Method Validation of Protein Content in Aquatic Feeds การตรวจสอบความใช้ได้ของวิธีทดสอบปริมาณโปรตีนในอาหารสัตว์น้ำ Method Validation of Protein Content in Aquatic Feeds ลัดดาวัลย์ ครองพงษ์ จิรวิช จุลบุษปะ ธนิกานต์ จันทงาม ประวิลดา วิกรัยพัฒน์ และ ไพรัตน์ กอสุธารักษ์

Research and Aquatic Feed Quality Control Section Introduction Department of Fisheries Inland Fisheries Research and Development Bureau Inland Feed Research Institute (IFRI) สถาบันวิจัยอาหารสัตว์น้ำจืด Research and Aquatic Feed Quality Control Section Control the quality of aquatic feeds in Thailand based on the feed quality control Act 1982 (B.E. 2525) and additional 1999 (B.E. 2542) since 1992 (B.E.2535) สถาบันวิจัยอาหารสัตว์น้ำจืด รับผิดชอบกำกับ ดูแล ควบคุมคุณภาพอาหารสัตว์น้ำ ตามพระราชบัญญัติควบคุมคุณภาพอาหารสัตว์น้ำ พ.ศ.2525 และ 2542 ตั้งแต่ปี พ.ศ. 2535 เป็นต้นมา

Introduction At present, DOF control 8 aquatic feed :- Marine Fish Feed (Carnivorous) Catfish Feed Freshwater fish Feed (Herbivorous) Freshwater fish Feed (Carnivorous) ซึ่งอาหารสัตว์น้ำภายใต้การกำกับ ดูแล ของกรมประมง มีทั้งสิ้น 8 ชนิด ได้แก่... Freshwater prawn Feed Soft-shell turtle Feed Marine shrimp Feed Frog Feed

Distillation & Titration Introduction Protocol for Protein analysis Report Kjeldahl Method Distillation & Titration Testing Report Test no. XXX Date:00/00/00 Sample Protein (%) 20.56 30.89 45.36 Digestion Weighing ปริมาณโปรตีน เป็นหนึ่งในพารามิเตอร์ที่ใช้ควบคุมคุณภาพอาหารสัตว์น้ำ ซึ่งวิธีที่ใช้ในการตรวจวิเคราะห์ปริมาณโปรตีนในอาหารสัตว์ที่นิยมกันทั่วไปได้แก่ Kjeldahl method โดยมีขั้นตอนหลักอยู่ 3 ขั้นตอน ได้แก่ การย่อย การกลั่น และการไตเตรท % Protein = % Nitrogen x 6.25

Near-Infrared Reflectance Spectroscopic Method Introduction AOAC Official Method 968.06 Dumas Method AOAC Official Method 989.03 Near-Infrared Reflectance Spectroscopic Method “Analytical measurements should be made to satisfy an agreed requirement.” (i.e. to a defined objective) Good practice in analytical measurement Analytical measurements should be made using methods and equipment which have been tested to ensure they are fit for purpose.” AOAC Official Method 990.03 Combustion Method 5

Introduction AOAC Official Method 2001.11 Protein (Crude) in Animal Feed, Forage (Plant Tissue), Grain, and Oilseeds Block Digestion Method Using Copper Catalyst And Steam Distillation into Boric Acid ISO 5983-2 Animal feeding stuffs - Determination of nitrogen content and calculation of crude protein content - Block digestion/ steam distillation method ปริมาณโปรตีน เป็นหนึ่งในพารามิเตอร์ที่ใช้ควบคุมคุณภาพอาหารสัตว์น้ำ ซึ่งวิธีที่ใช้ในการตรวจวิเคราะห์ปริมาณโปรตีนในอาหารสัตว์ที่นิยมกันทั่วไปได้แก่ Kjeldahl method โดยมีขั้นตอนหลักอยู่ 3 ขั้นตอน ได้แก่ การย่อย การกลั่น และการไตเตรท

Introduction 0.3 g for all Protein, % Test portion, g 6 – 24 25 – 40 Recommended test portion weight by AOAC Protein, % Test portion, g 6 – 24 25 – 40 41 – 50 51 – 60 61 – 90 1.5 ± 0.1 1.0 ± 0.1 0.8 ± 0.1 0.7 ± 0.1 0.5 ± 0.01 >90 Weigh test portion equivalent to 50 mg N 0.3 g for all

Introduction What is expected result?

Introduction Why is method validation necessary? (a) Importance of Analytical Measurement Virtually every aspect of society is supported in some way by analytical measurement It is important to determine the correct result and be able to show that it is correct (b) The Professional Duty of the Analytical Chemist Method validation enables chemists to demonstrate that a method is ‘fit for purpose’ It must be sufficiently reliable that any decision based on it can be taken with confidence.

