Food Chemistry 157 (2014) 518–523

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Analytical Methods

Fast methodology of analysing major steviol glycosides from Stevia rebaudiana leaves Cándida Lorenzo, Jéssica Serrano-Díaz, Miguel Plaza, Carmen Quintanilla, Gonzalo L. Alonso ⇑ Cátedra de Química Agrícola, E.T.S.I. Agrónomos, Universidad de Castilla-La Mancha, Campus Universitario, 02071 Albacete, Spain

a r t i c l e

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Article history: Received 19 April 2013 Received in revised form 4 February 2014 Accepted 18 February 2014 Available online 28 February 2014 Keywords: Stevioside Rebaudioside A Gradient elution Ultrafiltration Eco-friendly

a b s t r a c t The aim of this work is to propose an HPLC method for analysing major steviol glycosides as well as to optimise the extraction and clarification conditions for obtaining these compounds. Toward this aim, standards of stevioside and rebaudioside A with purities P99.0%, commercial samples from different companies and Stevia rebaudiana Bertoni leaves from Paraguay supplied by Insobol, S.L., were used. The analytical method proposed is adequate in terms of selectivity, sensitivity and accuracy. Optimum extraction conditions and adequate clarification conditions have been set. Moreover, this methodology is safe and eco-friendly, as we use only water for extraction and do not use solid-phase extraction, which requires solvents that are banned in the food industry to condition the cartridge and elute the steviol glycosides. In addition, this methodology consumes little time as leaves are not ground and the filtration is faster, and the peak resolution is better as we used an HPLC method with gradient elution. Ó 2014 Elsevier Ltd. All rights reserved.

1. Introduction In recent years, consumers are more and more worried about health, seeking natural and dietetic products. In this sense, the interest in Stevia rebaudiana Bertoni has increased considerably, as this plant has a high concentration (approximately 4–20%) of sweet diterpene glycosides in its dry-leaf matter (Geuns, 2003; Ghanta, Banerjee, Poddar, & Chattopadhyay, 2007). The high sweetness of the steviol glycosides makes them an attractive sugar substitute for food industries (Crammer & Ikan, 1986). Moreover, these glycosides are non-caloric sweeteners that reduce blood glucose and protecting the organism from diseases such as diabetes and obesity, among others (Geuns, 2003; Anton et al., 2010). Moreover, the steviol glycosides are related to other benefits, such as anti-hyperglycaemic, anti-hypertensive, anti-inflammatory, antitumour, antidiarrheal, diuretic and immunomodulatory effect (Chatsudthipong & Muanprasat, 2009). For all of these reasons, the steviol glycosides are known as the ‘‘sweeteners of the future’’ (Esmat, Azza, & Ferial, 2010; Brahmachari, Mandal, Rajeev, Mondal, & Brahmachari, 2011; Lemus-Mondaca, Vega-Galvez, Zura-Bravo, & Ah-Hen, 2012; Rao, Reddy, Ernala, Sridhar, & Ravikumar, 2012). The main sweet components present in S. rebaudiana Bertoni are stevioside and rebaudioside A, representing 90 wt.% of all sweet glycosides in the leaves (Bergs, Burghoff, Joehnck, Martin, ⇑ Corresponding author. Tel.: +34 967 599310; fax: +34 967 599238. E-mail address: [email protected] (G.L. Alonso). http://dx.doi.org/10.1016/j.foodchem.2014.02.088 0308-8146/Ó 2014 Elsevier Ltd. All rights reserved.

