Validation of Loss on Drying Methods in Pet Foods Dr. Jane M. Caldwell 2018 AAFCO Annual Meeting Fort Lauderdale, FL July 31, 2018
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%)
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 930.15 LOD for Feeds 135 2 Convection Oven 33 8.03 a* 112 AOAC 935.29/945.15 LOD in Malt 104 3 Convection Oven 33 7.73 a 108 AOAC 950.46 LOD in Meat 125 4 Convection Oven 24 7.63 a 106 AOAC 2001.12 (I) Moisture in Feed, Grain & Forage (MEOH/Formamide) 50 n/a Titration (Karl Fischer) 24 7.18 ab 100 ASTM D6869-03 Moisture in Plastics 130 var Titration (Karl Fischer) 24 7.06 ab 98 AOAC 2001.12 (II) Moisture in Feed, Grain & Forage (boiling MEOH) 66 n/a Titration (Karl Fischer) 8 6.80 ab 95 AOAC 969.35/925.45A LOD in Sugars (Mod.) 70 4 Vacuum Oven 33 6.25 b 87 *Levels not connected by the same letter are significantly different (p > 0.05)
Moisture in Dry Pet Food Determined By Titration (Karl Fischer) and Loss-on-Drying Oven (LOD) Oven Methods (Study 2) Moisture Sample AOAC 2001.12 (I) ASTM D 6869-03 AOAC 950.45 B(b) AOAC 945.15 AOAC 969.35 N = 3 Karl Fischer (Feed) Karl Fischer (Plastic) Convection (125⁰C,4 h) Convection (105⁰C,3 h) Vacuum (70⁰C, 4 h) 1 6.57 a 6.79 a 6.46 a 7.03 5.55 2 6.97 a 7.31 a 7.14 a 7.60 5.79 3 7.50 a 7.93 7.69 a 8.41 7.18 a 4 7.63 a 8.19 a 7.70 a 8.99 7.67 a 5 6.17 a 5.78 6.07 a 6.23 a 4.49 6 6.03 a 5.31 5.70 6.03 a 3.79 7 7.80 6.86 7.53 7.46 5.80 8 7.20 a 6.31 7.30 a 7.06 a 5.50 9 4.66 a 3.93 4.28 a 4.09 2.58 10 9.47 a 9.16 a 10.00 9.97 8.45 11 7.20 a 6.73 a 7.16 a 7.07 a 5.61 12 7.33 a 7.23 7.34 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 2001.12 (I) mean.
Observations for LODs in Dry Pet Food KF (AOAC 2001.12) is LOD validation standard Convection 135⁰C too hot (118%) 125⁰C okay 104⁰C okay Vacuum: too low at 4 h
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 930.15 X 135 2 Convection Oven 8.72 a** 110 AOAC 930.15/925.10 Y 133 2 Convection Oven 8.42 ab 106 AOAC 930.15 Z 135 2 Convection Oven 8.18 ab 103 AOAC 991.02 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 935.29 X 104 3 Convection Oven 7.78 bcd 98 NFTA 2.2.2.5 Z 105 3 Convection Oven 7.76 c 98 AOAC 934.06/925.45 Y 70 Overnight Vacuum Oven 7.48 cd 94 ASTM D 6869-03 Z 130 n/a Titration (Karl Fischer) 7.24 de 91 AOAC 925.09 X 100 5 Vacuum Oven 6.75 ef 85 AOAC 969.35 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)
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 930.15 Z 135 2 Convection Oven 6.10 a** 111 AOAC 930.15 X 135 2 Convection Oven 5.98 ab 109 AOAC 930.15/925.10 Y 133 2 Convection Oven 5.82 abc 106 NFTA 2.2.2.5 Z 105 3 Convection Oven 5.74 bc 105 ASTM D 6869-03 Z 130 n/a Titration (Karl Fischer) 5.52 cd 101 AOAC 991.02 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 935.29 X 104 3 Convection Oven 5.17 de 94 AOAC 934.06/925.45 Y 70 Overnight Vacuum Oven 4.98 ef 91 AOAC 925.09 X 100 5 Vacuum Oven 4.70 fg 86 AOAC 969.35 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)
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 930.15 Z 135 2 Convection Oven 9.21 a** 126 AOAC 930.15 X 135 2 Convection Oven 9.12 ab 125 AOAC 930.15/925.10 Y 133 2 Convection Oven 8.90 ab 122 NFTA 2.2.2.5 Z 105 3 Convection Oven 8.52 bc 116 AOAC 935.29 X 104 3 Convection Oven 7.96 cd 109 ASTM D 6869-03 Z 130 n/a Titration (Karl Fischer) 7.63 d 104 AOAC 934.06/925.45 Y 70 Overnight Vacuum Oven 7.56 d 103 AOAC 991.02 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 925.09 X 100 5 Vacuum Oven 6.60 ef 90 AOAC 969.35 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)
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 930.15 Z 135 2 Convection Oven 8.45 a** 109 AOAC 930.15 X 135 2 Convection Oven 8.20 abc 106 NFTA 2.2.2.5 Z 105 3 Convection Oven 8.08 ac 105 AOAC 930.15/925.10 Y 133 2 Convection Oven 7.76 abcd 101 AOAC 991.02 Y n/a n/a Titration (Karl Fischer) 7.72 abcd 100 ASTM D 6869-03 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 935.29 X 104 3 Convection Oven 7.28 de 94 AOAC 934.06/925.45 Y 70 Overnight Vacuum Oven 6.99 def 91 AOAC 969.35 Z 70 4 Vacuum Oven 6.51 f 84 AOAC 925.09 X 100 5 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)
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 (103-110%) 104⁰C good (98%) Vacuum OVERNIGHT (94%) High Protein KFs all close (99-101%) 104⁰C good (94 & 105%) DID NOT RUN 125⁰C
Observations for LODs in Dry Pet Food; part 2 Potato 135⁰ too hot (122-126%) 104⁰C hot (109-116%) Vacuum ON (103%) It depends Economy 133⁰C great (101%) DID NOT RUN 125⁰C 104 & 105⁰C okay (94 & 105%) Validate with AOAC 2001.12
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 930.15 LOD for Feeds 135 2 Convection Oven 34 24.61 a* 120 AOAC 935.29 LOD in Malt 104 3 Convection Oven 34 24.08 a 118 ASTM D6869-03 Moisture in Plastics 130 var Titration (Karl Fischer) 34 21.12 b 103 AOAC 969.35/925.45A LOD in Sugars 70 4 Vacuum Oven 34 20.75 b 102 AOAC 991.02 Moisture in Semi- Moist Pet Treats n/a n/a Methanol Extraction Titration (Karl Fischer) 25 20.43 b 100 *Levels not connected by the same letter are significantly different (p > 0.05) Glycerin and other humectants in treats. AOAC 991.02 titrates for moisture in soft-moist and semi-moist pet foods containing 20-30 moisture and other volatile materials. Gold standard for this matrix. Samples range from 17 to 26% moisture.
