Hilic Columns for the Analysis of Steviol Glycosides - eustas en

Hilic Columns for the Analysis of
Steviol Glycosides
Benno F. Zimmermann Ursula Wölwer-Rieck
Institut Prof. Dr. Georg Kurz GmbH, Bonn University
Cologne, Germany
and
Bonn University
1

Steviol Glycosides
Rebaudioside A, B, C, D, E, F
Stevioside
Rubusoside
Steviolbioside
Dulcoside A
minor steviol glycosides
2

Hilic
Hydrophilic
Interaction
Liquid
Chromatography
3

Hilic a (relatively) new stationary phase for HPLC
Hydrophilic
Interaction
Liquid
Chromatography
Selectivity: orthogonal to RP (more or less)
Gradient: 93 % acetonitrile ➔ 80 % acetonitrile
Eluents: water, acetonitrile, additives: formic acid, ammonium formate …
4

alculate Standard mixture the percentage of total steviol glycosides (sum the nine
rcentages).
Column: Capcell pak C18 MG II
250 x 4.6 mm, 5 µm
8.0e-2
6.0e-2
4.0e-2
2.0e-2
2.35
3.42
Rebaudioside D
7.60
Rebaudioside A
8.08
Stevioside
Rebaudioside F
Rebaudioside C
9.72
Dulcoside A
10.72
11.73
Isocratic: 32:68 Acetonitrile:Water, pH 2.6
Flow: 1.0 mL/min
JECFA, 2010
Rubusoside
15.62
23.23
21.57
0.0
Tim
0.00 5.00 10.00 15.00 20.00 25.00 30.00
Rebaudioside B
Steviolbioside
5

mAU
1800
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
accepted
1600
JECFA method, which utilizes NH -columns under isocratic conditions 2 1 and an improved method developed by the authors, which utilizes a Phenomenex Synergi Hydro-RP column and a linear
gradient2 , for the HPLC analysis of Rebaudioside A and related diterpene glycosides found in Stevia rebaudiana.
The improved 1400 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.
hello
Figure 1
Comparative study of HPLC methods for the Analysis of Diterpene
Glycosides 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
Experimental 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, Steven F. Baugh, Sylesh K. Venkataraman
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: Agilent 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 of Stevia glycosides is illustrated by the improved
method (Figure 1) and the JECFA method (Figure 2).
• The improved method (Figure 1: detail, Figure 3: full time scale)
displays better peak shape, greater sensitivity and resolution for
the Stevia glycosides while reversing the elution order.
• In the JECFA analysis of the mixed standard (Figure 2):
- Steviol and Isosteviol show poor resolution and retention by
eluting in the void volume,
- baseline is not achieved between compounds 3-8,
- As the 3-minute-wide Rebaudioside D peak elutes well after
Rebaudioside A, it may interfere with subsequent injections.
• Figures 1-5 utilize 10 µL injections of the same mixed standard.
• As Stevia glucosides are not readily soluble in acetonitrile or
JECFA mobile phase, the mixed standard was prepared using a
mixture of methanol and water.
• All UV signals are displayed at 210 nm to allow a fair comparison
to the JECFA method. However, sensitivity significantly improves
by utilizing 202 nm (peak heights are approximately 50% greater).
• Figures 3-7 show the robust compatibility of the improved method
with additional detection techniques (Figures 3-5) and modification
of the aqueous mobile phase (Figures 6, 7).
• The JECFA method is not ELSD or MS compatible due to the use
of phosphoric acid in the mobile phase and diluent.
• Limited ionization of compounds 10-12 was observed using
negative electrospray ionization (Figure 5); however, these
compounds showed excellent ionization in positive mode.
• The standard shown in Figures 6-10 lacks steviol glucuronide.
• Several aqueous mobile phase modifiers were evaluated including
formic acid, acetic acid, TFA, and isopropyl alcohol.
• Analyses over several years utilizing different instrumentation,
columns and standard concentration show only nominal
changes to retention times and resolution (see Graz 2007
poster 2 ).
OH
9
HO
OH
O O
OH
HO
O HO OH
O OH
OH
O
Steviolbioside Steviol glucuronide Isosteviolmonoside Steviol Isosteviol
HO
O
O
O
OH
O 8
O O 9
O O 10
11 12
O
HO
HO
OH
OH
HO
OH
Equipment
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.; Analysis of Sweet Diterpene Glycosides from Stevia rebaudiana: Improved HPLC Method. J. Agric. Food Chem.
2001, 49, 4538-4541.
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
11
11
12
12
min
HPLC: Agilent 1100 series equipped with a vacuum
degasser, an autosampler injection system, a
thermostated column oven, and a binary pump
with a quaternary low pressure mixing valve.
MS: Agilent 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
Phenomenex C18(2)-HST 100 x 2.00 mm, 2.5 µm particle size
Phenomenex Fusion-HST 100 x 2.00 mm, 2.5 µm particle size
• Figures 8-10 represent preliminary results from further method
development work. The results suggest that optimization of the
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 Agilent 1100 or
equivalent (fitted with low flow components).
Ethel Aardvark - Wiki Commons Sten Porse - Wiki Commons
6

