AOAC2016-03ERPRECOMMENDATION

ERP SUMMARY FOR FIRST TO FINAL ACTION METHOD RECOMMENDATION

AOAC 2016.03

Chloride in Milk, Milk Powder, Whey Powder, Infant Formula and Adult  Nutritionals Potentiometric titration

GUIDANCE FOR AOAC ERPS ‐ APPENDIX G 1 Considered? Comments/Reference if applicable Method Applicability  ERP First Action to Final Action  recommendations & improvements  Yes  ERP reports dated: 3/18/2015; 9/29/2015;  3/16/2016; 3/16/2017; 4/26/2017  Draft Final Action method reviewed by ERP Yes  November 2017 – approved by ballot  Safety Concerns  Yes  ReferenceMaterials  Yes  Single Laboratory Validation  Yes  Reproducibility/Uncertaintyand  Probability of Detection  Yes   MLT submitted for publication;   Comparison to SMPR ( SMPR criteria met?) Yes  Feedback from Users of Method  Yes 

DOCUMENTATION Safety Evaluation 

Available?

Comments

ERP reports dated: 3/18/2015; 9/29/2015;   3/16/2016; 3/16/2017; 4/26/2017 

Yes 

SPIFAN SLV Kit 

ReferenceMaterials 

Yes  Yes 

SLVs:  JAOAC v.99, 1113 (2016); JAOAC v.99, 198  (2016); JAOAC v98, 1390 (2015); for First Action  status  OMA Appendix L and SPIFAN MLT Protocol  ERP reports dated: 3/18/2015; 9/29/2015;   3/16/2016; 3/16/2017; 4/26/2017  AOAC 2016.03 ; AOAC 2015.08; AOAC 2015.07 

SLV or PTM 

Approved Validation Protocols 

Yes  Yes 

Statistics Review 

Method Published in OMA 

Yes  Yes  Yes  Yes  Yes  Yes 

SMPR 2014.015  

Method Performance vs SMPR criteria 

Also discussed in ISO  

Feedback Information  Additional Recognition(s) 

  Recommended to CCMAS by CCNFSDU 

From 3/2015 through 4/2017 

ERP Reports 

SLVs:  JAOAC v.99, 1113 (2016); JAOAC v.99, 198  (2016); JAOAC v98, 1390 (2015);   MLT publication in progress  The ERP approved the method only for the  SPIFAN related matrices.  The Final Action  method will not include the IDF matrices;  however, this information will be published  as part of the manuscript.  The title does not  change; however, the term “milk products”  have been used to categorize certain SPIFAN  matrices. 

Manuscript(s) Published in JAOAC 

ERP Method Recommendation   (Final Action/Repeal/Continuation) 

Final Action  Recommend ation 

1 Official Methods of Analysis of AOAC INTERNATIONAL , Appendix G: Procedures and Guidelines for the Use of AOAC Voluntary Consensus Standards  to Evaluate Characteristics of a Method of Analysis, p.3 “ First Action to Final Action Methods: Guidance for AOAC Expert Review Panels.” 

TABLE OF CONTENTS  AOAC ERP FOR SPIFAN NUTRIENT METHODS  RECOMMENDATION FOR FINAL ACTION OMA STATUS FOR AOAC 2016.03 

3 

AOAC 2016.03 – FIRST ACTION Method recommended by ERP (and manuscript) 

27  31  36  39  44  46  52  54  59  65 

AOAC 2016.03 as published in the eOMA  Published Manuscript for AOAC 2016.03  AOAC 2015.08 as published in the eOMA  Published Manuscript for AOAC 2015.08  AOAC 2015.07 as published in the eOMA  Published Manuscript for AOAC 2015.07 

ERP Report (4/2017)  ERP Report (3/2017)  ERP Report (3/2016)  ERP Report (9/2015)  ERP Report (3/2015) 

71  Standard Method Performance Requirements (SMPR®) for Determination of Chloride  in Infant Formula and Adult/Pediatric Nutritional Formula  79  SPIFAN Multi‐Lab Testing Protocol   80  OMA, Appendix L – Recommended Guidelines for SPIFAN Single‐Laboratory  Validation  85  SPIFAN II SLV Kit  86

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Chloride in Milk, Milk Powder, Whey Powder, Infant Formula and Adult Nutritionals Potentiometric titration - AOAC 2016.03: Collaborative study

First Action 2016 Final Action 2017

Greg Jaudzems 1 Nestle Quality Assurance Center, Dublin, OH; Fengxia Zhang, Test Center of Chinese Academy of Inspection and Quarantine: WU Bolong, Test Center of Chinese Academy of Inspection and Quarantine: Lei Bao, Nestle Food Safety Institute, Nestle R&D (China) Ltd.: Jing Xiao, China National Center for Food Safety Risk Assessment

