5.Hoekstra_-_Stevia_MM8gV

Stevia
(Stevia rebaudiana)
Developing Standards for New Food
Ingredients: The Stevia story
29 April 2010
Presented by:
Brant Hoekstra,
Group Leader of Reference Materials

10005 Muirlands Blvd., Suite G | +1-949-419-0288 | sales@chromadex.com | www.chromadex.com
New, Natural, Green & Organic
• Rebaudioside A (rebiana, RebA, RA)
• Stevia: minor constituents
• Blends with other sweeteners such as erythritol and inulin
• Diterpene or triterpene glycosides:
– Luo Han Guo: Mogrosides
– Licorice saponins
• What does the future of
natural sweeteners look like?

Overview of Technical Issues
• Analytical testing games – the data might not represent
the facts
• Standards that are used are often NOT “Standards”
• Water is a real problem
• Solubility issues
• Consistency issues
• Formulating with natural products can be challenging
• Taste profiles can be different between two materials of
the same purity

State of the Stevia Industry
• Stevia: 97% Rebaudioside A materials - GRAS
• Stevia: 95% total ‘named’ Steviol glycosides - JECFA
• Definitions for quality terms are used interchangeably
• What does 97% or 99% really mean?
• How pure is your Rebaudioside A?
• Everyone is using different analytical methods
• JECFA method – or is it the JECFA method?
• Laboratory shopping & testing into compliance

Stevia Analytical Problems
• Stevia-related glycosides have poor UV chromaphore
• Separation of these glycosides can be challenging
• Accurate reporting of impurities is important
• Amino chemistry is a real problem (JECFA & USP)
– Retention time degradation
– Solubility problems with sample prep
Many companies that claim to be following the JECFA or
USP method are not following it as it is written

Analytical Gaming
• Analytical problems help create confusion
• There are many ways to game these methods
– Co-elution inflates values
– Run time can be cut off early
– Analysis at low concentration minimizes impurities
– Sample solvent influences solubility & results
min0 5 10 15 20 25 30 35 40 45
mAU
0
5
10
15
20
25
30
DAD1 D, Sig=210,2 Ref=off (K0909K0909_59.D)
Area:46.7978Area:17.1066
Area:23.5566Area:400.936Area:54.5461
Area:8.79451
Area:170.458
Area:2.79294
7.436-Rubu+Stevbio
9.830-DulcosideA
10.616-RebaudiosideB
12.891-Stevioside
14.978-RebaudiosideC
15.648-RebaudiosideF
19.405-RebaudiosideA
39.701-RebaudiosideD

Example of Chromatogram Clipping
min0 5 10 15 20 25 30 35 40 45
mAU
0
1
2
3
4
5
6
Area:20.5833
Area:8.58517
Area:7.59773
Area:94.8485Area:21.2405
Area:2.47628
Area:52.1358
7.444-Rubu+Stevbio
9.878-DulcosideA
12.974-Stevioside
min0 2.5 5 7.5 10 12.5 15 17.5 20
mAU
0
1
2
3
4
5
6
Area:20.5833
Area:8.58517Area:7.59773
Area:94.8485
Area:21.2405
Area:2.47628
Area:52.1358
7.444-Rubu+Stevbio
9.878-DulcosideA
12.974-Stevioside

Example of Sample Solvent Effect
min0 5 10 15 20 25 30
mAU
0
200
400
600
800
1000
1200
DAD1 B, Sig=202,8 Ref=600,100 (K1209K1209_03.D)
Area:646.877
Area:6473.09
Area:5360.22
Area:183.215
Area:1263.68
Area:123.385
Area:267.222
Area:1433.76
Area:244.832
17.150-RebD
19.298-RebA19.435-Stevioside
19.861-RebF
20.042-RebC
20.278-DulcosideA
20.932-Rubusoside
21.428-RebB
21.691-Steviolbioside
min0 5 10 15 20 25 30
mAU
0
100
200
300
400
500
600
700
DAD1 B, Sig=202,8 Ref=600,100 (C:HPCHEMZEEMANDATA2009H0409H0409_03.D)
17.309-RebaudiosideD
19.463-RebaudiosideA19.612-Stevioside
20.232-DulcosideA
20.478-Rebaudioside
21.565-Steviolbioside
24.791
28.201
30.285
Methanol
Water

Example of Injection Concentration

Problems with Standards
• Analytical results are often NOT traceable to a
reference standard or reference material
• “Standards” that are used are often NOT in fact
Standards
• First batch of incoming raw material becomes
the “standard” for identity and purity
• Purity of “standards” are determined in the same
lab using the standard, generally by peak area.
• “Standard” used to determine purity or identity
came from the same supplier of the raw material
or product.

