This presentation describes the utilisation of microfluidic chromatography coupled with high resolution mass spectrometry incorporating ion mobility for separation, detection and identification of steviol glycoside isomers. Moving into the routine environment we then will show a simple, cost-effective solution for the determination of stevioside, Rebaudioside A and other non-nutritive sweeteners in a variety of food products.

1. ©2015 Waters Corporation 1
Stevia - analytical LC/MS methods from
research to routine

2. ©2015 Waters Corporation 2
What is ‘Stevia’?
 Stevia is a sweetener and sugar substitute extracted from the
leaves of the plant species Stevia rebaudiana Bertoni native to
regions of South America.
 The active compounds of interest in stevia are steviol glycosides
(mainly stevioside and rebaudioside), some of which have up to
150 times the sweetness of sugar, are heat-stable, pH-stable,
and not fermentable. Others may have taste profiles that are
undesirable in food products such as bitterness or undesirable
aftertaste.

3. ©2015 Waters Corporation 3
‘Stevia’ – health benefits and legislation
 Health benefits
– With incidence growth in diabetes and obesity, there has been a
move within the food and beverage industry to reformulate products,
and replace sugars with sweeteners to reduce calorie intake without
impacting on taste (sweetness).
– These steviosides also have a negligible effect on blood glucose
which makes stevia attractive to people on carbohydrate controlled
diets.
 Legislation and legal status
– The status of stevia extracts as food additives and supplements
varies from country to country. In the United States, stevia was
banned in 1991 after early studies found that it might be
carcinogenic; after additional studies, the FDA approved some
specific glycoside extracts for use as food additives in 2008. The
European Union approved stevia additives in 2011, and in Japan,
stevia has been widely used as a sweetener for decades.

4. ©2015 Waters Corporation 4
Analysis of Stevia - steviol glycosides
 The Joint FAO/WHO Expert Committee on Food Additives (JECFA)
established regulations for steviol glycosides requiring a purity level of at
least 95% of the seven chemically defined steviol glycosides.
 Food products containing steviol glycosides as sweeteners in Europe are
common. These include smoked/dried fish, fruit juice based drinks, cocoa
based confectionary, sweet and sour preserves, soy bean sauces, breakfast
cereals, chewing gum and reduced sugar products.
 Mean dietary exposure to steviol glycosides is expressed in terms of steviol
equivalents. The EFSA (European Food Safety Authority) report details the
revisions of acceptable use levels in a wide variety of food commodities and
also the acceptable daily intake (ADI) of 4mg/kg bw(body weight)/day for
toddlers.

5. ©2015 Waters Corporation 5
Analysis of Stevia - steviol glycosides
 To address the analytical requirements for steviol glycosides it is therefore
ideal to have methods to (1) detect the range of steviol glycosides and
their isomers for to assess purity. In addition it is essential to (2) add
detection and quantification of the most commonly used active steviol
glycosides (typically stevioside and rebaudioside A) to routine methods
for the analysis of non-nutritive sweeteners in final food products.
 This presentation will describe methods for (1) using negative mode
microflow UPLC coupled to Ion Mobility Mass Spectrometry (UPLC-IM-MS)
and (2) using conventional UPLC with MS Detection (UPLC-MS).

6. ©2015 Waters Corporation 6
Collaboration with Severine Goscinny,
WIV-ISP, Belgium
Applicability of ionKey Ion Mobility LCMS
Technology for Non-targeted Screening of Steviol
Glycosides

7. ©2015 Waters Corporation 7
Technology: IonKey Source and iKey
IonKey Source and iKey which incorporates Acquity BEH
C18, stationary phase in a 150µm diameter column
(100mm or 50mm), with ionisation emitter.
Post-Column Addition Channel
Analytical Channel
PCA iKey Design

8. ©2015 Waters Corporation 8
Technology - SYNAPT G2-Si High Definition
MS (HDMS) - instrument schematic
Size
Shape
Charge
1. Increased
sensitivity
2. Ion mobility
3. Accurate
mass
measurement
Orthogonal acceleration QToF