Abstract In September of 2015 both AOAC 2015.07 and 2015.08 chloride methods single laboratory validations were reviewed against SMPR 2014.015 (Standard Method Performance Requirements) by the SPIFAN ERP (Stakeholder Panel for Infant Formula and Adult Nutritional Expert Review Panel). Looking at the similarity and uniqueness of the two methods the authors agreed, as advised by the SPIFAN-ERP, to work together to merge the two methods into one. This combined method was assigned AOAC 2016.03. In order to determine the repeatability and reproducibility figures of the AOAC First Action 2016.03 method (Milk, milk products, infant formula and adult nutritionals — Determination of chloride — Potentiometric titration method), a collaborative study was organized.

1 To whom correspondence should be addressed. Tel. 614-537-5357. E-mail: greg.jaudzems@us.nestle.com

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The study was divided in two parts: method set up and qualification of participants (part 1) and collaborative study participation (part 2). During part 1 each laboratory was asked to analyze two practice samples. The laboratories providing results within a range of expected levels were qualified for part 2, during which they analyzed 25 samples in blind duplicates. The results were compared to the Standard Method Performance Requirements (SMPR 2014.015) established for Chloride. The precision results (repeatability and reproducibility) were within the requirements stated in the SMPR. In general, the precision results (repeatability and reproducibility) are well within the limits stated in the SMPR. Repeatability ranged from 0.4 to 1.9 %, in accordance with data obtained during SLV, with reported RSDr from 0.03 - 1.6 %. Meanwhile reproducibility ranged from 0.6 to 4.0 %. Finally the HorRat values are all below 1, from 0.2 to 0.9 %. The SPIFAN ERP determined that the data presented met the SMPR and hence recommended the method to be granted Final Action status.

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Introduction Sodium chloride (salt) is the main source of chloride in food. On September 26, 1980 the Infant Formula Act of 1980 was signed into law. This bill, which resulted from reports during 1979 that more than 100 infants became seriously ill as a consequence of using soybean-based formulas marketed with an insufficient amount of chloride, establishes a statutory requirement that formula manufacturers include chlorides as well as other essential elements in each infant formula preparation sold. It also gives the Secretary of Health and Human Services (HHS) authority to adjust nutritional standards to conform to the best available scientific knowledge. In addition, the bill requires manufacturers to test infant formulas on a periodic basis and to notify the Secretary promptly whenever formulas do not meet nutritional requirements. On May 28, 2013, World Health Assembly adopted the WHO Action Plan for the Prevention and Control of Non-communicable Diseases. One of the measures involves product reformulation. Target: 30% relative reduction in mean population intake of salt/sodium, by reducing level of salt/sodium added to food. Several Official Methods exist for the analysis of chloride in foods, and in particular in infant formula. A merger of AOAC 2015.07 and 2015.08 was proposed to the SPIFAN (Stakeholder Panel for Infant Formula and Adult Nutritional) and was approved as a First Action AOAC 2016.03 in 2016, with a recommendation to advance to multi-laboratory collaborative study. This paper presents the results of the collaborative study. Samples The study took place using SPIFAN matrices, which represent most of the products in the scope of the project (Infant Formula and Adult Nutritionals made from any combination of milk, soy, rice, whey, hydrolyzed protein, starch and aminoacids, with and without intact

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protein). In addition, IDF (International Dairy Federation) matrixes were added to this MLT in planning to be able to expand the scope to include dairy. All samples were blinded and codified before sending to participating laboratories.

Method Chloride in Milk, Milk Powder, Whey Powder, Infant Formula and Adult Nutritionals Potentiometric titration. Caution: Refer to Material Safety Data Sheets prior to use of chemicals. Use appropriate personal protective equipment when performing testing. A. Principle This document specifies one method (combined [6-9]) for the determination of chloride in milk, milk products, infant formula and adult nutritionals by potentiometry, with an analytical range of 0.35 mg chloride/100 g to 1060 mg chloride/100 g reconstituted product, or ready-to-feed liquids. Reconstitute powder samples by dissolving 25 g powder sample in 200 g warm water (40 ° C), while ready-to-feed products (RTF) are used as-is. Butter and cheese are prepared for liberation of chloride. Precipitate proteins by adding precipitation reagents I and II and centrifuge. Acidify the supernatant with nitric acid solution. Titrate chloride ions against standardized silver nitrate solution, 0,1 mol/l, potentiometrically using a silver electrode to detect the end point.

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B. Apparatus Usual laboratory equipment and, in particular, the following.

(a) Analytical balances , precision 0,01 mg and 0,1 mg.

(b) Centrifuge , table-top with rotor for 50 ml conical tubes, capable of operating at ≥ 12 000 g .

