1. Darrell Clinton and H. Dorota Inerowicz Purdue University
Preliminary Sugar Study in Animal Feeds by HPLC with Refractive Index Detection
Darrell Clinton and H. Dorota Inerowicz Purdue University
West Lafayette, IN

2. Introduction
OISC Feed Lab began testing sugars in animal feeds starting with lactose.
Official Method – AOAC – Purity of Lactose (Liquid Chromatographic Method)

3. Lactose Sample Preparation
Weigh sample and transfer into 200 mL volumetric flask.
Add Milli-Q H2O with magnetic stirrer.
Stir for 1-hr.
Add H2O to volumetric line, mix and allow solids to settle.
Transfer to LC vials by filtering through 0.45 µm syringe filters.
Standard Concentrations – 2, 4, 6, 8 and 10 mg/mL.

4. Interest in Sugars
OISC is interested in expanding its LC test for sugars in animal feeds to include Fructose, Glucose, Sucrose and Maltose.
Sugar content in animal feeds have gained much interest as animals, particularly companion pets, are subject to obesity and diabetes as with humans.
AOAC method gives the standard method to extract sugars from Presweetened cereals with RI detection.

5. Methods Used in Sugars
Comparative studies on animal feeds found variations typically dependent on extraction mixtures and solvents.
High Performance Anion Exchange Chromatography with PAD.
HPLC with ELSD.
TMS derivatization with GC-FID.
HPLC post column derivatization with FL detection.
At present, no official AOAC method for sugar analyses in animal feeds.

6. Ellington, Anderson and Berg Journal of AOAC International Vol. 99, No
Studying a method to test sugars in animal feeds using High Performance Anion Exchange Chromatography with PAD.
Studied various solvent extraction combinations in four different feed matrices:
100 % H2O, H2O/MeOH, H2O/EtOH, H2O/ACN.
Conclusion: 50/50 H2O/EtOH gave the greatest solubility while maintaining stability in all sugars.

7. Animal Feed Sugars at OISC Using Lactose Column
Adopted 50/50 H2O/EtOH solvent extraction mixture studied by Ellington et al.
Waters Alliance e2695 HPLC with Waters 2414 differential refractometer.
Mobile Phase: 75/25 ACN/H2O (premixed)
Column / Detector Temp: 30 °C
Lactose Column: Phenomenex Shodex Asahipak-NH2, 5 m, 4.6mm x 250mm with aminopropyl guard column.

8. Lactose vs. Maltose (4.0 mg/mL STD, 1.5 mL/min)
Fructose
Sucrose
Glucose
Maltose & Lactose

9. Disaccharide Structures

10. Lactose vs. Maltose (6.0 mg/mL STD, 1 mL/min)
Fructose
Sucrose
Glucose
Maltose & Lactose

11. New Sugar Column (Luna-NH2, 5 m, 4.6mm x 250mm)
Mobile phase: 80/20 ACN/H2O.
5.0 mg/mL STD, 2.0 mL/min.
Fructose
Sucrose
Maltose & Lactose?
Glucose

12. Sugars Test Plan (Phenomenex Luna-NH2 column)
Study fructose, glucose, sucrose and maltose.
5.0 mg/mL STD, 1.5 mL/min.
Fructose
Sucrose
Glucose
Maltose

13. Potato Powder vs. Poultry Feed (5.0 mg/mL STD, 1.5 mL/min)
Standard
Poultry Feed
Potato Powder

14. Current Lactose Testing
Use standard AOAC method and Asahipak Amino column to test for lactose.
Lactose test is generally used to test dairy type products or milk replacer samples.
What to do about possible feed samples that contain both lactose and maltose?????
Waters Corp. is currently in the process of trying to develop a column to separate maltose and lactose.

15. Animal Feed Sugar Preparation
Weigh 5.0g sample and transfer into 250 mL E. flask.
Add 200mL of 50/50 EtOH/H2O.
Stir for 1-hr.
Transfer to centrifuge tube, centrifuge at 1500g for 15 minutes.
Transfer to LC vials by filtering through 0.45 µm syringe filters.
Standard Concentrations – 0.08, 0.2, 0.5, 1.0 and 2.0 mg/mL.

