Development and Validation of a Reversed-Phase High-Performance

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Development and Validation of a Reversed-Phase High-Performance Liquid Chromatography Method for Routine Identification and Purity Assessment of High-Purity Steviol Glycoside Sweeteners Tsion Bililign, Jeffrey C. Moore,* Shane Tan, and Allan T. Leeks Research and Development and Food Standards, United States Pharmacopeial Convention, 12601 Twinbrook Parkway, Rockville, Maryland 20852, United States S Supporting Information *

ABSTRACT: The widespread application of stevia-based sweeteners in food products has resulted in the need for reliable analytical methods for measuring the purity and identity of high-purity steviol glycoside ingredients. The objective of this research was to develop and validate a new reversed-phase separation method capable of separating and quantifying nine steviol glycosides present in typical high-purity stevia extract ingredients. Results of the study established the linearity of the method at a correlation factor of 1.000 for the two major components and other minor components of this food ingredient. Method accuracy values were in the range of 99.1−100.9%. The percent relative standard deviation for six independent assay determinations was 1.0%. The method was determined to be robust for minor changes in column temperature, initial acetonitrile content, flow rate, and wavelength. The validated high-performance liquid chromatography method was found to be suitable to be included by USP as a Food Chemicals Codex compendial standard for steviol glycosides. KEYWORDS: high-purity steviol glycosides, rebaudioside A, validation, HPLC−UV, degradation

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INTRODUCTION High-purity stevia-derived sweeteners are being increasingly used in food products worldwide because of their high-intensity sweetening properties and low caloric contents. Other marketable qualities include remarkable stability in numerous processes, favorable flavor profile, and regulatory approvals for use as a sweetener in foods in numerous countries. Consumer demand for botanically derived sweeteners is on the rise, and stevia-derived sweeteners currently dominate this market segment. They are commercially produced from the leaves of the Stevia rebaudiana (Bertoni) plant using a variety of multistep extraction and purification processes. Commercial products are known to contain up to nine structures that include rebaudioside A and stevioside as the major components and rebaudiosides B−D and F, dulcoside A, rubusoside, and steviolbioside as minor components (Figure 1). The resulting ingredients can vary in composition but must contain at least 95% of the nine named steviol glycosides on a dry weight basis and be composed primarily of rebaudioside A and/or stevioside to conform to the current Joint FAO/WHO Expert Committee on Food Additives (JECFA) standard for steviol glycosides.1 Reliable analytical standards (analytical methods and reference materials) for raw ingredient testing are needed by buyers and sellers to evaluate the authenticity, or the identity and purity, of ingredients in global trade. Steviol glycoside products are known to contain a mixture of highly hydrophilic analytes carrying multiple sugar moieties, whereas other components exhibit the hydrophobic nature of the diterpenoid backbone common to all of the glycosides (Figure 1). A variety of analytical approaches for analyzing complex raw ingredients, such as high-purity steviol glycoside sweeteners, have been reported and reviewed previously, including high-performance © 2014 American Chemical Society

liquid chromatography (HPLC) with ultraviolet (UV), mass spectrometry, charged aerosol detection, or evaporative light scattering detection, capillary electrophoresis, thin-layer chromatography, and nuclear magnetic resonance.2−5 HPLC with UV detection is the most common and suitable method for use in routine quality assurance testing of raw ingredients.2 Several HPLC−UV methods have been established for steviol glycosides testing standards in the past few years; however, insufficient selectivity and rapidly degrading column performance have been reported for most. Early standardized HPLC−UV methods, such as the Chinese Guobiao (GB) 1999, the JECFA 2008 monograph, and the Food Chemical Codex (FCC) 2009 Rebaudioside A monograph, used amino columns with isocratic elution.6−8 These amino column methods provided sufficient separation of most glycosides of interest but suffered from short column lives and poor retention time stability and required long column equilibration times.2,3,9 JECFA recently revised their HPLC−UV method in 2010 to use a C18 column with isocratic elution to overcome some of these issues.1 This method has been criticized for its potential insufficient resolution (R < 1.5) of rebaudioside A and stevioside, the two main glycosides in commercial ingredients, as well as rebaudioside C and dulcoside.2,3,9 Additionally, no data with regard to the selectivity of the JECFA 2010 HPLC− UV method have been reported for the nine steviol glycoside analytes in the presence of potential degradation products and related compounds, limiting its potential application for Received: Revised: Accepted: Published: 1384