(c) Centrifuge tubes , 50 ml, conical, polypropylene.

(d) Pipettes , 1 ml, 10 ml, 20 ml, 50 ml and 100 ml, glass, volumetric or automatic, Class A in accordance with ISO 1042 [2].

(e) One-mark volumetric flasks , 50 ml, 100 ml, 500 ml, and 1 000 ml glass, Class A in accordance with ISO 1042 [2].

(f) Graduated cylinders , 25 ml, 100 ml and 500 ml, glass.

(g) Autosampler beaker , e.g. 120 ml, depending on the titrator used.

(h) pH-meter or mV-meter , with a scale covering ± 700 mV, and burette, 20 ml or 25 ml.

(i) Automatic titrator , (autosampler, motorized piston burette, with remote-control dispensing and filling).

Mettler T50, Roundo Tower autosampler, MettlerLabX 3.1 software or Metrohm 862 Compact Titrosampler, 800 Dosino, 10 ml Exchange Unit, or equivalent. Alternatively a

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semi-automated (e.g. MetrohmTitrado 905/907, with MetrohmTiamo™ software) or equivalent. Or a manual titrator (using a burette with accuracy of 0,01 ml) may be used. (j) Combined ring silver electrode , e.g. Mettler DM 141 or DMi145-SC, Metrohm Ag Titrode 6.0430.100 or equivalent, alternatively a silver electrode with reference electrode.

(k) Magnetic stirrer , Heidolph MR 3000, or Metrohm 804Ti Stand with 802 Rod Stirrer or equivalent.

(l) Water bath , Labotech DWM 16 or equivalent.

(m) Ultrasonic cleaner , Tianjin Auto Science AS2060B or equivalent.

(n) Disposable syringe , 3 ml, with handspike and 0,45 μm disposable syringe filter.

(o) Blender , capable to hold and blend 100 ml volume.

C.

Chemicals and Reagents

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(a) Water, purified, Type I (EMD Millipore) Corp., Billerica, MA, USA, or equivalent).

(b) Sodium chloride (NaCl), purity ≥ 99,5 %, certified reference material for titrimetry, Sigma Aldrich #71387 or equivalent.

(c) Silver nitrate (AgNO 3 ), meets analytical specification of European Pharmacopoeia ( Ph. Eur), British Pharmacopoeia ( BP), United States Pharmacopeia (USP), assay 99,8 % -100,5 %, Sigma-Aldrich 10220, or equivalent.

(d) Potassium ferrocyanide trihydrate (K 4 Fe(CN) 6 · 3H 2 O), puriss, p.a., ACS reagent, reag. ISO, reag. Ph. Eur., ≥ 99 %, Sigma-Aldrich # 31524 or equivalent.

(e) Zinc acetate dihydrate Zn(CH 3 COO) 2 · 2H 2 O, ACS reagent puriss p.a., ≥ 99,0 %, Sigma Aldrich # 96459 or equivalent.

(f) Nitric acid (HNO 3 ), minimum 65 % p.a., Merck #100452 or equivalent.

(g) Standardized AgNO 3 solution, substance concentration c = 0,1 mol/l, Titripur® Reag. Ph. Eur. Reag. USP. # 1,09081,1000 or EM3214-1, or ready-to-use standardized titrant prepared according to GB/T 601 [1], or equivalent.

(h) Sodium chloride (NaCl) standardized solution, c = 0,1 mol/l, Alfa Aesar, # 35616, (Ward Hill, MA, USA), or equivalent,

(i) Glacial acetic acid, 100 %, anhydrous for analysis EMSURE® ACS, ISO, Reag. Ph. Eur., MERCK, # 100063 or equivalent.

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(j) Potassium nitrate, (KNO 3 ), for analysis EMSURE® ISO,Reag, Ph. Eur., MERCK, # 105063 or equivalent.

(k) Acetone, for cleaning of the electrode, Honeywell, # 010-4, (Muskegon, MI, USA) or equivalent.

(l) Dimethylpolysiloxane, defoaming agent, Sigma-Aldrich, #DMPS2C or equivalent.

D.

Solutions

(a) Standardized silver nitrate (AgNO 3 ) solution, c = 0,1 mol/l.

If ready-to-use AgNO 3 standard solution (C. g) is not available, then weigh 16,9890 g ± 0,0005 g AgNO 3 (C. c) previously dried for 2 h at 120 °C ± 2 °C. Dissolve in water and make up to the mark in a 1000 ml volumetric flask. Store in a brown reagent bottle. After preparation, check the titer by titration of 5.0 ml with exactly 0.1 mol/l NaCl (C. h, or D. b). solution. For either commercial or in-house solution verify the titer on a regular basis following Formula 1 or 2 in Section I. If outside 0.095-0.105 mol/L investigate. The standardized AgNO 3 solution shall be protected from light, and can be stored for up to 2 months.