400 proved Standard method mixture offers greater sensitivity, greater resolution of minor constituents, Isocratic: maintains 80:20 resolution Acetonitrile:Water, over lifetime of the 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 been evaluated using standards of 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
Steviolbioside
8
7
OH

What’s Next? ACN Usage = 3.5 mL/run
Synergi Hydro-RP HST 100 x 2.00 mm, 2.5 µm particle size
mAU
80
70
60
50
40
30
20
10
0
mAU
80
70
60
50
40
30
20
10
0
mAU
70
60
50
40
30
20
10
0
DAD1 D, Sig=210,8 Ref =600,100 (X:\XAVIER\DATA\2009\H0109\H0109_23.D)
1
2
3
4
5
6
7
8
Figure 8
Mill-Q water
UV, 210 nm
8 9 10 11 12
Phenomenex C18(2)-HST 100 x 2.00 mm, 2.5 µm particle size
DAD1 D, Sig=210,8 Ref =600,100 (X:\XAVIER\DATA\2009\H0109\H0109_13.D)
1
1
2
2
3
3
4
4
5
5
6
6
7
7
8
8
7 8 9 10 11
DAD1 D, Sig=210,8 Ref =600,100 (X:\XAVIER\DATA\2009\H0109\H0109_27.D)
1
1
2
2
3
3
4
4
5
5
6
6
7
7
8
8
10
10
10
10
10
11
12
Figure 9
Milli-Q water
UV, 210 nm
Phenomenex Fusion-HST 100 x 2.00 mm, 2.5 µm particle size
8 9 10 11 12
11
11
12
12
Figure 10
Milli-Q water
UV, 210 nm
11
11
12
12
min
min
min
Hoekstra et al., 2009 (Chromadex)
8

RP by Geuns
Grace Alltima C18
150 mm x 4.6 mm, 5 µm
two columns in series
Reb A
Stevioside
9

RP by Geuns
Grace Alltima C18
150 mm x 4.6 mm, 5 µm
two columns in series
Reb A
Stevioside
Hilic by Zimmermann
column: M+N Nucleodur Hilic
125 mm x 2.0 mm, 3 µm
flow: 0.45 mL/min
gradient: 4–10 min: 10–15 % water in ACN
with 0.1 % formic acid
Stevioside
Reb A
10

RP by Geuns
Elution order
Reb A
Stevioside
Hilic by Zimmermann
1
2
3
4
Stevioside
5
6
7
Reb A
8
11

RP by Geuns
Elution order is roughly inverse
Reb A
8
Stevioside
5
7
6
3
1
4
2
Hilic by Zimmermann
1
2
3
4
Stevioside
5
6
7
Reb A
8
12

Hilic
➔ solves Reb A / Stevioside problem
13

Hilic
to be optimised:
Column
Gradient
(pH: basic or acidic analytes)
Buffer concentration
14

Hilic Columns
Kinetex (Phenomenex) 150 x 2.1 2.6 µm silica
Luna (Phenomenex) 150 x 3 3 µm diol
Nucleodur (Macherey + Nagel) 125 x 2 3 µm zwitterionic
TSKgel Amide-80 (Tosoh) 150 x 2 3 µm carbamoyl
15

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Hilic Columns
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Kinetex
7.8 %, 5 mM
0.66 mL / min
Luna
11 %, 5 mM
0.68 mL / min
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Sbi
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Dul A
Reb B
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Ste
Reb C
Reb F
����
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Reb A
Reb E
Luna
11 %, 5 mM
0.68 mL / min
Nucleodur
14 %, 10 mM
0.60 mL / min
Reb D
TSKgel
17.5 %, 5 mM
0.45 mL / min
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Hilic Columns
Kinetex (Phenomenex) 150 x 2.1 2.6 µm silica
Luna (Phenomenex) 150 x 3 3 µm diol
Nucleodur (Macherey + Nagel) 125 x 2 3 µm zwitterionic
TSKgel Amide-80 (Tosoh) 150 x 2 3 µm carbamoyl
17