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and molecular weight correction factors.1 This approach assumes that all nine measured steviol glycosides have equal molar UV absorptivities. Reports supporting the validity of this assumption are lacking and are necessary to ensure the accuracy of the approach. The relative response factor approach is a wellestablished quantitative approach and was established for this method.12 In this work, a reversed-phase HPLC−UV method with acceptable selectivity for typical steviol glycoside products was developed and validated, and optimal separation of the critical pair rebaudioside A and stevioside was established. The versatility of the method was demonstrated by evaluating forced degradation samples of rebaudioside A, a primary component in commercial samples. Additionally, reference standards and criterion-based system suitability tests were developed for the HPLC method to ensure efficient and reliable performance for quantitative measurements. Finally, the research provides validation data for establishing the linearity, accuracy, precision, and ruggedness of the developed HPLC method.

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MATERIALS AND METHODS

Reagents and Chemicals. Reagent-grade potassium dihydrogen phosphate (KH2PO4), 85% phosphoric acid (H3PO4), concentrated hydrochloric acid (HCl, 12.1 M), a sodium hydroxide (NaOH) solution [50% (w/w) in water], and pH 2.00 and 4.00 buffer solutions were purchased from Fisher-Scientific (Pittsburgh, PA). A pH 4.01 buffer solution was also obtained from Thermo-Scientific (Waltham, MA). HPLC-grade acetonitrile (ACN) was obtained from Merck KGaA (Darmstadt, Germany) and Fisher Scientific. Water (H2O) was purified using the Millipore (Billerica, MA) Advantage A10 system. A peroxide (H2O2) solution (30% in H2O) was purchased from SigmaAldrich (St. Louis, MO). Karl Fisher reagents Hydranol solvent and Titrant 5 were purchased from Fluka (Trademark of Sigma-Aldrich). United States Pharmacopeia (USP, Rockville, MD) primary reference standards were used for rebaudioside A, B, and D, rubusoside, stevioside, and steviolbioside. Reference standards for rebaudioside C and F, dulcoside A, isosteviolmonoside, steviol, and isosteviol were purchased from ChromaDex (Irvine, CA). A USP system suitability reference standard containing the nine steviol glycosides (Figure 1) to be measured with this method was used for performing system suitability checks. Four commercial samples of high-purity steviol glycoside ingredients used in this study were generous donations from three different suppliers. Standard and Sample Solutions. Potassium phosphate buffer (5 mM, pH 3.0) was prepared by dissolving KH2PO4 in H2O, and the pH was adjusted to 3.0 using H3PO4 on a Thermo-Scientific Orion 3 Star benchtop pH meter. The buffer solution was vacuum filtered by being passed through a filter membrane with a 0.45 μm pore size. The diluent solution was prepared by mixing 5 mM potassium phosphate buffer (pH 3.0) with ACN in a 65:35 (v/v) ratio. For quantitative measurements, standard and sample materials were equilibrated at ambient temperature for ≥24 h because of their hygroscopic nature. The moisture (H2O) content was determined by Karl Fisher titration using a Mettler-Toledo (Columbus, OH) DL38 titrator. Materials were accurately weighed on a Mettler-Toledo analytical balance (AT201), and solutions were routinely prepared in volumetric flasks. A brief sonication step (2−5 min) was used to dissolve materials. Rebaudioside A Forced Degradation Studies. Sample solutions of rebaudioside A (5 mM) were tested following several different types of degrading paradigms to test system stability and specificity. A 5.0 mL aliquot of a rebaudioside A solution (10 mM in diluent) was mixed with 5.0 mL of NaOH in one experiment, and HCl solutions (500 mM in H2O) in a separate experiment, both conducted at ambient temperature. The monitored reaction times were 2, 7, and 24 h. The reaction mixtures were neutralized with an equal molar ratio of acid or base and diluted 10-fold for analysis (0.5 mM rebaudioside

Figure 1. Structures of the nine components of JECFA’s steviol glycoside food ingredient.

