Validation of an HPLC Method for Pretreatment of Steviol Glycosides in Fermented Milk
Abstract
:1. Introduction
2. Materials and Methods
2.1. Reagents and Samples
2.2. Identification of the Optimal Pretreatment Method
2.3. HPLC Analysis
2.4. Evaluation of Steviol Glycoside Content
2.5. Monitoring Test
2.6. Statistical Analysis
3. Results and Discussion
3.1. Optimization of the Pretreatment Conditions
3.2. Separation of Steviol Glycosides
3.3. Calibration Curves, LOD, and Quantification
3.4. Accuracy
3.5. Monitoring Test for Various Foods
4. Conclusions
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Conflicts of Interest
References
- Carakostas, M.C.; Prakash, I.; Kinghorn, A.D.; Wu, C.D.; Soejarto, D.D. Steviol glycosides. In Alternative Sweeteners, 4th ed.; O’Brien-Nabors, L., Ed.; CRC Press: Boca Raton, FL, USA, 2016. [Google Scholar]
- Crammer, B.; Ikan, R. Sweet glycosides from the stevia plant. Chem. Br. 1986, 22, 915–917. [Google Scholar]
- Can, Z.; Baltas, N. Bioactivity and enzyme inhibition properties of Stevia rebaudiana. Curr. Enzym. Inhib. 2016, 12, 188–194. [Google Scholar] [CrossRef]
- Shukla, S.; Mehta, A.; Bajpai, V.K. Phytochemical screening and anthelmintic and antifungal activities of leaf extracts of Stevia rebaudiana. J. Biol. Act. Prod. Nat. 2013, 3, 56–63. [Google Scholar]
- Geuns, J.M.C. Stevioside. Phytochemistry 2003, 64, 913–921. [Google Scholar] [CrossRef]
- López, V.; Pérez, S.; Vinuesa, A.; Zorzetto, C.; Abian, O. Stevia rebaudiana ethanolic extract exerts better antioxidant properties and antiproliferative effects in tumour cells than its diterpene glycoside stevioside. Food Funct. 2016, 7, 2107–2113. [Google Scholar] [CrossRef]
- Ritu, M.; Nandini, J. Nutritional composition of Stevia rebaudiana, a sweet herb, and its hypoglycaemic and hypolipidaemic effect on patients with non-insulin dependent diabetes mellitus. J. Sci. Food Agric. 2016, 96, 4231–4234. [Google Scholar] [CrossRef]
- Ruiz-Ruiz, J.C.; Moguel-Ordoñez, Y.B.; Segura-Campos, M.R. Biological activity of Stevia rebaudiana Bertoni and their relationship to health. Crit. Rev. Food Sci. Nutri. 2017, 57, 2680–2690. [Google Scholar]
- Brusick, D.J. A critical review of the genetic toxicity of steviol and steviol glycosides. Food Chem. Toxicol. 2008, 46, S83–S91. [Google Scholar] [CrossRef]
- Momtazi-Borojeni, A.A.; Esmaeili, S.A.; Abdollahi, E.; Sahebkar, A. A review on the pharmacology and toxicology of steviol glycosides extracted from Stevia rebaudiana. Curr. Pharm. Des. 2017, 23, 1616–1622. [Google Scholar] [CrossRef] [PubMed]
- Gardana, C.; Scaglianti, M.; Simonetti, P. Evaluation of steviol and its glycosides in Stevia rebaudiana leaves and commercial sweetener by ultra-high-performance liquid chromatography-mass spectrometry. J. Chromatogr. A 2010, 1217, 1463–1470. [Google Scholar] [CrossRef] [PubMed]
- Kennelly, E.J. Sweet and non-sweet constituents of Stevia rebaudiana (Bertoni) Bertoni. In Stevia, the Genus Stevia. Medicinal and Aromatic Plants—Industrial Profiles; Kinghorn, A.D., Ed.; Taylor and Francis: London, UK, 2002; pp. 68–85. [Google Scholar]
- Lemus-Mondaca, R.; Vega-Gálvez, A.; Zura-Bravo, L.; Kong, A.H. Stevia rebaudiana Bertoni, source of a high-potency natural sweetener: A comprehensive review on the biochemical, nutritional and functional aspects. Food Chem. 2012, 132, 1121–1132. [Google Scholar] [CrossRef] [PubMed]
- Yildiz, M.; Karhan, M. Characteristics of some beverages adjusted with stevia extract, and persistence of steviol glycosides in the mouth after consumption. Int. J. Gastron. 2021, 24, 100326. [Google Scholar]
- Urban, J.D.; Carakostas, M.C.; Brusick, D.J. Steviol glycoside safety: Is the genotoxicity database sufficient? Food Chem. Toxicol. 2013, 51, 386–390. [Google Scholar] [CrossRef] [PubMed]
- European Food Safety Authority (EFSA). Commission Regulation (EU) No 1131/2011 of 11 November 2011 amending Annex II to Regulation (EC) No 1333/2008 of the European Parliament and of the Council with regard to steviol glycosides. Off. J. Eur. Union 2011, L295, 205–211. [Google Scholar]
- World Health Organization (WHO). Evaluation of Certain Food Additives: Sixty-Ninth Report of the Joint FAO/WHO Expert Committee on Food Additives. Available online: https://apps.who.int/iris/bitstream/handle/10665/44062/WHO_TRS_952_eng.pdf?sequence=1&isAllowed=y (accessed on 8 September 2010).