9. ©2015 Waters Corporation 9
Steviosides - Isomer Pairs
Structures and Masses
O
OH
OH
OH
O
OH
O
OH
OH
OH
O
OH
H
CH3
H
CH3
O
CH2
Rubusoside
M-H=641.3173 Da
O
O
O
OH
OH
OH
OH
OH
OH
OH
O
CH3
CH2
CH3
O
OH
H
H
Steviolbioside
M-H=641.3173 Da
O
O
O
OH
OH
OH
OH
O
O
OH
OH
O
OH
OH OH
OH
CH3
CH2
CH3
O
OH
H
H
Rebaudioside B
M-H=803.3701 Da
O
O
O
O
O
O
OH
OH
OH
O
OH
OH
OH
OH
OH
OH
OH
OH
CH3
CH3
CH2
H
H
Stevioside
M-H=803.3701 Da
Rebaudioside A
M-H=965.4230 Da
Rebaudioside E
M-H=965.4230 Da
CH2
CH3
CH3
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
O
O
O
O
O
O
O
O
O
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
O
O
O
O
O
O
O
OH
OH
O
OH
OH
O
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
CH3
CH3
CH2
H
H

10. ©2015 Waters Corporation 10
Steviosides
Structures and Masses
Dulcoside A
[M-H]-=787.3752 Da
Steviol
[M-H]-=317.2117 Da
OH
O
OH
CH3
CH3
CH2
H
H
Rebaudioside D
[M-H]-=1127.4758 Da
Rebaudioside F
[M-H]-=935.4124 Da
Rebaudioside C
[M-H]-=949.428 Da
CH2
CH3
CH3
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH O
O O
O
O
O
O
O
O
H
H
H
H
H
H
H H
H
H
H
H
H
H
H
HH
H
H
H
H
CH2
CH3
CH3
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
O
O
O
O
O
O
O
O
O
O
O
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H H
H
H
H
H
CH2
CH3
CH3
CH3
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
O
O
O
O
O
O
O
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
CH2
CH3
CH3
CH3
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
O
O
O
O
O
O
O
O
O
H H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
HH
H
H
H
H

11. ©2015 Waters Corporation 11
Expected retention times and
expected CCS values for steviol and
steviosides – from standards
Compound Formula [M-H]- Expected
Rt
Expected
TWCCSN2 (Å2)
Rebaudioside E C44H70O23 965.4230 6.60 289.2
Rebaudioside D C50H80O28 1127.4758 6.68 321.75
Rebaudioside F C43H68O22 935.4124 7.32 293.18
Rubusoside C32H50O13 641.3173 7.56 241.31
Rebaudioside B C38H60O18 803.3701 7.77 261.19
Steviolbioside C32H50O13 641.3173 7.81 235.78
Stevioside C38H60O18 803.3701 7.20 269.64
Rebaudioside A C44H70O23 965.4230 7.17 299.48
Steviol C20H30O3 317.2117 9.48 173.38
Rebaudioside C C44H70O22 949.4280 7.37 299.49
Dulcoside A C38H60O17 787.3752 7.40 270.75

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Stevioside ≤ 1pg/μL in Chocolate
Spread Extract Component Summary
920fg
1pg
1pg
1pg
680fg
640fg
1pg
760fg
720fg
700fg
800fg
• Three pairs of isomers indicated by coloured stars
• Differentiation by CCS values

13. ©2015 Waters Corporation 13
Stevioside ≤ 1pg/μL in Chocolate
Spread Extract (extracted mass chromatogram)
Steviolbioside 0.72pg
Rubusoside 0.68pg
Rebaudioside B 0.7pg
Stevioside 1pg
Rebaudioside D 0.80pg
Rebaudioside E 0.92pg
Rebaudioside F 0.76pg
Dulcoside A 1pg
Rebaudioside A 1pg
Rebaudioside C 1pg
Steviol 0.64pg
Stevioside and Rebaudoside coelute at 7.18mins

