1 The world leader in serving science

Kassandra I. Oates, Lillian Chen, Brian De Borba, and Jeffrey S. Rohrer 245th ACS National Meeting & Exposition April 7, 2013

OT70620_E 04/13S

Determination of Anions in Dried Distillers Grains with Solubles

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Overview

• Introduction to Dried Distillers Grains with Solubles (DDGS) • Nutrient Composition, Dietary Formulation, Processing

• Background on Target Anions

• Method 1: A rapid (<10 min) method for the determination of chloride, sulfate, and phosphate in DDGS

• Method 1 Results

• Method 2: A reliable method for the determination of inositol phosphates in DDGS using IC and postcolumn derivatization

• Method 2 Results

• Conclusion

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Introduction

Distillers Grains • Are a coproduct of fuel ethanol

production. • Have been used as an alternative

feed in ruminant diets since the early 1900s.

• Are an excellent source of: • Energy • Fat • Protein • Essential Minerals

• Provide three times the amount of nutrients available from grain alone.

Dooley, F.J.; Cox, M.; Cox, L. Distillers Grain Handbook: A Guide for Indiana Producers to Using DDGS for Animal Feed [Online]; Dept. of Agric. Econ., Purdue Univ., West Lafayette, IN, 2008; http://incorn.org/index.php?option=com_content&task=view&id=43 (accessed Mar. 27, 2013).

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DDGS Analysis

• For Trade Purposes • Moisture • Crude Protein • Crude Fat • Crude Fiber

• Residual Components • Mycotoxins • Antibiotics

• Nutrient Composition • Moisture, Crude Protein, Crude Fat,

Crude Fiber • Detergent Fiber • Ash (inorganic/total mineral content) • Trace Elements

• Essential for optimum animal heath (Mineral Tolerances of Animals, 2005)

• Amino Acids • Starch

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Nutrient Composition of DDGS

• 30% Protein • 10% Oil • 5% Ash • 55% Fiber • Amino Acids • Vitamins • Nonfermented sugars

(e.g., xylose, arabinose) • Low molecular weight

organic acids (e.g., lactic acid, acetic acid)

• Minerals • Major (0.05–1.15%): Ca, P, K,

Mg, S, Na, Cl • Minor (6–150 ppm): Zn, Mn, Cu,

Fe, Al, Se

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Dietary Formulation

• On a dry matter (DM) ration basis: • Beef Cattle: 10–40% • Dairy Cattle: 20–25% • Sheep: 10% • Poultry: 10–20% • Swine: ≥ 20%

• To prevent underfeeding, producers formulate diets under the assumption that mineral concentrations are low. • Supplements such as dicalcium

phosphate and calcium sulfate are added to the feed.

• This leads to nutritional disorders and excess waste.

DDGS replace corn grain or soybean meal and result in equal or better performance.

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DDGS Process Schematic from Whole Corn Kernels

DDGS WDGS

CDS

WDG

Parkin, G.; Weyer P.; Just C.L. Riding the Bioeconomy Wave: Smooth Sailing or Rough Water for the Environment and Public Health? Proceedings of the 2007 lowa Water Conference—Water and Bioenergy, March 6, 2007, Iowa State Center, Ames, IA.

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Sulfate

• Naturally occurring sulfur is present at 0.1% in the corn kernel and 0.2% in DDGS. • Organic sulfur (cystine, methionine)

• Reported sulfur up to1.0% in DDGS • Inorganic sulfur up to 0.8%

• Sulfuric acid • Water

Why does sulfur need to be regulated in DDGS? • Total sulfur per requirements of the National Research Council

for beef and cattle is 0.4% of the dry matter in rations. • Excess sulfur can lead to Polioencephalomalacia (PEM) in

ruminants, reduces the availability and absorption of other minerals, and can cause blindness, seizures, comas, and death.

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Phosphate and Inositol Phosphates

• Total phosphorus concentrations range from 0.5–1.0%. • Inorganic (phosphate) • Organic

• Phytate • Other (DNA, RNA, proteins, lipids, starch)

• Not all forms are digestible • Only ruminants have the enzymes needed to digest phytate

• Digestible phosphorus • Corn: ~21% • DDGS: ~59%

• Phosphate level is indicative of the extent of the hydrolysis of phytates and can be used to determine phosphorus bioavailability to nonruminants.