comprehensive quality assessments of commercial products. Potential new HPLC−UV conditions for improving the chromatography of steviol glycosides have been reported, including the use of gradient elution with C18 columns or the use of HILIC columns with isocratic elution, but none have been sufficiently evaluated or validated for use as standardized methods.2,10 The new chromatographic method presented here not only shows an improved and acceptable chromatographic performance but also features significant improvements of three other analytical elements of current HPLC analysis standards for steviol glycosides. Included are the addition of system suitability tests, the HPLC peak identification approach, and the use of modern and efficient quantitation approaches for measuring individual glycosides. System suitability tests are used to verify whether a chromatography system is fit for the analysis it will undertake on the day of analysis for critical parameters such as precision and resolution.11 Such tests are missing in current standardized methods of analysis for steviol glycosides and are critical and essential to ensure their reliable use in quality control laboratories. Peak identification in the current HPLC method established by JECFA uses a mixed standard solution prepared with nine individual standards.1 This approach is not practical for routine analysis, given the current cost of purchasing high-purity reference materials. Quantitation of individual steviol glycosides in the current JECFA methods is conducted using a quantitative stevioside reference standard 1385

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A). A 5.0 mL aliquot of a rebaudioside A solution (10 mM in diluent) was incubated with 5.0 mL of 30% H2O2 at ambient temperature. After incubation for 2 h, 1 day, and 5 days, 1.0 mL aliquots of the reaction mixture were removed and diluted as described above for analysis. Control reaction mixtures containing all components except degradant were analyzed, along with chromatographic controls containing all components except rebaudioside A. Rebaudioside A powder was exposed to UV (total of 1958 W h−1 m−2) and white light (total of 2302 klx/h) using a Caron (Marietta, OH) photostability chamber (model 6545-2). Light stability studies were conducted for up to 72 h, and samples were removed at 24, 48, and 72 h for analysis at a concentration of 1.2 mg/mL. Similarly, rebaudioside A powder was incubated at 60 °C in a Yamato (Santa Clara, CA) vacuum oven for up to 8 days. Customarily, photodiode array (PDA) peak purity analysis of rebaudioside A was attempted to provide evidence of the spectral purity of the main peak after forced degradation. Because of a lack of a unique UV spectrum for the main component (λmax = 210 nm), peak purity analysis could not be applied to identify a co-elution. However, mass balance to control reactions was evaluated to determine the amount or lack of significant degradation. Chromatographic Conditions. Agilent (Wilmington, DE) 1200 and Waters (Milford, MA) Alliance 2965 HPLC systems consisting of a degasser, a quaternary pump, an autosampler, a column compartment, and a PDA detector were used for analysis. Both of the HPLC systems were controlled using Waters Empower 2 software. The method used a YMC (Kyoto, Japan) ODS-AQ column (250 mm × 4.6 mm, 5 μm) and mobile phase consisting of solvent A [5 mM potassium dihydrogen phosphate buffer (pH 3.0)] and solvent B (ACN). Two step gradients with ACN were used to elute analytes of interest as follows: 10 to 35% solvent B over 10 min, held for 15 min at 35% solvent B, 35 to 75% solvent B over 10 min, and held for 15 min at 75% solvent B. The column was re-equilibrated to the starting condition of 10% solvent B and 90% solvent A for 15 min. The column was maintained at 32 °C with a flow rate of 0.5 mL/min. The injection volume was 15 μL for both sample and standard solutions, which were kept under ambient conditions in the autosampler. UV detection at 210 nm with a 4 nm bandwidth was used for analysis. Other tested columns for method development purposes included Shiseido (Tokyo, Japan) Capcell Pak C18 MGII (250 mm × 4.6 mm, 5 μm, JECFA, 2010) and Imtakt USA (Philadelphia, PA) Scherzo SMC18 (250 mm × 4.6 mm, 3 μm) columns. System Suitability Tests. A solution of the USP system suitability reference standard was prepared at a concentration of 1.2 mg/mL, and five replicate injections of the solution were performed to assess the HPLC system performance in terms of critical pair separation, system precision, and detector sensitivity. The following acceptance criteria were defined per USP general chapter ⟨621⟩11 and intended use of method: a resolution of rebaudioside A and stevioside of ≥1.5, a percent relative standard deviation (%RSD) of the rebaudioside A peak area responses for the five injections of ≤1.5%, and %RSD of the rubusoside peak responses of ≤2.5%. Total Assay Determination. Standard solutions of rebaudioside A were prepared at concentrations of 0.5 mg/mL (S1) and 0.03 mg/ mL (S2) to quantify major and minor analyte peaks in samples. The standard solutions were prepared in duplicate and confirmed to have weight-corrected UV responses (response factors) within 2.5% of each other. The nominal concentration for sample solutions was established at 1.2 mg/mL, which provided adequate detection of major and minor peaks. Separately, equal volumes of sample solutions at the nominal concentration were injected. The chromatograms from the system suitability solutions were used to identify the nine steviol glycosides in the samples. The percentages of each component in the sample were calculated as follows:

