Dedicated Analytical Solutions Michelle Walter FNA Technical Manager

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Dedicated Analytical Solutions - Michelle Walter: FNA Technical Manager 1 DUMAS OR KJELDAHL FOR

Dedicated Analytical Solutions - Michelle Walter: FNA Technical Manager 1 DUMAS OR KJELDAHL FOR REFERENCE ANALYSIS ? Comparison and considerations for Nitrogen/Protein analysis of food and feed

Dedicated Analytical Solutions 2 DIFFERENT INDUSTRIES USING NITROGEN/PROTEIN ANALYSIS RAW MILK TESTING DAIRY GRAIN,

Dedicated Analytical Solutions 2 DIFFERENT INDUSTRIES USING NITROGEN/PROTEIN ANALYSIS RAW MILK TESTING DAIRY GRAIN, MILLING & OILS WINE, SPIRITS & BREWING MEAT FEED & FORAGE ENVIRONMENTAL & OTHER INDUSTRIES LABORATORIES

Dedicated Analytical Solutions WHY REFERENCE ANALYSIS ? The answer is related to the use

Dedicated Analytical Solutions WHY REFERENCE ANALYSIS ? The answer is related to the use of lab results: Trading and import/export of raw materials and finished goods in Food and Feed Quality control in Food/Feed industry processes (ensure manufacturing specification) Documentation to comply with legal requirements Nutritional formulation Accurate food labeling Are there any of these areas where you would accept lower quality of lab results, and still claim that it doesn’t have any impact? The Lab result is used as a basis for important decisions in production, trading, and in documenting compliance in legal matters. IMPT: If the quality is not appropriate by the method chosen, consequences will be crucial and costly! 3

Dedicated Analytical Solutions MOST COMMON PROTEIN REFERENCE METHODS Kjeldahl method from 1883 For Nitrogen

Dedicated Analytical Solutions MOST COMMON PROTEIN REFERENCE METHODS Kjeldahl method from 1883 For Nitrogen /Protein Distillation after digestion q Dumas method 1980’s q For Nitrogen /Protein q Combustion of sample into NOx q NIR/FTIR methods 1980’s q For Crude Protein 4

Dedicated Analytical Solutions Kjeldahl q Classical Wet Chemistry according to official standards (ISO; AOAC

Dedicated Analytical Solutions Kjeldahl q Classical Wet Chemistry according to official standards (ISO; AOAC etc. ) q Involves man power, glassware and a balance q Specificity depends on lab preparations, (follow official methods) 5 METHOD CONSIDERATIONS -DIRECT VS. INDIRECT Dumas q Combustion according to official standards (ISO; AOAC etc. ) q Based on NIR/NIR detectors means less sample/chemical handlings q Involves some man power, capsules and a balance q Involves no man power (push button) q Specificity (for N) independent of preparations, and preparations q Specificity depends on instruments, conditions, calibrations q Per definition “Accurate” GOLD STD-No need for calibration q Accuracy secured from standards and calibration checks vs. ref. methods q Precision influenced by operator and preparations q Precision favored (homogenous samples) q Automation reduce Operator dependency, and spent time Direct NIR/NIT/FTIR Direct/ Indirect q Accuracy secured from calibrations developed using direct method correlations q Precision favored (homogenous samples) q Fast (few minutes) and Operator independent Indirect

Dedicated Analytical Solutions WHAT DO WE MEAN BY PROTEIN? Parameter Principle Recovery Kjeldahl protein

Dedicated Analytical Solutions WHAT DO WE MEAN BY PROTEIN? Parameter Principle Recovery Kjeldahl protein 3 steps Kjeldahl: Ammonia Dumas protein Combustion: HPLC protein NIR protein Chromatography: Amino acids NIR Spectra: We know what proteins are… – but how do we measure them in the lab? 6 Nitrogen Atomic bound spectroscopy

Dedicated Analytical Solutions PROTEIN METHODS - WHAT IS BEING MEASURED? Parameter Differentiators Kjeldahl protein

