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*

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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 (stevioside, mg of the sample was transferred into a 40 mL VOA vial. Then, 25 dulcoside A, rubusoside, steviobioside, and rebaudioside A−F, I, M, mL of acetonitrile/water (30%:70%) was added and the mixture was N) reference materials were provided by ChromaDex (Irvine, CA). sonicated for 30 ± 5 min and vortexed. The solution was filtered HPLC-grade acetonitrile and formic acid were purchased from Fisher through a 0.45 μm poly(tetrafluoroethylene) (PTFE) filter into an Scientific (Pittsburgh, PA). Reference materials used in the accuracy amber autosampler vial. Then, the vial was capped and analyzed using study were purchased from USP (Rockville, MD) and PhytoLab HPLC. Other samples’ weights were adjusted based on the sample’s (Vestenbergsgreuth, Germany). expected level and the mid-level of the calibration curve

concentration of mid‐× level of calibration solution ( mg/mL) final volume of diluent ( mL) sample (except for high‐‐ purity single compound ingredient) weight (mg) = sample estimated level ( % w/w)

2.4. Column Selection and HPLC Analysis. Seven Phenomenex Phenomenex Kinetex XB-C18 100 Å (2.6 μm, 150 mm × 4.6 mm) columns with different column chemistries were used for the column with a SecurityGuard Ultra Cartridge (Phenomenex, Torrance, CA) screen during the initial method development (Table 2S, Supporting was determined to be optimal and was used for the method validation. Information). The details of each column’s characteristics could be HPLC (Agilent 1260; Santa Clara, CA) equipped with a binary pump found on the Phenomenex’s website: https://www.phenomenex.com/ and a diode-array detector (190−640 nm) was employed for the hplc-column. analyses. The optimal instrument conditions were as follows: injection

B https://dx.doi.org/10.1021/acs.jafc.0c03453 J. Agric. Food Chem. XXXX, XXX, XXX−XXX Journal of Agricultural and Food Chemistry pubs.acs.org/JAFC Article