& Schembecker, 2012). Moreover, they have the most sweetness compared to sucrose (stevioside between 270 and 280 times more and rebaudioside A between 350 and 450). Probably for these reasons stevioside is the most studied glycoside (Montoro et al., 2013; Catharino & Santos, 2012) followed by rebaudioside A. Other diterpene glycosides present in lower concentrations are steviolbioside, rebaudioside B, C, D, F, dulcoside A and rubusoside. The analytical determination of single diterpene glycosides by chromatography is a difficult task because of the chemical structures of the glycosides (Fig. 1) are similar (FAO, 2010), and because the aqueous extracts of Stevia leaf have many impurities such as proteins, resins, organic acids, pigments and sesquiterpene lactones, among others (Kovylaeva et al., 2007). Many and varied extraction solvents and methods for steviol glycosides of Stevia leaves have been described in the literature: with chloroform and methanol (Kolb, Herrera, & Uliana, 2001), supercritical fluid extraction (Pól et al., 2007), by means of microwaves (Jaitak, Singh, & Kaul, 2009) and ultrasonics and enzymatic extraction (Jaitak et al., 2009; Liu, Li, & Tang, 2010; Puri, Sharma, & Tiwari, 2011; Puri, Sharma, Barrow, & Tiwari, 2012). In addition, there is abundant literature available with respect to the clarification and purification of these extracts (Zhang, Kumar, & Kutowy, 2000; Vanneste et al., 2011; Chhaya Sharma, Mondal, Majumdar, & De, 2012; Rao et al., 2012; Li, Chen, & Di, 2012), and several methods have also been reported for these extracts’ analysis: Kovylaeva et al. (2007), Henderson and Berry (2009), Gardana, Scaglianti, and Simonetti (2010), Woelwer-Rieck, Lankes,

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Fig. 1. Molecular structure of stevioside (a) and rebaudioside-A (b).

Wawrzun, and Wüst (2010), Bergs et al. (2012). However, for the application of the sweeteners as food additives, several restrictions and specifications must be taken into account (JECFA, 2007). For example, the use of solvents should be avoided as much as possible. The aim of this work was to propose a method for the analysis of the major steviol glycosides in S. rebaudiana leaves. Towards this goal, the optimum conditions for the extraction of these glycosides are proposed; in addition extract clarification is accomplished by microfiltration and ultrafiltration (UF) and quantification by HPLC-DAD, avoiding where possible the use of dangerous solvents that are environmentally damaging.

2. Materials and methods 2.1. Samples Different brands of commercial samples with several steviol glycosides concentrations, which were described on their labels, were used: Majota pill and powder (60% and 90% steviol glycosides), Masso 60% and 98% steviol glycosides, Azelis 95% and 98%, Cargill 98% and pure Stevia 99% steviol glycosides. Dried leaves of S. rebaudiana Bertoni from Paraguay, supplied by Insobol, S.L., were used. The samples were dried at room temperature to a moisture level between 5% and 6% as determined with a halogen lamp moisture balance model XM-120T (Cobos, Barcelona, Spain) at 105 °C. When the moisture loss was less than 0.1% in 180 s, it was considered that the samples had reached a constant mass.

Stevioside and rebaudioside A with purities P99.0% were purchased from Phytolab (Vestenbergsgreuth, Bavaria, Alemania). The calibration solutions for the HPLC analysis of stevioside and rebaudioside A were prepared by diluting the analytes with acetonitrile/water (8:2 v/v). 2.3. HPLC conditions The HPLC conditions were fixed with the standards cited above and diluted with acetonitrile/water (8:2 v/v) to 50 mg/L. The liquid chromatographer used was an Agilent 1200 HPLC (Palo Alto, CA, USA). Two columns were tested: Develosil ODSHG 140 Å 250 mm  4.6 mm i.d., 5 lm and Luna HILIC 150 mm  4.6 mm i.d., 5 lm Phenomenex (Le Pecq Cedex, France). Isocratic and gradient elution modes were tested. The eluents were water (A) and acetonitrile (B) in both systems, trying 80% B–20% A in the isocratic elution and several ramps in the gradient, as well as different elution times. The oven was thermostated at 36 °C. The flow rate was 1 mL/ min, and the sample injection volume was 20 ll. The DAD detector (Hewlett Packard, Waldbronn, Germany) was set at 210, 256, 330, 360 and 450 nm. To confirm the chemical structure of the compounds, a Mass Spectrometer 6130 Quadrupole LC/MS, G1956 (SL) multimode electrospray and atmospheric pressure chemical ionisation (MMESI/APCI-MS) system was used, coupled to an Agilent Chem Station (version b.03.01) data-processing station. The parameters employed for MM-ESI–MS were dry gas, N2, 10 mL/min; drying temperature, 350 °C; vaporizer temperature, 200 °C; nebulizer, 55 psi; capillary, 200 V (negative ionisation mode).

2.2. Reagents and standards

2.4. Method evaluation

HPLC-grade acetonitrile was used from Panreac Química, SAU (Castellar del Vallés, Barcelona, España). Ultra-high-purity water was produced using a Milli-Q System from Millipore (Bedford, MA), and PVDF filters (13 mm, 0.45 lm) were also purchased from Millipore.