Note solution S1 was used to quantify rebaudioside A and stevioside, which were the two major components, and solution S2 was used to quantify all other minor components. It is noteworthy that if rU for rebaudioside A or stevioside was less than 12% of rS in solution S1, then solution S2 was used for a more accurate quantification. The sum of percentages of the nine measured steviol glycosides was determined as the total assay. Method Validation. The USP general chapter ⟨1225⟩ validation of compendial procedures was used to design validation experiments.13 The validation was limited to the nine named steviol glycosides in the JECFA monograph because of their established significance as components of the food ingredient.1 The linearity range for the procedure was evaluated at a concentration level of 0.01−1.2 mg/mL for the two major components, rebaudioside A and stevioside. The range for the seven minor components was 0.01−0.06 mg/mL, except for rebaudioside C, which was evaluated up to 0.25 mg/mL because of its higher natural abundance in evaluated commercial samples. Linear regression analysis was performed to determine the correlation coefficient. The RRF values for each of the eight steviol glycosides with reference to rebaudioside A were determined at a single concentration (0.05−0.08 mg/mL) with duplicate preparations. The accuracy of the method was assessed by conducting the total assay determinations at the 80, 100, and 120% levels of the nominal assay concentration using a commercial product with an established assay value determined using an alternative procedure. Triplicate samples at each level were prepared and analyzed (n = 9). The precision of the method was determined by evaluating the %RSD of assay measurements with six sample preparations (n = 6) at the nominal concentration. The intermediate precision (ruggedness) was examined by performing assay determinations with six sample preparations by a different analyst using a different column and HPLC system, on a different day. The difference of the means was determined at the 90% confidence interval. The robustness of the method was evaluated by introducing small deliberate changes to key chromatographic conditions and assessing the effect upon critical pair separation and system precision. The modifications included the monitored wavelength at ±3 nm, a column temperature of ±2 °C, and a relative change of ±10% of the initial ACN content and flow rate.

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RESULTS AND DISCUSSION Method Development. A reversed-phase HPLC−UV method with acceptable selectivity for typical steviol glycoside products was developed for its ease and widespread use of methodology. The method was also capable of the separation and identification of steviol and isosteviol, the 2-unglycosylated aromatic aglycons, and a rarely occurring monoglycosylated structure known as isosteviolmonoside. Figure 2 shows a chromatogram of a solution composed of individual reference standards for each analyte combined at the concentration listed in Table 1. The analytes were eluted with an ACN gradient that was the key parameter affecting the selectivity of the method

% assay = (rU /rS) × (CS/C U) × (1/F ) × 100 where rU is the peak area for the analyte in the sample, rS is the peak area for rebaudioside A in S1 or S2, CS is the concentration of USP rebaudioside A RS in S1 or S2 corrected for water and purity, CU is the concentration in the sample solution corrected for water content, and F is the relative response factor (RRF) (see Table 2).