16. Calibration Curve Linearity (Acquired 3x During Run)
2.0
1.0
0.50
0.20
0.08

17. Calibration Curve – Fructose & Glucose

18. Calibration Curve – Sucrose & Maltose

19. Fructose Working Standard Suitability (WS = 0.5 mg/mL)

20. Glucose Working Standard Suitability (WS = 0.5 mg/mL)

21. Maltose Working Standard Suitability (WS = 0.5 mg/mL)

22. Sucrose Working Standard Suitability (WS = 0.5 mg/mL)

23. Limit of Detection (LOD)
Lowest concentration of analyte that can be detected (not quantitated).
Typically determined when s/n ratio > 3.

24. Experimental Procedure
Repeated five (5) injections using four (4) different standards concentrations.
STD concentrations : 0.1, 0.075, 0.05 and mg/mL.

25. Chromatograms
0.025
0.05
0.075
0.10

26. LOD – Fructose & Glucose s/n Ratio
Conc. %
0.1
0.075
0.05
0.025
LOD
0.2
injection 1
11.451
8.255
4.244
2.252
injection 2
10.349
7.924
4.615
1.959
injection 3
13.228
6.563
3.764
1.723
injection 4
8.916
10.446
5.251
1.395
injection 5
10.369
8.354
4.228
1.72
Mean
8.3084
4.4204
1.8098
Glucose
0.3
6.624
4.89
2.626
1.173
5.872
4.305
2.237
1.028
7.395
3.802
1.929
0.63
5.073
5.945
2.717
0.675
6.079
4.602
2.468
0.849
6.2086
4.7088
2.3954
0.871

27. LOD - Sucrose & Maltose s/n Ratio
Conc. %
0.1
0.075
0.05
0.025
LOD
0.2
injection 1
7.637
5.477
2.753
1.276
injection 2
7.101
5.098
3.607
1.212
injection 3
9.061
4.151
2.357
1.029
injection 4
5.684
6.968
3.629
0.678
injection 5
7.049
5.483
2.653
1.287
Mean
7.3064
5.4354
2.9998
1.0964
Maltose
0.3
4.265
2.894
0.997
0.515
4.257
3.114
1.968
0.467
4.828
2.626
1.132
0.619
3.525
4.196
1.613
0.234
4.368
3.586
1.772
0.717
4.2486
3.2832
1.4964
0.5104

28. Limit of Quantitation (LOQ)
Lowest concentration of analyte that can be quantitated.
LOQ = 10*(s/a)
s = standard deviation of y-intercept.
a = mean of slope.

29. LOQ Calculation Y-Intercept Fructose Glucose Sucrose Maltose Curve 1
Curve 2
Curve 3
Curve 4
Curve 5
SD =
Slope
Mean =
LOD
0.0257
0.0065
0.0104
0.0210
LOQ
0.0779
0.0196
0.0316
0.0636
Sample Wt (g)
5.0000
Extract Volume (mL)
% LOQ
0.311
Mean LOQ
0.193 %
0.078
0.126
0.255

30. AOAC Standard Method Performance Requirements for Sugars in Animal Feeds
Published by AOAC, March 12, 2018.
Analytical Range (%):
<5-50
>50-100
Recovery %:
90-110
95-105
97-103
% RSDr ≤7 ≤5 ≤3

31. Standard Addition Recovery Studies
Spike samples were tested at 1.0 % and 0.5%.
Spike samples consisted of adding highest concentration STD to five (5) prepared replica samples.
Spike samples were studied using a feed sample that contained no detectable sugars (AAFCO – Association of American Feed Control Officials sample ).

32. 1.0% Standard Addition Chromatograms
Spike 5
Original
Spike 4
Spike 3*
Spike 2
Spike 1

33. 1.0% Standard Addition Results
Original sample showed no detectable sugars.
Maltose Trial 3 was omitted.
Replica 1 2 3 4 5
Fructose
101.09
104.73
97.43
101.64
104.27
Glucose
97.99
98.89
104.78
99.45
107.34
Maltose
101.92
103.6 --
104.75
106.49
Sucrose
95.2
91.95
106.16
95.24
102.48

34. 0.5% Standard Addition Chromatograms
Spike 5
Spike 4
Spike 3
Spike 2
Spike 1
original

35. 0.5% Standard Addition Results
Original sample showed % sucrose (<LOQ).
No other sugars were detected.
Recovery results are within the acceptable range for SMPR requirements.
Replica 1 2 3 4 5
Fructose
101.21
101.08
104.59
101.16
105.35
Glucose
97.65
94.59
107.32
105.34
Maltose
101.40
106.79
97.40
94.53
102.88
Sucrose
100.95
105.45
105.53
97.31

