Validation of Loss on Drying Methods in Pet Foods. Dr. Jane M. Caldwell 2018 AAFCO Annual Meeting Fort Lauderdale, FL July 31, 2018

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1 Validation of Loss on Drying Methods in Pet Foods Dr. Jane M. Caldwell 2018 AAFCO Annual Meeting Fort Lauderdale, FL July 31, 2018

2 Pet Food Categories Dry pet food (<15%) Semi-moist pet treats (>15%) Wet pet food (>30%) Freeze-dried pet treats (<8%) Dried Beef Sticks (~13%)

3 Moisture in Dry Pet Food Determined by Titration (Karl Fischer) and Loss-on-Drying (LOD) Oven Methods (Study 1) Method Number Official Name Temp (⁰C) Time (hr) Equipment/Procedure N Mean Moisture Relative Recovery AOAC LOD for Feeds Convection Oven a* 112 AOAC / LOD in Malt Convection Oven a 108 AOAC LOD in Meat Convection Oven a 106 AOAC (I) Moisture in Feed, Grain & Forage (MEOH/Formamide) 50 n/a Titration (Karl Fischer) ab 100 ASTM D Moisture in Plastics 130 var Titration (Karl Fischer) ab 98 AOAC (II) Moisture in Feed, Grain & Forage (boiling MEOH) 66 n/a Titration (Karl Fischer) ab 95 AOAC /925.45A LOD in Sugars (Mod.) 70 4 Vacuum Oven b 87 *Levels not connected by the same letter are significantly different (p > 0.05)

4 Moisture in Dry Pet Food Determined By Titration (Karl Fischer) and Loss-on-Drying Oven (LOD) Oven Methods (Study 2) Moisture Sample AOAC (I) ASTM D AOAC B(b) AOAC AOAC N = 3 Karl Fischer (Feed) Karl Fischer (Plastic) Convection (125⁰C,4 h) Convection (105⁰C,3 h) Vacuum (70⁰C, 4 h) a 6.79 a 6.46 a a 7.31 a 7.14 a a a a a 8.19 a 7.70 a a a a 6.23 a a a a a 7.06 a a a a 9.16 a a 6.73 a 7.16 a 7.07 a a a 7.48 a 6.19 a Means within a row with the same letter are not statistically different (p > 0.05) from Karl Fischer AOAC (I) mean.

5 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

6 Moisture in Dry Pet Food (Grain-free) Determined by Titration (Karl Fischer) and Loss-on-Drying (LOD) Oven Methods* Method Lab Temp (⁰C) Time (hr) Equipment/Procedure Mean Moisture Relative Recovery AOAC X Convection Oven 8.72 a** 110 AOAC / Y Convection Oven 8.42 ab 106 AOAC Z Convection Oven 8.18 ab 103 AOAC Y n/a n/a Titration/Methanol extraction (Karl Fischer) 7.93 bc 100 AOCS Ca 2e-84 X n/a n/a Titration (Karl Fischer) 7.79 bcd 98 AOAC X Convection Oven 7.78 bcd 98 NFTA Z Convection Oven 7.76 c 98 AOAC / Y 70 Overnight Vacuum Oven 7.48 cd 94 ASTM D Z 130 n/a Titration (Karl Fischer) 7.24 de 91 AOAC X Vacuum Oven 6.75 ef 85 AOAC Z 70 4 Vacuum Oven 6.24 f 79 *Total N = 34; Lab X & Y had 2 N/method; Lab Z had 5N/method **Levels not connected by the same letter are significantly different (p > 0.05)