Figure 2. Representative chromatogram of a mixture of several reference standards of steviol glycosides in solution. 1386

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Table 1. Elution Profile of Steviol Glycosidesa peak name

concn (mg/mL)

RT (min)

RT (% RSD)

resolution (R)

rebaudioside D rebaudioside A stevioside rebaudioside F rebaudioside C dulcoside A rubusoside rebaudioside B steviolbioside isosteviolmonoside steviol isosteviol

0.6 0.9 0.6 0.7 0.6 0.6 0.8 0.6 0.6 0.2 0.6 0.6

17.5 21.9 22.4 24.2 25.2 26.4 31.5 34.8 35.3 39.8 46.6 51.9

0.1 0.2 0.2 0.2 0.2 0.2 0.2 0.1 0.0 0.0 0.0 0.0

− 20.3 2.0 5.9 3.0 3.3 11.9 10.4 3.0 28.2 29.4 15.0

solution of rebaudioside A (5 mM) was tested following several different types of degrading paradigms to test system stability and specificity. Solutions depicted were subjected to degradation in the following conditions: base (250 mM NaOH, 2 h), peroxide (15% in H2O2, 5 days), acid (250 mM HCl, 7 h), heat (60 °C, 8 days), and UV and white light (1958 W h−1 m−2 and 2302 klx/h, 72 h). Heat exposure and UV and white light exposure were used for rebaudiside A powder, while base, peroxide, and acid incubations were used in solution. Hydrolysis of the ester-linked sugar leading to rebaudioside B was observed within 2 h of incubation in a strong base [RT = 34.8 min (Figure 3)]. In addition, an unknown peak at a RT of 20.3 min appeared to be well-resolved (R = 5.9) from rebaudioside A and rebaudioside D. An acid-induced degradant eluted near rebaudioside A within 7 h of incubation (RT = 22.7; R = 3.3). As shown in Figure 3, several minor unknown peaks that eluted close to rebaudioside D were also observed during the prolonged oxidative condition (5 days). Rebaudioside D, however, was resolved from the degradation peaks at R values of ≥1.7. Rebaudioside A powder exposed to UV and white light for up to 72 h and incubation at 60 °C for up to 8 days did not lead to known or unknown structures as compared to an unexposed sample. Stability-indicating HPLC methods provided the ability to discover compromises to product quality more efficiently. System Suitability Tests. Validation of the system suitability criteria was obtained via five independent experiments conducted on different days, by two different analysts, and on two different HPLC instrument setups. The assessments established that the resolution of the critical pair, rebaudioside A and stevioside, was observed at 1.8−2.1 with a criterion of ≥1.5. The system precision was monitored by evaluating the %RSD of the rebaudioside A peak area response from five replicate injections, which was determined at 0.12− 0.46% with a criterion of ≤1.5%. The %RSD of the rubusoside peak response was used to evaluate detector sensitivity as the smallest peak (0.004 AU) in the mixture. The variation in peak area response was detected at a %RSD of 1.3−1.8% with a criterion of ≤2.5%. The acceptable performance of the HPLC method against the evaluated system suitability criteria

a

Individual characteristics of reference standards in solution tested in Figure 2.

under reversed-phase conditions. Two step gradients were introduced; the first served to resolve the hydrophilic components at an acceptable resolution [R of ≥2.0 (Table 1)], and the second one allowed for efficient elution of the hydrophobic diterpenoids. The %RSD for retention time [RT (Table 1)] was obtained from five replicate injections of the mixture solution from three independent experiments on different days, which showed minimal shifts in retention time (%RSD, ≤0.2%), and well-resolved peaks (R ≥ 2.0) were consistently achieved. The optimal separation of the critical pair rebaudioside A and stevioside was also established by evaluating up to 10 commercial samples with various ratios of the critical pair that demonstrated R values in the range of 1.7−2.2. The worstcase scenario (R of 1.7) was observed for a commercial product containing 95% rebaudioside A and 1% stevioside. In addition, aside from the primary column, C18 columns from two other manufacturers with slight differences in particle packing and size were tested that provided R of ≥1.9 for the critical pair. The stability-indicating capability of the newly developed method was determined by conducting forced degradation studies of rebaudioside A. As shown in Figure 3, a sample

Figure 3. Overlaid chromatograms of degraded sample solutions of rebaudiside A (Reb A). 1387

