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Rapid and Economic Determination of 13 Steviol Glycosides In pubs.acs.org/JAFC Article Rapid and Economic Determination of 13 Steviol Glycosides in Market-Available Food, Dietary Supplements, and Ingredients: Single-Laboratory Validation of an HPLC Method Zhiyan Liu, Kangzi Ren, Ye Feng, Tommy Uong, Scott Krepich, and Hong You* Cite This: https://dx.doi.org/10.1021/acs.jafc.0c03453 Read Online ACCESS Metrics & More Article Recommendations *sı Supporting Information ABSTRACT: Steviol glycosides, obtained from leaves of Stevia rebaudiana Bertoni (stevia) or produced via bioconversion and biosynthesis, are diterpenes used by the food/dietary supplement industry as zero-calorie sweeteners derived from natural sources. JECFA 2017 is the most updated international standardized method but it runs for 80 min per sample with suboptimal separations on several critical pairs for its high-performance liquid chromatography-ultraviolet (HPLC-UV) determination. We developed and validated a rapid and economic HPLC-UV method using the superficially porous particle column to determine 13 steviol glycosides (stevioside, dulcoside A, rubusoside, steviobioside, and rebaudioside A−F, I, M, and N). Baseline separation with a minimum resolution of 1.5 for 13 steviol glycosides was achieved within only 14 min of separation time. The hydrocarbon stationary phase with additional steric interactions from the isobutyl side chains on the C18 ligand was shown to be an important contributor to chromatographic selectivity of several critical pairs of steviol glycosides. The method was proven to perform suitably on columns from three different manufacturers and two HPLC instruments. The method was further used to perform a single-lab validation on eight food and supplement products with multiple matrices. The results ranged from 0.05% w/w rebaudioside A for a hard-candy finished product to 100.8% w/w purity for a rebaudioside M raw ingredient. The validation test results showed that the method was linear, suitable, specific, accurate, and precise. The method is therefore suitable to be considered as a new industrial standard for quality control analysis for stevia products. KEYWORDS: steviol glycosides, HPLC, superficially porous particle column, food analysis, dietary supplement 1. INTRODUCTION Pharmacopeia Food Chemicals Codex (USP FCC) mono- Steviol glycosides are zero-calorie sweeteners obtained from graph method used a 67 min gradient HPLC-UV method and utilized response factors for the individual compound the leaves of Stevia rebaudiana Bertoni (stevia) or via 16 bioconversion and biosynthesis.1,2 Stevia products are in determination. Several ultra-high-performance liquid chro- great demand in the food and dietary supplement industry3,4 matography (UHPLC) methods have also been developed for 17,18 Downloaded via Hong You on September 8, 2020 at 01:01:54 (UTC). with an expected global market of >1 billion US dollars by the rapid analysis of the major steviol glycosides. 2021.5,6 High-purity steviol glycosides have been evaluated by The taste and physical characteristics of steviol glycosides various global scientific and regulatory bodies to be safe for can be further improved via innovative processing such as consumption by the general population and may be effective in biosynthesis and bioconversion, of which technologies have led improving sugar/calorie reduction, weight management, dental to an increasing market availability of high-purity non-naturally See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles. health, etc.5,7,8 abundant minor steviol glycosides such as rebaudioside E, I, M, From the product quality control perspective, the industry and N.3,4,19,20 Because major steviol glycosides are often the needs a rapid, economic, and scientifically valid analytical substrates or impurities of the novel steviol glycoside method to identify and quantify steviol glycosides to meet productions, an analytical method to separate and quantify 9,10 regulatory requirements. Liquid chromatography (LC) is both major and minor steviol glycosides is needed for the 11−13 the technique-of-choice for testing steviol glycosides. product quality control and processing development trouble- Most of the analytical methods focused on separating nine shooting. The most updated standardized method for steviol major steviol glycosides that are naturally abundant in stevia glycosides analysis is JECFA 2017,13 which runs a gradient leaves: stevioside, rubusoside, steviobioside, dulcoside A, and elution for 80 min for the accurate quantification of 13 steviol rebaudioside A, B, C, D, and F.2,12,14 For example, the method in the Joint FAO/WHO Expert Committee on Food Additives (JECFA) 2010 monograph used C18 columns and ran an Received: June 1, 2020 isocratic separation for 30 