Comparative study of HPLC methods for the Analysis of Diterpene

Glycosides from Stevia rebaudiana

Brant C. Hoekstra, Keith A. Chamberlain, James S. Traub, Steven F. Baugh, Sylesh K. Venkataraman
ChromaDex, Inc., 2830 Wilderness Place, Boulder, Colorado 80301, USA.
mAU

Abstract
1800 & Introduction
Extracts of Stevia rebaudiana have demonstrated sweetness up to 300 times greater than table sugar, and have recently been granted approval through the GRAS certification process for use in commercial food
and beverage products. With the growing commercial use of Stevia extracts there is an increased need for testing for the known ‘impurities’ of Stevia extracts. A comparative study is made between the industry
1600JECFA method, which utilizes NH -columns under isocratic conditions1 and an improved method developed by the authors, which utilizes a Phenomenex Synergi Hydro-RP column and a linear
accepted 2
gradient , for the HPLC analysis of Rebaudioside A and related diterpene glycosides found in Stevia rebaudiana.
2

1400
The improved method offers greater sensitivity, greater resolution of minor constituents, maintains resolution over lifetime of the column and reduces the amount of 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 of JECFA
method. The two methods have been evaluated using standards of rebaudiosides A, B, C, D and F, dulcoside A, isosteviol, isosteviol monoside, steviol, steviol glucuronide, stevioside and steviolbioside.

OH OH OH OH OH

hello ADC1
HO
AOH
, ADC1 CHANNE
HO OH
L A (R:\INGOLD\DATA\2009\H0309\H0309_02.D)
HO OH HO OH HO OH

mAU HO
O O OH
HO
O O OH OH OH
HO
O O
HO
O O OH
HO
O O OH OH OH
HO O O HO O O HO O O HO O O HO O O HO O O HO O O HO O O

900 HO HO HO HO HO HO HO HO
O HO OH O HO OH O HO OH O HO OH O HO OH O HO OH O HO OH O HO OH

O OH O OH O OH O OH O OH O OH O OH O OH OH
O
800 Rebaudioside D Rebaudioside A Stevioside Rebaudioside F Rebaudioside C Dulcoside A Rebaudioside B Steviolbioside Steviol glucuronide Isosteviolmonoside Steviol Isosteviol
O O O O O O HO HO O O O
O O 1 O O 2 O O 3 O O 4 O O 5 O O 6 O 7 O 8 O O 9 O O 10 11 OH
12
O
HO HO HO HO HO HO HO HO
700 O
OH
OH OH OH OH OH OH OH
HO HO HO HO HO HO HO HO
OH O OH OH OH OH OH OH OH
HO HO
OH
600 HO O O
OH

500

400
Figure 1

300

200

1 2 3 4 5 6 7 8 9 10 11 12
100

18 20 22 24 26 28 30 min
mAU
D AD 1 C, Sig=210, 8 R ef =600, 100 (H 0309\ H 0309_07. D )
m AU

11
80

12
Figure 2

60

8 6 3
40
7 5 9
10 4 2
1
20

0 5 10 15 20 25 30 35 40 45 m in

Experimental ACN Usage = 21.5 mL/run JECFA ACN Usage = 30 mL/run Equipment
Column: Synergi Hydro-RP 250 x 4.6 mm, 4 µm particle size Column: Agilent Zorbax NH2 250 x 4.6 mm, 5 µm particle size HPLC: Agilent 1100 series equipped with a vacuum
Flow Rate: 1.0 mL/minute Flow Rate: Adjust such that Rebaudioside A is retained ~21 min. degasser, an autosampler injection system, a
Temperature: 60 °C Temperature: 40 °C thermostated column oven, and a binary pump
Detection: UV, 210 nm with a quaternary low pressure mixing valve.
Time, Min. %Aqueous* %Acetonitrile Isocratic 80:20 Acetonitrile:Water, adjusted to pH 3.0 with phosphoric MS: Agilent 1100 SL series Ion Trap
0 95 5 acid and filtered prior to use. ELSD: Alltech 200ES ELSD
3 95 5
38 5 95
40 5 95 Discussion What’s Next? ACN Usage = 3.5 mL/run
43 95 5
• HPLC analysis of Stevia glycosides is illustrated by the improved Synergi Hydro-RP HST 100 x 2.00 mm, 2.5 µm particle size
51 95 5 DAD1 D, Sig=210,8 Ref =600,100 (X:\XAVIER\DATA\2009\H0109\H0109_23.D)
3
method (Figure 1) and the JECFA method (Figure 2). Figure 8
mAU

80

*Aqueous Mobile Phase is Milli-Q water with or without modifier (see below). 6 Mill-Q water
• The improved method (Figure 1: detail, Figure 3: full time scale)
70

60 8 UV, 210 nm
mAU
ADC1 A, ADC1 CHANNEL A (V:\INGOLD\DATA\2009\H0309\H0309_02.D)

displays better peak shape, greater sensitivity and resolution for 50 2 4

5

the Stevia glycosides while reversing the elution order.

