and UV detection was carried out at a wavelength of 210 nm. The injection volume was 2 l. Electrospray conditions were as follows: drying temperature 365 C; drying gas nitrogen, flow 9 l/min; nebuliser gas nitrogen, pressure 40 psi; capillary voltage 4 kV. Experimental procedure The extraction step was started by heating the extractor to a selected temperature. In all experiments, the block for accommodating the extraction vessel and the preheating unit were heated to the same temperature. Meanwhile, dried and ground Stevia rebaudiana leaves were weighed (approximately 100 mg), mixed with sand, and directly put into the extraction cartridge. Then, the extraction cartridge was inserted into the extraction device and the whole system was immediately purged with nitrogen for 2 min (switching by valve V1) to remove residual air. This prevented potential oxidation of a matrix, target analytes, and materials used for setup of the apparatus. The static mode of extraction was controlled only by valve V3, valve V2 was open permanently and valve V4 was switched to the stainless steel capillary outlet. At the

beginning of this mode, valve V3 was closed and the pump was initiated to deliver the solvent. A pressure and temperature programme was applied during this step and the details are in Extraction. When the pressure reached the set value at the set temperature, the pump automatically turned off. This point was considered as the start of the static extraction. The static extraction mode was ended by careful opening of valve V3, causing slow depressurisation and release of the extract into the vial by a stream of nitrogen. In the dynamic mode of extraction, valves V2 and V3 were permanently open and valve V4 was switched to the fused silica capillary outlet (length 70 cm, inner diameter 50 m). The pump started to deliver the solvent employing a flow rate of 8 ml/min until the set pressure was reached. Then the flow rate was changed to be the same as the outlet flow rate from the fused silica capillary. Similarly to the static mode of extraction, this point was defined as the start of the extraction step. The dynamic mode was terminated by turning the pump off and switching valve V4 to the stainless steel capillary outlet. The extract was transferred into the vial by a stream of nitrogen. The extract was made up to 50-ml volume and subsequently injected into the liquid chromatograph. The experiments to test the degradation of stevioside at elevated temperatures were performed in a similar manner. The extraction cartridge was filled with glass balls (diameter 1 mm) and then an aqueous standard solution of stevioside was dosed in. The extraction cartridge was placed in the extraction device and the extraction step was initiated as described above.

Results and discussion In the development of this comparative study of PFE and PHWE, liquid-chromatographic separation with UV and mass-spectrometric detection was optimised first by analysis of the aqueous solution of the stevioside standard. The extraction methods were optimised in terms of the extraction time and temperature. The extraction performances of water and methanol for the isolation of stevioside were compared as well as the influence of the extraction media employed, and the effects on the thermal degradation of stevioside were also compared. Liquid-chromatograhic analysis LC was used with both UV and ion-trap MS detectors. The calibration curve was constructed from five points (0.1031 1.0317 mg/ml) according to the typical concentration of stevioside in the extract. For UV detection, a linear calibration line was used (R =0.9999). For MS a nonlinear (quadratic) dependence was employed (R =0.9999). The

