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 47907-2063  

Introduction OISC Feed Lab began testing sugars in animal feeds starting with lactose. Official Method – AOAC 984.22 – Purity of Lactose (Liquid Chromatographic Method)

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.

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 982.14 gives the standard method to extract sugars from Presweetened cereals with RI detection.

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.

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.

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.

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

Disaccharide Structures

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

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

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

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

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.

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.

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

Calibration Curve – Fructose & Glucose

Calibration Curve – Sucrose & Maltose

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

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

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

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

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

Experimental Procedure Repeated five (5) injections using four (4) different standards concentrations. STD concentrations : 0.1, 0.075, 0.05 and 0.025 mg/mL.

Chromatograms 0.025 0.05 0.075 0.10

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 10.8626 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

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

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.

LOQ Calculation Y-Intercept Fructose Glucose Sucrose Maltose Curve 1 -2704.0482 335.3401 -1445.6495 6033.9163 Curve 2 3961.8661 1081.0983 1427.5625 -963.7537 Curve 3 -10377.3340 1154.5853 -2282.1233 -1029.2857 Curve 4 -2006.7311 2964.0479 2474.5892 -1462.5821 Curve 5 -2115.3283 2626.2564 -1523.8140 5824.1486 SD = 5102.2309 1115.3073 2086.7037 3883.8138 Slope 628058.5827 591616.9570 686422.6367 594798.7464 575362.6555 565617.1304 624504.3684 702549.8057 785409.7730 607549.6597 707088.0158 601702.5799 635892.0146 545811.8940 595773.9872 677411.0859 651427.8534 535754.5915 687315.3920 475448.8178 Mean = 655230.1759 569270.0465 660220.8800 610382.2071 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) 200.0000 % LOQ 0.311 Mean LOQ 0.193 % 0.078 0.126 0.255

AOAC Standard Method Performance Requirements for Sugars in Animal Feeds Published by AOAC, March 12, 2018. Analytical Range (%): 0.1 - 5 <5-50 >50-100 Recovery %: 90-110 95-105 97-103 % RSDr ≤7 ≤5 ≤3

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 201743).

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

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

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

0.5% Standard Addition Results Original sample showed 0.058 % 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

Potato Powder Chromatogram

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)

Turkey, Equine and Calf Feed Chromatogram

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

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 0.008 102.724 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 0.137 145.907 0.149 0.104 0.103 0.158 0.214 0.046 0.146 31.453

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

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.