40
1000 11
30 1 7 10
3
800
Figure 3
5
6
10
• In the JECFA analysis of the mixed standard (Figure 2): 20 12

600
0.1% TFA 1 4
7
9 - Steviol and Isosteviol show poor resolution and retention by
10

ELSD 2
8
0

eluting in the void volume,

8 9 10 11 12 min
400

12 Phenomenex C18(2)-HST 100 x 2.00 mm, 2.5 µm particle size

200
11 - baseline is not achieved between compounds 3-8, DAD1 D, Sig=210,8 Ref =600,100 (X:\XAVIER\DATA\2009\H0109\H0109_13.D)
3
Figure 9
mAU

- As the 3-minute-wide Rebaudioside D peak elutes well after

80
0 8
5 10 15 20 25 30 35 40 min

DAD1
DAD1D,D,
Sig=210,8
Sig=210,8
Ref
Ref
=600,100
=600,100(H0109\H0109_07.D)
(H0109\H0109_07.D)
70 6
Milli-Q water
mAU
300 Rebaudioside A, it may interfere with subsequent injections. 60

4 UV, 210 nm
2
• Figures 1-5 utilize 10 µL injections of the same mixed standard.
200 50
5
7
100
3 6 40

0
1
2 4
5
78
9 • As Stevia glucosides are not readily soluble in acetonitrile or 30
1 10 11
12

-100 Figure 4 10
JECFA mobile phase, the mixed standard was prepared using a 20

-200 0.1% TFA 11

12
10

-300
UV, 210 nm mixture of methanol and water. 0

7 8 9 10 11 min
-400
• All UV signals are displayed at 210 nm to allow a fair comparison
-500 Phenomenex Fusion-HST 100 x 2.00 mm, 2.5 µm particle size
0 5 10 15 20 25 30 35 40 min
to the JECFA method. However, sensitivity significantly improves DAD1 D, Sig=210,8 Ref =600,100 (X:\XAVIER\DATA\2009\H0109\H0109_27.D)

3
Figure 10
mAU
Intens.
x107 9
by utilizing 202 nm (peak heights are approximately 50% greater).
1.50
8
Milli-Q water
70
6

• Figures 3-7 show the robust compatibility of the improved method

1.25

UV, 210 nm
60

Figure 5 50 2 4

with additional detection techniques (Figures 3-5) and modification

1.00
5

0.75
0.1% TFA 6 8 40
7
11
3
LC/MS, TIC
4
5
7
of the aqueous mobile phase (Figures 6, 7). 30 1 10

0.50 2 20

1
12 • The JECFA method is not ELSD or MS compatible due to the use 10
12

0.25
10

0.00
of phosphoric acid in the mobile phase and diluent. 0

8 9 10 11 12 min

• Limited ionization of compounds 10-12 was observed using

0 5 10 15 20 25 30 35 40 T ime [min]

mAU
DAD1 D, Sig=210,8 Ref =600,100 (H0409\H0409_05.D)
3 • Figures 8-10 represent preliminary results from further method
175

6 negative electrospray ionization (Figure 5); however, these development work. The results suggest that optimization of the
method on HST columns could reduce run times to ~20 minutes
150

compounds showed excellent ionization in positive mode.

125
Figure 6 2
4
8 (including wash & re-equilibration) while maintaining resolution.
100
Milli-Q water
5
• The standard shown in Figures 6-10 lacks steviol glucuronide.
UV, 210 nm 10 • HST chromatography can be run on an Agilent 1100 or
1
• Several aqueous mobile phase modifiers were evaluated including
75

7
50
11 equivalent (fitted with low flow components).
25 12 formic acid, acetic acid, TFA, and isopropyl alcohol.
0
• Analyses over several years utilizing different instrumentation,
0 5 10 15 20 25 30 35 40 min

mAU
*DAD1
*DAD1
D, Sig=210,8
D, Sig=210,8
RefRef
=600,100
=600,100
(H0509\H0509_11.D)
(H0509\H0509_11.D)
- DAD1
- DAD1
D,D,
Sig=210,8
Sig=210,8
Ref
Ref
=600,100
=600,100
(H0509\H0509_29.D)
(H0509\H0509_29.D)
3 columns and standard concentration show only nominal
200
6
changes to retention times and resolution (see Graz 2007
5

Figure 7 1
7
8 poster2).
150 2 4
1.0% IPA 10
Ethel Aardvark - Wiki Commons Sten Porse - Wiki Commons

100
UV, 210 nm
11
1. Kolb, N., Herrera, J.L., Ferreyra, D., Uliana, R.; Analysis of Sweet Diterpene Glycosides from Stevia rebaudiana: Improved HPLC Method. J. Agric. Food Chem.
50
12
2001, 49, 4538-4541.
0

0 5 10 15 20 25 30 35 40 min
2. “An Improved HPLC Method for the Analysis of Diterpenoid Glycosides in Stevia rebaudiana,” Brant C. Hoekstra, Brian T. Schaneberg, Poster, 55th International
Congress & Annual Meeting of the Society for Medicinal Plant Research, Graz, Austria, EU, Sept 2-6, 2007.
3. Steviol Glycosides; 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