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Food Additives & Contaminants: Part A Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/tfac20
Classification of stevia sweeteners in soft drinks using liquid chromatography and time-of-flight mass spectrometry a
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a
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Y. Kakigi , T. Suzuki , T. Icho , A. Uyama & N. Mochizuki
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Research Laboratories for Food Safety Chemistry, Ibaraki, Japan Accepted author version posted online: 17 Sep 2013.Published online: 29 Oct 2013.
To cite this article: Y. Kakigi, T. Suzuki, T. Icho, A. Uyama & N. Mochizuki (2013) Classification of stevia sweeteners in soft drinks using liquid chromatography and time-of-flight mass spectrometry, Food Additives & Contaminants: Part A, 30:12, 2043-2049, DOI: 10.1080/19440049.2013.843101 To link to this article: http://dx.doi.org/10.1080/19440049.2013.843101
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Food Additives & Contaminants: Part A, 2013 Vol. 30, No. 12, 2043–2049, http://dx.doi.org/10.1080/19440049.2013.843101
Classification of stevia sweeteners in soft drinks using liquid chromatography and time-of-flight mass spectrometry Y. Kakigi, T. Suzuki, T. Icho, A. Uyama and N. Mochizuki* Research Laboratories for Food Safety Chemistry, Ibaraki, Japan
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(Received 2 July 2013; accepted 7 September 2013) The aim of this study was to develop a comprehensive analytical method for the characterisation of stevia sweeteners in soft drinks. By using LC and time-of-flight MS, we detected 30 steviol glycosides from nine stevia sweeteners. The mass spectral data of these compounds were applied to the analysis to determine steviol glycosides in nine soft drinks. On the basis of chromatographic data and principal-component analysis, these soft drinks were classified into three groups, and the soft drinks of each group, respectively, contained high-rebaudioside A extract, normal stevia extract or alfa-glucosyltransferase–treated stevia extract. Keywords: stevia sweetener; steviol glycoside; soft drink; LC-TOFMS; principal-component analysis
Introduction Stevia rebaudiana Bertoni is a perennial plant that belongs to the Compositae family, distributed in Brazil and Paraguay (Soejarto et al. 1982; Ohta et al. 2010). Historically, leaves of this plant have been used for making Mate in Paraguay for centuries (Hanson & De Oliveira 1993). Nowadays, stevia sweetener, produced by the extraction of these leaves by hot water and powdering, is commercially used in Japan, China, the United States, Australia and other countries. Sweetening compounds in stevia sweetener are steviol glycosides, which consist of a steviol aglycone and sugar units. The representative compounds are stevioside (Stv) and rebaudioside A (RebA), and analytical standards of nine compounds including Stv and RebA are commercially available (Figure 1). These steviol glycosides are 50–400 times sweeter than sucrose, and the quality of the taste also varies among the compounds. For instance, Stv has slight bitterness and astringency in addition to sweetness. In contrast, RebA has more pure sweetness than does Stv, and the taste is said to be comparatively similar to that of sucrose (Kolb et al. 2001; Pól et al. 2007). Stevia sweetener is principally classified into stevia extract and alfa-glucosyltransferase (AGT)-treated stevia extract (Nihon Shokuhin Tenkabutsu Kyokai 2007). In addition, stevia extract contains relatively high proportions of RebA, especially called high-RebA extract. We refer to the stevia extract other than the high-RebA extract as normal stevia extract in this article. AGT-treated stevia extract is a product of enzyme reaction between stevia extract and sugar sources such as dextrin to improve the sweet taste, but this extract is not allowed to be added to
foods in many countries except for Japan and the United States (Tanimura 2007; Ohta et al. 2010). In such a situation, a method to confirm the type of stevia sweetener contained in foods is necessary for the food regulation. In the present study, we developed a simple method for the analysis of steviol glycosides in soft drinks to estimate the types of stevia sweeteners they contain. Methods for the analysis of steviol glycosides in stevia extracts and foods by using LC-ultraviolet detection (Ozawa et al. 2000, 2003) and LC-quadrupole MS (Yasumura et al. 2004; Yang & Chen 2009; Ujike et al. 2011; Shah et al. 2012) have been previously reported, but the target compounds were limited to commercially available compounds. We considered that other compounds need to be determined because of the presence of AGTtreated stevia extract, which contains alfa-glucosyl steviol glycosides. By using the LC-ultraviolet method or the LCquadrupole MS method, it is difficult to determine the compounds without analytical standards because there are many raw materials and other ingredients in soft drinks. Hence, we performed the analysis by using LCtime-of-flight MS (TOFMS), which enables us to qualify the compounds with high selectivity and accuracy. Materials and methods Samples and reagents Nine stevia sweeteners were obtained from stevia suppliers or purchased via the Internet: four high-RebA extracts (H1–H4), three normal stevia extracts (N1–N3), and two AGT-treated stevia extracts (A1 and A2). Nine soft drinks labelled with “stevia” (S1–S9) were purchased
*Corresponding author. Email: [email protected] © 2013 Taylor & Francis
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Figure 1.
Y. Kakigi et al.
Compound
Abbr.