14. ©2015 Waters Corporation 14
Ion Mobility separated Stevioside and
coeluting Rebaudioside A fragment
differentiated by CCS
B: CCS=269.48Å2
Stevioside
m/z 803.3707
A: CCS=261.15Å2
Rebaudioside A
Fragment
m/z 803.3707
A B

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Spectral cleanup – RT and DT aligned
Stevioside and Rebaudioside A
Stevioside
Rebaudioside A
RT – retention time
DT – drift time

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Steviosides Screening Summary
 ionKey/MS with ion mobility offers some unique advantages for profiling
complex matrices
– Sensitivity in combination with high resolution full spectra acquisition. Spectral clean up,
Collision cross section to provide unique selectivity and added confidence in
identification.
 ionKey/MS with ion mobility has been used to profile steviol glycosides, it has
been possible to detect all steviol glycosides at ≤1pg on column, in a complex
matrix (chocolate spread).
 Chromatographically coeluting isomeric species (stevioside and rebaudioside A
fragment) have been separated.
 3 pairs of steviol glycoside isomeric species have also been distinguished using
ion mobility and CCS measurement.

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Application Overview
Routine Aanalysis of Sweeteners
Analytical Challenges:
 Product labelling requirements
 Multiple LC methods to detect and quantify various
sweeteners
 Some sweeteners are UV transparent
 Complex food and beverage matrices
Waters Solution:
ACQUITY UPLC H-Class & ACQUITY QDa Detector
 Identification and quantification using a single analysis method
 No need for complete chromatographic separation
 Significantly improves analytical throughput

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Technology - ACQUITY UPLC H-Class
Analytical benefits compared to HPLC
 Enhanced analytical performance
– Greater resolution, throughput &
sensitivity
– Increase lab productivity
 Improve peak resolution
– Less co-eluting peaks
– More accurate integration ->
better RSDs
 Document no. of injections on
each column
– ACQUITY UPLC
Columns have eCords
UltraPerformance LC (UPLC)

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Why Mass Detection with the
ACQUITY QDa Detector?
Detector concept
Adjustment-free & pre-optimized
Automated calibration
Familiar software
Easy to set up - can be up and
running within a very short space
of time run the samples
Ease of use and maintenance,
especially for new users
The push-button simplicity is
proven to remove the obstacles to
introducing MS data into
chromatography labs
Ease of qualitative & quantitative
data interpretation
Better accuracy, better
reproducibility, faster
turnaround for our clients

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 Variety of sweeteners are used in sugar-free products
– Traditionally detected by multiple optical detectors (including UV, ELSD
and fluorescence)
Analysis of Sweeteners in Foods and
Beverages
Rebaudioside A
(RebA)
Acesulfame K SaccharinCyclamate
Sucralose
Stevioside
NeotameAspartame

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Mass detection of Sweeteners
Chromatograms of standard

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Sweeteners in Food & Beverages
Increased selectivity of MS Detection
Saccharin
Ace K
Aspartame
Neotame
AU
QDa
SIRs
PDA
Data

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Sweeteners in Food & Beverages
Increased scope using MS DetectionAU
Cyclamate
UV
transparent
Sucralose
UV
transparent
QDa
SIRs
PDA
Data

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Sweeteners in Food & Beverages
Increased Selectivity of MS Detection
Stevioside
Reb A
Co-elution
AU
QDa
SIRs
PDA
Data

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Quantification of Stevioside &
Rebaudioside A

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Calibration ranges and levels of
sweeteners in real samples

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Stevioside & Rebaudioside A in real
sample

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Routine analysis of sweeteners in food
Conclusions
 The combination of mass detection and chromatographic separation provides
increased selectivity in identifying analytes of interest, and reducing false peak
identification.
 Complementary to traditional detectors, allowing for improved selectivity,
including detection of co-eluting compounds
 Reliable detection of UV- transparent analytes
 Simplified method development