- European Food Safety Authority (EFSA). Scientific opinion on the safety of steviol glycosides for the proposed uses as a food additive. EFSA J. 2010, 8, 1537. [Google Scholar]
- Roberts, A.; Renwick, A.G. Comparative toxicokinetics and metabolism of rebaudioside A, stevioside, and steviol in rats. Food Chem. Toxicol. 2008, 46, S31–S39. [Google Scholar] [CrossRef] [PubMed]
- Toews, I.; Lohner, S.; de Gaudry, D.K.; Sommer, H.; Meerpohl, J.J. Association between intake of non-sugar sweeteners and health outcomes: Systematic review and meta-analyses of randomised and non-randomised controlled trials and observational studies. BMJ 2019, 364, 11. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Bergs, D.; Burghoff, B.; Joehnck, M.; Martin, G.; Schembecker, G. Fast and isocratic HPLC-method for steviol glycosides analysis from Stevia rebaudiana leaves. J. Verbr. Lebensm. 2012, 7, 147–154. [Google Scholar] [CrossRef]
- Bayraktar, M.; Naziri, E.; Karabey, F.; Akgun, I.H.; Bedir, E.; Röck-Okuyucu, B.; Gürel, A. Enhancement of stevioside production by using biotechnological approach in in vitro culture of Stevia rebaudiana. Int. J. Sec. Metab. 2018, 5, 362–374. [Google Scholar] [CrossRef]
- Puri, M.; Sharma, D.; Barrow, C.J.; Tiwary, A.K. Optimisation of novel method for the extraction of steviosides from Stevia rebaudiana leaves. Food Chem. 2012, 132, 1113–1120. [Google Scholar] [CrossRef] [PubMed]
- Chao, Y.Y.; Chen, Y.L.; Lin, H.Y.; Huang, Y.L. Rapid screening of basic colorants in processed vegetables through mass spectrometry using an interchangeable thermal desorption electrospray ionization source. Anal. Chim. Acta 2018, 1010, 44–53. [Google Scholar] [CrossRef]
- Li, J.; Chen, Z.; Di, D. Preparative separation and purification of Rebaudioside A from Stevia rebaudiana Bertoni crude extracts by mixed bed of macroporous adsorption resins. Food Chem. 2012, 132, 268–276. [Google Scholar] [CrossRef]
- Liu, Y.; Di, D.; Bai, Q.; Li, J.; Chen, Z.; Lou, S.; Ye, H. Preparative separation and purification of rebaudioside a from steviol glycosides using mixed-mode macroporous adsorption resins. J. Agric. Food Chem. 2011, 59, 9629–9636. [Google Scholar] [CrossRef]
- Cacciola, F.; Delmonte, P.; Jaworska, K.; Dugo, P.; Mondello, L.; Rader, J.I. Employing ultra high pressure liquid chromatography as the second dimension in a comprehensive two-dimensional system for analysis of Stevia rebaudiana extracts. J. Chromatogr. A 2011, 1218, 2012–2018. [Google Scholar] [CrossRef]
- Fayaz, S.; Sharma, R.; Rajput, Y.S.; Mann, B.; Lata, K. Estimation of steviol glycosides in food matrices by high performance liquid chromatography. J. Food Sci. Technol. 2018, 55, 3325–3334. [Google Scholar] [CrossRef]
- Rodenburg, D.L.; Alves, K.; Perera, W.H.; Ramsaroop, T.; Carvalho, R.; McChesney, J.D. Development of HPLC analytical techniques for diterpene glycosides from Stevia rebaudiana (Bertoni) Bertoni: Strategies to scale-up. J. Braz. Chem. Soc. 2016, 27, 1406–1412. [Google Scholar]
- Chen, B.; Li, R.; Chen, X.; Yang, S.; Li, S.; Yang, K.; Chen, G.; Ma, X. Purification and preparation of rebaudioside A from steviol glycosides using one-dimensional hydrophilic interaction chromatography. J. Chromatogr. Sci. 2016, 54, 1408–1414. [Google Scholar] [CrossRef] [PubMed]
- Fu, Q.; Zhang, H.; Dai, Z.; Jiang, D.; Sun, M.; Ke, Y.; Jin, Y.; Liang, X. A ternary eluent strategy to tune the peak shape of steviol glycosides in reversed-phase liquid chromatography. J. Chromatogr. B 2021, 1173, 122673. [Google Scholar] [CrossRef] [PubMed]
- FAO/WHO Expert Committee on Food Additives (JECFA). Steviol Glycosides from Stevia rebaudiana Bertoni. Available online: http://www.fao.org/3/BU297en/bu297en.pdf (accessed on 15 June 2017).