Figure 2. Final method HPLC-UV chromatogram of 13 steviol glycosides reference material mixture on the Phenomenex Kinetex XB-C18 column. Analytes: 1, Rebaudioside E; 2, Rebaudioside D; 3, Rebaudioside N; 4, Rebaudioside M; 5, Rebaudioside I; 6, Rebaudioside A; 7, Stevioside; 8, Rebaudioside F; 9, Rebaudioside C; 10, Dulcoside A; 11, Rubusoside; 12, Rebaudioside B; 13, Steviolbioside. volume of 5.0 μL, column temperature at 60 °C, flow rate at 0.7 mL/ 2.5.4. MDL and LOQ. The method detection limit (MDL) and min, and detection wavelength at 210 nm. The gradient program with limit of quantification (LOQ) tests were designed and conducted 0.1% formic acid in water as mobile phase A and acetonitrile as similar to previous work of our group.28 The MDL was defined as the − − mobile phase B is listed below: 0 5.1 min, from 27 to 32% B; 5.1 6 minimum concentration of a substance that can be measured and min, hold 32% B; 6−9.4 min, from 32 to 35.6% B; 9.4−11.1 min, fi − − reported with 99% con dence that the analyte concentration is greater from 35.6 to 42% B; 11.1 12 min, hold 42% B; 12 12.1 min, from than zero. Two sets of seven replicates containing low concentrations 42 to 27% B; 12.1−17 min, hold 27% B. The separation time was 14 of the mixed reference solutions were analyzed, and the MDL was min and the total run time was 17 min. determined as the product of the standard deviation of the replicates To demonstrate the adaptability and robustness of the method, two α − superficially porous particle reversed-phase C18 columns from other and the t-statistic value at = 0.01 and n 1 degree of freedom. The fi manufacturers (Restek Raptor ARC-18, 4.6 mm × 150 mm, 2.7 μm LOQ (limit of quanti cation) was calculated as 10 times the standard and Agilent InfinityLab Poroshell 120 SB-C18, 4.6 mm × 150 mm, deviation of the replicates. 2.7 μm) were tested using the optimized method. 2.5.5. Accuracy. To demonstrate method accuracy, placebo 2.5. Single-Laboratory Validation Parameters. 2.5.1. Specific- samples were spiked with two (low and medium levels for dulcoside ity (Selectivity). Method specificity was demonstrated by running A, rubusoside, steviobioside, and rebaudioside B, C, F, I, N) or three “ ” Placebo samples expected to be free of steviol glycosides. The (low, medium, and high levels for stevioside) different levels of placebo was to mimic a dietary supplement excipient mixture and was analyte reference material and prepared in triplicate (n = 3) for each formulated as follows: 50% maltodextrin, 42% hydroxypropyl level. Then, the diluent (acetonitrile/water, 30%:70%) was added to methylcellulose, 5% stearic acid, 2% magnesium stearate, and 1% make the total extraction solution volume to 10 mL. To test the silicon dioxide. No peaks presented in the chromatographic region of method accuracy of high-purity raw ingredient matrices, the certified interest in both placebo and blank solvent solutions should have a ff response of >10% of the lowest-level analyte reference material. For reference materials for rebaudioside A, D, M, and E from di erent compound identifications in study samples, the chromatographic peak suppliers (USP and PhytoLab) were tested. Recovery rates were purity was verified for each analyte in each sample to confirm that calculated by comparing the actual test results against the theoretical there were no coeluted interferences. The UV−vis spectrum of values (spike recovery testing) or the values in the certificates of analyte in samples was compared against those of the corresponding analysis (certified reference material testing). reference materials. Spectrum matching factor >950 (a function of 2.5.6. Precision. Eight dietary ingredients and supplements in Agilent ChemStation data processing software) was used as the different matrices were tested to demonstrate the repeatability and fi fi passing criterion for the steviol glycoside identi cation con rmation. reproducibility of the method. All of the eight dietary ingredients and 2.5.2. System Suitability. To demonstrate the overall chromato- fi supplements examined for repeatability were tested in quadruplicate graphic system suitability, ve replicate injections of the mid-level (n = 4) on day 1 by analyst #1 on instrument #1. The repeatability reference material mixture solution were performed for all 13 steviol RSD of within-day results was calculated for all of the materials glycosides. Peak retention time, peak area, and peak shape were r analyzed. Percent relative standard deviation (%RSD) of peak area, % investigated. RSD on retention time, USP tailing factor, and the relative retention For evaluating the method reproducibility, intermediate precision fi time of each steviol glycoside were calculated. As the quality control tests were performed another two times by running ve samples. The # # criteria for system suitability, reference material peak area and full precision test design was as follows: Run 1: Day 1, Analyst 1, retention time %RSD must be ≤2.5. The USP tailing factor of the Instrument#1; Run 2: Day#2, Analyst#1, Instrument#1; Run 3: reference material peak must be less than 2.0 for all analyte peaks. The Day#3, Analyst#2, Instrument#2. Both instrument #1 and instrument peak resolution between steviol glycosides of interests must be ≥1.5. #2 are HPLCs (Agilent 1260; Santa Clara, CA) equipped with a 2.5.3. Linearity. Six calibration solutions were injected at the binary pump and a diode-array detector (190−640 nm). The beginning of each injection sequence. The calibration curves of the between-day results, reproducibility (RSD) values, were documented. − method covered the range of approximately 0.01 1 mg/mL for 13 Dry-basis results were shown for all raw ingredient samples. ffi steviol glycosides. The correlation coe cients, calibration equation 2.6. Data Analysis. Agilent ChemStation was used to automati- slopes, and y-intercepts were automatically generated by the HPLC cally determine individual steviol glycosides as follows data processing software (e.g., Agilent ChemStation). The blank was not considered as a part of the calibration curve. Each calibration % w/w analyte in sample curve was made up of six data points, and the square of correlation of 2 area(sample)− calibration equation intercept coefficient R for all curves must exceed the requirement of NLT = × 100 0.999. calibration equation slope×[ sample concentration ( mg/mL) ]

C https://dx.doi.org/10.1021/acs.jafc.0c03453 J. Agric. Food Chem. XXXX, XXX, XXX−XXX Journal of Agricultural and Food Chemistry pubs.acs.org/JAFC Article

