EUSTAS Round-Robin Testing of Steviol glycosides

Prof. Jan M.C. Geuns Lab Functional Biology, KULeuvenKasteelpark Arenberg 31, MB 24363001 Heverlee-LeuvenBELGIUMJan.Geuns@bio.kuleuven.be

Abbreviations

• SV: steviol, hence: SV gly and SV glu

• ST: stevioside

• Reb A – G: rebaudioside A - G

• Rub: rubusoside

• DulA: dulcoside A

Problems

- Different steviol glycosides (10)- Different molecular weights- Difference of a few % is very important!- Validation: each step in analysis should be

validated- Very pure standards are required of (all)

steviol glycosides- Often an UV detector is used at 210 nm- Often absorption spectra taken in EtOH

Problems

• Extinction coefficients of all compounds are not known

• Methods used so far were not validated, and are often based on the assumption that extinction coefficients are the same for all compounds

• If extinction coefficients are the same, it follows that the slopes of calibration curves should be the same (plotted as mM conc), eg. ST (804), RebA (966), Rub (642)

Proof of similar slopesCalibration curves

yRub = 1479,5xR2 = 0,9985

yST = 1432,2xR2 = 0,9989

yRebA = 1375,8xR2 = 0,9989

0

500

1000

1500

2000

2500

3000

0 0,5 1 1,5 2

mM

Are

a

ST RebA Rub

Linear (Rub) Linear (ST) Linear (RebA)

Problems

• Nearly everybody claims that their method is the best one and is accurate.....

Problems

• Nearly everybody claims that their method is the best one and is accurate.....

• So do we !

Extinction coefficients

• Extinction coefficients measured in water, EtOH, 80% AcCN and 35% AcCN

• Measured at 31 and 62 µM• λmax = 205 (EtOH)

196 (H2O)199 (80% AcCN)197 (35%AcCN)

• Molar Extinction coefficient = A/l.c

Extinction coefficients

• ε-values in H2O and AcCN mixtures much larger than in EtOH (± 7500 > ± 4000)

• ε-values measured at λmax much larger than at λ210

• This is also reflected in slopes of calibration curves

Calibration Stevioside 190 and 210 nm

Stevioside at 190 and 210 nm

y = 2,8769xR2 = 0,9991

y = 4,0277xR2 = 0,9976

0

1000

2000

3000

4000

5000

6000

-100,0 100,0 300,0 500,0 700,0 900,0 1100,0 1300,0

Conc. µM

Are

a

UV 190 nm UV 210 nm

Problems

• Often percentual composition is taken as percentage purity! These are not related at all!

• Proof: measurement of SVgly in presence/absence of 90% glucose!

1 mg/mL Measurement in water

1 mg/mL plus 10 mg glu/mL

Measurement in presence of impurities (claim: >95% purity)

Sample 505171 Ip0906 Nv2060301

Loss on drying 2.3 % 2.8% 5%

Stevioside

Reb A

Reb C

Dulcoside A

Other

31.29%

14.67

4.89

1.63

0.65

52.44%

23.93

7.21

2.95

0.98

56.44

28.22

6.51

3.26

3.26

Total = Purity 53.13% 87.51% 97.68%

Examples of % composition

Examples of % composition

RT Height Area Percentage 5.772 7.283 9.200 10.315 12.787

4429.755 10673.043 177817.469 17543.112 62830.125

72049.688 257330.981 3852938.000 175961.281 1828652.375

1.1107 3.9669 59.3951 7.3372 28.1898

Total 273293.801 6486932.328 100

Examples of % composition

• Percentual composition was given

• Calculation: peak areas without correction for MW!

• Different MW were not taken into account!

