eustas round-robin testing of steviol glycosides prof. jan m.c. geuns lab functional biology,...
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EUSTAS Round-Robin Testing of Steviol glycosides
Prof. Jan M.C. Geuns Lab Functional Biology, KULeuvenKasteelpark Arenberg 31, MB 24363001 [email protected]
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Abbreviations
• SV: steviol, hence: SV gly and SV glu
• ST: stevioside
• Reb A – G: rebaudioside A - G
• Rub: rubusoside
• DulA: dulcoside A
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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
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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)
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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)
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Problems
• Nearly everybody claims that their method is the best one and is accurate.....
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Problems
• Nearly everybody claims that their method is the best one and is accurate.....
• So do we !
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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
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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
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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
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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!
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1 mg/mL Measurement in water
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1 mg/mL plus 10 mg glu/mL
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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%
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Examples of % composition
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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
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Examples of % composition
• Percentual composition was given
• Calculation: peak areas without correction for MW!
• Different MW were not taken into account!
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Examples of % composition
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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
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Same sample analysed
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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
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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
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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
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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?
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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
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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).
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Sample 1, gradient: 25 min
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Sample 1, isocratic 33%; 60 min
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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
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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
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Flows of 1 and 0.8 mL/min
Flow Area RebA 1 mL/min 5020 0.8 mL/min 7143 (+42.3%)
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Conclusion flow rate
• Variation in flow rate (even a few %) might produce huge errors in quantification!
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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
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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
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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 %
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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???
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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.
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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
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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 %
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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.
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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)
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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
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Possible errors in (G)LP
• Purity of standards:– absence of other SV gly– drying to constant weight– always validate quality of standards!!!
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Purity of standards: ST: claim: 98.6% purity!
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Purity of standards: % composition: ST only 94.97
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Purity of standards: on dry wt. basisonly 93.54 % purity (wt loss: 3.1%)
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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 %
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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
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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
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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!
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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.
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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”