Table 2. Accuracy Test Results Summary for Each Targeted Steviol Glycoside

high level (spike recovery + verify certified low level (spike recovery) medium level (spike recovery) reference materials) mean mean recovery RSD mean mean recovery RSD mean mean recovery RSD results (% w/w) (%) (%) (% w/w) (%) (%) (% w/w) (%) (%) rebaudioside E 0.034 94.58 6.58 6.537 96.6 1.21 88.79a 100.0 N/A rebaudioside D 0.030c 90.60 5.58 6.076 96.27 0.22 91.13a 99.0 N/A rebaudioside N 0.039 98.58 1.25 7.313 97.03 1.24 N/A N/A N/A rebaudioside M 0.037 96.55 5.14 7.055 97.9 0.84 84.54a 101.0 N/A rebaudioside I 0.028 90.33 3.18 5.754 97.68 1.04 N/A N/A N/A rebaudioside A 0.038 98.21 3.50 7.165 97.87 1.48 99.97b 101.0 N/A stevioside 0.035 96.74 3.31 6.801 97.98 1.62 84.86 100.6 0.46 rebaudioside F 0.037 96.12 6.96 7.235 98.07 0.40 N/A N/A N/A rebaudioside C 0.030 93.61 4.62 6.000 97.91 1.19 N/A N/A N/A dulcoside A 0.036 94.36 5.37 7.041 97.22 1.63 N/A N/A N/A rubusoside 0.031 93.84 6.55 6.101 97.7 1.70 N/A N/A N/A rebaudioside B 0.033 93.97 5.81 6.386 96.83 1.67 N/A N/A N/A steviolbioside 0.038 95.49 3.41 7.226 96.4 1.15 N/A N/A N/A aVerified method accuracy on this matrix by testing a Phytolab-certified reference material. bVerified method accuracy on this matrix by testing a USP-certified reference material. cN/A: Not available.

3. RESULTS AND DISCUSSION rebaudioside A and stevioside under the final method 3.1. Column Selection and Comparison. conditions. In botanical fi analysis, multiple phytochemicals are simultaneously separated Two super cially porous particle columns from other brands − fi within a short period of time.29 31 The critical challenge of this (Restek Raptor ARC-18 and Agilent In nityLab Poroshell 120 steviol glycosides separations is the resolution between the two SB-C18) were tested and were shown to have a similar main steviol glycosides, rebaudioside A and stevioside, separation as the Phenomenex Kinetex XB-C18 column (Figures 2S and 3S, Supporting Information). The method followed by the resolution of early-eluted rebaudioside E, D, ff N, and M. We screened seven Phenomenex Kinetex super- was shown to be successfully reproduced on two di erent ficially porous particle columns with different column HPLC instruments (as shown in the intermediate precision study) and columns from three brands (Phenomenex, Restek, chemistry (XB-C18, EVO C18, C18, Polar C18, C18 Positive and Agilent). Surface charge, Phenyl−Hexyl, Biphenyl) (Table 2S, Support- 3.2. Method Validation. The high-purity steviol glyco- ing Information). Phenomenex Kinetex XB-C18 100 Å (2.6 sides are hygroscopic and can readily absorb and lose water μm, 150 mm × 4.6 mm) obtained the best separation for 13 from the atmosphere. Therefore, it was important to allow steviol glycosides. Rebaudioside D, rebaudioside E, and samples to acclimate to the environment and equilibrate to rebaudioside N have a low resolution between each other constant weight and to be simultaneously tested for water under the isocratic condition that can separate rebaudioside A content using a Karl Fischer Titrator. and stevioside (Figure 1S, Supporting Information). Gradient fi We used mass spectrometry-friendly formic acid as the elution was therefore used to nd optimal and rapid mobile phase additive so that the method has the potential to separation. Among the seven columns screened, Kinetex XB- be directly used for mass spectrometry testing for minor steviol C18 was the only column that can achieve baseline separation glycoside determination. A relatively high column temperature with a minimum resolution of 1.5 (Figure 2 and Table 6S), (60 °C) was used to shorten the separation time. Chaturvedula suggesting that the additional steric interactions from the et al. have proven 60 °C to be an acceptable column isobutyl side chains on the C18 ligand are an important temperature that does not degrade steviol glycosides during contributor to chromatographic selectivity of this critical pair. the analysis.15 Hydrocarbon stationary phases, such as C8s and C18s, are To demonstrate method specificity, the chromatograms of a fl traditionally low in steric selectivity given the uidity of linear reference material mixture solution (Figure 2) and eight test hydrocarbons. By incorporating isobutyl chains at the base of samples (Figure 4S, Supporting Information) were shown. A the ligand, it results in overall the same general C18 type of UV−vis spectrum library (Figure 6S, Supporting Information) selectivity, but adds some rigidness, restricting the fluidness of was established in the HPLC Agilent ChemStation software to the ligands and yielding some additional steric/shape capture the UV−vis spectra of corresponding reference selectivity as more planar shapes will be able to interact materials. Peak purities were checked for each analyte in more within the C18 groups and more bulky molecules only each sample that was determined in specificity, precision, and on the surface. Given the structural commonality of the steviol accuracy tests. The result chromatograms indicated that the glycoside molecules, sharing the three-ringed hydrocarbon method was specific for the 13 steviol glycoside analysis in backbone, the additional steric selectivity proved useful, as the validated matrices because no interference was observed, and level of planarity vs bulkiness of each in three dimensions is all identified peaks passed the UV−vis spectrum verification influenced by the axial R1 and R2 variable groups for a tertiary criteria listed in Section 2.5.1. The replicate injection of five interaction in addition to hydrophobicity. Although some of mid-level reference material solutions showed that all system the other columns can separate rebaudioside D and rebaudio- suitability criteria were met (Tables 3S−6S, Supporting side N with higher resolution, they could not baseline-separate Information). For the linearity test, all calibration curves