R1
R2
rebaudioside B
RebB
H
β-Glc-β-Glc(2-1) β-Glc(3-1)
rebaudioside C
RebC
β-Glc
β-Glc-α-Rha(2-1) β-Glc(3-1)
rebaudioside D
RebD
β-Glc-β-Glc(2-1)
β-Glc-β-Glc(2-1) β-Glc(3-1)
rebaudioside F
RebF
β-Glc
β-Glc-β-Xyl(2-1) β-Glc(3-1)
dulcoside A
Dul
β-Glc
β-Glc-α-Rha(2-1)
rubsoside
Rub
β-Glc
β-Glc
steviolbioside
Stb
H
β-Glc-β-Glc(2-1)
Chemical structures of commercially available steviol glycosides.
on the Japanese market. Analytical standards for Stv, RebA, rebaudioside B (RebB) and rubsoside were purchased from Wako Pure Chemical Industries (Osaka, Japan). Rebaudioside C (RebC), rebaudioside D (RebD), rebaudioside F (RebF), dulcoside A and steviolbioside were purchased from ChromaDex (Irvine, CA, USA). Stock solutions of analytical standards except for RebD were prepared in acetonitrile/water (80/20 v/v), and RebD was prepared in acetonitrile/water (40/60 v/v). Standard solution for analysis was prepared by mixing and diluting stock solutions to the desired concentrations with acetonitrile/water (90/10 v/v). Sample preparation Each stevia sweetener (10 mg) was dissolved in 50 mL of acetonitrile/water (90/10 v/v) by sonication. Then, the solution was filtered with a 0.2-μm membrane filter and used in LC-TOFMS analysis. Each soft drink (1 mL) was suspended in acetonitrile (9 mL) and centrifuged at 3000 rpm for 10 min. The supernatant was filtered with a 0.2-μm membrane filter and analysed by using LCTOFMS.
Instruments and analytical conditions The analyses were performed by using a LC-TOFMS system. A Nexera system consisting of a binary pump, an auto-sampler and a column heater (Shimadzu, Kyoto, Japan) was used. Chromatographic separation was carried out on an Asahipak NH2P-50 2D column, 150 × 2.1 mm inner diameter, 5 μm (Showa Denko, Tokyo, Japan). Solvent A was 1% formic acid in water, and solvent B was acetonitrile. The linear gradient was formed as follows: 0–10 min (97% B), 25 min (95% B), 30–60 min (85% B) and 61–70 min (97% B). The flow rate was set at 0.5 mL/min, and the column temperature was 40°C. An auto-sampler was used to inject 10-μL volumes. The mass spectra in the negative mode were acquired by using a Triple TOF 5600 system (AB Sciex, Framingham, MA, USA) with ESA. The capillary voltage was set at –4500 V, and the temperature of the ion source was set at 500°C. Collision energies of –10 V for full-scan and –80 V for informative data acquisition were applied. The scan range was between m/z 100 and 2000. After adding Irganox 1010 (Tokyo Chemical Industry, Tokyo, Japan), APCI negative calibration solution (AB Sciex) was used to calibrate the accurate mass with higher accuracy.
Food Additives & Contaminants: Part A Principal-component analysis LC-TOFMS analysis of each soft drink was performed in triplicate. Then, the obtained raw full-scan data were filtered and aligned by using Marker View software (AB Sciex). After extracting the signals derived from specific ions of steviol glycosides, we normalised the signal intensities in order that the maximum value of each compound in all soft drinks was equal. Afterwards, we conducted principal-component (a)
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analysis (PCA) by using Excel Statistics 2012 software (Social Survey Research Information, Tokyo, Japan). Results and discussion Specific ions of steviol glycosides in stevia sweeteners Firstly, we acquired mass spectra of detected peaks in nine stevia sweeteners through the LC-TOFMS full-scan
12
Intensity (cps)
2.0 × 10–6
1.0 × 10–6
0
10
20
30 40 Time (min)
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0
10
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30 40 Time (min)
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0
10
20
30 40 Time (min)
50
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(b)
Intensity (cps)
3.0 × 10–6
2.0 × 10–6
1.0 × 10–6
(c)
2.0 × 10–6 Intensity (cps)
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3.0 × 10–6
1.0 × 10–6
Figure 2. LC-TOFMS chromatograms of stevia sweeteners. (a) High-RebA extract (H1), (b) normal stevia extract (N1) and (c) AGTtreated stevia extract (A1). AGT, alfa-glucosyltransferase; RebA, rebaudioside A; TOF, time of flight.
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(a)
(b) 1011.4301 [M+HCOO]
–5
–4
Intensity (cps)
8.0 × 10
965.4246 [M-H]
–4
6.0 × 10
–4
4.0 × 10
803.3711 [M-C6H11O5]
2.0 × 10–4 0
200
400
600
Intensity (cps)
–3
1.2 × 10
–3
1.0 × 10
–3
8.0 × 10
–2
6.0 × 10
–2
4.0 × 10
–2
800 1000 1200 1400 1600 1800 m/z
803.3711
641.3182
CH O 317.2142 479.2660 C6H10O5 6 10 5 C6H10O5
200 300 400 500 600 700 800 900 1000 m/z (d)
(c)
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1.4 × 10
2.0 × 10–2
2.0 × 10
–4
1.6 × 10
–4
1.2 × 10
–4
8.0 × 10
–3
1011.4284 [M+HCOO] 965.4227 [M-H]
803.3691 [M-C6H11O5]
4.0 × 10–3 0
200
400
600
800 1000 1200 1400 1600 1800 m/z
Intensity (cps)
Intensity (cps)
1.0 × 10
1.0 × 10
–3
8.0 × 10
–2
6.0 × 10
–2
4.0 × 10
–2
2.0 × 10
–2
803.3701 641.3168 479.2645 317.2129 C6H10O5
C6H10O5
C6H10O5
200 300 400 500 600 700 800 900 1000 m/z
Figure 3. Mass spectra of peak no. 12 in high-RebA extract (H1) and RebA analytical standard. (a) Full-scan mass spectrum of peak no. 12, (b) product ion mass spectrum of the deprotonated molecule of peak no. 12, (c) full-scan mass spectrum of RebA analytical standard and (d) product ion mass spectrum of the deprotonated molecule of RebA analytical standard. RebA, rebaudioside A.