- Kolb, N.; Herrera, J.L.; Ferreyra, D.J.; Uliana, R.F. Analysis of sweet diterpene glycosides from Stevia rebaudiana: Improved HPLC method. J. Agric. Food Chem. 2001, 49, 4538–4541. [Google Scholar] [CrossRef]
- Zimmermann, B.F. Beaming steviol glycoside analysis into the next dimension. Food Chem. 2018, 241, 150–153. [Google Scholar] [CrossRef] [PubMed]
- Abdellatif, A.A.H.; El Hamd, M.A.; Salman, K.H.; Abd-El-Rahim, A.M.; El-Maghrabey, M.; Tawfeek, H.M. Integrative physicochemical and HPLC assessment studies for the inclusion of lornoxicam in buffalo’s milk fat globules as a potential carrier delivery system for lipophilic drugs. Microchem. J. 2020, 152, 104321. [Google Scholar] [CrossRef]
- Pajewska-Szmyt, M.; Buszewski, B.; Gadzała-Kopciuch, R. Supported ionic liquid adsorbent and ELSD–HPLC method as an alternative procedure for exogenous fatty acid analysis in breast milk. Microchem. J. 2020, 157, 104961. [Google Scholar] [CrossRef]
- Ha, M.S.; Ha, S.D.; Choi, S.H.; Bae, D.H. Assessment of Korean consumer exposure to sodium saccharin, aspartame and stevioside. Food Addit. Contam. Part A 2013, 30, 1238–1247. [Google Scholar] [CrossRef] [PubMed]
- Ruiz-Rodriguez, A.; Reglero, G.; Ibañez, E. Recent trends in the advanced analysis of bioactive fatty acids. J. Pharm. Biomed. Anal. 2010, 51, 305–326. [Google Scholar] [CrossRef] [Green Version]
- Chemat, F.; Vian, M.A.; Cravotto, G. Green extraction of natural products: Concept and principles. Int. J. Mol. Sci. 2012, 13, 8615. [Google Scholar] [CrossRef] [Green Version]
- Milani, G.; Curci, F.; Cavalluzzi, M.M.; Crupi, P.; Pisano, I.; Lentini, G.; Clodoveo, M.L.; Franchini, C.; Corbo, F. Optimization of microwave-assisted extraction of antioxidants from bamboo shoots of Phyllostachys pubescens. Molecules 2020, 25, 215. [Google Scholar] [CrossRef] [Green Version]
- Caputo, L.; Quintieri, L.; Cavalluzzi, M.M.; Lentini, G.; Habtemariam, S. Antimicrobial and antibiofilm activities of citrus water-extracts obtained by microwave-assisted and conventional methods. Biomedicines 2018, 6, 70. [Google Scholar] [CrossRef] [Green Version]
- Lorenzo, C.; Serrano-Díaz, J.; Plaza, M.; Quintanilla, C.; Alonso, G.L. Fast methodology of analysing major steviol glycosides from Stevia rebaudiana leaves. Food Chem. 2014, 157, 518–523. [Google Scholar] [CrossRef]
- Uhler, B.; Yang, Z. Rebaudioside A and other unreported steviol glycoside isomers found in the sweet tea (Rubus suavissimis) leaf. Phytochem. Lett. 2018, 28, 93–97. [Google Scholar] [CrossRef]
- International Standard, Milk and Milk Products—Determination of Lactose Content by High-Performance Liquid Chromatography (Reference Method). Available online: https://cdn.standards.iteh.ai/samples/36384/b364b06284d44e579169d109b3815bbe/ISO-22662-2007.pdf (accessed on 15 September 2007).