D https://dx.doi.org/10.1021/acs.jafc.0c03453 J. Agric. Food Chem. XXXX, XXX, XXX−XXX Journal of Agricultural and Food Chemistry pubs.acs.org/JAFC Article

Table 3. Precision Test Results Summary for Each Targeted Steviol Glycoside

repeatability intermediate precision

sample analyte mean (% w/w) RSDr HorRatr RSDR HorRatR 110a rebaudioside D 0.41 4.82 1.07 4.79 1.06 rebaudioside A 7.63 2.32 0.79 1.67 0.57 stevioside 2.96 3.51 1.03 2.67 0.79 rebaudioside F 0.25 4.59 0.93 7.49 1.53 rebaudioside C 1.12 2.94 0.74 5.68 1.45 total 12.37 2.55 0.93 1.97 0.72 165 rebaudioside D 0.25 1.08 0.22 4.09 0.83 rebaudioside N 0.42 0.66 0.14 7.06 1.55 rebaudioside M 0.42 0.52 0.11 2.23 0.49 rebaudioside I 1.44 0.60 0.16 2.31 0.61 rebaudioside A 1.25 0.75 0.20 1.94 0.50 stevioside 1.24 1.34 0.35 1.48 0.38 rebaudioside F 0.21 1.98 0.40 2.52 0.50 rebaudioside C 0.49 2.84 0.64 2.55 0.57 dulcoside A 0.07 2.53 0.43 4.95 0.84 rubusoside 0.03 2.23 0.33 3.95 0.58 rebaudioside B 0.12 2.18 0.40 4.24 0.78 steviobioside 0.11 2.47 0.44 4.36 0.78 total 6.06 0.84 0.28 1.42 0.47 484 rebaudioside D 0.02 2.23 0.32 7.63 1.10 rebaudioside A 1.66 4.72 1.28 6.45 1.74 stevioside 0.73 4.62 1.11 4.71 1.13 rebaudioside C 0.04 2.69 0.42 7.43 1.16 rebaudioside B 0.07 4.03 0.68 3.99 0.67 total 2.53 4.47 1.30 5.52 1.59 289 rebaudioside A 0.05 2.81 0.45 7.13 1.13 029a rebaudioside D 0.94 2.98 0.74 5.88 1.46 rebaudioside N 0.56 3.93 0.90 5.07 1.16 rebaudioside Mb 0.38 4.93 1.06 5.42 1.17 rebaudioside A 29.42 0.50 0.21 0.69 0.29 stevioside 39.59 0.30 0.13 0.86 0.38 rebaudioside F 2.22 2.17 0.61 3.98 1.12 rebaudioside C 11.10 0.83 0.30 1.63 0.59 dulcoside A 1.00 0.95 0.24 7.35 1.84 rubusoside 1.50 4.26 1.12 3.34 0.88 rebaudioside B 0.46 2.03 0.45 6.81 1.51 steviobioside 0.43 3.56 0.80 5.83 1.30 total 87.60 0.37 0.18 0.66 0.32 333a rebaudioside D 95.48 1.46 0.72 N/A N/A 291a rebaudioside M 100.78 0.65 0.33 N/A N/A 729a rebaudioside A 98.37 0.27 0.14 N/A N/A aReported dry-basis results after moisture correction. bN/A: Not available. were shown to have R2 > 0.999 in the range of 0.01−1 mg/mL provided by the product manufacturer (13−16% w/w, Table (Figure 5S, Supporting Information). The MDL for the 13 1). As per the AOAC appendix K guidelines for recovery,27 the steviol glycoside ranged from 0.001 to 0.003 mg/mL and the acceptance range for this leaf product’s (sample 110) total LOQ ranged from 0.004 to 0.008 mg/mL (Table 1S, steviol glycosides is 12.35−16.8% w/w. Therefore, all of the Supporting Information). The accuracy test results are above results fulfilled the AOAC appendix K guidelines for summarized in Table 2. The low level (0.03% w/w) and recovery (Accuracy). The precision study samples were medium level (6% w/w) of accuracy tested were performed for finished products (liquid, powder, and hard candy) and raw all 13 steviol glycosides. The mean recovery ranged from 90.6 ingredients (dry extract and whole leaf) containing steviol to 98.2% (Table 2). The high-level method accuracy of five glycosides. The results ranged from 0.05% w/w purity for a steviol glycosides were demonstrated by performing spike hard candy (sample 289) to 100.8% w/w purity for a recovery for stevioside and testing certified reference materials rebaudioside M raw ingredient (sample 291). Repeatability of rebaudioside A, D, E, and M from PhytoLab and USP. The results (Table 3) showed that the total and individual steviol mean recovery ranged from 99 to 101% (Table 2). The glycoside values (only accounted for analytes > LOQ) of eight method accuracy on the leaf sample matrix can be further validation samples met the AOAC international guidelines for ≤ demonstrated by comparing the precision result of sample 110 repeatability RSDr ( 5%). The Horwitz ratio (HorRat) was (12.37% w/w, Table 3) against its estimated level that was used to evaluate the method performance based on the ratios