data to extract specific ions of steviol glycosides (Figure 2). The mass spectra with/without collisioninduced dissociation of a representative peak no. 12 in stevia sweetener H1 are shown in Figure 3(a). From fullscan spectrum of peak no. 12, the presence of the deprotonated molecule that has accurate mass of 965.4246 Da is indicated. The chemical formula is considered to be [C44H69O23]– with mass accuracy of 1.1 ppm. Signal of m/z 803.3691 indicates the in-source decay of the deprotonated molecule, and m/z 1011.4284 means the formic acid adducted ion. The product ion spectrum by collisioninduced dissociation of the deprotonated molecule of 965.4246 Da is shown in Figure 3(b). Signal of m/z 317.2142 indicates the presence of C20H29O3, and this strongly suggests the presence of steviol aglycone. Equally spaced signals from m/z 317.2142 to 803.3711 indicate the presence of C6H10O5 fragments, which is highly likely to be four hexose units. In this case, the mass spectra and the retention time closely resembled those of RebA analytical standard (Figure 3c and d). Thus, we identified peak no. 12 as RebA. In the same manner, we eventually qualified the 30 steviol glycosides in nine stevia sweeteners (Table 1).
Profiling of steviol glycosides in soft drinks Secondly, nine soft drinks were tested by using the method described in the previous section, and an extracted ion chromatogram, which enables us to compare the steviol glycoside profile without interference from other ingredients or raw materials, was drawn according to the obtained specific deprotonated ions. As a result, RebA and RebB were detected in soft drinks S1–S4, S6 and S8 and the signal ratio of RebA was prominently high (Figure 4a). In addition, the chromatographic patterns were similar to those of high-RebA extracts. Thus, we considered that these soft drinks contain high-RebA extracts as ingredients. In soft drinks S5and S7, various steviol glycosides identified as Stv, RebA, RebB, RebC and steviolbioside were detected (Figure 4b). The signal ratio of Stv was relatively high. Moreover, the chromatographic pattern was closely similar to that of normal stevia extracts. Then, we considered that these drinks contained normal stevia extracts as ingredients. As for soft drink S9, because some peaks were not identified by comparison with analytical standards, we assigned these peaks on the basis of mass spectra by the scheme described above. As a result, strong signals of steviol
37.1 37.7 39.3
43.9 45.3
29 30
C68H109O43 C68H109O43
C62H99O38 C62H99O38 C68H109O42 2.5 2.5
2.2 3.3 4.6
RebD
RebC RebF RebA
Stv RebB
Rub Stb Dul
Identification 2Glc 2Glc 2Glc, Rha 2hex, pen 2hex, pen 3hex 3Glc 3Glc 3hex 3Glc, Rha 3Glc, pen 4Glc 4hex 4hex 4hex 4hex 4hex 5hex 5Glc 5hex 5hex, deshex 6hex 6hex 6hex 6hex, deshex 7hex 7hex 7hex, deshex 8hex 8hex
Sugar bonds
+
+ +
+ +
+ +
H1
+
+ + +
+ +
+
H2
+
+
+
+ + +
+ +
+
H3
+ + +
+ +
H4
+ +
+ + +
+ + + +
+
+ + + + +
+ +
+ + +
+
A1
+
+
+
+ +
+ +
+
+ +
+
A2
Stevia sweetener
+ + +
+ + +
N1 + + + +
+
+
+ + +
+ + +
+
N2 +
+ +
+ + +
+ +
N3 + + + + +
Note: +, detected; deshex, desoxyhexose; Dul, dulcoside A; hex, hexose; pen, pentose; RebA, rebaudioside A; RebC, rebaudioside C; RebD, rebaudioside D; RebF, rebaudioside F; Rub, rubsoside; Stb, steviolbioside; Stv, stevioside.
1613.6388 1613.6388
1451.5852 1451.5868 1597.6461
3.6 1.8 2.4 2.6
26 27 28
1289.5338 1289.5315 1289.5322 1435.5902
C56H89O33 C56H89O33 C56H89O33 C62H99O37
33.0 33.2 33.7 34.7
22 23 24 25
−2.5 −2.5 −2.4 −1.9 −0.7 4.1 1.8 2.5 2.6 −0.8 3.2 0.6 3.2 −0.7 0.6 −0.3 −3.0 1.7 2.5 0.7 0.6
Mass error (ppm)
C32H49O13 C32H49O13 C38H59O17 C37H57O17 C37H57O17 C38H59O18 C38H59O18 C38H59O18 C38H59O18 C44H69O22 C43H67O22 C44H69O23 C44H69O23 C44H69O23 C44H69O23 C44H69O23 C44H69O23 C50H79O28 C50H79O28 C50H79O28 C56H89O32
2.4 4.2 4.8 5.3 6.0 6.6 8.9 11.4 12.7 13.2 15.5 22.2 25.9 27.2 28.1 28.4 28.8 30.5 30.9 31.2 31.6
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21
641.3163 641.3163 787.3739 773.3575 773.3585 803.3740 803.3721 803.3727 803.3728 949.4294 935.4159 965.4241 965.4266 965.4228 965.4241 965.4232 965.4206 1127.4782 1127.4792 1127.4771 1273.5399
Rt. Measured mass Estimated formula (min) (Da) [M-H]−
Peak assignment of detected peaks in nine stevia sweeteners.