(b) Sodium chloride solution, c = 0,1 mol/l.

If ready-to-use NaCl (C. h) standard solution is not available, weigh 5,8440 g ± 0,0005 g NaCl (C. b), previously dried for 2 h at 110 °C ± 2 °C. Dissolve in water and make up to the mark in a 1000 ml volumetric flask. This solution is stable for up to 1 month.

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(c) Precipitating solution (Carrez) I.

Weigh 106 g potassium ferrocyanide trihydrate, (C. d) dissolve in an appropriate amount of water and transfer into a 1000 ml volumetric flask. Make up to the mark using water. Mix well.

(d) Precipitating solution (Carrez) II.

Weigh 220 g zinc acetate dihydrate (C. e) and transfer into a 1000 ml volumetric flask. Dissolve with an appropriate amount of water, and add 30 ml glacial acetic acid. Make up to the mark using water. Mix well.

(e) Nitric acid solution, c = 4 mol/l.

With care, add 100 ml concentrated nitric acid (C. f) to 300 ml water. Mix well.

(f) Wash solution.

According to autosampler/titrator manufacturer’s instructions, (e.g. acetone, nitric acid solution (D. e), or other).

(g) AgNO 3 solution, c = 0,025 mol/l (optional).

Into a 1 000 ml volumetric flask, pipet 250 ml AgNO 3 solution, 0,1 mol/l (C. g) or (D. a). Make up to the mark with water. Prepare freshly before use. Then check the titer by titration of 25 ml against 0,025 mol/l NaCl solution.

(h) NaCl solution, c = 0,025 mol/l (optional).

Into a 100 ml volumetric flask pipet 25 ml NaCl solution, 0,1 mol/l (C .h or D. b). Make up to the mark with water. Prepare freshly before use.

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(i) KNO 3 solution, c = 1 mol/l.

Weigh 10.11 g potassium nitrate (C. j) into a 100 ml volumetric flask. Add about 80 ml water and place it in an ultrasonic cleaner (B. m) to dissolve with ultrasound and heating until dissolved thoroughly. Cool down to room temperature and make up to the mark with water. Filter using a 0,45 membrane disposable syringe before use.

E.

Sample preparation

(a) Powders, for milk, milk products, and infant formula powders

Mix well to ensure that the sample is homogeneous, Reconstitute powder samples by dissolving 25 g powder sample in 200 ml warm water (40 °C).

(b) Cheese, for hard or rinded cheese

Prior to analysis, remove the rind or smear or mouldy surface layer of the cheese, in such a way as to provide a sample representative of the cheese as it is usually consumed. Grind or grate the sample by means of an appropriate device. Mix the ground or grated mass quickly, and if possible grind or grate a second time and again mix thoroughly. If the sample cannot be ground or grated, mix it thoroughly by intensive stirring and kneading. Transfer the sample to an airtight container to await analysis, which should be carried out as soon as possible after grinding. If delay is unavoidable, take all precautions to ensure proper preservation of the sample and to prevent condensation of moisture on the inside surface of the container. The storage temperature should be 10 °C to 12 °C.

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(c) Butter

If the sample is visibly not homogeneous, or if the history of the sample (age, storage conditions) is such that inhomogeneity is expected, homogenize the sample as follows. Warm the sample in the original unopened container, which should be from one-half to two-thirds full, to a temperature at which the sample will be soft enough to facilitate thorough mixing to a homogeneous state (either by a mechanical shaker or by hand). Take care that the mixing temperature does not exceed 30 °C. Cool the sample to ambient temperature with constant mixing until cooling is complete. As soon as possible thereafter, open the sample container and stir briefly (no longer than 10 s) with a suitable device, for example a spoon or spatula, before weighing.

F.

EXTRACTION

(a). Cheese

Weigh 2 g to 5 g of the prepared sample (E. b) into the vessel. For processed cheese weigh 2.5 g. Add 30 ml of water at about 55 °C. Suspend the sample using a blender, Rinse the blender with approximately 10 ml of water, collecting the rinsing in the vessel. Add 2 ml to 3 ml of the nitric acid solution of c = 4 mol/l (D. e). Proceed with (F. g).

(b). Butter

Weigh 2 g to 4 g of the prepared sample (E. c) into the vessel. For salted butter weigh 2.5 g. Add 100 ml of boiling water and heat to boiling to dissolve the test portion. Cool the obtained suspension to below 55 °C. Proceed with (F. g).

