stevia - analytical lc/ms methods from research to routine - waters corporation food quality

©2015 Waters Corporation 1

Stevia - analytical LC/MS methods from

research to routine


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.


‘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.


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.


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).


Collaboration with Severine Goscinny,

WIV-ISP, Belgium

Applicability of ionKey Ion Mobility LCMS

Technology for Non-targeted Screening of Steviol

Glycosides


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


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


Steviosides - Isomer Pairs Structures and Masses

O

OH

OH

OH

O

OH

O

OH

OH

OH

O

OH

H

CH3

HCH3

O

CH2

Rubusoside M-H=641.3173 Da

O

OO

OH

OH

OH

OH

OH

OH

OH

O

CH3CH2

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

CH3CH2

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

CH2CH3

CH3

OH

OH

OH

OHOH

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

OO

O

OH

OH

O

OH

OH

O

OH

OH

OH

OH

OH

OH

OH

OH

OH

OH

CH3

CH3

CH2H

H


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

OO

O

O

HH

H

H

H

H

H H

H

H

H

H

H

H

H

HH

H

H

H

H

CH2CH3

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

OHO

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

OHOH

OH

OH

OH

OHO

OO

O

O

O

O

O

O

H H

H

H

H

H

H

H

H

H

HH

H

H

H

H

HH

H

H

H

H


Expected retention times and expected CCS values for steviol and steviosides – from standards

Compound

Formula

[M-H]-

Expected

Rt

Expected

TWCCSN2 (Å

2)

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


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


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


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


Spectral cleanup – RT and DT aligned Stevioside and Rebaudioside A

Stevioside

Rebaudioside A

RT – retention time DT – drift time


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.


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


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)


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


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 Saccharin Cyclamate

Sucralose

Stevioside

Neotame Aspartame


Mass detection of Sweeteners Chromatograms of standard


Sweeteners in Food & Beverages Increased selectivity of MS Detection

Saccharin

Ace K

Aspartame

Neotame

AU

QDa SIRs

PDA Data


Sweeteners in Food & Beverages Increased scope using MS Detection

AU

Cyclamate

UV transparent

Sucralose

UV transparent

QDa SIRs

PDA Data


Sweeteners in Food & Beverages Increased Selectivity of MS Detection

Stevioside

Reb A

Co-elution

AU

QDa SIRs

PDA Data


Quantification of Stevioside & Rebaudioside A


Calibration ranges and levels of sweeteners in real samples


Stevioside & Rebaudioside A in real sample


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