Peak no.
Table 1.
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12 2.5 × 10–4
Intensity (cps)
2.0 × 10–4 1.5 × 10–4 1.0 × 10–4 5.0 × 10–3
8 10
(b)
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30 40 Time (min)
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7 5.0 × 10–3
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Intensity (cps)
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4.0 × 10–3
**
7
3.0 × 10–3
** 10
2.0 × 10–3 1.0 × 10–3
8 0
10
*** * 12 20
* *
30 30 40 Time (min)
50
60
Figure 4. Overlaid 14 extracted ion chromatograms of steviol glycosides in soft drinks. m/z 641.3173, 787.3752, 773.3596, 803.3701, 949.4280, 935.4124, 965.4230, 1127.4763, 1289.5292, 1273.5342, 1435.5865, 1451.5820, 1597.6399 and 1613.6348. (a) Soft drink S1, (b) S5 and (c) S9 (square means extended figure around retention time 30 min). Peak identification, numbers, see Table 1. Note: *Steviol glycosides with four hexose units; **Steviol glycosides with five hexose units; ***Steviol glycosides with six hexose units.
glycosides with four hexose units other than RebA, moderate signals of steviol glycosides with five hexose units other than RebD and trace-levelled signals of steviol glycosides with six hexose units were qualified (Figure 4c). Existence of such steviol glycosides has
been hardly reported and is thought to be rare in nature. Moreover, the accurate masses and retention times of these detected peaks were corresponding with those of AGT stevia extract A1. Consequently, soft drink S9 was considered to contain AGT stevia extract.
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applied to the analysis of various food products containing stevia sweeteners.
0.6 S9 0.4 group C 0.2
PC2 (19.4%)
S4 –1.0
0.0 0.0
–0.5
0.5
–0.2
References
S1 S3 S8 1.0
S6
1.5
S2 group A
–0.4 –0.6 S7 –0.8
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S5
group B –1.0 PC1 (63.4%)
Figure 5. PCA plots of nine soft drinks. Nine soft drinks (S1–S9) are indicated as black rhombusess (n = 3 for each). Three soft drink groups (groups A, B and C) are indicated as circles. PCA, principal-component analysis.
Principal-component analysis We performed PCA to visualise the profiled results of all tested soft drinks. PCA plots were obtained with high accuracy and high cumulative contribution rate (Figure 5). On the basis of score plots, nine soft drinks were divided into three groups (groups A, B and C). Six soft drinks (S1–S4, S6 and S8) that were considered to contain high-RebA extracts belong to group A. Two soft drinks (S5 and S7) that contain normal stevia extracts belong to group B. Then, one drink (S9) considered to contain AGT-treated stevia extract belongs to group C. Consequently, we estimated the types of stevia sweetener contained as ingredients in nine soft drinks by using LC-TOFMS and represented the total results by PCA. This method can be adapted to the surveillance of stevia sweeteners in soft drinks for the purpose of food regulation. And with further research, this method will be
Hanson JR, De Oliveira BH. 1993. Stevioside and related sweet diterpenoid glycosides. Nat Prod Rep. 10:301–309. Kolb N, Herrera JL, Ferreyra DJ, Uliana RF. 2001. Analysis of sweet diterpene glycosides from Stevia rebaudiana: improved HPLC method. J Agric Food Chem. 49:4538–4541. Nihon Shokuhin Tenkabutsu Kyokai. 2007. Shokuhin Tenkabutsu Kouteisho. 8th ed. Tokyo: Nihon Shokuhin Tenkabutsu Kyokai. α-Glucosyltransferase Treated Stevia, Stevia Extract; 326–328;451–452. Ohta M, Sasa S, Inoue A, Tamai I, Fujita I, Morita K, Matsuura F. 2010. Characterization of novel steviol glycosides from leaves of Stevia rebaudiana Morita. J Appl Glycosci. 57:199–209. Ozawa H, Hirokado M, Shimamura Y, Nakajima K, Kimura K, Yasuda K. 2000. An analysis method for Stevia sweeteners in food by high performance liquid chromatography. Tokyo Eisei Kenkyu zyo Nenpo. 51:75–79. Ozawa H, Hirokado M, Taguchi N, Kobayashi C, Yamazima Y, Saito K. 2003. Simultaneous determination of glycyrrhizinic acid, stevioside and rebaudioside A in food by solid phase extraction and HPLC. Tokyo Kenko Anzen Cent Nenpo. 54:93–98. Pól J, Hohnová B, Hyötyläinen T. 2007. Characterisation of Stevia rebaudiana by comprehensive two-dimensional liquid chromatography time-of-flight mass spectrometry. J Chromatogr A. 1150:85–92. Shah R, De Jager LS, Begley TH. 2012. Simultaneous determination of steviol and glycosides by liquid chromatography-mass spectrometry. Food Addit Contam A. 29:1861–1871. Soejarto DD, Kinghorn AD, Fransworth NR. 1982. Potential sweetening agents of plant origin. III. Organoleptic evaluation of stevia leaf herbarium samples for sweetness. J Nat Prod. 45:590–599. Tanimura A. 2007. Shokuhin Tenkabutsu Kouteisho. 8th ed. Kaisetsusho. Tokyo: Hirokawa Shoten. α-Glucosyltransferase Treated Stevia; D1014–D1022. Ujike A, Mori K, Yasunaga M, Ishikawa J, Nishioka C. 2011. Study on use of sweeteners in food products (II). Kagawa ken Kankyo Hoken Kenkyu Cent Shoho. 10:80–84. Yang D-J, Chen B. 2009. Simultaneous determination of nonnutritive sweeteners in foods by HPLC/ESI-MS. J Agric Food Chem. 57:3022–3027. Yasumura K, Ibuki S, Tanaka T, Kitada Y. 2004. Analysis of stevia components in food by LC/MS/MS. Nara ken Hoken Kankyo Kenkyu Cent Nenpo. 38:97–98.