E https://dx.doi.org/10.1021/acs.jafc.0c03453 J. Agric. Food Chem. XXXX, XXX, XXX−XXX Journal of Agricultural and Food Chemistry pubs.acs.org/JAFC Article of the observed relative standard deviation (RSD) to the USP tailing factor throughout five replicate reference corresponding predicted relative standard deviation. The material solution injections (Table 5S); critical reso- ∧ − predicted RSDs were calculated as RSDr =C ( 0.15) and lution factor to the closest related compound (Table ∧ − RSDR =2C ( 0.15). The AOAC single-laboratory validation 6S); chromatogram of a steviol glycosides standard mix guidelines accept a HorRat ranging from 0.5 to 2. A HorRat on Phenomenex XB-C18 using the isocratic method value <0.5 was considered acceptable in this study due to the (Figure 1S); final method HPLC-UV chromatogram of excellent method performance under tightly controlled 13 steviol glycosides reference material mixture on conditions. In our method, the HorRatr value ranged from Restek Raptor ARC-18 column (Figure 2S); final 0.11 to 1.46. Intermediate precision test results (Table 3) method HPLC-UV chromatogram of 13 steviol glyco- fi showed that all ve samples have their reproducibility RSDR sides reference material mixture on Agilent InfinityLab ≤ acceptable by the AOAC guidelines ( 8%). The HorRatR Poroshell 120 SB-C18 column (Figure 3S); Chromato- value ranged from 0.29 to 1.84. The method was therefore grams of eight samples (110, 165, 484, 289, 029, 333, considered as precise. 291, 729) in precision session (Figure 4S); calibration 3.3. Further Investigation and Future Work. During curves of individual steviol glycosides (Figure 5S); UV− the development and validation of this method, our group was vis spectrum of 13 steviol glycosides (Figure 6S); invited to join an International Stevia Council Task Force for chromatogram of a modified method to baseline- proposing an analytical method to be adopted as a new separate 15 steviol glycosides on Agilent 1260 HPLC standardized method for JECFA 2020 monograph. We slightly instrument using 0.1% formic acid as mobile phase A optimized the method to expand its scope by adding and 0.1% formic acid in acetonitrile as mobile phase B rebaudioside O and isorebaudioside A as the targeted steviol (Figure 7S); chromatogram of a modified method to glycosides. The gradient and temperature were adjusted to baseline-separate 15 steviol glycosides on Agilent 1260 separate the two extra steviol glycosides. The method was as HPLC instrument using 10 mmol/L sodium phosphate follows: injection volume of 5.0 μL; column temperature at 55 buffer (pH 2.6) as mobile phase A and acetonitrile as °C; flow rate at 0.7 mL/min; and detection wavelength at 210 mobile phase B (Figure 8S); chromatogram of a nm. The gradient program with 0.1% formic acid as mobile modified method to baseline-separate 15 steviol glyco- phase A and 0.1% formic acid in acetonitrile as mobile phase B sides on Agilent 1100 HPLC instrument using 0.1% is listed below: 0−6 min, from 25.5 to 32% B; 6−9.5 min, from formic acid as mobile phase A and 0.1% formic acid in 32 to 35.5% B; 9.5−11 min, from 35.5 to 42% B; 11−13 min, acetonitrile as mobile phase B (Figure 9S) (PDF) hold 42% B; 