Dedicated Analytical Solutions PROTEIN METHODS - WHAT IS BEING MEASURED? Parameter Differentiators Kjeldahl protein Only digestible and distilled nitrogen as Amino acids, NH 3, prep separation for NPN NOT: NO 2/NO 3 or Heterocyclic N Dumas protein HPLC protein NIR protein Include all N – nitrogen oxides Just specific amino acids Calibrated (trained) specific Depending on the need … – but you have to choose according to that need! 7

J u n e Dedicated Analytical Solutions 4 , 2 0 2 1 8

J u n e Dedicated Analytical Solutions 4 , 2 0 2 1 8 MACRO – KJELDAHL

Dedicated Analytical Solutions KJELDAHL PRINCIPLE Digestion Acid hydrolysis breaks CN bonds in organic sample

Dedicated Analytical Solutions KJELDAHL PRINCIPLE Digestion Acid hydrolysis breaks CN bonds in organic sample and reduces nitrogen to NH 4+ Distillation Converts & liberates NH 4+ to NH 3 Transfers by distillation NH 3 as gas Traps NH 3 as [NH 4][H 2 BO 3] in aq. solution Titration Titrates to obtain amount of nitrogen Calculate figures as %N and %Protein 9

THE KJELDAHL METHOD -ADVANTAGES AND DISADVANTAGES Dedicated Analytical Solutions Advantages • A historical method

THE KJELDAHL METHOD -ADVANTAGES AND DISADVANTAGES Dedicated Analytical Solutions Advantages • A historical method to isolate and accurately quantify nitrogen in simple or complex mixtures • Officially accepted as the reference method, all over the world • Versatile - All types of samples & larger sample sizes • Accurate - No need for instrument calibration Disadvantages 10 • Hazardous Chemicals • Longer analysis time

Dedicated Analytical Solutions DUMAS/COMBUSTION PRINCIPLE Combustion of a sample in the presence of catalysts

Dedicated Analytical Solutions DUMAS/COMBUSTION PRINCIPLE Combustion of a sample in the presence of catalysts into oxides Resulting oxides are reduced to elemental nitrogen with water and carbon dioxide as by-products Helium (example) is used as a carrier gas to transport resulting gases (NOx, CO 2, H 2 O) from the combustion reactor through various filters/traps to the detector – TCD (thermal conductivity detector) The total resulting gases cause a change in thermal conductivity of the carrier gas, which is registered by the TCD The TCD signal (area or top height) corresponds to the amount of Nitrogen in the combusted sample. Nitrogen content is calculated by linear standard calibration. A conversion to protein concentration using product specific factors can be used. 11

Dedicated Analytical Solutions THE DUMAS METHOD -ADVANTAGES/DISADVANTAGES Advantages Much faster than the Kjeldahl method

Dedicated Analytical Solutions THE DUMAS METHOD -ADVANTAGES/DISADVANTAGES Advantages Much faster than the Kjeldahl method (under 4 minutes per measurement, compared to 1 -2 hours for Kjeldahl) No hazard chemicals or catalyst Samples are measured automatically Disadvantages High initial cost Maintenance downtime Not true protein content analyzed (separation of NPN fractions as Kjeldahl method) 12 Difficult to obtain a representative sample due to small sampler size

Dedicated Analytical Solutions CONSIDERATIONS - WHY TO CONSIDER EITHER ALTERNATIVES? Cost & throughput Shortened

Dedicated Analytical Solutions CONSIDERATIONS - WHY TO CONSIDER EITHER ALTERNATIVES? Cost & throughput Shortened analysis time Reduction in Cost per test Ease of operation Automation Quality – Accuracy and precision Official status & acceptance approvals Increased safety – Reduced use of chemicals Waste handling Optimized use of labs – Foot print, lack of fume hoods 13