Food Additives & Contaminants: Part A Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/tfac20
Classification of stevia sweeteners in soft drinks using liquid chromatography and time-of-flight mass spectrometry a
a
a
a
Y. Kakigi , T. Suzuki , T. Icho , A. Uyama & N. Mochizuki
a
a
Research Laboratories for Food Safety Chemistry, Ibaraki, Japan Accepted author version posted online: 17 Sep 2013.Published online: 29 Oct 2013.
To cite this article: Y. Kakigi, T. Suzuki, T. Icho, A. Uyama & N. Mochizuki (2013) Classification of stevia sweeteners in soft drinks using liquid chromatography and time-of-flight mass spectrometry, Food Additives & Contaminants: Part A, 30:12, 2043-2049, DOI: 10.1080/19440049.2013.843101 To link to this article: http://dx.doi.org/10.1080/19440049.2013.843101
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http:// www.tandfonline.com/page/terms-and-conditions
Food Additives & Contaminants: Part A, 2013 Vol. 30, No. 12, 2043–2049, http://dx.doi.org/10.1080/19440049.2013.843101
Classification of stevia sweeteners in soft drinks using liquid chromatography and time-of-flight mass spectrometry Y. Kakigi, T. Suzuki, T. Icho, A. Uyama and N. Mochizuki* Research Laboratories for Food Safety Chemistry, Ibaraki, Japan
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(Received 2 July 2013; accepted 7 September 2013) The aim of this study was to develop a comprehensive analytical method for the characterisation of stevia sweeteners in soft drinks. By using LC and time-of-flight MS, we detected 30 steviol glycosides from nine stevia sweeteners. The mass spectral data of these compounds were applied to the analysis to determine steviol glycosides in nine soft drinks. On the basis of chromatographic data and principal-component analysis, these soft drinks were classified into three groups, and the soft drinks of each group, respectively, contained high-rebaudioside A extract, normal stevia extract or alfa-glucosyltransferase–treated stevia extract. Keywords: stevia sweetener; steviol glycoside; soft drink; LC-TOFMS; principal-component analysis
Introduction Stevia rebaudiana Bertoni is a perennial plant that belongs to the Compositae family, distributed in Brazil and Paraguay (Soejarto et al. 1982; Ohta et al. 2010). Historically, leaves of this plant have been used for making Mate in Paraguay for centuries (Hanson & De Oliveira 1993). Nowadays, stevia sweetener, produced by the extraction of these leaves by hot water and powdering, is commercially used in Japan, China, the United States, Australia and other countries. Sweetening compounds in stevia sweetener are steviol glycosides, which consist of a steviol aglycone and sugar units. The representative compounds are stevioside (Stv) and rebaudioside A (RebA), and analytical standards of nine compounds including Stv and RebA are commercially available (Figure 1). These steviol glycosides are 50–400 times sweeter than sucrose, and the quality of the taste also varies among the compounds. For instance, Stv has slight bitterness and astringency in addition to sweetness. In contrast, RebA has more pure sweetness than does Stv, and the taste is said to be comparatively similar to that of sucrose (Kolb et al. 2001; Pól et al. 2007). Stevia sweetener is principally classified into stevia extract and alfa-glucosyltransferase (AGT)-treated stevia extract (Nihon Shokuhin Tenkabutsu Kyokai 2007). In addition, stevia extract contains relatively high proportions of RebA, especially called high-RebA extract. We refer to the stevia extract other than the high-RebA extract as normal stevia extract in this article. AGT-treated stevia extract is a product of enzyme reaction between stevia extract and sugar sources such as dextrin to improve the sweet taste, but this extract is not allowed to be added to
foods in many countries except for Japan and the United States (Tanimura 2007; Ohta et al. 2010). In such a situation, a method to confirm the type of stevia sweetener contained in foods is necessary for the food regulation. In the present study, we developed a simple method for the analysis of steviol glycosides in soft drinks to estimate the types of stevia sweeteners they contain. Methods for the analysis of steviol glycosides in stevia extracts and foods by using LC-ultraviolet detection (Ozawa et al. 2000, 2003) and LC-quadrupole MS (Yasumura et al. 2004; Yang & Chen 2009; Ujike et al. 2011; Shah et al. 2012) have been previously reported, but the target compounds were limited to commercially available compounds. We considered that other compounds need to be determined because of the presence of AGTtreated stevia extract, which contains alfa-glucosyl steviol glycosides. By using the LC-ultraviolet method or the LCquadrupole MS method, it is difficult to determine the compounds without analytical standards because there are many raw materials and other ingredients in soft drinks. Hence, we performed the analysis by using LCtime-of-flight MS (TOFMS), which enables us to qualify the compounds with high selectivity and accuracy. Materials and methods Samples and reagents Nine stevia sweeteners were obtained from stevia suppliers or purchased via the Internet: four high-RebA extracts (H1–H4), three normal stevia extracts (N1–N3), and two AGT-treated stevia extracts (A1 and A2). Nine soft drinks labelled with “stevia” (S1–S9) were purchased
*Corresponding author. Email: [email protected] © 2013 Taylor & Francis
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Figure 1.
