Sugar Method Development for
Feeds and Pet Foods
Dan Berg
26 July 2014
Confidential
Background
Method needs statement:
Past studies: Multiple labs, extraction techniques, analytical instruments
Pure standards with no pet food extraction to get agreement between labs
Project goal: identify reasons for differences and move towards consensus on
best practices
1) Hold analysis constant and evaluate extraction
2) Hold extraction constant and find analysis that meets performance
requirements and most adaptable for stakeholders
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Sugar Method Development for Feeds and Pet Foods
Confidential
Methods
Materials
Dry cat food
Dry dog food
Horse feed pellets
Sample preparation
Ground in blender
2 g sample
Extracted into 50 ml solvent for 60 minutes at room temp
Bring to volume 100 mL
Centrifuge 15 mins at 3000 rpm
Dilute to standard curve levels
Filter through 0.45um PTFE syringe filter
Analysis
Dionex ICS-5000 HPAEC-PAD
25 uL injection 30° C column
PA1 guard (4mm X 50 mm) and analytical column (4mm X 250mm)
47 min gradiant UPW to 600 mM NaOH
Gold Electrode using triple potential waveform
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Sugar Method Development for Feeds and Pet Foods
Confidential
Extraction Solvent Comparison (1:1 with water)
Cat Food
Dog Food
Horse Feed
4
Average of triplicate analysis
Confidential
Sugar Method Development for Feeds and Pet Foods
Water Extraction
Triplicate analysis of each
Glucose RSD
Horse feed in water 37%
Cat food in water 19%
Horse and cat food in 3 other solvents 1-4%
Sucrose Accuracy Spikes
60 and 66% Recoveries of sucrose spikes on horse feed
Inversion to fructose and glucose
Inconsistent glucose measurements
Active enzymes, probiotics and other bacteria
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Sugar Method Development for Feeds and Pet Foods
Confidential
What is the best % ethanol / water ratio?
Balance of stability and solubility
Experiments of 30 to 80% ethanol
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Sugar Method Development for Feeds and Pet Foods
Confidential
Horse Feed Extraction
3.00%
2.50%
2.00%
Galactose
Glucose
Fructose
Sucrose
Lactose
Maltose
1.50%
1.00%
0.50%
0.00%
30
8
40
50
60
70
% Ethanol in Extraction
Sugar Method Development for Feeds and Pet Foods
Confidential
80
Cat Food Extraction
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Sugar Method Development for Feeds and Pet Foods
Confidential
Dog Food Extraction
2.50%
2.00%
Galactose
Glucose
Fructose
Sucrose
Lactose
Maltose
1.50%
1.00%
0.50%
0.00%
30
10
40
50
60
70
% Ethanol in Extraction
Sugar Method Development for Feeds and Pet Foods
Confidential
80
Spike accuracy
Dog Food spiked at 100% of innate
50% Ethanol extraction
Replicate Galactose Glucose
Fructose
Sucrose
Lactose
Maltose
Innate
content:
<0.005%
0.150%
0.197%
2.02%
0.0733%
0.0110%
1
98
103
107
103
111
93
2
99
100
103
101
106
91
3
97
98
102
98
104
91
Average
98
100
104
101
107
92
Spiked 2% Lactose and Maltose to confirm recovery at higher level
Lactose: 94% and 97% recovery
Maltose: 93% and 96% recovery
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Sugar Method Development for Feeds and Pet Foods
Confidential
Extraction Conclusions
Sugars may not be stable in pure water extractions
50/50 Ethanol/water appears to provide good balance of
stability and solubility
Optimized extraction technique and HPAEC-PAD
detection gave consistent results, even at LOQ, and
accurate spike recoveries
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Sugar Method Development for Feeds and Pet Foods
Confidential
Next steps: Evaluate instruments
HPAEC-PAD chromatography
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Sugar Method Development for Feeds and Pet Foods
Confidential
GC chromatography overlays
Glucose
Fructose
Sucrose
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Confidential
HPLC-RI chromatography
Sucrose
Fructose
Glucose
Maltose
Lactose
Standards
Sucrose
Dog Food Sample
Lactose??
And Galactose??
Salt
Glucose
Fructose
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Confidential
Maltose
Next Steps
Evaluate analysis instrument options
1) HPLC – sample clean up, optimized column
2) HPLC with post column derivatization
3) HPAEC-PAD
4) GC - derivatized samples
Cross lab study using same method
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Sugar Method Development for Feeds and Pet Foods
Confidential
Acknowledgements
Phil Anderson
David Ellingson
Darryl Sullivan
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Sugar Method Development for Feeds and Pet Foods
Confidential