7 Moisture in Dry Pet Food (High Protein/Meat) Determined by Titration (Karl Fischer) and Loss-on-Drying (LOD) Oven Methods* Method Lab Temp (⁰C) Time (hr) Equipment/Procedure Mean Moisture Relative Recovery AOAC Z Convection Oven 6.10 a** 111 AOAC X Convection Oven 5.98 ab 109 AOAC / Y Convection Oven 5.82 abc 106 NFTA Z Convection Oven 5.74 bc 105 ASTM D Z 130 n/a Titration (Karl Fischer) 5.52 cd 101 AOAC Y n/a n/a Titration (Karl Fischer) 5.48 cd 100 AOCS Ca 2e-84 X n/a n/a Titration (Karl Fischer) 5.44 cd 99 AOAC X Convection Oven 5.17 de 94 AOAC / Y 70 Overnight Vacuum Oven 4.98 ef 91 AOAC X Vacuum Oven 4.70 fg 86 AOAC Z 70 4 Vacuum Oven 4.40 g 80 *Total N = 34; Lab X & Y had 2 N/method; Lab Z had 5N/method **Levels not connected by the same letter are significantly different (p > 0.05)

8 Moisture in Dry Pet Food (Potato) Determined by Titration (Karl Fischer) and Loss-on-Drying (LOD) Oven Methods* Method Lab Temp (⁰C) Time (hr) Equipment/Procedure Mean Moisture Relative Recovery AOAC Z Convection Oven 9.21 a** 126 AOAC X Convection Oven 9.12 ab 125 AOAC / Y Convection Oven 8.90 ab 122 NFTA Z Convection Oven 8.52 bc 116 AOAC X Convection Oven 7.96 cd 109 ASTM D Z 130 n/a Titration (Karl Fischer) 7.63 d 104 AOAC / Y 70 Overnight Vacuum Oven 7.56 d 103 AOAC Y n/a n/a Titration (Karl Fischer) 7.32 de 100 AOCS Ca 2e-84 X n/a n/a Titration (Karl Fischer) 7.24 de 99 AOAC X Vacuum Oven 6.60 ef 90 AOAC Z 70 4 Vacuum Oven 6.44 f 88 *Total N = 34; Lab X & Y had 2 N/method; Lab Z had 5N/method **Levels not connected by the same letter are significantly different (p > 0.05)

9 Moisture in Dry Pet Food (Economy) Determined by Titration (Karl Fischer) and Loss-on-Drying (LOD) Oven Methods* Method Lab Temp (⁰C) Time (hr) Equipment/Procedure Mean Moisture Relative Recovery AOAC Z Convection Oven 8.45 a** 109 AOAC X Convection Oven 8.20 abc 106 NFTA Z Convection Oven 8.08 ac 105 AOAC / Y Convection Oven 7.76 abcd 101 AOAC Y n/a n/a Titration (Karl Fischer) 7.72 abcd 100 ASTM D Z 130 n/a Titration (Karl Fischer) 7.50 bd 97 AOCS Ca 2e-84 X n/a n/a Titration (Karl Fischer) 7.37 bcd 95 AOAC X Convection Oven 7.28 de 94 AOAC / Y 70 Overnight Vacuum Oven 6.99 def 91 AOAC Z 70 4 Vacuum Oven 6.51 f 84 AOAC X Vacuum Oven 6.45 ef 84 *Total N = 34; Lab X & Y run duplicate reps/method; Lab Z ran quintuplicate reps/method **Levels not connected by the same letter are significantly different (p > 0.05)

10 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

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

12 Moisture in Semi-Moist Pet Treats Determined by Titration (Karl Fischer) and Loss-on-Drying (LOD) Oven Methods Method Number Official Name Temp (⁰C) Time (hr) Equipment/Procedure N Mean Moisture Relative Recovery AOAC LOD for Feeds Convection Oven a* 120 AOAC LOD in Malt Convection Oven a 118 ASTM D Moisture in Plastics 130 var Titration (Karl Fischer) b 103 AOAC /925.45A LOD in Sugars 70 4 Vacuum Oven b 102 AOAC Moisture in Semi- Moist Pet Treats n/a n/a Methanol Extraction Titration (Karl Fischer) b 100 *Levels not connected by the same letter are significantly different (p > 0.05) Glycerin and other humectants in treats. AOAC titrates for moisture in soft-moist and semi-moist pet foods containing moisture and other volatile materials. Gold standard for this matrix. Samples range from 17 to 26% moisture.