InsP0-5 + PO4

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Method 1

Equipment • Thermo Scientific™ Dionex™ ICS-2100 Integrated Reagent-Free™

IC (RFIC™) system including: • Pump • Degasser • Eluent Generator • Column Heater • Autosampler

Data Analysis • Thermo Scientific™ Dionex™ Chromeleon™ Chromatography Data

System software 7.1

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Method 1 Conditions

Column: Thermo Scientific™ Dionex™ IonPac™ AG11/AS11, 2 mm Eluent: 3–15 mM KOH 0–5 min, step at 5 min to 25 mM Eluent Source: Dionex ICS-2100, Thermo Scientific™ Dionex™ EGC III KOH with Thermo Scientific™ Dionex™ CR-ATC II Flow Rate: 0.5 mL/min Inj. Volume: 5 µL Temp: 30° C Detection: Thermo Scientific™ Dionex™ ASRS™ 300 Anion

Self-Regenerating Suppressor (2 mm), recycle mode

Sample Preparation Anions were extracted from a 1 g sample of DDGS by ultrasonic-assisted extraction in 100 mL of DI water, followed by centrifugation and dilution.

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Column

• The Dionex IonPac AS11 column is designed to separate a wide range of small organic acids from common anions which are often present in these types of samples.

• It has the ability to separate strongly retained polyvalent anions, such as phosphate.

• It is stable between pH 0–14.

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Anions in DDGS

1

2

3

0 1.5 3.0 4.5 6.0 7.5 -5

24.2

min

µS µS

-5.1

21.4

A

B

Column: Dionex IonPac AG11, AS11, 2 mm Eluent: 3–15 mM KOH 0–5 min, step at 5 min to 25 mM Eluent Source: Dionex EGC III KOH with Dionex CR-ATC Flow Rate: 0.5 mL/min Inj. Volume: 5 µL Temp: 30 ºC Detection: Dionex ASRS 300 (2 mm), recycle mode

Samples: A. Prepared DDGS sample B. Calibration standard Peaks: A B c 1. Chloride 1.3 mg/L 0.13 %* 5 mg/L 2. Sulfate 15.4 1.54 40 3. Phosphate 11.2 1.12 40 * calculated amount

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Linearity, Limits of Detection (LODs) and Limits of Quantification (LOQs)

1 Seven calibration levels, each injected in triplicate 2 LOD calculated as 3 × S/N 3 LOQ calculated as 10 × S/N

Analyte Calibration

Range (mg/L)

Linearity1 (r2)

LOD2 (µg/L)

LOQ3 (µg/L)

Chloride 0.15–5 0.9999 1.7 5.7 Sulfate 1.25–40 0.9999 3.3 10.9 Phosphate 1.25–40 0.9999 5.7 18.8

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Precision

Analytes Measured in Different Starting Grain Weights

(n = 7) Chloride Sulfate Phosphate

Amount Sampled

(g)

Average Amount Found

(%)

Peak Area RSD

Average Amount Found

(%)

Peak Area RSD

Average Amount Found

(%)

Peak Area RSD

0.75 0.13 0.41 1.52 0.44 1.11 0.43

1.00 0.13 0.40 1.49 0.50 1.08 0.48

1.25 0.13 0.59 1.49 0.63 1.09 0.69

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Accuracy

Chloride, Sulfate, and Phosphate Recovery in Spiked DDGS Samples

Chloride Sulfate Phosphate

Final Spiked Concn (mg/L)

Amount Recovered

(%)

Final Spiked Concn (mg/L)

Amount Recovered

(%)

Final Spiked Concn (mg/L)

Amount Recovered

(%)

0.27 107 3.13 102 2.30 104

0.55 100 5.87 99.2 4.59 97.5

1.16 100 12.4 101 9.15 99.0

1.49 97.5 15.5 98.4 11.5 96.2

1.71 97.9 18.5 98.8 13.7 96.5

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Method 2

Equipment • Dionex ICS-5000 RFIC system including:

• SP Single Pump or DP Dual Pump • DC Detector/Chromatography Compartment • AS-AP Autosampler

• Thermo Scientific Dionex ICS-5000+ Series VWD Variable Wavelength Detector with Analytical Flow Cell

• Thermo Scientific Dionex AXP Auxiliary Pump • Thermo Scientific Dionex EGC Carbonate Mixer Kit • Knitted Reaction Coil, 750 µL • Manifold, 3-way mixing tee