min for the high-performance liquid Revised: August 3, 2020 chromatography-ultraviolet (HPLC-UV) determination of Accepted: August 13, 2020 nine major steviol glycosides.13 Chaturvedula, et al. used a Published: August 13, 2020 30 min isocratic method to test the separation of the nine compounds under various temperatures.15 The United States © XXXX American Chemical Society https://dx.doi.org/10.1021/acs.jafc.0c03453 A J. Agric. Food Chem. XXXX, XXX, XXX−XXX Journal of Agricultural and Food Chemistry pubs.acs.org/JAFC Article glycosides in HPLC-UV with optional mass spectrometry Table 1. Samples Used in the Repeatability Study (MS) determination for the estimation of 34 steviol glycosides. sample estimated level (w/w Other methods are available to simultaneously analyze multiple name matrices form purity) steviol glycosides using HPLC-fluorescence detector (FLD),11 21 110 dried stevia whole leaf raw total steviol high-performance thin-layer chromatography (HPTLC), ingredient glycosides 13−16% liquid chromatography with tandem mass spectrometry (LC/ 165 stevia organic liquid sweetener finished unknown MSMS),22,23 and NMR.24,25 The disadvantages of JECFA containing cane alcohol product 2017 and the other above methods include suboptimal 484 organic stevia sweetener blend finished unknown separation, long elution time, long sample preparation time, containing erythritol Product 289 hard candy finished unknown limited types of steviol glycosides being analyzed, or the need product for expensive equipment, which might make them less ideal to 029 stevia leaf dry extract powder raw total steviol be considered as the standardized method for the food and ingredient glycosides 90% dry dietary supplement industry. basis The objective of this study was to develop and validate a 333 ingredient dry powder raw rebaudioside D > rapid and economic HPLC method that could separate 13 ingredient 95% dry basis market-available steviol glycosides including stevioside, dulco- 291 ingredient dry powder raw rebaudioside M > − ingredient 95% dry basis side A, rubusoside, steviobioside, and rebaudioside A F, I, M, 729 ingredient dry powder raw rebaudioside A > and N (Figure 1). To achieve this goal, several columns, ingredient 95% dry basis Eight samples in the forms of finished products (liquid, powder, and hard candy) and raw ingredients (dry extract and whole leaf) were assigned by random ID codes and used for the validation study (Table 1). Samples were acquired from iHerbs.com, Amazon.com, or via donation. Samples were stored at room temperature prior to testing. 2.2. Preparation of Standard Solutions. The reference material stock solution was prepared by accurately weighing and transferring 5 ± 0.5 mg of the reference material into a 20 mL VOA vial and then diluting with 5.0 mL of acetonitrile/water (30%:70%). The reference material stock solution was diluted to make a six-point calibration curve for each steviol glycoside of interest. The range of standard concentrations was between 0.01 and 1 mg/mL in the solution. The six-point calibration curves were utilized for the determination of linearity. Figure 1. Chemical structures of steviol glycosides. Glc, Rha, and Xyl All high-purity raw ingredient samples were to be analyzed against a represent glucose, rhamnose, and xylose, respectively. one-point calibration curve that was derived from the average value of five replicate injections of the reference material stock solution. The % relative standard deviation (%RSD) of five replicate injections must separation schemes, and mobile phases were screened and be less than 1.5. optimized. After the optimized method has been developed, it 2.3. Sample Preparation. Because stevia dry extract raw was tested for its specificity, system suitability, linearity, ingredients are hygroscopic, dry-basis results are often used to method detection limit/limit of quantification, accuracy, and establish product specifications. For stevia dry extract ingredients, precision. The International Conference on Harmonisation approximately 1−2 g of sample was distributed into a thin layer (not (ICH) method validation guideline Q2(R1) and the AOAC more than 6.4 mm thickness) in a suitably sized weighing boat and (Association of Analytical Communities) appendix K were allowed to sit on a workbench for not less than 15 h. Intermittent used to design the single-laboratory validation study and set up stirring was conducted to ensure uniform moisture absorption. The 26,27 water content of the equilibrated sample at the time of weighing was the pass/fail criteria of each test. determined using Karl Fischer titration (Mettler-Toledo, LLC, Columbus, OH). 2. MATERIALS AND METHODS For high-purity single-compound ingredient samples, 25.0 ± 2.5 2.1. Reagents and Materials. Steviol glycosides
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