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limit of detection defined for a signal-to-noise ratio of 3 was 30 ng for UV detection and 2 ng for ion-trap MS. Stevioside has two absorption maxima in the UV region, 210 and 405 nm. The more intense maximum of the two 210 nmwas used for UV detection. The reproducibility of five-times repeated injection was 2.1%. The mass spectrometer was operated in the negative ion mode with universal detection of the characteristic fragment ion of stevioside (m/z 641) and it was used for quantification. The details of the stevioside mass spectra are discussed in the next section. LC-MS analysis of stevioside standard solution The standard of stevioside that is available on the market has only a 97 mol% purity. Other sweet glycosides present in Stevia, the source of stevioside, were also observed in the LC-MS chromatogram (Fig. 2). Stevioside was the most abundant peak in both UV and total ion chromatography traces (6.26.3 min). MS/MS of the deprotonated molecular ion of stevioside (m/z 803) resulted in three fragments (m/z 641, m/z 479, and m/z 317) that referred to the loss of one, two, and three molecules of glucose, respectively (Fig. 3). m/z 641 and m/z 479 were also observed as fragments coming from the ion source in single MS mode. The peak at 7.5 min with intense m/z 611 might refer to a deprotonated structure consisting of a molecule of steviol with two molecules of sucrose. Rebaudioside C was identified as a deprotonated molecular ion (m/z 949) at 7.2 min. MS/MS of this precursor produced fragment m/z 787
Fig. 2 Chromatogram traces of stevioside standard; total ion chromatography (TIC), UV at 210 nm, electrostatic ion chromatography (EIC) m/z 479, EIC m/z 611, EIC m/z 641, EIC m/z 787, EIC m/z 803, EIC m/z 949, and EIC m/z 965. Stevioside is presented at 6.2 6.3 min as [M-H]- at m/z 803 and [M-glucose]- at m/z 641
Intens. x10 7 4 2 0 2 0 x10 5 2 1 0 x10 5 1.5 1.0 0.5 0.0 x10 7 2 1 0 x10 5 1.0 0.5 0.0 x10 5 4 2 0 x10 4 6 4 2 0 x10 5 4 2 0 2 4

(observed also as an ion source fragment in single MS mode), indicating the loss of one molecule of glucose, and MS3 showed the steviol fragment, thus proving rebaudioside C was present. Rebaudioside A was eluted at 6.1 min and it was characterised by a deprotonated molecular ion at m/z 965. Rebaudioside B has the same molecular formula as and a very similar structure to stevioside, and therefore it is difficult to separate the two glycosides employing singlecolumn LC-MS. In addition, we suppose that the MS/MS spectra of both compounds would have very similar patterns owing to their structural similarity (three glucose units on the steviol skeleton). The percentage of sweet glycosides in the plant depends on the growing conditions, and it has been reported to be 4380% for stevioside and 00.02% for rebaudioside B [20]. The content of rebaudioside B is negligible in comparison with that of stevioside, and therefore the peak at 6.26.3 min was considered to pertain to stevioside only. Structures of the sweet glycosides present in Stevia are shown in Fig. 4. In general, the MS/MS experiments are known to improve the sensitivity and selectivity. Unfortunately, in the present case the MS/MS mode did not result in any significant advantage over the simple MS mode. Under the low-energy fragmentation on the spherical ion trap, the isobaric compounds stevioside and rebaudioside B which are coeluted under the conditions used in our experiments would have similar fragmentation patterns consisting of sequential cleavage of three glucose units. However, this is only a hypothesis that could not be verified because of the absence of the rebaudioside B
6.2 004-0401.D: TIC All

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standard on the market. Since the experimental setup and optimisation of the MS/MS experiments is much more complicated and time-consuming than that of simple MS, we finally decided to use the simple MS mode for the quantitation. Extraction In some static PFE experiments described in the literature, the extraction cell was heated only after it had been filled with the sample and pressurised with the (cold) solvent. This procedure can result in severe overpressurising of the cell during heating. To relieve the pressure, it may be necessary to release some solvent from the cell to the collection vial even before the cell has reached the operating temperature. Obviously, this results in an uncertain composition of the final extract because different portions of the extract correspond to different extraction conditions. Such an uncertainty is highly undesirable, especially if analyte degradation processes are to be studied. In this work, therefore, a different procedure was employed. After filling the cell with the sample and purging it with nitrogen, the cell was pressurised to about a quarter of the operating pressure, then it was heated to the operating temperature for 2 min and carefully pressurised to the operating pressure. In this way, the composition uncertainty mentioned above was avoided. Since large differences exist between PFE and PHWE as regards the solvent properties, special attention was paid to