Examples of % composition

Examples of % composition;Claim: >96% RebA

RT Height Area % ST RebC RebA

8.728 9.782 12.058

8798.549 2342.823 180234.266

160006.984 38351.609 5027667.500

3.0617 0.7339 96.2044

191375.638 5226026.094 100.000

Same sample analysed

Same sample, claim >96% purity

% composition Purity (% Dry wt) RebA ST RebF RebC DulA RebG Rub RebB

89.759 5.282 0.799 2.279 0.471 0.253 0.094 1.063

81.931 4.821 0.729 2.080 0.430 0.231 0.086 0.971

100 91.279

Impurities in samples (C18 column)Always inject blanks (below)

[min.]Time

2 4 6 8 10

[mAU]

Abs

orba

nce

0

10

20

30

40

50

60

STRA98-080603-GS95 - S 2500: Channel 1

STRA60-080701-SQ95 - S 2500: Channel 1

Round-robin testing

• 10 labs

• Each lab: use of own standards and methods

• 2 unknown samples

- sample 1: 96.2% purity

- sample 2: 80:20 dilution of sample 1 with NaHCO3

AIM of Round-robin testing

- check as many parameters as possible

- drying process (NaHCO3)

- peak integration

- calculations

- weighing........

- try to explain the differences in analyses

- how to improve analysis?

Round-robin testing

• Most labs: NH2 columns, a few C18,

1 SILIC; 250 x 4.6 mm; 5µm; UV 205 or 210 nm

• Solvents:

- AcCN:H2O or AcCN:diluted NH4OAc or phosphoric acid (isocratic or gradient)

- C18: 2 x Alltech Alltima with gradient AcCN:0.1 mM phosphoric acid

Round-robin testing• In theory, both HPLC methods should be

equivalent and give similar results after proper calibration.

• Use of only 1 early eluting standard for calibration combined with an isocratic solvent, might give an underestimation of slowly eluting compounds (due to flattening of peaks, partly disappearing in base line).

Sample 1, gradient: 25 min

Sample 1, isocratic 33%; 60 min

Difference gradient vs. isocratic

HPLC RebA ST RebF RebC DulA RebG Rub RebB SB Gradient 22.08 60.559 1.492 7.382 3.039 0.671 1.418 0.839 2.520 33% isocr. 22.078 61.891 1.004 7.180 2.980 0.444 1.372 0.808 2.273 Ratio 1.000 0.978 1.486 1.028 1.020 1.511 1.033 1.038 1.108

Difference gradient vs. isocratic

• Later peaks are systematically smaller than early eluting peaks

• Combination of “disappearance in baseline” and greater signal due to longer time in detector, as demonstrated by a chromatogram at 1 mL/min and 0.8 mL/min

Flows of 1 and 0.8 mL/min

Flow Area RebA 1 mL/min 5020 0.8 mL/min 7143 (+42.3%)

Conclusion flow rate

• Variation in flow rate (even a few %) might produce huge errors in quantification!

Round-robin testing: Sample 1Lab Purity # Corr. Cor

purity

1

2**

3

4

5*

6*

7

8*

9

10

80.1

87.0

95.4

91.7

96.2

92.0

79.8

91.1

86.0

4

5

4

8

9

5

5

9

4

6.0

5.7

6.0

0.66

0.34

5.73

5.73

0.48

6.0

86.1

92.7

101.4

92.36

96.5

97.73

85.53

91.58

92.0

Sample 1

• Only 3 labs analysed 8 or 9 compounds

• Only 2 labs found purity > 95%

• After correction for compounds not analysed: only 3 labs purity >95%. However, lab 3 excessive values for RebC and DulA

Sample 2: Weight loss• 20 % NaHCO3

• 100 mg: 80 mg sample 1 + 20 mg NaHCO3

• Heating at 105°C, several hours:

2 NaHCO3 Na2CO3 + CO2 + H2Oloss of 36.9 %!

100 mg sample 2: contains 80 mg “wet” sample 1. Wt loss is 4.8 mg (6 %)

20 mg NaHCO3: loss of 7.38 mgTotal: 4.8 + 7.38 = 12.18 mg or 12.18 %

Sample 2: Weight loss

• Reported weight loss: between 4.9 and 12.7%

• Insufficient drying! Drying should be done to constant weight.

• Weak point in JECFA recommendation (2 h at 105°C, wt loss< 6%).

