mAU 1800 Extracts <strong>of</strong> Stevia rebaudiana have demonstrated sweetness up to 300 times greater than table sugar, and have rec<strong>en</strong>tly be<strong>en</strong> granted approval through <strong>the</strong> GRAS certification process <strong>for</strong> use in commercial food and beverage products. With <strong>the</strong> growing commercial use <strong>of</strong> Stevia extracts <strong>the</strong>re is an increased need <strong>for</strong> testing <strong>for</strong> <strong>the</strong> known ‘impurities’ <strong>of</strong> Stevia extracts. A comparative study is made betwe<strong>en</strong> <strong>the</strong> industry accepted 1600 JECFA method, which utilizes NH -columns under isocratic conditions 2 1 and an improved method developed by <strong>the</strong> authors, which utilizes a Ph<strong>en</strong>om<strong>en</strong>ex Synergi Hydro-RP column and a linear gradi<strong>en</strong>t2 , <strong>for</strong> <strong>the</strong> HPLC analysis <strong>of</strong> Rebaudioside A and related diterp<strong>en</strong>e glycosides found in Stevia rebaudiana. The improved 1400 method <strong>of</strong>fers greater s<strong>en</strong>sitivity, greater resolution <strong>of</strong> minor constitu<strong>en</strong>ts, maintains resolution over lifetime <strong>of</strong> <strong>the</strong> column and reduces <strong>the</strong> amount <strong>of</strong> acetonitrile consumed during analysis. The improved method is also readily compatible with additional detection techniques including mass spectrometry (LC/MS) and Evaporative Light Scatting Detection (ELSD), overcoming a limitation <strong>of</strong> JECFA method. The two methods have be<strong>en</strong> evaluated using standards <strong>of</strong> rebaudiosides A, B, C, D and F, dulcoside A, isosteviol, isosteviol monoside, steviol, steviol glucuronide, stevioside and steviolbioside. hello Figure 1 Comparative study <strong>of</strong> HPLC methods <strong>for</strong> <strong>the</strong> <strong>Analysis</strong> <strong>of</strong> Diterp<strong>en</strong>e <strong>Glycosides</strong> from Stevia rebaudiana Abstract & Introduction mAU 900 800 HO 700 O OH HO OH O OH 600 HO OH 500 400 300 200 100 Figure 2 mAU OH OH OH OH HO OH HO OH HO OH HO OH ADC1 A, ADC1 CHANNEL A (R:\INGOLD\DATA\2009\H0309\H0309_02.D) HO O O HO 1 1 O O 11 12 HO OH O O HO HO OH 10 8 Time, Min. %Aqueous* %Acetonitrile 6 7 0 95 5 3 95 5 38 5 95 40 5 95 43 95 5 51 95 5 *Aqueous Mobile Phase is Milli-Q water with or without modifier (see below). Figure 3 0.1% TFA ELSD Figure 4 0.1% TFA UV, 210 nm Figure 5 0.1% TFA LC/MS, TIC Figure 6 Milli-Q water UV, 210 nm Figure 7 1.0% IPA UV, 210 nm HO OH O O OH HO HO HO O HO OH O HO OH O HO OH HO HO HO OH OH OH O O OH 2 2 3 3 4 4 5 5 6 6 7 7 8 8 9 9 10 10 11 11 12 12 18 20 22 24 26 28 30 3 HO HO O O HO HO OH OH 5 4 O O 2 HO HO HO OH OH OH O O HO HO HO HO O HO OH O HO OH O HO OH O HO OH HO HO O O O OH O OH O OH O OH O OH O OH O OH Rebaudioside D Rebaudioside A Stevioside Rebaudioside F Rebaudioside C Dulcoside A Rebaudioside B O O O O O O HO O O 1 O O 2 O O 3 O O 4 O O 5 O O 6 O 7 Experim<strong>en</strong>tal ACN Usage = 21.5 mL/run Column: Synergi Hydro-RP 250 x 4.6 mm, 4 µm particle size Flow Rate: 1.0 mL/minute Temperature: 60 °C 3 3 5 5 6 6 10 7 7 1 1 4 4 9 2 2 8 8 3 1 2 4 56 3 1 2 4 9 78 10 56 3 1 2 4 9 78 10 56 9 78 10 6 6 8 8 3 3 7 7 5 5 2 2 4 4 1 1 1 1 3 3 6 6 4 4 2 2 5 5 8 8 3 3 7 7 6 6 9 9 10 10 10 10 5 5 7 7 1 1 8 8 2 2 4 4 10 10 Brant C. Hoekstra, Keith A. Chamberlain, James S. Traub, Stev<strong>en</strong> F. Baugh, Sylesh