36. Potato Powder Chromatogram

37. Potato Powder 1-Day Trial
Fructose
Day A B C D E s x
% RSD 1
2.690
2.697
2.679
2.645
2.699
0.022
2.682
0.825
Glucose
19.644
20.024
19.797
19.662
19.777
0.152
19.781
0.768
Sucrose
4.435
4.857
4.327
4.941
4.578
0.265
4.628
5.725
Maltose
8.991
9.350
9.302
9.282
9.316
0.146
9.248
1.578
As expected, sugar % were high.
% RSD was < 7 (<5% glucose/maltose)

38. Turkey, Equine and Calf Feed Chromatogram

39. Turkey Feed 3-Day Replica Trial
Fructose
Day A B C D E s x
% RSD 1
0.401
0.457
0.416
0.371
0.386
0.033
0.406
8.119 2
0.359
0.313
0.305
0.358
0.325
0.025
0.332
7.596 3
0.368
0.373
0.339
0.018
0.354
5.072
Glucose
(< LOQ)
0.086
0.075
0.056
0.076
0.073
0.011
14.821
0.026
0.029
0.042
0.041
0.012
0.039
30.788
0.074
0.079
0.052
0.054
0.069
0.066
18.375
Sucrose
2.915
2.863
2.884
2.930
2.880
0.027
2.894
0.945
2.932
2.859
2.921
2.946
2.798
0.062
2.891
2.138
3.168
3.060
3.062
3.137
3.163
0.053
3.118
1.711
Maltose
0.250
0.421
0.430
0.466
0.452
0.088
0.404
21.741
0.096
0.097
0.050
0.072
0.103
0.022
0.084
26.567
0.068
0.085
26.247

40. Equine Feed 3-Day Replica Trial
Fructose
Day A B C D E s x
% RSD 1
0.640
0.606
0.639
0.657
0.681
0.027
0.645
4.264 2
0.650
0.649
0.695
0.595
0.036
5.498 3
0.638
0.658
0.668
0.661
0.012
0.654
1.870
Glucose
(< LOQ)
0.010
0.000
0.019
0.008
0.018
0.013
0.009
99.609
0.061
0.044
0.062
0.053
0.139
0.038
0.072
53.285
Sucrose
1.895
2.001
1.930
1.958
1.932
0.039
1.943
2.023
1.945
1.963
1.934
2.070
0.056
1.971
2.858
1.973
1.998
2.027
1.867
1.991
3.115
Maltose
0.107
0.166
0.082
0.060
0.092
65.136
0.049
0.033
0.137
0.149
0.104
0.103
0.158
0.214
0.046
0.146
31.453

41. Calf Feed 3-Day Replica Trial
Fructose
Day A B C D E s x
% RSD 1
0.486
0.515
0.501
0.491
0.403
0.044
0.479
9.185 2
0.381
0.341
0.366
0.354
0.373
0.016
0.363
4.351 3
0.484
0.459
0.445
0.438
0.450
0.018
0.455
3.916
Glucose
(< LOQ)
0.233
0.232
0.204
0.177
0.164
0.031
0.202
15.525
0.028
0.061
0.058
0.023
0.032
0.040
43.952
0.097
0.118
0.133
0.112
0.114
0.013
0.115
11.247
Sucrose
1.810
1.626
1.681
1.666
1.625
0.076
1.682
4.512
1.502
1.491
1.462
1.504
1.499
0.017
1.492
1.158
1.812
1.803 --
1.799
1.739
0.033
1.788
1.861
Maltose
0.176
0.074
0.205
0.311
0.220
0.085
0.197
43.296
0.048
0.073
0.057
0.056
0.011
20.052
0.053
0.069
0.077
16.126

42. Conclusions – RI Detection for Sugars in Feed Samples
Preparation method works very well for feed samples with sugar concentrations > 1 %.
STD linearity ≥ 0.999xxx.
Working STD system suitability < 2.5 %RSD.
STD Addition recovery 91 – 107 %.
LOQ is ~ 0.2 %.
Replica studies for fructose / sucrose ≤ 7 %.
OISC Feed Lab will implement method for in-house feed sample analysis.