Dedicated Analytical Solutions WHY IS ”KJELDAHL PROTEIN” NOT THE SAME AS ”DUMAS PROTEIN”? VISUAL

Dedicated Analytical Solutions WHY IS ”KJELDAHL PROTEIN” NOT THE SAME AS ”DUMAS PROTEIN”? VISUAL Dumas determines Total N, including inorganic fractions like NO 2/NO 3 Kjeldahl determines Organic N plus NH 3 For many samples the difference might be negligible – but you have to check! (apples to apples comparison) 14

Dedicated Analytical Solutions 15 CONSIDERATIONS - CHEMISTRY METHOD LIMITATIONS SUMMARY Dumas Method (DM) determines

Dedicated Analytical Solutions 15 CONSIDERATIONS - CHEMISTRY METHOD LIMITATIONS SUMMARY Dumas Method (DM) determines total nitrogen, including inorganic fractions like nitrate and nitrite, lending to possible sensitivity issues for protein conversions so could be susceptible to adulteration by other type organic and inorganic compounds that contain nitrogen (i. e. melamine) Neither the Kjeldahl Method (KM) or Dumas Method (DM) can give a measure of true protein directly, although NPN recoveries can be identified by preparation steps and separation of filtration fractions by either method Both methods need correction factors based off of specific algorithms used to offset different amino acid sequences within product matrices-PATTERNS MIGHT MATTER Even after method optimizations, DM and KM might lend to different results dependent on the NPN content of the analyzed sample type. EX. A lettuce sample with elevated nitrate content of 33. 000 mg/kg dry matter will correspond to 0. 75% added nitrogen or 4. 7% crude protein (factor 6. 25) when run by DM in comparison to KM

Dedicated Analytical Solutions MORE CONSIDERATIONS - KJELDAHL VS DUMAS OPERATIONS Kjeldahl Dumas $2. 25

Dedicated Analytical Solutions MORE CONSIDERATIONS - KJELDAHL VS DUMAS OPERATIONS Kjeldahl Dumas $2. 25 $1. 93 Initial investment Analytical flexibility Cost per sample * Automation Unattended operation Throughput Repeatability Accuracy Time per sample Environment / waste Health and safety 16 * Kjeltec 8420 cost of USD 3. 25 per sample v. USD 1. 93 for comparable Dumatec 8000. Based on chemicals/reagents including gas. No depreciation or labour cost Ø Kjeldahl enables broader and more versatile use for labs (commercial offerings) Ø Dumas preferred by some labs when: Ø Narrow variation of sample types Ø Applications for grains and milk powder Ø Sample flexibility improves ROI Ø Market approvals are most important – confidence with good Dumas results and validations can influence choice Ø Dumas becoming standard in some FOSS segments - e. g. cereals, flour, dairy powders, animal feed Ø Kjeldahl remains the primary reference method in disputes and globally

Dedicated Analytical Solutions 17 CONSIDERATIONS -FINAL BULLET POINTS Need for True Reference: The actual

Dedicated Analytical Solutions 17 CONSIDERATIONS -FINAL BULLET POINTS Need for True Reference: The actual need for the reference data : Dumas is gaining in acceptance, but Kjeldahl remains the recognized historical reference. Ie. Official Standards Operations: Dumas is faster with more easy to operate than any other protein traditional lab method (Kjeltec, HPLC). NIR indirect is easier Lab setup: Dumas can be installed without any wet chemicals involved (fume hoods, waste). However gas installations is high cost consumable Operator skills: Operators can handle Dumas without much chemistry insight Sample throughput: Dumas can handle more samples with less attention Sensitivity: Matrix and sample size needs

Dedicated Analytical Solutions COMPARISON STUDIES - RESULTS AND PERFORMANCE General Results from Ring Tests

Dedicated Analytical Solutions COMPARISON STUDIES - RESULTS AND PERFORMANCE General Results from Ring Tests Reported Validation Trials –Non FOSS Application Validation – FOSS Labs 18