K. V<strong>en</strong>kataraman ChromaDex, Inc., 2830 Wilderness Place, Boulder, Colorado 80301, USA. 12 11 11 12 11 11 11 11 12 12 12 12 12 HO HO OH OH O O HO OH HO OH HO O O O O JECFA ACN Usage = 30 mL/run Column: Agil<strong>en</strong>t Zorbax NH 2 250 x 4.6 mm, 5 µm particle size Flow Rate: Adjust such that Rebaudioside A is retained ~21 min. Temperature: 40 °C Detection: UV, 210 nm Isocratic 80:20 Acetonitrile:Water, adjusted to pH 3.0 with phosphoric acid and filtered prior to use. Discussion • HPLC analysis <strong>of</strong> Stevia glycosides is illustrated by <strong>the</strong> improved method (Figure 1) and <strong>the</strong> JECFA method (Figure 2). • The improved method (Figure 1: detail, Figure 3: full time scale) displays better peak shape, greater s<strong>en</strong>sitivity and resolution <strong>for</strong> <strong>the</strong> Stevia glycosides while reversing <strong>the</strong> elution order. • In <strong>the</strong> JECFA analysis <strong>of</strong> <strong>the</strong> mixed standard (Figure 2): - <strong>Steviol</strong> and Isosteviol show poor resolution and ret<strong>en</strong>tion by eluting in <strong>the</strong> void volume, - baseline is not achieved betwe<strong>en</strong> compounds 3-8, - As <strong>the</strong> 3-minute-wide Rebaudioside D peak elutes well after Rebaudioside A, it may interfere with subsequ<strong>en</strong>t injections. • Figures 1-5 utilize 10 µL injections <strong>of</strong> <strong>the</strong> same mixed standard. • As Stevia glucosides are not readily soluble in acetonitrile or JECFA mobile phase, <strong>the</strong> mixed standard was prepared using a mixture <strong>of</strong> methanol and water. • All UV signals are displayed at 210 nm to allow a fair comparison to <strong>the</strong> JECFA method. However, s<strong>en</strong>sitivity significantly improves by utilizing 202 nm (peak heights are approximately 50% greater). • Figures 3-7 show <strong>the</strong> robust compatibility <strong>of</strong> <strong>the</strong> improved method with additional detection techniques (Figures 3-5) and modification <strong>of</strong> <strong>the</strong> aqueous mobile phase (Figures 6, 7). • The JECFA method is not ELSD or MS compatible due to <strong>the</strong> use <strong>of</strong> phosphoric acid in <strong>the</strong> mobile phase and dilu<strong>en</strong>t. • Limited ionization <strong>of</strong> compounds 10-12 was observed using negative electrospray ionization (Figure 5); however, <strong>the</strong>se compounds showed excell<strong>en</strong>t ionization in positive mode. • The standard shown in Figures 6-10 lacks steviol glucuronide. • Several aqueous mobile phase modifiers were evaluated including <strong>for</strong>mic acid, acetic acid, TFA, and isopropyl alcohol. • Analyses over several years utilizing differ<strong>en</strong>t instrum<strong>en</strong>tation, columns and standard conc<strong>en</strong>tration show only nominal changes to ret<strong>en</strong>tion times and resolution (see Graz 2007 poster 2 ). OH 9 HO OH O O OH HO O HO OH O OH OH O <strong>Steviol</strong>bioside <strong>Steviol</strong> glucuronide Isosteviolmonoside <strong>Steviol</strong> Isosteviol HO O O O OH O 8 O O 9 O O 10 11 12 O HO HO OH OH HO OH Equipm<strong>en</strong>t 1 1 1 1 1 3 6 2 4 5 3 3 6 6 4 4 2 2 5 5 3 3 6 6 2 2 4 4 5 5 HO OH 8 7 8 8 7 7 8 8 7 7 1 10 10 10 10 10 HO O HO O Figure 8 Mill-Q water UV, 210 nm 11 12 Figure 9 Milli-Q water UV, 210 nm 11 11 12 12 Figure 10 Milli-Q water UV, 210 nm 1. Kolb, N., Herrera, J.L., Ferreyra, D., Uliana, R.; <strong>Analysis</strong> <strong>of</strong> Sweet Diterp<strong>en</strong>e <strong>Glycosides</strong> from Stevia rebaudiana: Improved HPLC Method. J. Agric. Food Chem. 2001, 49, 4538-4541. 