- Ministry of Food and Drug Safety (MFDS). Available online: https://foodsafetykorea.go.kr/foodcode/01_03.jsp?idx=317 (accessed on 29 June 2021).
- Ministry of Food and Drug Safety (MFDS). Food Code. Available online: https://foodsafetykorea.go.kr/foodcode/01_03.jsp?idx=10031 (accessed on 29 June 2021).
- Bartholomees, U.; Struyf, T.; Lauwers, O.; Ceunen, S.; Geuns, J.M.C. Validation of an HPLC method for direct measurement of steviol equivalents in foods. Food Chem. 2016, 190, 270–275. [Google Scholar] [CrossRef]
- AOAC. AOAC Guidelines for Single Laboratory Validation of Chemical Methods for Dietary Supplements and Botanicals; Association of Official Analytical Chemists: Gaithersburg, MD, USA, 2002. [Google Scholar]
- Morlock, G.E.; Meyer, S.; Zimmermann, B.F.; Roussel, J.M. High-performance thin-layer chromatography analysis of steviol glycosides in Stevia formulations and sugar-free food products, and benchmarking with (ultra) high-performance liquid chromatography. J. Chromatogr. A 2014, 1350, 102–111. [Google Scholar] [CrossRef] [PubMed]
Standard | LOD 1 (mg/kg) | LOQ 2 (mg/kg) | r2 | Linear Regression |
---|---|---|---|---|
Rebaudioside D | 0.19 | 0.57 | 0.9997 | y = 1607.4x + 1984.8 |
Rebaudioside A | 0.18 | 0.55 | 0.9996 | y = 2662.4x + 730.18 |
Stevioside | 0.19 | 0.57 | 0.9993 | y = 2171.5x + 693.09 |
Rebaudioside F | 0.12 | 0.37 | 0.9994 | y = 1463.3x + 389.16 |
Rebaudioside C | 0.11 | 0.33 | 0.9996 | y = 2694.3x +1466.4 |
Dulcoside A | 0.56 | 1.69 | 0.9997 | y = 1290.5x + 41.995 |
Rubusoside | 0.13 | 0.41 | 0.9996 | y = 3260.3x + 270.18 |
Rebaudioside B | 0.33 | 1.00 | 0.9995 | y = 2099.6x + 66.253 |
Steviobioside | 0.19 | 0.57 | 0.9996 | y = 3351.6x + 675.03 |
Range | 0–50 mg/mL |
Spiked Level 1 | Non-Fermented Milk | |||||||||
Reb D | Reb A | SV | Reb F | Reb C | Dul A | RS | Reb B | SB | ||
6.25 mg/kg | Recovery (%) | 99.37 | 96.61 | 88.34 | 101.68 | 83.96 | 85.57 | 84.59 | 104.31 | 83.57 |
RSD 2 (%) | 1.86 | 0.86 | 0.97 | 1.45 | 1.45 | 2.35 | 0.46 | 0.83 | 0.78 | |
SD 3 | 1.85 | 0.83 | 0.85 | 1.48 | 1.22 | 2.01 | 0.39 | 0.86 | 0.65 | |
12.5 mg/kg | Recovery (%) | 99.58 | 104.84 | 90.63 | 95.70 | 87.75 | 94.05 | 90.56 | 88.28 | 87.14 |
RSD (%) | 1.23 | 0.22 | 0.79 | 1.24 | 1.27 | 1.10 | 0.22 | 0.54 | 0.44 | |
SD | 1.23 | 0.23 | 0.71 | 1.19 | 1.12 | 1.03 | 0.20 | 0.47 | 0.38 | |
25 mg/kg | Recovery (%) | 100.84 | 98.79 | 101.20 | 101.26 | 91.54 | 88.74 | 90.55 | 91.84 | 89.60 |
RSD (%) | 1.45 | 1.58 | 0.98 | 0.77 | 1.17 | 0.48 | 1.41 | 1.37 | 0.59 | |