GENERAL COMPARISON OF - ”PROTEIN CONTENT” Dedicated Analytical Solutions Sample Type Kjeldahl Dumas Amino

GENERAL COMPARISON OF - ”PROTEIN CONTENT” Dedicated Analytical Solutions Sample Type Kjeldahl Dumas Amino Acid AAFCO 200921 Chicken 17, 29 (0, 15) 17, 64 (0, 33) 14, 22 (0, 17) AAFCO 200922 Pig starter 23, 94 (0, 33) 24, 51 (0, 39) 19, 73 (1, 18) AAFCO 200923 Chow 12, 3 (0, 52) 12, 51 (0, 65) 7, 16 (0, 19) Sum of 18 reported AA calculated as protein (F 6. 25): Significant differences from Non-Protein Nitrogen (Ammonia, Urea…) and Incomplete recovery in AA SD for reported results between KT & DM are comparible , but DM recovery values are higher 19 Non FOSS Alanine, Arginine, Aspartic Acid, Cystein. Glutamic acid, Glycine, Histidine, Iso-Leucine, Methionine, Phenylalanine, Proline, Serine, Threonine, Tryptophane, Tyrosine, Valine

Dedicated Analytical Solutions COLLABORATIVE STUDY ON -WHEAT & BARLEY Method % CP ICC 105/2

Dedicated Analytical Solutions COLLABORATIVE STUDY ON -WHEAT & BARLEY Method % CP ICC 105/2 (Kjeldahl, Cu) 12, 46 ISO 5983 -2 (Kjeldahl, Cu) 12, 45 ISO 20483 (Kjeldahl, Cu/Ti) 12, 39 ISO 16634 (Dumas/Combustion) 12, 55 Small differences in results: Less than 1 standard deviation 20 Non FOSS

Dedicated Analytical Solutions COLLABORATIVE STUDY ON WHEAT Non FOSS During 5 harvest years (2000

Dedicated Analytical Solutions COLLABORATIVE STUDY ON WHEAT Non FOSS During 5 harvest years (2000 -2004) the Max Rubner Institute in Germany performed a study with more than 800 wheat samples comparing DM and KM and found that 2% of Dumas protein was not determined by Kjeldahl They presented the following relationship between DM and KM protein values: KM = 0. 959 * DM + 0. 258 Method differences attributed to both growing years and conditions so not convertable* 21

Dedicated Analytical Solutions A. H. Simonne, RR Eitenmiller and CP Cresman: January 1997 Journal

Dedicated Analytical Solutions A. H. Simonne, RR Eitenmiller and CP Cresman: January 1997 Journal of the Science of Food and Agriculture, Volume 73, Issue 1_”Could the Dumas Method Replace Kjeldahl Digestion for Nitrogen and Crude Protein Determination in Foods? ” This study selected food groups for analysis and found that product specific and appropriate coefficients needed to be used They suggested correction factors below when calculating crude protein for: 1. 01 for Dairy, 1. 00 for oilseeds, 0. 99 for feeds, 0. 98 for infant formulas, 0. 95 for cereals, 0. 94 for meats, 0. 89 for vegetables, 0. 80 for fish, and 0. 73 for fruits are used Many think that complication overestimation is too hard to manage with calculated analytics and avoid conflicts by multiple method choice by matrix. * 22 Non COLLABORATIVE STUDY ON FOOD GROUPS FOSS

PROFICIENCY TESTING - AAFCO PTS (300+ PARTICIPANTS) Dedicated Analytical Solutions Method Sample KT (Cu/Ti.