Reference Standard Types
There are different types of standards
• A Standard is defined by level of
characterization, not its purity
• Reagent Grade – little if any
characterization, not quantitative
• Secondary – purity by area% only
(insufficient for Steviol glycosides)
• Primary – much more complete
characterization
• Certified – more analytics including
uncertainty

Reference Materials Needed
Analysis of Stevia glycosides is not easy and
different types of Reference materials are needed
• Primary or Certified Standards
• Laboratory Control Sample Materials
• Proficiency Materials
• Matrix Reference Materials
- Food matrices
- Beverage matrices
- Other representative matrices

Reference Standards Available
• Stevia Leaf BRM
• Rebaudioside A
• Stevioside
• Rebaudioside B
• Rebaudioside C
• Rebaudioside D
• Dulcoside A
• Steviolbioside
• Rubusoside
• Steviol - Aglycone
• Isosteviol - Aglycone
• Coming soon – Rebaudiosides E and F
O
O
O
OH
OH
HO
HO
H
H
O
O
O O
OO
HO
HO
HO OH
OHHO
OH
HO
H
H
OH
OH
H

Chinese Standard
• Purity Claim: 97.2%
• % by in-house standard
• Supporting Documents

Japanese Standard
• Purity Claim: ?
• Loss on Drying: 1.1%
• Purity by HPLC: 98.3%
• Supporting Documents

• Purity Claim: 93.46%
• HPLC Purity: 97.9%
• Purity recently confirmed
ChromaDex Standard

Comparison
Vendor Chinese Japan ChromaDex
Purity Claim 97.2% 98.3% 93.46%
HPLC % 96.2% 95.8% 97.9%
Res. Solv 0.0% 0.0% 0.0%
Water (KF) 9.6% 6.0% 4.2%
Adjusted % 87.0% 90.0% 93.8%
*Adjusted purity is calculated: (100% - % Water - % Residual Solvents) x % HPLC purity

Quantifying Standards
Vendor Chinese Japan ChromaDex
Purity Claim 97.2%
Using the above purity claim, we have quantified
the Rebaudioside A content of the other materials.
Adjusted % 97.2% 100.6% 104.8%

Why is this important?

Stevia Water Problems
• Rebaudioside A is very hygroscopic (studies have shown
as much as 2 to 10% water uptake in 1 hour)

Stevia Water Problems
Water content will have an impact on purity
• Method of reporting water content is critical
• Loss on Drying (LOD) is not a sufficient measure
of water, as it does not account for the
“water of crystallization” or differentiate solvent
• Studies have shown that all of the water will not
be removed (as much as 1% to 2%)
• Karl Fischer (KF) titration is much more accurate

Stevia Solubility Problems
Solubility can vary
• Example: two samples of RA97 with
confirmed purities over 97%
• one sample is 100% soluble while
the other gives a turbid solution
Why?
• Drying technique can influence
crystal form of Rebaudioside A
• Different crystalline forms may have different solubility

Stevia Consistency Issues
• Consistency is critical (batch-to-batch variability)
• Related to taste profile variances
• Purity of product
• Impurity profile in the product
• Water content
• Solubility
• All of these will have an impact on consistency
and taste

Summary
• Yes, there are challenges in selecting and formulating
with Stevia
• Understanding, monitoring and controlling these issues
is possible – and critical
• Setting specifications and implementing a rigorous
quality control program is crucial
• Despite these issues there are companies that have
solved these problems
• Despite these issues there are high quality Stevia
materials available…you just have to find them

10005 Muirlands Blvd., Suite G | +1-949-419-0288 | sales@chromaDex.com | www.chromadex.com
Thank you
Brant Hoekstra
Group Leader, Reference Materials
ChromaDex, Inc.
10005 Muirlands Blvd., Suite G
Irvine, CA 92618 USA
Phone: +1-949-419-0288
Fax: +1-949-419-0294
Email: BrantH@chromadex.com
Web: www.chromadex.com

5.Hoekstra_-_Stevia_MM8gV

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