• Moisture calibration of standard and sample: possible eg. RebA. What about all other compounds: hygroscopic properties???

Purity of sample 2

• Amount of SV gly in sample 2:

- purity of sample 1= 96.2% or 90.4% before correction for weight loss (= 6 %)

- 100 mg sample 2 contains 80 % of sample 1 or 90.4 x 80 % = 72.32 mg.

- Theoretical purity of sample 2: 72.32/0.8782 = 82.35 mg or %. This is 85.6% of purity of sample 1.

Purity of sample 2Lab Purity

% of

Sple1

#

ana

Corr. Corr

purity

1

2**

3

4

5*

6*

7

8*

9

10

59.0

66.1

72.8

80.1

81.8

71.2

58.1

73.3

64.4

73.6

75.9

76.3

87.3

85.0

77.4

72.8

80.5

74.9

4

5

4

8

9

5

5

9

4

5.09

4.85

5.09

0.5

0.26

4.85

4.85

0.4

5.09

64.0

70.95

77.88

80.6

82.06

76.05

62.95

73.67

69.50

Purity of sample 2

• Only 3 labs analysed 8 or 9 compounds

• Purity should be 85.6% of that of sample 1.

• Only 2 labs found about this value (85 and 87.3) and a wt loss of 12.1 and 12.4 %.

• Even after correction for compounds not analysed, purity reported by several labs was far below 82.35 %

Accuracy of measurement• Only way to know what is the exact

measurement

• Standard addition method: at least 3 known amounts are added of ultra-pure and dry standards

• Extrapolation gives intercept with Y-axis. This value should be the same as that of an unspiked sample.

Standard addition of Sample 1Sample 1: Standard Addition Reb A

y = 6202,4x + 1277,5

R2 = 0,9996

-1000

0

1000

2000

3000

4000

5000

6000

7000

8000

-0,4 -0,2 0 0,2 0,4 0,6 0,8 1

conc. added

Are

a

Reb, A Linear (Reb, A)

Standard addition of Sample 1

• Intercept gave a value of 1277.5 for RebA

• unspiked sample: value of 1309

• Difference: 2.5 %, acceptable as expt. was done only once

Possible errors in (G)LP

• Purity of standards:– absence of other SV gly– drying to constant weight– always validate quality of standards!!!

Purity of standards: ST: claim: 98.6% purity!

Purity of standards: % composition: ST only 94.97

Purity of standards: on dry wt. basisonly 93.54 % purity (wt loss: 3.1%)

Possible errors (in %)

• Calibration with impure standards leads to an overestimation of purity of samples!

• ST: claimed purity 98.6%, real purity: 93.54%

Claimed Found (wet)

Found

(dry wt.)

“100 %” vs. wet corr

98.6 90.4 93.54

(100 %) Δ 9 % Δ 5.4 % Δ 10.6 %

Conclusions• HPLC method, NH2 vs. C18 less important than

GLP!• Purity of standards: utmost importance

– No other compounds present

– Moisture content

– To be validated by each lab

• Drying of standards and samples to constant weight

• Use of gradients might avoid underestimation of small peaks eluting at the end in isocratic HPLC

Conclusions

• Weighing process itself

- Weigh enough sample/standard (at least 50 mg) on a sensitive balance- Weighing of solution (eg. mg/g) might decrease errors- if using automatic pipettes: do not trust them and calibrate regularly! and again and again.... check quality of tips

Conclusions

• Dissolution of sample! Check that sample is well dissolved

• Inject enough. Large peaks should be > 500 mV. This reduces relative standard errors of smaller peaks.

• Check base-line in peak integrations!

Conclusions

• Check accuracy by standard addition method!

• More round-robin tests are required to improve and unify the analysis of steviol glycosides.

• Cooperation between different companies would be good for the “Stevia Industry” as a whole.

Acknowledgements

• Hilde Verlinden, Bert Demarsin for their excellent help

• Peter Grosser, Medherbs, Wiesbaden, Germany for financial support

• My wife Christine for being a “Stevia Widow”