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(c). Milk, milk powders, infant formula and adult nutritional products

For high protein samples requiring additional protein precipitation than that accomplished by addition of nitric acid solution, proceed with (F. d-f), Otherwise proceed with (g). (d).Weigh an appropriate (well mixed) aliquot of RTF or reconstituted (E. a) powder (e.g. 25 g accurate to 0.1 mg) into a 50 ml centrifuge tube. For samples with a high chloride content, weigh a smaller test portion e.g. 5 g of reconstituted or RTF product. (e). Transfer 2.5 ml precipitating solution I (D. c) and 2,5 ml precipitating solution II (D. d) into the tube. Complete to 50 ml with water. Mix well. If foam impacts the constant volume, add one or two drops of defoaming agent (C. l).

(f). Centrifuge at 12000 g for 5 min at 4 °C. Then equilibrate to room temperature.

(g).Accurately transfer either 10 ml supernatant from steps (D. d-f) or weigh an appropriate aliquot of RTF or reconstituted (E. a) powder (e,g, 25 g accurate to 0.1 mg). For samples with a high chloride content, weigh a smaller test portion e.g. 5 g of reconstituted or RTF product into a 120 ml sample beaker or autosampler cup. Add 5 ml nitric acid solution (D. e), and 50 ml of water before titration. Add a magnetic stirring rod (if the

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titrator does not have a build-in rod stirrer). Place the autosampler cup or beaker onto a magnetic stirrer and stir until dissolved or finely suspended.

(h).The pH of the test solution shall be below 1.5, In case of any doubts, check by means of a pH-meter and, if necessary, add a little more nitric acid solution (D. e).

G.

INSTRUMENT OPERATING CONDITIONS

(a). Check and maintenance of the combined silver electrode

Rinse electrode with deionized water and wipe before use. Renew the electrolyte c = 1 mol/l KNO 3 (D. i) periodically per manufacturer’s recommendations. If fat sticks to the electrodes during a series of analyses, then eliminate it by briefly immersing the electrode in acetone. Store the silver electrode in KNO 3 solution (D. i) after appropriate cleaning. NOTE Instead of the combined silver electrode, separate silver and reference electrodes may also be used. Connect the combined silver electrode to the titration apparatus, according to the manufacturer's indications. Ensure that the titration vessels are correctly placed on the autosampler and there are enough reagents, both nitric acid solution (if added automatically) (D. e) and silver nitrate solution (C. g or D. a). If no autosampler is available, place the sample solutions manually under the titration equipment. (b). Titration

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Put the wash solution (D. f) in the washing position if an auto sampler is used. Ensure that the volume of wash solution is adequate. Under continuous stirring and without touching the electrode, titrate the sample solution automatically with silver nitrate solution (C. g or D. a), up to the end potential, The consumption of silver nitrate solution (C. g or D. a) should be recorded automatically and can be read from the titrator software, or documented in the titrator operating records. For manual titration using a burette, add standardized silver nitrate solution (C. g or D. a), until the end potential has nearly been met. Continue to titrate slowly until the end point is met as observed on the meter with two small (about 0,05 ml) additions of silver nitrate solution.

(c). Determination of very low amounts of chloride

When determining low chloride (e.g. <10-20 mg/ 100g) concentrations, like in desalted whey powder, for greater precision a standardized 0,025 mol/l AgNO 3 (D. g) solution should be used for the titration,

(d). Blank test

To determine the reagent background content of chloride, perform a blank test using reagents substituting water (C. a) for the sample portion, The titrant consumption of the blank test obtained at the endpoint shall be less than 0.05 ml while using the standardized silver nitrate solution (D. a) and 0.2 ml when using the 0.025 mol/l standardized silver nitrate solution (D. g). Otherwise check the reagents and water involved in the procedures, and perform the blank test again until the criteria is achieved.

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H.

SYSTEM SUITABILITY TEST

Prior to use, transfer 5 ml of NaCl solution (D. h or D. b) into a 120 ml sample beaker, If 0.025 mol/l AgNO 3 titrant is required, use 1 ml NaCl solution. Add 5 ml nitric acid solution (D. e) and 50 ml water. Place the wash solution (D. f) in the washing position of the auto sampler. Titrate using an automatic, semi-automatic or manual titrator. Repeat in triplicate Calculate concentration of the silver nitrate solution according to Section I. The difference between the calculated concentration and the certified value should be within 0.5 %. If outside the acceptance value, check the experimental procedures and titration system. If issue is not resolved, then use fresh standardized silver nitrate. If fresh standardized silver nitrate does not provide an acceptable result, replace the electrolyte of the electrode and check the operating condition of the dosing unit. I. CALCULATIONS Calculate the concentration of silver nitrate, c snc , in mol/l, for system suitability verification and report to 4 decimal places. If using the prepared sodium chloride solution (D. b), use Formula (1): = 1 5,844× 1 ×10 1 = mass of sodium chloride in 5 ml or 1 ml of (D. b) standard solution (in mg); where

