1. Validation of Loss on Drying Methods in Pet Foods
Dr. Jane M. Caldwell
37th Annual Midwest Section AOAC Meeting
Lincoln, Nebraska
June 6, 2018
Seemingly simple method
Taken for granted
Important because it affects every other downstream analyte measured

2. AAFCO Guidelines on Moisture in Animal Feeds
Data lacking in pet foods: No official LOD methods
Moisture Best Practices WG requested collaborative data
Midwest Labs responded

3. Pet Food Categories Dry pet food (<15% moisture)
Semi-moist pet treats (>15% moisture)
Wet pet food (>30% moisture)
Freeze-dried pet treats (<8% moisture)
Liquid ingredients
Chew bones

4. Review of Methods Titration – Karl Fischer (“True” Moisture)
Convection Oven (LOD)
Forced air ( ⁰C)
Two step drying – partial & lab drying (55⁰C ON; 105⁰C)
Vacuum Oven (LOD)
< 2” Hg (60-70⁰C)
Sweet liquids – quartz sand or no sand
Excessive heat can release volatiles such as organic acids, vitamins, terpenes, alcohols, esters, ketones and aldehydes. What we might call aromas or fragrances.
Maillard rxn begins at 140 C: Combination of sugar carbonyl group with protein amino group releases at least three molecules of water. Freeing covalently bound water.

5. Karl Fischer (Moisture by Titration)
GOLD STANDARD: Reaction of iodine with water.
Used for TRUE MOISTURE & NIR calibration
Not free of procedural issues……..
AOAC (I & II) (2005) – Moisture in Animal Feed, Grain and Forage (1-15%)
AOAC (1995) – Moisture in Soft-Moist & Semi-Moist Pet Treats (20-30%)
ASTM D (2011) - Moisture in Plastics ( %)

6. AOAC 2001.12 (I) Moisture in Animal Feed, Grain and Forage (1-15%)
High-speed homogenization in vessel?
Pre-homogenization outside vessel
MEOH/Formamide extraction (50⁰C)
Time in titration vessel not a factor
Run in triplicate
Comparable to ASTM D

7. AOAC 2001.12 (II) Moisture in Animal Feed, Grain and Forage (1-15%)
Boiling MEOH (66⁰C)
No obvious advantages over (I).
Small volume of water easy to release?
Vapor pressure issues
Never used

8. AOAC 991.02 – Moisture in Soft-Moist & Semi-Moist Pet Treats (20-30%)
MEOH extraction with titration
Shake 15 room temperature
10 g
Results always low
Limited by time

9. ASTM D6869-03 - Moisture in Plastics (0.005-100%)
Used by global pet food giants
0.10 to 1.0 g (<0.25 g)
Heated to 130 to 140⁰C
Rapid: < 15 min
Small sample size
Maillard reaction begins at 140 C
Rapid and automatic
Stromboli!

10. Experimental Design Design 1: Compare methods by product category
Design 2: Look at reps of individual samples within categories/methods

12. Why is 104 higher than 125? High thru-put lab environments.
400+ proximate samples per day.
Different labs with different balances, analysts and equipment.

14. Observations for LODs in Dry Pet Food
KF (AOAC ) is LOD validation standard
Convection
135⁰C too hot (118%)
125⁰C okay
104⁰C okay
Vacuum: too low at 4 h

15. AOCS Ca 2e-84 is a methanol extraction (RT) approved for oils and fats.

19. Observations for LODs in Dry Pet Food; part 2
Did NOT use KF standard for animal feed (1-15%)
It depends…LAB and INGREDIENT variation
Grain-Free
135⁰C might work ( %)
104⁰C good (98%)
Vacuum OVERNIGHT (94%)
High Protein
KFs all close (99-101%)
104⁰C good (94 & 105%)
DID NOT RUN 125⁰C

20. Observations for LODs in Dry Pet Food; part 2
It depends…
Potato
135⁰ too hot ( %)
104⁰C hot ( %)
Vacuum ON (103%)
Economy
133⁰C great (101%)
DID NOT RUN 125⁰C
104 & 105⁰C okay (94 & 105%)
Validate with AOAC