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⟨621⟩11 and are used to demonstrate the acceptable separation of neighboring peaks. Average method accuracy values were 99.1, 99.9, and 100.9% at 80, 100, and 120%, respectively, of the nominal concentration of 1.2 mg/mL. The %RSD for six independent assay determinations at the nominal concentration was 1.0%. A second analyst repeated the precision experiment with a different HPLC system and column, on a different day. The difference of means at the 90% confidence interval was within 2.0%, which was deemed an acceptable equivalence for the intended purpose. The method was determined to be robust for minor changes in column temperature, initial ACN content, and flow rate. A significant difference in the peak area response was observed when the detection wavelength changed ±3 nm. Specifically, a 40% decline in the peak area response was measured at 213 nm for rebaudioside A and rubusoside during system suitability evaluations. However, the %RSD for the two affected analyte peaks and the resolution of the critical pair met the established acceptance criteria, indicating a negligible impact on analysis. The validation results met the established acceptance criteria for linearity, accuracy, precision, and ruggedness. The acceptance criteria for the validation experiments were developed using industry-accepted guidance14,15 and were based on the intended use of the method for quantification of steviol glycosides within JECFA’s monograph specification of ≥95.0%. Total Assay Determinations. Standard solutions of rebaudioside A were prepared at concentrations of 0.5 mg/ mL (S1) and 0.03 mg/mL (S2) to generate a two-point calibration curve. Duplicate preparations of standard solutions showed response factors in the range of −0.1 to 2.2% throughout the validation experiments. Figure 4 illustrates profiles of select commercial samples, which showed the major components to be rebaudioside A and stevioside, with rebaudioside C observed at 3−8%. The other minor components were observed at 0.8−1.8% of the total assay. Assay determinations of individual and total steviol glycosides for commercial purposes were in close agreement with the manufacturer’s certificate of analysis (data available in Table S1 of the Supporting Information) and within the JECFA Steviol Glycosides monograph specification of ≥95.0% (JECFA, 2010). The acceptable results of the analysis added further evidence to

established that the method was suitable for quantitative measurements. Method Validation. The linearity of the UV response for each of the analytes of interest was evaluated independently at concentration ranges commonly observed in commercial samples. Results of the study established the linearity of the method at a correlation coefficient of 1.000 with a negligible intercept (≤2%). The limit of quantification was determined to be 1% of the nominal concentration of 1.2 mg/mL (∼0.01 mg/ mL) established as the lowest concentration point of the linearity curve for each of the glycosides. RRF values were determined at a single concentration level with duplicate preparations within the linear range for all analytes. In addition, the ratio of the slopes from the linear regression studies provided equivalent values. Table 2 lists the RRF values and Table 2. Chromatographic Profile of Steviol Glycosides with Respect to Rebaudioside A name

relative retention time (RRT)

USP resolution

relative response factor (RRF)

rebaudioside D

0.80

0.83

rebaudioside A stevioside rebaudioside F rebaudioside C dulcoside A rubusoside rebaudioside B steviolbioside

1.00 1.03 1.10 1.15 1.21 1.44 1.59 1.61

not available 21.3 2.1 5.6 3.1 3.5 8.6 12.2 3.0

1.00 1.22 1.02 0.98 1.16 1.06 1.07 1.38

relative retention times (RRTs) determined for the eight other steviol glycosides with reference to rebaudioside A in the USP system suitability reference standard solution. The RRF values were intended to correct for intrinsic differences in the UV response among steviol glycosides.12 This was especially important for assay determinations, where rebaudioside A was used for quantification of all other related analytes in uncharacterized samples. Similarly, the RRT values served to easily identify analytes in uncharacterized samples. USP resolution values are defined in USP General Chapter

Figure 4. Overlaid chromatograms of sample analysis that illustrate the varying ratios of the critical pair rebaudioside A and stevioside in commercial samples. Traces are offset to illustrate peak differences. 1388