(1)

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1 = volume of 0.1 mol/l or 0.025 mol/l AgNO 3 solution consumed at titration endpoint (in ml); 5.844 = sodium chloride mass in μg corresponding to 1 ml of 0.1 mol/l AgNO 3 ; 10 = mass conversion from titer to the concentration of titrant. Or if using a purchased standard grade 0.10 mol/l sodium chloride solution (C. h), use Formula (2): = 0,1× 3 1 (2) where V 3 = volume of purchased standard grade 0.10 mol/l sodium chloride added (in ml); 1 = volume of 0.1 mol/l or 0.025 mol/l AgNO 3 solution consumed at titration endpoint (in ml), Calculate the chloride mass fraction, w cl , in mg/100 g in the sample and report to 3 significant digits, using Formula (3): = 35,45× ×( 2 − 0 )× ×100 (3) where m = the sample mass (in g); c = the certified concentration of silver nitrate titrant (0.1000 mol/l or standardized concentration (Formula 1); V 2 = the volume of AgNO 3 solution consumed at titration endpoint (in ml);

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= the volume of AgNO 3 solution consumed at titration endpoint for blank (G. d) (in ml); = the dilution factor for preparation of reconstituted powder, RTF or concentrate, For samples requiring the protein precipitation step (F. d-f) an additional factor (e.g. for 25 g sample f = 2) is needed;

V 0

f

35.45 = chloride mass in µg corresponding to 1 ml of 1 mol/l AgNO 3 ; 100 = the mass conversion to mg/100 g.

J.PRECISION

(a). General Details of the interlaboratory test of the precision of the method are summarized in Table 1. . The values derived from the interlaboratory test may not be applicable to analyte concentration ranges and/or matrices other than those given in Table 1.

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Annex A (informative) Examples of titration end point determination

Key Y the voltage of Ag electrode detected during titration, in mV; X the volume of consumption of the standardized AgNO 3 titrant during titration, in ml; 1 titration curve; 2 first derivative of the titration curve drawn by voltage of electrode versus volume of titrant consumption. Figure A.1 — Automatic titration endpoint recognition using dynamic titration (DET U) mode on Methohm Titrodo 905

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Figure A.2 — Example of a titration curve from Mettler autotitrator

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Collaborative Study Protocol Part 1:

All participant laboratories received two practice samples and were asked to analyze each of them in duplicate (two extractions from each reconstituted sample). Any deviation from the written method was to be recorded and reported. Results were communicated to the Study Director using the electronic template provided with the protocol. The participants were asked to report chloride results, titration volumes for standards and samples, and the different masses used during sample preparation. After review by the Study Director, results within a range of expected levels (average ± 2 x standard deviation of reproducibility) identified the laboratories qualified for the second part of the study. Part 2: All qualified laboratories received approval for the second phase of the study containing fifty coded products, corresponding to twenty five products in blind duplicates. The samples were a set of infant formula and adult nutritional products plus milk based matrixes, representing a wide range of commercially available products. The laboratories were asked to analyze all the samples (single extraction from each liquid or reconstituted powder) or milk based matrix.. The blind duplicates were assigned to be analyzed the same day. Results were communicated to the Study Director using an electronic template similar to the one used in Part 1. Two of the samples (Child Formula Powder Milk Based - Placebo and Adult Nutritional RTF High Fat -Placebo) failed to meet acceptance criteria during Part 2. Due to a suspicion on the integrity of the samples (some laboratories reported product spoilage), and the fact that they have reached their expiration date; they were excluded from review.

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Statistical evaluation: After data collection, outliers were detected using Cochran and Grubbs tests. Average concentrations, standard deviations and relative standard deviations of repeatability (S r and RSD r ) were estimated from the blind duplicates. Standard deviations of reproducibility (S R ), relative standard deviations of reproducibility (RSD R ) and HorRat (Horwitz ratio) values (RSD R /predicted RSD R ) were also estimated. Details on statistical analysis can be found in the “Appendix D: Guidelines for Collaborative Study Procedures To Validate Characteristics of a Method of Analysis” of the Official Methods of Analysis of AOAC [4]. Results and Discussion Twenty six laboratories initially agreed to participate to the collaborative study. One laboratory dropped out during Part 1 due to time availability. Nine laboratories were un- able to receive samples due to customs. The remaining 16 laboratories set up the method as described in the protocol for both Part 1 and 2. Four laboratories used the additional protein precipitation steps (F. d-f) based on regional requirements. After data compilation, average and standard deviation of repeatability and reproducibility were calculated. Conclusions The precision figures obtained during this collaborative study show that method 2016.03 complies with the requirements set in the corresponding SMPR, and thus, it is fit for purpose for the analysis of chloride in milk, milk products, infant formula and adult/pediatric nutritionals. These data were submitted to the SPIFAN ERP (Expert Review

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Panel) for review. The ERP determined that the data presented met the SMPR set by SPIFAN and hence recommended the method to be granted Final Action status.