Objectives To investigate the validity of protein determination by using partial modified method To ensure that partial modified method are fit for protein analysis in aquatic feeds “Analytical measurements should be made to satisfy an agreed requirement.” (i.e. to a defined objective) Good practice in analytical measurement Analytical measurements should be made using methods and equipment which have been tested to ensure they are fit for purpose.” To perform that protein measurement by IFRI lab consistent with those measure elsewhere (Accredited under International Standard ISO/IEC 17025)

1 2 3 4 Methodology Protocol & equipment check Determination for protein content in Reference material 2 Measurement protein content in aquatic feeds 3 4 Participate in Proficiency Testing Program

Materials and Methods (NH4)2SO4 Accuracy of protocol and equipment performance check Nitrogen loss % Recovery Theoretical yield, % N of (NH4)2SO4 = 21.2 Acceptable criteria ≥ 99%

Materials and Methods Accuracy of protocol and equipment performance check Digestion efficiency Acetanilide (C8H9NO) % Recovery Acceptable criteria ≥ 98 % Theoretical yield, % N of C8H9NO = 10.36

Materials and Methods Accuracy of protocol and equipment performance check Distillation and titration efficiency (NH4)2SO4 % Recovery Acceptable criteria ≥ 99.5 %

Acceptable Criteria1 (%) Results Accuracy of protocol and equipment performance check Results for recovery of nitrogen express accuracy of protocol and equipment performance Performance Checks Rep. Recovery (%) Acceptable Criteria1 (%) Nitrogen loss Digestion efficiency Distillation and titration efficiency 14 18 6 100.37 99.04 100.46 ≥ 99 ≥ 98 ≥ 99.5 Note 1AOAC, 2005

Materials and Methods Validity of Test Method for Protein in Reference Material (RM) Accuracy & Precision of Protein content in RM Testing for protein content 10 replications x Means Standard deviations, sd Evaluate Bias by t-test for accuracy Compare RSDlab vs. RSDHorwitz for precision Repeat analysis by same analyst at different time 2.56 x 6.25 = 16.0

Results Validity of Test Method for Protein in Reference Material (RM) Accuracy & Precision of Protein content in RM Accuracy and precision of protein content in reference material (RM) from different time of analysis by one analyst Statistic parameters Testing time 1 (n = 14) 2 (n = 16) 3 (n = 14) Mean sd texp *tcrit %RSDLab %RSDHorwitz HORRAT 16.02 0.27 0.03 2.160 1.68 1.74 0.96 16.35 0.26 0.66 2.131 1.59 1.73 0.92 16.30 0.18 0.56 1.10 0.63 Note *The critical values of t for a two-tailed test (p = 0.05)

Homogeneity check by F-test Testing for Protein by 2 Analysts Materials and Methods Validity of Test Method for Protein in Reference Material (RM) Precision of Protein content in Aquatic Feed Duplication analysis Homogeneity check by F-test Grind & Sieve Commercial crumble shrimp feed Grind & sieve through 1.0 mm then kept in zip bag Sampling sample for protein content Duplication analyzed in each sample Variance comparison of 2 data set for homogeneity by F-test Testing for protein content in shrimp feed by 2 analyst compare mean by t-test Testing for Protein by 2 Analysts Compare means of 2 Analysts by t-test

Results Validity of Test Method for Protein in Reference Material (RM) Precision of Protein content in Aquatic Feed Results of protein analysis in shrimp feed by 2 analysts Protein content (g/100 g) Analyst I Analyst II n Mean sd Variance 18 42.75 0.13 0.0169 17 42.86 0.19 0.0361 F 0.0169/0.0361 = 0.468 t-test for means compare 1.88 Note F-Critical for a one-tailed test, 17 and 16 df is equal to 2.70 at 95% confidential level t-Critical for a two-tailed test, 33 df is equal to 2.035 at 95% confidential level

Materials and Methods Precision of protein analysis in aquatic feeds Intermediate Precision of protein content in shrimp feed Analyzed shrimp feed in duplicate for 20 day Precision of protein content in aquatic feeds at different protein levels Range from 15 – 60 % Protein

Results Precision of protein analysis in aquatic feeds Intermediate Precision of protein content in shrimp feed Analyzed shrimp feed in duplicate for > 20 day Sample No. of Testing Protein (g/100g) RSDlab RSDHorwitz HORRAT Ῡ1 Ῡ2 Mean 1 2 19 28 42.37 37.88 42.42 37.89 42.40 1.23 0.76 1.50 1.53 0.82 0.50

Sample of aquatic feeds with different protein levels (%) Results Precision of protein analysis in aquatic feeds Precision of protein content in aquatic feeds at different protein levels Relative standard deviation (RSD) values and predicted Horwitz RSD values of individual protein content in aquatic feeds with different protein content Sample of aquatic feeds with different protein levels (%) 15.5 25 30 40 50 57 n Mean sd RSD Predicted Horwitz RSD 10 15.49 0.107 0.69 1.75 24.90 0.189 0.76 1.63 30.97 0.207 0.67 1.57 39.56 0.174 0.44 1.52 50.91 0.392 0.58 1.46 57.16 0.389 0.68 1.44 HORRAT 0.39 0.47 0.43 0.29 0.40

Materials and Methods Control the quality of test report z-score Participated in the Proficiency testing scheme Performed protein analysis of PT program by FAPAS (Food Analysis Performance Assessment Scheme, England) Joined PT program provided by DSS (Department of Science Service, Thailand) Evaluated the level of satisfactory by z-scores z-score Acceptable criteria | z | < 2 2 < | z | <3 | z | > 3 satisfactory questionable unsatisfactory

Results Control the quality of test report Participated in the Proficiency testing scheme Year Providers Sample Assigned value (g/100g) Lab result Z-score 2007-2008 2008 2009 FAPAS DSS Poultry Feed Pig Feed Fishmeal 15.100 ± 0.401 19.354 ± 0.281 62.365 ± 0.635 15.02 19.64 62.44 -0.30 0.73 0.12

Conclusion IFRI modified method by applying test sample weight of 0.3 g for protein content determination in reference material is accurate and precise IFRI modified method is fit for analyzing protein content in aquatic feeds Testing results of protein content by IFRI lab are consistent with those analyzed elsewhere and meet the general requirements of International Standard ISO/IEC 17025

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