decompression of the extracts. In PFE with organic solvents, the solubility of extracted nonpolar analytes is usually sufficient even after decompression and cooling of the extract to ambient pressure and ambient temperature. In this case, the analytes are quantitatively transferred into the collection vial. In PHWE, however, the decompression process is more complicated because the aqueous solubilities of nonpolar analytes fall dramatically as the temperature decreases. After decompression and cooling of the aqueous extract, the analytes can precipitate on their way from the extraction vessel to the collection vial. This usually results in nonquantitative transfer of the analytes from the extraction vessel or, in the worst case, in plugging of the transfer capillaries. Therefore, as mentioned in Extraction apparatus, a series of two thermostatted decompression units were placed between the outlet of the extraction vessel and the collection vial (Fig. 1). The purpose of the units was to avoid possible precipitation of the analytes and to prevent boiling of the extract at the extractor outlet. The temperature of each unit was electronically controlled and could be adjusted according to the extraction solvent employed. The temperatures of the two units were 110 and 95 C for PHWE and 110 and 60 C for PFE with methanol, respectively. Therefore, the downstream unit (no. 8 in Fig. 1) was always kept at a temperature below the normal boiling point of the extraction solvent. Also, precipitation was thereby prevented even in nonpolar constituents of the matrix that could be extracted from the sample. After cooling to room temper-

Fig. 3 Tandem mass spectrometry fragmentation spectra of the deprotonated molecular ion of stevioside [M-H]- m/z 803.2 indicating the loss of three glucose units (m/z 162)

Intens. x10 4 641.3

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479.2 65.9 0.2 317.1 413.3 0.0 100 200 300 400 500 600 700 800 m/z 18.1 803.2 18.1

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ature, no precipitation was observed in the Stevia extracts from both PFE and PHWE. The static and the dynamic mode, both lasting 10 min, were compared for both solvents at 50 bar and 110 C. The same extraction yield was achieved in both modes. Further, we decided to use the simpler static mode in the investigations described below. For both PFE and PHWE, several approaches to the preparation of Stevia leaves before the extraction were

tested. The leaves were ground and mixed with sand or glass balls or were just untreated and then inserted into the extraction cartridge. Extractions performed under the same conditions (50 bar, 110 C, 10 min) resulted in very similar extraction yields for all the pretreatments. In the subsequent experiments, the untreated Stevia leaves were extracted directly; this procedure shortened the total time of the analysis and also eliminated the source of possible contamination of the sample.

Fig. 4 Structures of steviol, stevioside, and other glycosides. The constituents attached to the base structure of steviol are glucose (Glc), rhamnose (Rha), and xylose (Xyl)

1854 Fig. 5 Degradation of stevioside in water (circles) and methanol (squares) as a function of temperature. The experiment was repeated three times with a relative standard deviation of 35%. Relative recovery of 100% is equal to the concentration of stevioside in the standard solution

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Fig. 6 EIC traces of stevioside standard treated at 110 C: m/z 479, m/z 641, m/z 803, and m/z 965

Anal Bioanal Chem (2007) 388:18471857 Fig. 7 EIC traces of stevioside standard treated at 160 C: m/z 479, m/z 641, m/z 803, and m/z 965
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Degradation of stevioside Thermal degradation of stevioside in aqueous solution has been reported; the maximum temperatures employed were 80 C [24] and 100 C [25] and the degradation products have not been identified. The study of stevioside degradaFig. 8 Extraction efficiency of stevioside from Stevia rebaudiana leaves as a function of temperature and extraction solvent; water (circles) and methanol (squares)

tion at elevated temperatures and the determination of the degradation products provides important guidelines to carry out the PFE and PHWE experiments. A 1-ml aliquot of stevioside standard solution (1 mg/ml) in water was dosed into an empty extraction cartridge. Then, the normal extraction procedure followed. The water

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effluent was analysed by LC-MS for stevioside and degradation products. A very similar decrease in the concentration of stevioside with increasing temperature was observed in both solvents (both in water and in methanol) (Fig. 5). On the other hand, there was an increase in the area of the peak at a retention time of 4.8 min starting at 110 C (Fig. 6), and this continuously increased up to 160 C (Fig. 7). The molecular mass and the MS/MS spectra of this chromatographic peak were the same as those of stevioside. A rearrangement of the stevioside molecule probably occurred under the high-temperature treatment, so the molecular mass remained the same but the retention in LC changed owing to different interaction with the stationary phase. Unfortunately, the changed structure could not be described with the current instrumentation. Extraction with methanol The extraction temperatures tested ranged from 70 to 160 C, and the extraction was run in static mode lasting 10 min. The pressure was kept at 50 bar. Figure 8 shows the gradual increase of extraction yield with temperature within the whole profile. The suggested temperature for carrying out further experiments was 110 C because (1) the extraction yield of stevioside reached an almost quantitative relative recovery plateau and (2) the degradation of stevioside was not yet significant. A higher temperature would result in a slightly higher extraction yield but also in significant degradation of