Acknowledgments The authors would like to specially thank Covance Laboratories (Madison, WI, USA) and MUVAkempten (Kempten, Germany) for the dispatch of the samples to the participants. The authors greatly thank the following participating laboratories:

Country

Laboratory

Nestle Shah Alam Nestlé Malaysia

Malaysia

Nestle - São Paulo

Brazil

ASKEATON,Wyeth Nutrition

Ireland

FrieslandCampina Laboratory and quality services

Netherlands

Nestle – Dublin, OH

USA

Covance NCFS Singapore Mead Johnson Nutrition

Singapore

USA

Eurofins CLF Specialised Nutrition Testing Services GmbH Eurofins Nutrition Analysis Center -Des Moines IA

Germany

USA

Ausnutria Hyproca

Netherlands Netherlands New Zealand

Qlip

Eurofins New Zealand Laboratory Services Ltd.

Guangzhou Quality Supervision and Testing Institute(GQT) Shandong Technical Center of Inspection&Quarantine

China China

Analysis Center Laboratory of Inner Mongolia Mengniu Dairy (Group) Co., Ltd. Technical Center For Animal Plant And Food Inspection And Quarantine in Shanghai Entry-Exit Inspection and Quarantine Bureau

China

China

Inner Mongolia Yili Industrial Group Co.,Ltd.

China

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2016.03 (Nov. 2017) - Chlor-02/04 Final Action Review FOR ERP USE ONLY DO NOT DISTRIBUTE

Summarized results of the full MLT set of samples can be found in Table 1 below.

≤ 2 %

≤ 4 %

Requirements (SMPR 2014.015)

Mean

RSD r (%)

RSD R (%)

HorRat (%)

Sample

n

Adult Nutritional Powder Low Fat

16 10 16 16 16 15 14 15 15 15 14 13 16 16 11 11 16 16 12 12 10 13 11 8

385.5 162.6 154.2 701.1 478.6 510.3 413.7 330.4 386.1 380.2 357.3 443.6 372.5 42.4

0.58 1.05 1.94 0.72 0.87 0.94 1.12 1.76 1.12 0.73 0.61 1.00 0.44 0.83 1.49 0.52 1.68 1.28 0.56 1.37 0.68 1.5 1.12

3.46 2.54 3.01 2.44 3.76 2.34 2.01 3.49 2.54 4.08 2.27 3.67 3.56 3.90 3.50 2.24 3.47 3.85 2.57 2.72 3.57 2.8 3.89

0.75 0.48 0.57 0.58 0.84 0.36 0.45 0.76 0.54 0.88 0.49 0.78 0.79 0.84 1.09 0.60 3.11 0.49 0.48 0.75 0.64 0.74 0.61 0.48

Adult Nutritional RTF High Fat

Adult Nutritional RTF High Protein

SRM 1849a

Toddler Formula Powder Milk Based

Infant Formula RTF Milk Based

Infant Formula Powder Soy Based Infant Formula Powder Milk Based Infant Formula Powder FOS/GOS Based Infant Formula Powder Part Hyd Soy Based Infant Formula Powder Part Hyd Milk Based

Infant Formula Powder Milk Based Child Formula Powder Milk Based

Infant Elemental Powder

Adult Nutritional RTF High Fat – Placebo

35.4 40.2 32.4 22.5

3.14*

7.21*

Adult Nutritional RTF High Protein – Placebo Child Formula Powder Milk Based – Placebo

12.4*

20.8*

Infant Formula RTF Milk Based – Placebo

Infant Elemental Powder – Placebo

358.9 375.5 562.2 169.5 711.6

IDF – Butter IDF- Cheese

IDF - Whey Protein Concentrate

IDF – Whole Milk Powder

IDF – Whole Milk

95.3

IDF – Whey Powder

9

281.3

0.74 _______ _______ _________

n = number of laboratories (after outliers removal) RSD r : relative standard deviation of repeatability RSD R : relative standard deviation of reproducibility

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2016.03 (Nov. 2017) - Chlor-02/04 Final Action Review FOR ERP USE ONLY DO NOT DISTRIBUTE