SD | 1.47 | 1.56 | 0.99 | 0.78 | 1.07 | 0.43 | 1.28 | 1.26 | 0.53 | |
Spiked Level | Fermented Milk | |||||||||
Reb D | Reb A | SV | Reb F | Reb C | Dul A | RS | Reb B | SB | ||
6.25 mg/kg | Recovery (%) | 99.47 | 96.11 | 88.53 | 100.09 | 85.57 | 86.31 | 87.03 | 103.87 | 84.71 |
RSD (%) | 0.42 | 0.93 | 0.20 | 1.43 | 1.66 | 0.78 | 2.83 | 0.37 | 1.19 | |
SD | 0.41 | 0.89 | 0.18 | 1.43 | 1.42 | 0.67 | 2.46 | 0.39 | 1.01 | |
12.5 mg/kg | Recovery (%) | 100.02 | 103.98 | 91.34 | 95.50 | 88.63 | 95.04 | 90.41 | 88.59 | 88.17 |
RSD (%) | 1.88 | 0.77 | 1.32 | 0.29 | 0.86 | 1.67 | 0.52 | 0.41 | 1.02 | |
SD | 1.89 | 0.80 | 1.21 | 0.28 | 0.77 | 1.59 | 0.47 | 0.36 | 0.90 | |
25 mg/kg | Recovery (%) | 101.15 | 98.70 | 99.92 | 100.07 | 91.94 | 88.27 | 90.93 | 91.67 | 89.14 |
RSD (%) | 0.69 | 0.67 | 1.12 | 1.51 | 0.50 | 1.07 | 0.42 | 0.16 | 0.45 | |
SD | 0.70 | 0.66 | 1.12 | 1.52 | 0.46 | 0.94 | 0.38 | 0.15 | 0.40 |
Sample Name | Reb D 1 | Reb A 2 | SV 3 | Reb C 4 | |||||
---|---|---|---|---|---|---|---|---|---|
Tested Value (mg/L) | RSD 5 | Tested Value (mg/L) | RSD | Tested Value (mg/L) | RSD | Tested Value (mg/L) | RSD | ||
Non-fermented milk | T-1 | 427.89 ± 0.58 | 0.14 | 1.34 ± 0.05 | 4.01 | 5.68 ± 0.09 | 1.53 | ||
Fermented milk | T-2 | 99.38 ± 1.20 | 1.19 | 32.93 ± 0.05 | 0.14 | 20.40 ± 0.34 | 1.65 | 3.87 ± 0.09 | 2.19 |
T-3 | 204.94 ± 4.29 | 2.09 | 58.92 ± 0.11 | 0.18 | 21.62 ± 0.04 | 0.16 | |||
T-4 | 91.12 ± 0.32 | 0.35 | 120.65 ± 0.88 | 0.73 | 18.51 ± 0.22 | 1.19 | 1.83 ± 0.05 | 2.77 | |
T-5 | 217.23 ± 1.64 | 0.76 | 122.30 ± 0.98 | 0.80 | 21.03 ± 0.72 | 3.44 | |||
T-6 | 363.81 ± 3.82 | 1.05 | 43.00 ± 0.18 | 0.43 | 18.43 ± 0.23 | 1.25 | |||
T-7 | 124.16 ± 1.31 | 1.05 | 55.10 ± 0.33 | 0.59 | 20.79 ± 0.47 | 2.26 | |||
T-8 | 173.81 ± 2.74 | 1.57 | 27.64 ± 0.29 | 1.05 | 20.68 ± 0.32 | 1.56 | |||
T-9 | 63.79 ± 1.09 | 1.72 | 49.85 ± 0.13 | 0.27 | 19.47 ± 0.34 | 1.73 |
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Kim, J.-M.; Koh, J.-H.; Park, J.-M. Validation of an HPLC Method for Pretreatment of Steviol Glycosides in Fermented Milk. Foods 2021, 10, 2445. https://doi.org/10.3390/foods10102445
Kim J-M, Koh J-H, Park J-M. Validation of an HPLC Method for Pretreatment of Steviol Glycosides in Fermented Milk. Foods. 2021; 10(10):2445. https://doi.org/10.3390/foods10102445
Chicago/Turabian StyleKim, Jin-Man, Jong-Ho Koh, and Jung-Min Park. 2021. "Validation of an HPLC Method for Pretreatment of Steviol Glycosides in Fermented Milk" Foods 10, no. 10: 2445. https://doi.org/10.3390/foods10102445