Y. Kakigi et al.
Compound
Abbr.
R1
R2
rebaudioside B
RebB
H
β-Glc-β-Glc(2-1) β-Glc(3-1)
rebaudioside C
RebC
β-Glc
β-Glc-α-Rha(2-1) β-Glc(3-1)
rebaudioside D
RebD
β-Glc-β-Glc(2-1)
β-Glc-β-Glc(2-1) β-Glc(3-1)
rebaudioside F
RebF
β-Glc
β-Glc-β-Xyl(2-1) β-Glc(3-1)
dulcoside A
Dul
β-Glc
β-Glc-α-Rha(2-1)
rubsoside
Rub
β-Glc
β-Glc
steviolbioside
Stb
H
β-Glc-β-Glc(2-1)
Chemical structures of commercially available steviol glycosides.
on the Japanese market. Analytical standards for Stv, RebA, rebaudioside B (RebB) and rubsoside were purchased from Wako Pure Chemical Industries (Osaka, Japan). Rebaudioside C (RebC), rebaudioside D (RebD), rebaudioside F (RebF), dulcoside A and steviolbioside were purchased from ChromaDex (Irvine, CA, USA). Stock solutions of analytical standards except for RebD were prepared in acetonitrile/water (80/20 v/v), and RebD was prepared in acetonitrile/water (40/60 v/v). Standard solution for analysis was prepared by mixing and diluting stock solutions to the desired concentrations with acetonitrile/water (90/10 v/v). Sample preparation Each stevia sweetener (10 mg) was dissolved in 50 mL of acetonitrile/water (90/10 v/v) by sonication. Then, the solution was filtered with a 0.2-μm membrane filter and used in LC-TOFMS analysis. Each soft drink (1 mL) was suspended in acetonitrile (9 mL) and centrifuged at 3000 rpm for 10 min. The supernatant was filtered with a 0.2-μm membrane filter and analysed by using LCTOFMS.
Instruments and analytical conditions The analyses were performed by using a LC-TOFMS system. A Nexera system consisting of a binary pump, an auto-sampler and a column heater (Shimadzu, Kyoto, Japan) was used. Chromatographic separation was carried out on an Asahipak NH2P-50 2D column, 150 × 2.1 mm inner diameter, 5 μm (Showa Denko, Tokyo, Japan). Solvent A was 1% formic acid in water, and solvent B was acetonitrile. The linear gradient was formed as follows: 0–10 min (97% B), 25 min (95% B), 30–60 min (85% B) and 61–70 min (97% B). The flow rate was set at 0.5 mL/min, and the column temperature was 40°C. An auto-sampler was used to inject 10-μL volumes. The mass spectra in the negative mode were acquired by using a Triple TOF 5600 system (AB Sciex, Framingham, MA, USA) with ESA. The capillary voltage was set at –4500 V, and the temperature of the ion source was set at 500°C. Collision energies of –10 V for full-scan and –80 V for informative data acquisition were applied. The scan range was between m/z 100 and 2000. After adding Irganox 1010 (Tokyo Chemical Industry, Tokyo, Japan), APCI negative calibration solution (AB Sciex) was used to calibrate the accurate mass with higher accuracy.
Food Additives & Contaminants: Part A Principal-component analysis LC-TOFMS analysis of each soft drink was performed in triplicate. Then, the obtained raw full-scan data were filtered and aligned by using Marker View software (AB Sciex). After extracting the signals derived from specific ions of steviol glycosides, we normalised the signal intensities in order that the maximum value of each compound in all soft drinks was equal. Afterwards, we conducted principal-component (a)
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analysis (PCA) by using Excel Statistics 2012 software (Social Survey Research Information, Tokyo, Japan). Results and discussion Specific ions of steviol glycosides in stevia sweeteners Firstly, we acquired mass spectra of detected peaks in nine stevia sweeteners through the LC-TOFMS full-scan
12
Intensity (cps)
2.0 × 10–6
1.0 × 10–6
0
10
20
30 40 Time (min)
50
60
0
10
20
30 40 Time (min)
50
60
0
10
20
30 40 Time (min)
50
60
(b)
Intensity (cps)
3.0 × 10–6
2.0 × 10–6
1.0 × 10–6
(c)
2.0 × 10–6 Intensity (cps)
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3.0 × 10–6
1.0 × 10–6
Figure 2. LC-TOFMS chromatograms of stevia sweeteners. (a) High-RebA extract (H1), (b) normal stevia extract (N1) and (c) AGTtreated stevia extract (A1). AGT, alfa-glucosyltransferase; RebA, rebaudioside A; TOF, time of flight.