Steviol glycosides were tentatively identified with the DAD detector by comparison with the corresponding UV–Vis spectra, on the basis of their molecular ion by electrospray ionisation mass spectrometry (MM-ESI–MS) (Fig. 1), and comparing with retention time of their pure standards in the chromatogram.

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The selectivity was determined by comparing the chromatograms obtained from the leaf samples with those of the standards solutions. For the linearity study, calibration graphs were performed by injecting standard solutions of stevioside and rebaudioside A with six different concentrations of each analyte. Each level of concentration was analysed in triplicate. The concentration ranges were from 25 to 500 mg/L. Qualitative determination was achieved by comparing the retention times and the UV–Vis spectrum of the standard solution with those of the samples. Quantification was possible by applying the calibration plot equations calculated by the least-squares method. The sensitivity of the method was determined with regard to the limit of detection (LOD) and the limit of quantification (LOQ), comparing the height of a sample peak and the height of a noise peak. The LOD is reached at a signal-to-noise ratio greater than three, and the LOQ is reached at a signal-to-noise ratio greater than 10 (IHC, 2005). The accuracy of the method was calculated with eight commercial samples spiked with different amounts (from 10% to 50% concentration increase) of the two steviol glycosides, which were diluted to different volumes with a mixture of acetonitrile/water (8:2, v/v) according to the glycoside concentration declared on the samples’ labels. The standard deviation for each compound (square root of the arithmetic mean of the variances) was calculated to obtain the repeatability (%RSD). The standard deviation of the three values for each compound multiplied by the square root of 3 was taken as the reproducibility value (if this value was higher than the repeatability; if not, this last value was also taken as the reproducibility) (Ortega, López, Cacho, & Ferreira, 2001). 2.5. Extraction conditions To choose the best extraction conditions for the steviol glycosides, we tried both milled (to 0.5 lm) and unmilled dry leaves. Other factors such as the temperature, agitation and extraction time were considered. On the basis of other authors (Pól et al., 2007; Vaneˇk, Nepovím, & Valícˇek, 2001; Woelwer-Rieck et al., 2010) and our own experiences, the temperatures chosen for testing were 37, 60 and 100 °C with and without agitation for 20 min. After setting the temperature, the extraction time was tested by analysing the sample extracts every 10 min until no increase in the steviol glycosides concentration was observed. Finally, 50 g of unmilled dry leaves were extracted with 2 L of distilled water. The extractor used was a Thermomix TM-31 de Vorwerk & Co. KG (Wuppertal, Germany). One millilitre of the extract was diluted to 10 ml with acetonitrile/water (8:2 v/v). This solution was filtered through a membrane filter (0.45 lm) before the HPLC analysis. All of the samples were analysing in triplicate. 2.6. Centrifugal UF treatment The extract was centrifugated in a Centronic BL-II centrifuge from JP Selecta S.A. (Abrera, Barcelona, Spain) at 4400 rpm for 10 min. The supernatant was centrifugated at 12,000 rpm for 5 min, and the new supernatant was subjected to successive filtrations in a vacuum with membranes of 10, 2.5 and 1 lm. The last extract was subjected to different centrifugal UF conditions: a 5000 Da (5 KDa) Centricon Plus-20 ultrafiltration membrane by centrifugation at 4400 rpm for 1 h; a 3000 Da (3 KDa) Centriplus YM-3 ultrafiltration membrane by centrifugation at 4400 rpm for 1 h; a 3000 Da Centriplus YM-3 ultrafiltration membrane by centrifugation at 4400 rpm for 30 min; and finally, a 3000 Da Centriplus YM-3 ultrafiltration membrane by centrifugation at 12,000 rpm for 5 min. All of these membranes were obtained from Merck Millipore Headquarters (Billerica, MA, USA).