PROFICIENCY TESTING - AAFCO PTS (300+ PARTICIPANTS) Dedicated Analytical Solutions Method Sample KT (Cu/Ti. O 2) Dumas % CP sd % AAFCO 0826 18, 2 0, 35 18, 2 0, 29 18, 4 0, 32 AAFCO 0827 12, 9 0, 36 12, 8 0, 46 13, 0 0, 46 AAFCO 0828 40, 8 0, 77 40, 0 0, 68 41, 0 0, 43 AAFCO 0829 23, 6 0, 42 23, 0 0, 58 23, 5 0, 33 AAFCO 0830 18, 2 0, 27 18, 2 0, 24 18, 5 0, 32 AAFCO 0831 27, 5 0, 40 27, 3 0, 64 27, 8 0, 46 23, 5 0, 43 23, 2 0, 48 23, 7 0, 39 Average 23 KT (Cu) Non FOSS

Dedicated Analytical Solutions APPLICATIONS - EXAMPLES AND RESULTS FOSS Typical applications – Validated at

Dedicated Analytical Solutions APPLICATIONS - EXAMPLES AND RESULTS FOSS Typical applications – Validated at FOSS Feed and Forage Cereal Grain and Oil Seeds Meat and meat products Dumatec 8000 Beer, wort and grains for breweries AAFCO Reference Milk and Dairy products Pet Foods Other applications: Soil Fertilizer 24

FOSS APPLICATION DEVELOPMENT - FEED & FORAGE AOAC 990. 03, DIN EN ISO 16634

FOSS APPLICATION DEVELOPMENT - FEED & FORAGE AOAC 990. 03, DIN EN ISO 16634 Dedicated Analytical Solutions 80 FOSS Dumatec & Kjeltec Mean value (n = 6) %CP 70 60 50 40 FOSS Dumatec 8000 30 FOSS Kjeltec 8400 AFFCO Combustion method 20 AAFCO Kjeldahl method 10 fc ee &B w rd gr o He e Da iry in Sw ilk al ni /fi er ei ot pr rn Co fm sh er . co n pe ts lo Be ef fe ed te ar st nc l cia er ow r/ gs ick en Ch In fa nt Pi er w ro gg Pi 25 gr ta pe rte lle t r 0

Dedicated Analytical Solutions Product Dumatec 8000 Pig Grower Pellet (AAFCO 201121) Infant Pig Starter

Dedicated Analytical Solutions Product Dumatec 8000 Pig Grower Pellet (AAFCO 201121) Infant Pig Starter (AAFCO 201123) Chicken starter/Grower (AAFCO 201126) Beef Feedlot Special (AAFCO 201128) 26 Corn Protein Concentrate (AAFCO 201131) Swine Grower/Finishe r (AAFCO 201132) Dairy Herd&Beef Calf Milk (AAFCO 201226) FEED AND FORAGE – KJELTEC VS DUMATEC Foss internal test Kjeldtec Differences 8400 (Combustion Kjeldahl) Combustion ring test FOSS AAFCO ring test Differences Kjeldahl Differences ring test (Combustion - ( Combustion Dumatec 8000 ) Kjeldahl) 18. 61 18. 59 0. 02 18. 76 18. 58 0. 15 24. 67 24. 72 -0. 05 25. 03 24. 81 0. 22 0. 36 19. 884 0. 00 19. 99 19. 79 0. 20 0. 11 44. 82 44. 31 0. 51 44. 83 44. 40 0. 43 0. 01 77. 47 77. 25 0. 22 78. 71 77. 42 1. 29 1. 05 20. 79 20. 56 0. 33 20. 85 20. 74 0. 11 0. 06 20. 20 19. 78 0. 42 20. 48 20. 13 0. 35 0. 28

APPLICATION DEVELOPMENT - CEREAL GRAIN AND OIL SEED AOAC 992. 23 Dedicated Analytical Solutions

APPLICATION DEVELOPMENT - CEREAL GRAIN AND OIL SEED AOAC 992. 23 Dedicated Analytical Solutions 20 18 FOSS Dumatec & Kjeltec Mean value (n = 5) %CP 16 14 12 10 FOSS Dumatec 8000 FOSS Kjeltec 8400 8 6 4 2 0 Sunflower Rape seeds seed 27 Barley Oat Wheat Rice Corn