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Y. Kakigi et al.
(a)
(b) 1011.4301 [M+HCOO]
–5
–4
Intensity (cps)
8.0 × 10
965.4246 [M-H]
–4
6.0 × 10
–4
4.0 × 10
803.3711 [M-C6H11O5]
2.0 × 10–4 0
200
400
600
Intensity (cps)
–3
1.2 × 10
–3
1.0 × 10
–3
8.0 × 10
–2
6.0 × 10
–2
4.0 × 10
–2
800 1000 1200 1400 1600 1800 m/z
803.3711
641.3182
CH O 317.2142 479.2660 C6H10O5 6 10 5 C6H10O5
200 300 400 500 600 700 800 900 1000 m/z (d)
(c)
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1.4 × 10
2.0 × 10–2
2.0 × 10
–4
1.6 × 10
–4
1.2 × 10
–4
8.0 × 10
–3
1011.4284 [M+HCOO] 965.4227 [M-H]
803.3691 [M-C6H11O5]
4.0 × 10–3 0
200
400
600
800 1000 1200 1400 1600 1800 m/z
Intensity (cps)
Intensity (cps)
1.0 × 10
1.0 × 10
–3
8.0 × 10
–2
6.0 × 10
–2
4.0 × 10
–2
2.0 × 10
–2
803.3701 641.3168 479.2645 317.2129 C6H10O5
C6H10O5
C6H10O5
200 300 400 500 600 700 800 900 1000 m/z
Figure 3. Mass spectra of peak no. 12 in high-RebA extract (H1) and RebA analytical standard. (a) Full-scan mass spectrum of peak no. 12, (b) product ion mass spectrum of the deprotonated molecule of peak no. 12, (c) full-scan mass spectrum of RebA analytical standard and (d) product ion mass spectrum of the deprotonated molecule of RebA analytical standard. RebA, rebaudioside A.
data to extract specific ions of steviol glycosides (Figure 2). The mass spectra with/without collisioninduced dissociation of a representative peak no. 12 in stevia sweetener H1 are shown in Figure 3(a). From fullscan spectrum of peak no. 12, the presence of the deprotonated molecule that has accurate mass of 965.4246 Da is indicated. The chemical formula is considered to be [C44H69O23]– with mass accuracy of 1.1 ppm. Signal of m/z 803.3691 indicates the in-source decay of the deprotonated molecule, and m/z 1011.4284 means the formic acid adducted ion. The product ion spectrum by collisioninduced dissociation of the deprotonated molecule of 965.4246 Da is shown in Figure 3(b). Signal of m/z 317.2142 indicates the presence of C20H29O3, and this strongly suggests the presence of steviol aglycone. Equally spaced signals from m/z 317.2142 to 803.3711 indicate the presence of C6H10O5 fragments, which is highly likely to be four hexose units. In this case, the mass spectra and the retention time closely resembled those of RebA analytical standard (Figure 3c and d). Thus, we identified peak no. 12 as RebA. In the same manner, we eventually qualified the 30 steviol glycosides in nine stevia sweeteners (Table 1).
Profiling of steviol glycosides in soft drinks Secondly, nine soft drinks were tested by using the method described in the previous section, and an extracted ion chromatogram, which enables us to compare the steviol glycoside profile without interference from other ingredients or raw materials, was drawn according to the obtained specific deprotonated ions. As a result, RebA and RebB were detected in soft drinks S1–S4, S6 and S8 and the signal ratio of RebA was prominently high (Figure 4a). In addition, the chromatographic patterns were similar to those of high-RebA extracts. Thus, we considered that these soft drinks contain high-RebA extracts as ingredients. In soft drinks S5and S7, various steviol glycosides identified as Stv, RebA, RebB, RebC and steviolbioside were detected (Figure 4b). The signal ratio of Stv was relatively high. Moreover, the chromatographic pattern was closely similar to that of normal stevia extracts. Then, we considered that these drinks contained normal stevia extracts as ingredients. As for soft drink S9, because some peaks were not identified by comparison with analytical standards, we assigned these peaks on the basis of mass spectra by the scheme described above. As a result, strong signals of steviol
37.1 37.7 39.3
43.9 45.3
29 30
C68H109O43 C68H109O43
C62H99O38 C62H99O38 C68H109O42 2.5 2.5
2.2 3.3 4.6
RebD
RebC RebF RebA
Stv RebB
Rub Stb Dul
Identification 2Glc 2Glc 2Glc, Rha 2hex, pen 2hex, pen 3hex 3Glc 3Glc 3hex 3Glc, Rha 3Glc, pen 4Glc 4hex 4hex 4hex 4hex 4hex 5hex 5Glc 5hex 5hex, deshex 6hex 6hex 6hex 6hex, deshex 7hex 7hex 7hex, deshex 8hex 8hex
Sugar bonds
+
+ +
+ +
+ +
H1
+
+ + +
+ +
+
H2
+
+
+
+ + +
+ +
+
H3
+ + +
+ +
H4
+ +
+ + +
+ + + +
+
+ + + + +
+ +
+ + +
+
A1
+
+
+
+ +
+ +
+
+ +
+
A2
Stevia sweetener
+ + +
+ + +
N1 + + + +
+
+
+ + +
+ + +
+
N2 +
+ +
+ + +
+ +
N3 + + + + +
Note: +, detected; deshex, desoxyhexose; Dul, dulcoside A; hex, hexose; pen, pentose; RebA, rebaudioside A; RebC, rebaudioside C; RebD, rebaudioside D; RebF, rebaudioside F; Rub, rubsoside; Stb, steviolbioside; Stv, stevioside.