13−13.1 min, from 42 to 25.5% B; 13.1−20 min, hold 25.5% B. The separation time was 14 min and the total ■ AUTHOR INFORMATION run time was 20 min. The method chromatogram is shown in Corresponding Author Figure 7S. In the 210 nm wavelength determination, sodium − fi fi phosphate buffer as the mobile phase resulted in an about 7% Hong You Euro ns Supplement Analysis Center, Euro ns fl Scientific, Inc., Petaluma, California 94954, United States; higher peak area and a atter baseline compared to the formic fi acid mobile phase. Therefore, 10 mmol/L sodium phosphate Euro ns Botanical Testing Inc. US, Brea, California 92821, ff United States; orcid.org/0000-0002-6665-1346; bu er (as mobile phase A) and acetonitrile (as mobile phase fi B) were also tested as an alternative plan for laboratories that Phone: +1-5157081576; Email: hongyou@euro nsus.com do not expect mass spectrometry method expansion. The Authors method was also shown to work on an old-fashion Agilent Zhiyan Liu − Eurofins Supplement Analysis Center, Eurofins 1100 HPLC instrument. The chromatograms of these trials are Scientific, Inc., Petaluma, California 94954, United States shown in Supporting Information, Figures 8S and 9S. Kangzi Ren − Eurofins Botanical Testing Inc. US, Brea, 3.4. Conclusions. To address the industrial needs for rapid California 92821, United States and economic analysis of steviol glycoside products, we Ye Feng − Eurofins Supplement Analysis Center, Eurofins developed and validated a gradient HPLC-UV method using fi fi Scienti c, Inc., Petaluma, California 94954, United States a super cially porous particle reversed-phase C18 column to Tommy Uong − Phenomenex, Inc., Torrance, California 90501- separate 13 steviol glycosides. The validation results showed fi 1430, United States that the proposed method was speci c, suitable, linear, Scott Krepich − ChromaDex, Inc., Irvine, California 92618- accurate, and precise. This method has the potential to be 2538, United States adapted as an industrial standard for the rapid and economic analysis for testing steviol glycosides. Complete contact information is available at: https://pubs.acs.org/10.1021/acs.jafc.0c03453 ■ ASSOCIATED CONTENT Notes *ı s Supporting Information The authors declare no competing financial interest. The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acs.jafc.0c03453. ■ ACKNOWLEDGMENTS Method detection limit (MDL) and LOQ (limit of The study received financial support through the intercompany quantification) for each targeted steviol glycoside (Table research & development funding. 1S); details of the Phenomenex columns screened (Table 2S); retention time precision throughout five ■ ABBREVIATIONS USED replicate reference material solutions injections (Table JECFA, Joint Food and Agriculture Organization/World fi 3S); peak area precision throughout ve replicate Health Organization Expert Committee on Food Additives; reference material solutions injections (Table 4S); HPLC-UV, high-performance liquid chromatography with

F https://dx.doi.org/10.1021/acs.jafc.0c03453 J. Agric. Food Chem. XXXX, XXX, XXX−XXX Journal of Agricultural and Food Chemistry pubs.acs.org/JAFC Article

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