Hilic Mechanism
mobile phase
stagnant water
enriched layer
silica backbone
OH
analytes
C3H5(OH)2
C3H 6 CN
C3H 6 NH2
C2H2ONH2
C2H4SO3H
18

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Kinetex
7.8 %, 5 mM
0.66 mL / min
Luna
11 %, 5 mM
0.68 mL / min
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Dul A
Reb B
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Ste
Reb C
����
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Reb A
Luna
11 %, 5 mM
0.68 mL / min
Nucleodur
14 %, 10 mM
0.60 mL / min
TSKgel
17.5 %, 5 mM
0.45 mL / min
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etention Retention factor Factor k k
10
1
0,1
Rubusoside 5 mM
Rubusoside 10 mM
Steviolbioside 5 mM
Hilic Columns
Steviolbioside 10 mM
L Rubusoside 5 mM
L Rubusoside 10 mM
L Steviolbioside 5 mM
L Steviolbioside 10 mM
T Rubusoside 5 mM
T Rubusoside 10 mM
T Steviolbioside 10 mM
T Rubusoside 5 mM
Nucleodur
Luna
0 5 10 15 20
% water % Water (isocratic)
TSKgel
20

Stevia Leaf Extract
dried and ground leaves
extracted with ACN / water 50 / 50
UV
MS
Stevioside
Reb A
21

Stevia Leaf
UV
MS
Stevioside
Stevioside
Reb A
Reb A
22

Stevia Leave
Stevioside
Reb A
UV
MS
Stevioside
Stevioside
Reb A
Reb A
23

Stevioside
Reb A
UV
MS
a
240
220
200
180
160
140
120
100
80
60
40
20
0
b
240
220
200
180
160
140
120
100
80
60
40
20
0
0
0
mAU
mAU
1
1
2
2
3
3
4
4
after SPE
Woelwer-Rieck et al., 2010
5
5
stevioside
Fig. 1 Chromatograms of a Stevia leaf extract (a) after and (b) 24
without SPE clean-up
6
without SPE
6
7
stevioside
7
rebaudioside A
8
8
9
9
10
rebaudioside A
10
11
11

Hilic
➔ solves Reb A / Stevioside problem
but:
➔ no retention of Steviol
➔ choice of internal standard
➔ sample to be dissolved in solvent with high
organic percentage
25

ACN / Water 50 / 50 Water
Hilic
Solubility
10 µL
5 µL
Injection volume
2 µL
26

ACN / Water 50 / 50 Water
Hilic
Solubility
10 µL
5 µL
2 µL
27

ACN / Water 50 / 50 Water
Hilic
Solubility
10 µL
5 µL
2 µL
28

Conclusion
➔ Hilic columns are suitable for steviol glycoside analysis
(different stationary phases)
➔ Hilic columns give enhanced resolution of
stevioside / rebaudioside A
➔ Optimization of separation by buffer concentration
more details:
Woelwer-Rieck et al. (2010), European Food Research and Technology
Zimmermann et al. (2011), Food Analytical Methods
Zimmermann (2011), Rapid Communications in Mass Spectrometry
29

%
1
641(1) Rubusoside
641(1) Steviobioside
Minor Steviol Glycosides
773(1)
787(1)
787(2) Dulcoside A
803(1)
773(4)
803(2) Reb B
803(3) Stevioside
949(1) Reb C
935(2) Reb F
Time
2.50 5.00 7.50 10.00 12.50 15.00 17.50 20.00
935(3)
965(1) Reb A
965(2) Reb E
1127(1)
1127(3)
1.61e6
30

Minor Steviol Glycosides
Ohta et al. (2010): 10 “new” glycosides: 7 %
Chaturvedula et al. (2011): 3 “new” glycosides
Zimmermann (2011): 7 “new” glycosides
10 “traditional” steviol glycosides:
Rebaudioside A, B, C, D, E, F
Stevioside
Rubusoside
Steviolbioside
Dulcoside A
31

Ohta et al., 2010
new
32

Ohta et al., 2010
new
33

Ohta et al., 2010
new
34

Conclusion
➔ Hilic columns are suitable for steviol glycoside analysis
(different stationary phases)
➔ Hilic columns give enhanced resolution of
stevioside / rebaudioside A
➔ Optimization of separation by buffer concentration
more details:
Woelwer-Rieck et al. (2010), European Food Research and Technology
Zimmermann et al. (2011), Food Analytical Methods
Zimmermann (2011), Rapid Communications in Mass Spectrometry
35