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extracts by 1H NMR spectroscopy. J. Agric. Food Chem. 2011, 59, 4378−4384. (6) People [sic] Republic of China. GB 8270. Food additive− steviosides. 1999 (http://www.freestd.us/soft/173907.htm) (accessed July 17, 2013). (7) JECFA. Steviol glycosides, monograph 5. In Food and Agricultural Organization of the United Nations, Combined Compendium of Food Additive Specifications; 2008 (http://www. fao.org/food/food-safety-quality/scientific-advice/jecfa/jecfaadditives/en/) (accessed July 17, 2013). (8) USP. Food Chemicals Codex. FCC Second Supplement to the Eighth edition; The United States Pharmacopeial Convention: Rockville, MD, 2009. (9) Markosyan, A. A. Stevia analysis: Current status overview and development outlook. Institute of Food Technologists Annual Meeting, New Orleans, LA, June 2011. (10) Hutapea, A. M.; Toskulkao, C.; Wilairat, P.; Buddhasukh, D. High-performance liquid chromatographic separation and quantitation of stevioside and its metabolites. J. Liq. Chromatogr. Relat. Technol. 1999, 22, 1161−1170. (11) United States Pharmacopeia and National Formulary (USP 36-NF 31); The United States Pharmacopeial Convention: Rockville, MD, 2012; pp 268−275. (12) Elmer, J.; Curgess, C.; Kleinschmidt, G.; Miller, J. H. M. Performance Parameters, Calculations and Tests. In Method Validation in Pharmaceutical Analysis: A Guide to Best Practice; Ermer, J., Miller, J. H. M., Eds.; Wiley-VCH,: Weinheim, Germany, 2005; pp 72−74. (13) USP. USP general Chapter ⟨1225⟩. In USP 36-NF 31; The United States Pharmacopeial Convention: Rockville, MD, 2012; pp 983−988. (14) Thompson, M.; Ellison, S. L. R.; Wood, R. Harmonized guidelines for single-laboratory validation of methods of analysis. Pure Appl. Chem. 2002, 74, 835−855. (15) Guidelines for Single Laboratory Validation of Chemical Methods for Dietary Supplements and Botanicals; Association of Official Analytical Chemists: Gaithersburg, MD, 2002.

the utility of the method for quality and strength determinations of commercial steviol glycoside products. Advantages. Steviol glycoside products are known to contain a mixture of highly hydrophilic analytes carrying multiple sugar moieties, whereas other compounds in the mixture exhibit the hydrophobic nature of the diterpenoid backbone common to all of the glycosides. The developed reversed-phase HPLC method was capable of separating the nine related structures known to be components of steviol glycoside products with exceptional reproducibility. In addition, the method was able to identify the 2-diterpenoid backbones found in the glycosylated structures and a rarely occurring glycoside. Moreover, the rebaudioside A forced degradation studies provided solid evidence that the developed reversedphase HPLC method could resolve known and unknown structures that may arise from extreme stress conditions of these products. The method validation results met the established acceptance criteria for linearity, accuracy, precision, and ruggedness. The method was also found to be robust for minor changes to LC parameters that may arise during routine analysis. The validated HPLC method was found to be suitable for quality and strength assessments and is to be included by USP in the Food Chemicals Codex compendial standard for steviol glycosides.

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ASSOCIATED CONTENT

S Supporting Information *

Commercial sample assay results. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Phone: (301) 816-8288. Fax: (301) 8168157. Notes

The authors declare no competing financial interest.

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ABBREVIATIONS %RSD, percent relative standard deviation; ACN, acetonitrile; CoA, certificate of analysis; FCC, Food Chemicals Codex; GB, Chinese Guobiao; JECFA, Joint FAO/WHO Expert Committee on Fodd Additives; PDA, photodiode array; RT, retention time; RRT, relative retention time; RRF, relative response factor; USP, United States Pharmacopeia

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REFERENCES

(1) JECFA. Steviol glycosides, monograph 10. In Food and Agricultural Organization of the United Nations. Combined Compendium of Food Additive Specifications; 2010 (http://www. fao.org/food/food-safety-quality/scientific-advice/jecfa/jecfaadditives/en/) (accessed July 17, 2013). (2) Zimmermann, B. F.; Woelwer-Reick, U.; Papagiannopoulos, M. Separation of steviol glycosides by hydrophilic liquid interaction chromatography. Food Anal. Methods 2011, 5, 266−271. (3) Woelwer-Rieck, U.; Lankes, C.; Wawrzum, A.; Wust, M. Improved HPLC method for the evaluation of the major steviol glycosides in leaves of Stevia rebaudiana. Eur. Food Res. Technol. 2010, 231, 581−588. (4) Pol, J.; Hohnova, B.; Hyotylainen, T. Characterization of Stevia rebaudiana by comprehensive two-dimensional liquid chromatography time-of-flight mass spectrometry. J. Chromatogr., A 2007, 1150, 85−92. (5) Pieri, V.; Belancic, A.; Morales, S.; Stuppner, H. Identification and quantification of major steviol glycosides in Stevia rebaudiana purified 1389

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