Observations for LODs in Semi-Moist Pet Treats LOD in Sugars (102%): How sweet it is! Vacuum oven 70⁰C 4 hours
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 2.2.1.1/2.1.4 AOAC 991.02 LOD for Forages, Silage, Wet Feeds (Two-step method) Moisture in Semi-Moist Pet Treats (Two-step method) 55 (105) ON (3) Convection Ovens 20 76.64 a 100 55 (n/a) ON (n/a) Convection Oven Methanol Extraction Titration (Karl Fischer) 20 76.54 a 100 AOAC 935.29 LOD in Malt 104 3 Convection Oven 20 75.39 a 98 AOAC 950.46 B(b) LOD in Meat 125 4 Convection Oven 20 75.37 a 98 AOAC 930.15 LOD for Feeds 135 2 Convection Oven 20 75.35 a 98 AOAC 991.02 Moisture in Semi-Moist Pet Treats n/a n/a Methanol Extraction Titration (Karl Fischer) 20 71.76 b 94 *Levels not connected by the same letter are significantly different (p > 0.05) AOAC 991.02 titrates for moisture in soft-moist and semi-moist pet foods containing 20-30 moisture and other volatile materials.
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.
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 2001.12 (I) Moisture in Feed, Grain & Forage (MEOH/Formamide) 50 n/a Titration (Karl Fischer) 7 3.31 a* 100 ASTM D6869-03 Moisture in Plastics 130 var Titration (Karl Fischer) 7 2.86 a 86 AOAC 935.29 LOD in Malt 104 3 Convection Oven 7 2.55 a 77 AOAC 969.35/925.45A AOAC 969.35/925.45D** LOD in Sugars 70 4 Vacuum Oven 7 0.77 b 23 LOD in Sugars 60 18 Vacuum Oven 7 0.77 b 23 *Levels not connected by the same letter are significantly different (p > 0.05) **No digest quartz sand used in D method.
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 930.15 LOD for Feeds 135 2 Convection Oven 7 5.95 a* 138 AOAC 950.46 B(b) LOD in Meat 125 4 Convection Oven 7 5.17 a 120 AOAC 2001.12 (I) Moisture in Feed, Grain & Forage (MEOH/Formamide) 50 n/a Titration (Karl Fischer) 7 4.30 a 100 *Levels not connected by the same letter are significantly different (p > 0.05)
Observations for LODs in Freeze-Dried Pet Food Treats Chewy.com <5 to <8% moisture Pure Meat?
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 2001.12 (I) Moisture in Feed, Grain & Forage (MEOH/Formamide) 50 n/a Titration (Karl Fischer) 7 13.66 a* 100 AOAC 930.15 LOD for Feeds 135 2 Convection Oven 7 13.37 a 98 AOAC 950.46 B(b) LOD in Meat 125 4 Convection Oven 7 11.54 a 84 *Levels not connected by the same letter are significantly different (p > 0.05)
Discussion of Pet Food LOD Results Review data Commentary period for labs/stakeholders Suggestions for further studies Can we make some conclusions/give guidance?
Dr. Jane Caldwell Dr. 402-829-9858 Jane M. Caldwell 402-979-1411 jcaldwell@midwestlabs.com jcaldwell@midwestlabs.com
Karl Fischer (Moisture by Titration) GOLD STANDARD: Reaction of iodine with water. Used for TRUE MOISTURE & NIR calibration Not free of procedural issues.. AOAC 2001.12 (I & II) (2005) Moisture in Animal Feed, Grain and Forage (1-15%) AOAC 991.02 (1995) Moisture in Soft-Moist & Semi-Moist Pet Treats (20-30%) ASTM D6869-03 (2011) - Moisture in Plastics (0.005-100%) AOCS Ca 2e-84 (1997) Moisture in Fats & Oils
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 D6869-03
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
AOAC 991.02 Moisture in Soft-Moist & Semi-Moist Pet Treats (20-30%) MEOH extraction with titration Shake 15 min @ room temperature 10 g Results always low Limited by time
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
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