APPLICATION DEVELOPMENT Dedicated Analytical Solutions - MEAT AND MEAT PRODUCTS AOAC 992. 15 25

APPLICATION DEVELOPMENT Dedicated Analytical Solutions - MEAT AND MEAT PRODUCTS AOAC 992. 15 25 %CP FOSS Dumatec & Kjeltec Mean value (n = 5) 20 15 FOSS Dumatec 8000 FOSS Kjeltec 8400 10 5 0 Medister Sausage 28 Liver Paté Minced meet Pork Minced meet Beef

APPLICATION DEVELOPMENT Dedicated Analytical Solutions FOSS - PET FOOD ACC. TO AOAC 992. 15

APPLICATION DEVELOPMENT Dedicated Analytical Solutions FOSS - PET FOOD ACC. TO AOAC 992. 15 35 FOSS Dumatec & Kjeltec Mean value (n = 5) %CP 30 25 20 FOSS Dumatec 8000 FOSS Kjeltec 8400 15 10 5 0 29 Cat food wet Cat food Dry Dog food wet Cat food Dry

APPLICATION DEVELOPMENT Dedicated Analytical Solutions - MILK & MILK PRODUCTS DIN EN ISO 14891

APPLICATION DEVELOPMENT Dedicated Analytical Solutions - MILK & MILK PRODUCTS DIN EN ISO 14891 FOSS 14 FOSS Dumatec & Kjeltec Mean value (n = 5) %CP 12 10 8 FOSS Dumatec 8000 FOSS Kjeltec 8400 6 4 2 0 30 Milk full, normal Milk full, high protein Curd cheese Sour cream

Dedicated Analytical Solutions DAIRY PRODUCTS – KJELTEC VS DUMATEC Product Milk full, normal Milk

Dedicated Analytical Solutions DAIRY PRODUCTS – KJELTEC VS DUMATEC Product Milk full, normal Milk full, high protein Sheep curd cheese Sour cream FOSS Dumatec 8000 Foss internal test Kjeltec 8400/8420 3. 44 3. 45 -0. 01 6. 07 5. 97 0. 10 11. 99 2. 38 11. 93 1. 82 0. 06 0. 56 Differences (Combustion Kjeldahl) Reference: DIN EN ISO 14891 Milk and milk products-determination of nitrogen content. Routine method using combustion according to Dumas principle 31

Dedicated Analytical Solutions SUMMARY & CONCLUSIONS - METHOD CONSIDERATIONS* Kjeldahl Method (Protein) Kjeldahl is

Dedicated Analytical Solutions SUMMARY & CONCLUSIONS - METHOD CONSIDERATIONS* Kjeldahl Method (Protein) Kjeldahl is still the most important reference and routine method for crude protein/protein fractions* with historical significance Dumas Method (Protein) Many international standards use Dumas, and Dumas results are accepted as proficiency test results Improved by catalyst and salt to increase yield, No “wet” hazardous chemicals increases safety and ease of operation considerably speed, safety New standards and automation reflect instrumental and methodological progress No alternatives can replace N based methods with regard to accuracy, precision, sample throughput and cost per analysis • EC confirms the Kjeldahl method as the community method for official controls (EC No 152/2009) – Regulatory Compliance 32 • Best solution depends on the lab environment, facilities, samples to be tested as there is no universal rule – there are many parameters that can change the picture Automation and data handling allows unattended operation and easy for calculations/reporting Dumas is the closest we can come to an alternative Kjeldahl reference in terms of quality, throughput and costs

Dedicated Analytical Solutions FNA CONTACTS Thank you for your attention! Available: White Paper "Dumas

Dedicated Analytical Solutions FNA CONTACTS Thank you for your attention! Available: White Paper "Dumas or Kjeldahl for reference analysis? ” & Webinar 33 FOSS web page: http: //www. foss. dk E-mail: mwalter@fossna. com or mrussell@fossna. com Contact: Michelle Walter_Technical Manager Marcy Russell_ISM, Laboratories