3. Results and discussion 3.1. HPLC conditions To choose the best HPLC method, two columns together with two elution types were tested, as it was described in Materials and methods Fig. 2(a and b) show the different chromatograms obtained with the column Luna HILIC (150  4.6 mm i.d., 5 lm) and Fig. 2(c and d) show those obtained with Develosil ODS-HG (140 Å 250  4.6 mm i.d., 5 lm). HPLC method proposed is based on Woelwer-Rieck et al. (2010) procedure and, as well, the best results were obtained for Luna HILIC column. In terms of elution gradient, several modifications were carried out to get a better peak resolution, as shown in Fig. 2. This Figure shows that using isocratic elution in both columns (b and d), the compounds stevioside and rebaudioside A are coeluted. However, they are clearly resolved when gradient elution was assayed in both columns (Fig. 2a and c), showing Luna column the best results. For these reasons, Luna column and gradient elution were chosen for the method. This finding is in accordance with the results shown by Hutapea, Toskulkao, Wilairat, and Buddhasukh (1999), who report that this type of column shows poor selectivity, which can be solved using gradient elution. After testing different elution gradients with water (A) and acetonitrile (B) as eluents, we obtained the best results with the following: 80% B, 0–10 min; 80–50% B, 10–12 min; 50–0% B, 12–14 min; 0–80% B, 14–16 min; 80% B, 16–18 min. The flow rate was 1 mL/min, and the sample injection volume was 20 ll. The wavelength used for quantification was 210 nm as this wavelength was the most adequate for our objectives. The evaluation of the method, performed by means of its selectivity, sensitivity and accuracy (Tables 1 and 2), showed that the method was adequate for our objectives. In Table 1, we can see the linearity of the proposed method by means of calibration graphs for stevioside (y = 0.2091x) and rebaudioside A (y = 0.2092x) in a wide concentration range from 25 to 500 mg/L. Note that the correlation coefficients in both steviol glycosides (stevioside and rebaudioside A) were higher than 0.99, which demonstrates an excellent linearity of the method. The sensitivity was similar in both compounds, which showed a LOD of 1.07 mg/L and LOQs of 3.55 and 3.56 mg/L for stevioside and rebaudioside A, respectively. We have not found information about the LOD and LQD in the method described by Woelver-Rieck et al. (2010), which we relied on, but these limits are similar to those obtained in the method proposed by Bergs et al. (2012). The accuracy of the experimental procedure was also evaluated by studying the reproducibility and repeatability. For the reproducibility of a method (%RSD) to be considered acceptable, its value should be less than 20%. As seen in the results summarised in Table 2, this parameter ranged from 3.33% to 5.39%. The same limit (20%) was taken to represent good repeatability; in this case, this value ranged from 5.76% to 9.32%. Therefore, the method proposed shows very good repeatability and reproducibility parameters. 3.2. Extraction conditions To choose the best extraction conditions, we studied the concentrations of the steviol glycosides in the different situations. The first major decision we made was not to grind the sample. This choice was because, in the extraction conditions used (50 g dry leaves, 2 L water, 60 °C for 1 h), grinding the sample did not significantly improve the extraction, whereas the filtration was much more tedious. That is to say, the increase in the efficiency of the extraction does not compensate for the higher cost of filtration.

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Fig. 2. Comparison between chromatograms of same Stevia leaf extract using Luna HILIC column (a: gradient elution; b: isocratic elution) and Develosil column (c: gradient elution; d: isocratic elution). (1) Stevioside, (2) rebaudioside A.

Table 1 Linearity of the method. Compound

Retention time (min)

Slope

Correlation coef. (r2)

Stevioside Rebaudioside A

5.15 6.15

0.2091 0.2092

0.999 0.996

Table 2 Precision of the method. Compound

LOD (mg/L)

LOQ (mg/L)

Reproducibility (%)

Repeatability (%)

Stevioside Rebaudioside A

1.07 1.07

3.55 3.56

3.33 5.39

5.76 9.32

The results obtained by comparing the concentrations we extracted at different temperatures over 20 min (37, 60 and 100 °C, with and without agitation) led us to set the extraction temperature at 100 °C, which was the same temperature used by other authors (Vaneˇk et al., 2001). Once the temperature was reached in the equipment (with water boiling), sample extracts were taken every 10 min and analysed to evaluate the influence of the