1613.6388 1613.6388
1451.5852 1451.5868 1597.6461
3.6 1.8 2.4 2.6
26 27 28
1289.5338 1289.5315 1289.5322 1435.5902
C56H89O33 C56H89O33 C56H89O33 C62H99O37
33.0 33.2 33.7 34.7
22 23 24 25
−2.5 −2.5 −2.4 −1.9 −0.7 4.1 1.8 2.5 2.6 −0.8 3.2 0.6 3.2 −0.7 0.6 −0.3 −3.0 1.7 2.5 0.7 0.6
Mass error (ppm)
C32H49O13 C32H49O13 C38H59O17 C37H57O17 C37H57O17 C38H59O18 C38H59O18 C38H59O18 C38H59O18 C44H69O22 C43H67O22 C44H69O23 C44H69O23 C44H69O23 C44H69O23 C44H69O23 C44H69O23 C50H79O28 C50H79O28 C50H79O28 C56H89O32
2.4 4.2 4.8 5.3 6.0 6.6 8.9 11.4 12.7 13.2 15.5 22.2 25.9 27.2 28.1 28.4 28.8 30.5 30.9 31.2 31.6
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21
641.3163 641.3163 787.3739 773.3575 773.3585 803.3740 803.3721 803.3727 803.3728 949.4294 935.4159 965.4241 965.4266 965.4228 965.4241 965.4232 965.4206 1127.4782 1127.4792 1127.4771 1273.5399
Rt. Measured mass Estimated formula (min) (Da) [M-H]−
Peak assignment of detected peaks in nine stevia sweeteners.
Peak no.
Table 1.
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12 2.5 × 10–4
Intensity (cps)
2.0 × 10–4 1.5 × 10–4 1.0 × 10–4 5.0 × 10–3
8 10
(b)
20
30 40 Time (min)
50
60
30 40 Time (min)
50
60
7 5.0 × 10–3
12
Intensity (cps)
4.0 × 10–3 3.0 × 10–3 2.0 × 10–3
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2 1.0 × 10–3
10 0
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(c) 2 5.0 × 10–3 19 Intensity (cps)
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4.0 × 10–3
**
7
3.0 × 10–3
** 10
2.0 × 10–3 1.0 × 10–3
8 0
10
*** * 12 20
* *
30 30 40 Time (min)
50
60
Figure 4. Overlaid 14 extracted ion chromatograms of steviol glycosides in soft drinks. m/z 641.3173, 787.3752, 773.3596, 803.3701, 949.4280, 935.4124, 965.4230, 1127.4763, 1289.5292, 1273.5342, 1435.5865, 1451.5820, 1597.6399 and 1613.6348. (a) Soft drink S1, (b) S5 and (c) S9 (square means extended figure around retention time 30 min). Peak identification, numbers, see Table 1. Note: *Steviol glycosides with four hexose units; **Steviol glycosides with five hexose units; ***Steviol glycosides with six hexose units.
glycosides with four hexose units other than RebA, moderate signals of steviol glycosides with five hexose units other than RebD and trace-levelled signals of steviol glycosides with six hexose units were qualified (Figure 4c). Existence of such steviol glycosides has
been hardly reported and is thought to be rare in nature. Moreover, the accurate masses and retention times of these detected peaks were corresponding with those of AGT stevia extract A1. Consequently, soft drink S9 was considered to contain AGT stevia extract.
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applied to the analysis of various food products containing stevia sweeteners.
0.6 S9 0.4 group C 0.2
PC2 (19.4%)
S4 –1.0
0.0 0.0
–0.5
0.5
–0.2
References
S1 S3 S8 1.0
S6
1.5
S2 group A
–0.4 –0.6 S7 –0.8
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S5
group B –1.0 PC1 (63.4%)
Figure 5. PCA plots of nine soft drinks. Nine soft drinks (S1–S9) are indicated as black rhombusess (n = 3 for each). Three soft drink groups (groups A, B and C) are indicated as circles. PCA, principal-component analysis.
Principal-component analysis We performed PCA to visualise the profiled results of all tested soft drinks. PCA plots were obtained with high accuracy and high cumulative contribution rate (Figure 5). On the basis of score plots, nine soft drinks were divided into three groups (groups A, B and C). Six soft drinks (S1–S4, S6 and S8) that were considered to contain high-RebA extracts belong to group A. Two soft drinks (S5 and S7) that contain normal stevia extracts belong to group B. Then, one drink (S9) considered to contain AGT-treated stevia extract belongs to group C. Consequently, we estimated the types of stevia sweetener contained as ingredients in nine soft drinks by using LC-TOFMS and represented the total results by PCA. This method can be adapted to the surveillance of stevia sweeteners in soft drinks for the purpose of food regulation. And with further research, this method will be
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