stevioside, and the presence of degradation products could result in errors and interferences in the analysis. The influence of the duration of the extraction step on the extraction yield was tested at 110 C and 50 bar for 10, 20, and 30 min. The extraction yield was same for all three times tested. Enhanced solvation power and diffusivity of subcritical methanol were probably sufficient for dissolving and releasing stevioside from the matrix within 10 min. The proposed extraction conditions for PFE with methanol are 110 C, 50 bar, and 10 min of static extraction time. Fragmentograms showing the distribution of main components in the extract obtained are presented in Fig. 9. Five-times repeated extraction of Stevia rebaudiana leaves was accomplished with a reproducibility of 5.2% for stevioside. Extraction with water Extraction with water was performed in a similar manner to the extraction with methanol. The extraction temperatures tested were from 70 to 160 C at 50 bar and the static extraction period lasted 10 min. The extraction yield of stevioside increased continuously up to 110 C and then a linear decrease was observed (Fig. 8). Stevioside has a polar character and its solubility probably decreases at temperatures above 110 C; therefore, the extraction yield is lower at temperatures higher than 110 C. Together with thermal degradation of stevioside, the extraction yield affects the shape of the extraction curve. The proposed

Fig. 9 Liquid-chromatographic analysis of PFE extract. m/z 479: 6.3 minfragment of stevioside, 8.0 minunknown compound; m/z 611: 7.5 min steviol with two sucrose molecules; m/z 625: unknown compound; m/z 641: 4.8 min compound with molecular mass 804 (possible structural isomer of stevioside), 6.2 minfragment of stevioside, 7.6 min unknown compound; m/z 787: 7.2 minfragment of rebaudioside C; m/z 803: 4.8 min compound with molecular mass 804 (possible structural isomer of stevioside, it gives fragment m/z 641), 6.1 minstevioside (molecular peak); m/z 949: 7.3 minrebaudioside C (molecular peak); m/z 965: 6.1 minrebaudioside A

Intens. x10 5 2 0 x10 5 2 1 0 x10 6 1.0 0.5 0.0 x10 7 1.0 0.5 0.0 x10 5 2 0 x10 6 6.1 0.5 0.0 x10 5 1 0 x10 5 4 6.1 2 0 2 4 6 8 4.8 4.8 7.6 6.2 7.4 6.3 8.0

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temperature for extraction of stevioside from Stevia rebaudiana leaves with water is 110 C. In the same way as for methanol, a static extraction step of 10-min duration proved to be sufficient for quantitative relative extraction recovery. The optimised parameters for PHWE of stevioside from Stevia rebaudiana leaves are 110 C, 50 bar, and 10 min of static extraction time, the same as for methanol. The reproducibility of PHWE evaluated from five-times repeated extraction was 4.7%.

References
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Conclusion A temperature of 110 C was determined to be optimal for extraction of stevioside from Stevia rebaudiana leaves using either water or methanol. An increased temperature resulted in significant degradation of stevioside in the environment of both solvents or in a decline in the extraction yield in water. Both solvents demonstrated stevioside extraction with very similar reproducibility and the proposed extraction parameters are the same for both methods. The total analysis time, including sample handling, extraction, and analysis, was 50 min. Water represents a more environmentally conscious and cheaper alternative to methanol. Compared with current extraction methods, our method is faster owing to the accelerated mass transfer in subcritical solvents. Extraction with pressurised hot water also presents a possibility for upscaling the extraction process and establishing a green method for isolation of stevioside sweetener from Stevia rebaudiana.
Acknowledgements Financial support from the Grant Agency of the Academy of Sciences of the Czech Republic (project no. B4031405) and from the Czech Science Foundation (project no. 203/05/2106) is gratefully acknowledged.

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