2. “An Improved HPLC Method <strong>for</strong> <strong>the</strong> <strong>Analysis</strong> <strong>of</strong> Diterp<strong>en</strong>oid <strong>Glycosides</strong> in Stevia rebaudiana,” Brant C. Hoekstra, Brian T. Schaneberg, Poster, 55th International Congress & Annual Meeting <strong>of</strong> <strong>the</strong> Society <strong>for</strong> Medicinal Plant Research, Graz, Austria, EU, Sept 2-6, 2007. 3. <strong>Steviol</strong> <strong>Glycosides</strong>; FAO JECFA Monographs 5 (2008) 10005 Muirlands Blvd. | Suite G | First Floor | Irvine, CA 92618 USA | Phone: +1 (949) 419-0288 | Fax: +1 (949) 419-0294 | www.chromadex.com 11 11 12 12 min HPLC: Agil<strong>en</strong>t 1100 series equipped with a vacuum degasser, an autosampler injection system, a <strong>the</strong>rmostated column ov<strong>en</strong>, and a binary pump with a quaternary low pressure mixing valve. MS: Agil<strong>en</strong>t 1100 SL series Ion Trap ELSD: Alltech 200ES ELSD What’s Next? ACN Usage = 3.5 mL/run Synergi Hydro-RP HST 100 x 2.00 mm, 2.5 µm particle size Ph<strong>en</strong>om<strong>en</strong>ex C18(2)-HST 100 x 2.00 mm, 2.5 µm particle size Ph<strong>en</strong>om<strong>en</strong>ex Fusion-HST 100 x 2.00 mm, 2.5 µm particle size • Figures 8-10 repres<strong>en</strong>t preliminary results from fur<strong>the</strong>r method developm<strong>en</strong>t work. The results suggest that optimization <strong>of</strong> <strong>the</strong> method on HST columns could reduce run times to ~20 minutes (including wash & re-equilibration) while maintaining resolution. • HST chromatography can be run on an Agil<strong>en</strong>t 1100 or equival<strong>en</strong>t (fitted with low flow compon<strong>en</strong>ts). E<strong>the</strong>l Aardvark - Wiki Commons St<strong>en</strong> Porse - Wiki Commons 6
400 proved Standard method mixture <strong>of</strong>fers greater s<strong>en</strong>sitivity, greater resolution <strong>of</strong> minor constitu<strong>en</strong>ts, Isocratic: maintains 80:20 resolution Acetonitrile:Water, over lifetime <strong>of</strong> <strong>the</strong> column pH and 3.0reduces th ed Column: method is Synergi also readily Hydro-RP compatible with additional detection techniques including Flow: 1.0 mass mL/min, spectrometry Temperature: (LC/MS) and Evaporative 60 °C Light Scatt d. The two methods have be<strong>en</strong> evaluated using standards <strong>of</strong> rebaudiosides A, B, C, D and F, dulcoside A, isosteviol, isosteviol monoside, steviol mAU 900 800 700 600 500 400 300 200 100 HO OH HO OH HO OH HO OH ADC1 A, ADC1 CHANNEL A (R:\INGOLD\DATA\2009\H0309\H0309_02.D) HO HO HO O HO HO OH O HO OH O O O O 250 x 4.6 mm, 4 μm O O OH O OH 1 1 O O HO OH OH OH Rebaudioside D 1 HO OH HO HO O HO OH OH O HO O O OH O O O O O HO OH OH Rebaudioside A OH O O O O 2 O O 3 O O 4 O O 5 O O 6 HO HO HO OH HO OH OH O O O O Stevioside HO OH OH OH 2 2 3 3 4 4 5 5 HO HO HO HO OH OH O HO OH O O O O O HO OH OH Rebaudioside F Hoekstra et al., 2009 (Chromadex) HO HO HO HO OH 6 6 7 7 8 8 9 9 10 10 18 20 22 24 26 OH O HO OH O O O O O HO OH OH Rebaudioside C HO HO HO OH HO OH OH O O O HO O HO OH Dulcoside A OH HO HO OH O HO HO O OH O O O O O HO OH OH Rebaudioside B 7 OH HO HO HO O OH O O O O HO OH OH <strong>Steviol</strong>bioside 8 7 OH
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