extraction time (Fig. 3). The maximum stevioside concentration was at 20 min, with a concentration of 2714.95 mg/L, representing 10.86% in plant weight. The highest quantity of rebaudioside A was reached at 30 min (683.72 mg/L), representing 2.73% in plant weight. These values are within the range of concentrations described by several authors for sweetening compounds (Geuns, 2003; Puri et al., 2011). Taking into account these results, we chose 30 min as the best extraction time for the major steviol glycosides (stevioside and rebaudioside A). We chose not to use solid-phase extraction to purify the sample as this technique requires solvents that are banned in the food industry to condition the cartridge and elute the steviol glycosides. For this reason, we tried to clarify the samples only by micro- and ultrafiltration. After removing solids by means of centrifugation, the clarification of the extracts was performed using different membranes (Fig. 4) for microfiltration (successive filtrations in vacuum: 10, 2.5 and 1 lm). After passing through the final membrane (1 lm), the steviol glycosides concentrations showed by the extracts were as follows: 3409.83 mg/L for stevioside and 1853.73 mg/L for rebaudioside A, representing 9.09% and 4.94%, respectively, of the total mass of the starting plant material. Therefore, these sweeteners represent approximately 14% of the plant mass, which is

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a

2800 2700 mg/L

2600 2500 2400 2300 2200 10

20

30

40

50

60

Extracon me (min)

b

700

mg/L

680

because most of the water passes through the membranes, diluting the final concentrations. This fact is a problem because there are losses of these compounds; we could therefore improve the efficiency by, for example, introducing wash steps of rejections in the final method. Other authors (Chhaya et al., 2012) tested different UF membranes to clarify Stevia extract and obtained approximately 45% recovery of stevioside as the most suitable choice. For all of these reasons, we can say that the use of the proposed membranes in this study is an adequate first step for the clarification of Stevia leaf extracts, although the process must be completed with nanofiltration, as is proposed by other authors (Vanneste et al., 2011; Rao et al., 2012). 4. Conclusions

660 640 620 600 10

20

30

40

50

60

Extracon me (min) Fig. 3. Influence of the extraction time in the concentration of stevioside (a) and rebaudioside A (b).

100% 90% 80% 70% 60%

The HPLC method proposed allows the major steviol glycosides to be quantified in an easy way with good selectivity, sensitivity and accuracy. We propose the best extraction conditions as follows: 100 °C for 30 min without agitation and with no grinding of the leaves. The clarification of the extracts by means of microand ultrafiltration allows an acceptable percentage of the major steviol glycosides to be extracted. The proposed methodology is adequate for determining, in a quick and easy procedure, that the major steviol glycosides (stevioside and rebaudioside A) are safer for health and have a lower environmental impact than other methodologies described in literature. This is of great relevance in the food industry, which are seeking substitutes for the artificial sweeteners widely used up to now, but whose consumption starts to be rejected by consumers increasingly concerned about their health.

50%

References

40% 30% 20% 10% 0% 5KDa 1h 4400 rpm

3KDa 1h 4400 rpm

3KDa 1/2h 4400 rpm

3KDa 5min 12000 rpm

Fig. 4. Percentages of stevioside rejection (above, light grey) and recovery (down, dark grey) when extract is passed through different membranes.

consistent with those concentrations describe by other authors (Geuns, 2003; Puri et al., 2011). To perform the ultrafiltration, the resultant extract was subjected to different centrifugal UF conditions to find the conditions that allow us to obtain the highest concentration of Stevia glycosides and the lowest concentration of undesirable substances: 5 KDa by centrifugation at 4400 rpm for 1 h; 3 KDa by centrifugation at 4400 rpm for 1 h; 3 KDa by centrifugation at 4400 rpm for 30 min; and finally, 3 KDa by centrifugation at 12,000 rpm for 5 min. The results obtained with the UF stages are satisfactory in terms of removing the vast impurity peak, but there is significant rejection of steviol glycosides (Fig. 3). Ultrafiltration with the 5 KDa membrane by centrifugation at 4400 rpm for 1 h showed the highest steviol glycosides recovery but the worst efficiency in removing impurity peak. Therefore, ultrafiltration is a necessary step when designing the clarification method, but it must be supplemented with other treatments. With 3 KDa membranes, we are able to remove the immense impurity peak. However, the percentage of rejection for steviol glycosides is 50% in the case of centrifugation at 4400 rpm both for 1 h and for 30 min, whereas in the case of centrifugation at 12,000 rpm for 5 min, the percentage of rejection is 75%. The higher percentage of compounds in the rejections is

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