13 Observations for LODs in Semi-Moist Pet Treats LOD in Sugars (102%): How sweet it is! Vacuum oven 70⁰C 4 hours

14 Moisture in Wet Pet Food Determined by Titration (Karl Fischer) and Loss-on-Drying (LOD) Oven Methods Method Number Official Name Temp (⁰C) Time (hr) Equipment/Procedure N Mean Moisture Relative Recovery NFTA /2.1.4 AOAC LOD for Forages, Silage, Wet Feeds (Two-step method) Moisture in Semi-Moist Pet Treats (Two-step method) 55 (105) ON (3) Convection Ovens a (n/a) ON (n/a) Convection Oven Methanol Extraction Titration (Karl Fischer) a 100 AOAC LOD in Malt Convection Oven a 98 AOAC B(b) LOD in Meat Convection Oven a 98 AOAC LOD for Feeds Convection Oven a 98 AOAC Moisture in Semi-Moist Pet Treats n/a n/a Methanol Extraction Titration (Karl Fischer) b 94 *Levels not connected by the same letter are significantly different (p > 0.05) AOAC titrates for moisture in soft-moist and semi-moist pet foods containing moisture and other volatile materials.

15 Observations for LODs in Wet Pet Food Two-step methods for KF and LOD 104, 125 & 135⁰C also 98% KF for semi-moist low (94%) More data needed.

16 Moisture in Freeze-Dried Pet Food Treats Determined by Titration (Karl Fischer) and Loss-on-Drying (LOD) Oven Methods Method Number Official Name Temp (⁰C) Time (hr) Equipment/Procedure N Mean Moisture Relative Recovery AOAC (I) Moisture in Feed, Grain & Forage (MEOH/Formamide) 50 n/a Titration (Karl Fischer) a* 100 ASTM D Moisture in Plastics 130 var Titration (Karl Fischer) a 86 AOAC LOD in Malt Convection Oven a 77 AOAC /925.45A AOAC /925.45D** LOD in Sugars 70 4 Vacuum Oven b 23 LOD in Sugars Vacuum Oven b 23 *Levels not connected by the same letter are significantly different (p > 0.05) **No digest quartz sand used in D method.

17 Moisture in Freeze-Dried Pet Food Treats Determined by Titration (Karl Fischer) and Loss-on-Drying (LOD) Oven Methods Study 2 Method Number Official Name Temp (⁰C) Time (hr) Equipment/Procedure N Mean Moisture Relative Recovery AOAC LOD for Feeds Convection Oven a* 138 AOAC B(b) LOD in Meat Convection Oven a 120 AOAC (I) Moisture in Feed, Grain & Forage (MEOH/Formamide) 50 n/a Titration (Karl Fischer) a 100 *Levels not connected by the same letter are significantly different (p > 0.05)

18 Observations for LODs in Freeze-Dried Pet Food Treats Chewy.com <5 to <8% moisture Pure Meat?

19 Moisture in Dried Beef Sticks Determined by Titration (Karl Fischer) and Loss-on-Drying (LOD) Oven Methods Method Number Official Name Temp (⁰C) Time (hr) Equipment/Procedure N Mean Moisture Relative Recovery AOAC (I) Moisture in Feed, Grain & Forage (MEOH/Formamide) 50 n/a Titration (Karl Fischer) a* 100 AOAC LOD for Feeds Convection Oven a 98 AOAC B(b) LOD in Meat Convection Oven a 84 *Levels not connected by the same letter are significantly different (p > 0.05)

20 Discussion of Pet Food LOD Results Review data Commentary period for labs/stakeholders Suggestions for further studies Can we make some conclusions/give guidance?

21 Dr. Jane Caldwell Dr Jane M. Caldwell

22 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 ( %) AOCS Ca 2e-84 (1997) Moisture in Fats & Oils

23 AOAC (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

24 AOAC (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

25 AOAC 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

26 ASTM D Moisture in Plastics ( %) Used by global pet food giants 0.10 to 1.0 g (<0.25 g) Heated to 130 to 140⁰C Rapid: < 15 min

27 AOCS Ca 2e-84 (1997) Moisture in Fats & Oils 5-25 g sample of fat or oil Chloroform/methanol extraction Electrometric end point No time restrictions

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