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Method 2: Conditions

Columns: Dionex CarboPac PA100 Analytical and Guard, 4 mm Eluent: A. DI water B. 0.5 M HCl Program: -15–0 min 5% B, 0–8 min 5–10% B, 8–25 min 10–35% B, 25–35 min 35–100% B, 35–42 min 100% B, 42–42.1 min 100–5% B Eluent Flow Rate: 1.0 mL/min Inj. Volume: 100 µL (full) Temperature: 30 ˚C Postcolumn Conditions PCR: 1% Fe(NO3)3 ∙ 9 H2O in 0.33 M HClO4 PCR Flow Rate: 0.4 mL/min Detection: UV absorbance, 290 nm Noise: 0.9 mAU

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The Dionex CarboPac PA100 Column

• Specifications • 8.5 µm EVB/DVB substrate agglomerated with 275 nm MicroBead

quaternary ammonium functionalized latex

• Able to separate InsP2-6 isomers because of its high ion-exchange capacity

• Isomers elute in order of increasing number of phosphate groups.

• Stable between pH 0–14

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Sample Preparation

• Inositol phosphates were extracted from DDGS by ultrasonic-assisted extraction in 0.5 M HCl, followed by centrifugation, and filtration.

• The sample was then treated with a Thermo Scientific™ Dionex™ OnGuard™ II RP and AG/H cartridges in series. • OnGuard treatment of the sample removes hydrophobic compounds and

chloride, which is necessary to reduce column overloading from the chloride in the sample.

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DDGS Sample vs In-House Reference Standard

Samples: a) DDGS sample b) In-house reference standard Peaks: 1–4: InsP2 5–13: InsP3 14–20: InsP4 21–24: InsP5 25: InsP6

0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 -65

0

600

min

mAU

12 13

14

15

16 17

18

19 20

21

22 23

24

25

1

2

3

4 6 8

9

11

PO4 Ox

SO4

a

b

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Linearity, LODs and LOQs

Analyte Calibration

Range (mg/L)

Linearity1

(r2) LOD2

(mg/L) LOQ3

(mg/L)

DL-Ins(1,2,5,6)-P4 3.1-100 >0.999 1.0 3.2

DL-Ins(1,2,4,5,6)-P5 3.1-100 >0.999 1.0 3.2

Ins(1,2,3,4,5,6)-P6 10-300 >0.999 1.0 3.2 1 Six calibration levels, each level injected in duplicate 2 LOD calculated as 3 × S/N 3 LOQ calculated as 10 × S/N

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To compare the amounts to the literature values, InsP amounts were determined on a dry matter (DM) basis.

Quantitation of Inositol Phosphate Isomers

InsP Analogue Prep # Peak Area

(mAU*min)

Peak Area RSD

Amount Found (mg/L)

Calculated Amount

(mg/g, DM)1

DM1 %

Average DM1 %

Ins(1,2,3,4,5,6)-P6 1 62.2 0.1 97.6 2.1 0.21

0.205 2 59.5 4.1 93.7 2.0 0.20

Ins(1,2,4,5,6)-P5 1 28.9 0.1 67.7 1.5 0.15

0.14 2 25.9 0.4 60.7 1.3 0.13

(1,2,5,6)-P4 1 18.9 1.3 33.0 0.68 0.07

0.07 2 17.4 0.3 30.4 0.66 0.07

1 Dry matter content was determined by drying 2 g of DDGS in an oven at 135 ˚C for 2 h. The sample contained 10.5% moisture (a dry matter value of 89.5%).

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Estimation of Total InsP2-6

Total InsP2-6 amounts were estimated using relative response factors.

Analogue Calculated

Amount (mg/g, DM)

% DM

InsP6 2.0 0.20

InsP5 2.3 0.23

InsP4 1.3 0.13

InsP3 1.3 0.13

InsP2 0.4 0.04

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Conclusion

• Method 1 provides a simple and accurate approach for determining water-soluble anions present in DDGS in less than 10 min on a Dionex IonPac AS11 column.

• Method 2 provides a rapid and reliable sample preparation procedure and chromatographic method to determine the phytate hydrolysis products in DDGS.

• For the determination of inositol phosphates, the Dionex OnGuard II RP and Ag/H cartridges simplified the sample preparation procedure when compared to the most commonly used capture and release methods.

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Thank you for your attention!