References [1] GB/T 601-2002 Chemical reagent – Preparations of Standard Volumetric Solutions [2] ISO 1042:1998, Laboratory glassware – One-mark volumetric flasks [3] ISO 5725-2:1994, Accuracy (trueness and precision) of measurement methods and results — Part 2: Basic method for the determination of repeatability and reproducibility of a standard measurement method [4] AOAC INTERNATIONAL. AOAC Official Methods Program, Associate Referee’s Manual on development, Study, Review, and Approval Process . Part IV AOAC Guidelines for Collaborative Studies, 1995, pp. 23–51 [5] AOAC SMPR 2014.015 - Standard Method Performance Requirements for Determination of Chloride in Infant and Adult/Pediatric Nutritional Formula [6] AOAC Official Method 2015.07, Chloride in infant formula and adult/pediatric nutritional formula: Potentiometric titration method, 1 st action 2015 [7] AOAC Official Method 2015.08, Chloride in infant formula and adult/pediatric formula: Potentiometry, 1 st action 2015 [8] ISO 15648 | IDF 179 - Butter — Determination of salt content —Potentiometric method [9] ISO 5943 | IDF 88 - Cheese and processed cheese products — Determination of chloride content Potentiometric titration method

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(n) Disposable syringe .—3 mL, with handspike and 0.45 μm disposable syringe filter. C. Chemicals and Reagents (a) Water, purified .—>18MΩ (EMD Millipore Corp., Billerica, MA, USA), or equivalent. (b) Sodium chloride (NaCl) .—Certified reference material for titrimetry, ≥99.5%, certified by the Federal Institute for Materials Research and Testing (Berlin, Germany) according to ISO 17025 (Cat. No. 71387; Sigma-Aldrich, St. Louis, MO, USA), or equivalent. (c) Silver nitrate (AgNO 3 ) .—Meets analytical reagent specification of the European Pharmacopoeia (Reag. Ph. Eur.), British Pharmacopoeia, and the United States Pharmacopeia (USP); assay 99.8–100.5% (Cat. No. 10220; Sigma-Aldrich); or equivalent. (d) Potassium ferrocyanide trihydrate [K 4 Fe(CN) 6 · 3H 2 O] .— Grade puriss p.a., American Chemical Society (ACS), International Organization for Standardization (ISO), and Reag. Ph. Eur.; ≥99% (Cat. No. 31524; Sigma-Aldrich); or equivalent. (e) Zinc acetate dihydrate [Zn(CH 3 COO) 2 2H 2 O] .—Grade ACS and puriss p.a., ≥99.0% (Cat. No. 96459; Sigma-Aldrich), or equivalent. (f) Nitric acid (HNO 3 ) .—Minimum 65% puriss p.a. (Cat. No. 100452; Merck, Darmstadt, Germany), or equivalent. (g) Standardized AgNO 3 solution .—0.1 mol/L (0.1 N) Titripur ® grade Reag. Ph. Eur. and USP (Cat. No. 1.09081.1000 or EM3214-1; Merck, Darmstadt, Germany) or ready-to-use standardized titrant prepared according to GB/T 601-2002 (1), or equivalent. (h) Sodium chloride ( NaCl) standardized solution, 0.1 M .—Cat. No. 35616 (Alfa Aesar, Ward Hill, MA, USA), or equivalent. (i) Glacial acetic acid, 100% . — Anhydrous for analysis; EMSURE ® grade ACS, ISO, and Reag. Ph. Eur. (Cat. No. 100063; Merck); or equivalent (j) Potassium nitrate (KNO 3 ) .—For analysis, EMSURE ® grade ISO and Reag. Ph. Eur. (Cat. No. 105063; Merck), or equivalent. (k) Acetone.— For cleaning of the electrode (Cat. No. 010-4; Honeywell, Muskegon, MI, USA), or equivalent. (l) Dimethylpolysiloxane .—Defoaming agent (Cat. No. DMPS2C; Sigma-Aldrich), or equivalent. D. Preparation of Solutions (a) Standardized AgNO 3 solution, 0.1 M. —If ready-to-use AgNO 3 standard solution, C ( c ), is not available, then weigh 16.9890 ± 0.0005 g AgNO 3 previously dried for 2 h at 120 ± 2°C. Dissolve in water and dilute to volume in a 1000 mL volumetric flask. Store in a brown reagent bottle. Note 1 : After preparation, check the titer by titration of 5.0 mLwith exactly 0.1 M NaCl solution. For either commercial or in-house solution, verify the titer on a regular basis. Note 2 : The standardized AgNO 3 solution must be protected from light, and can be stored for up to 2 months. (b) Sodium chloride solution, 0.1 M .—If ready-to-use NaCl standard solution is not available, weigh 5.8440 ± 0.0005 g NaCl, C ( b ), previously dried for 2 h at 110 ± 2°C. Dissolve in water and dilute to volume in a 1000 mL volumetric flask. Note : This solution is stable for up to 1 month. (c) Precipitating solution (Carrez) I .—Weigh 106 g potassium ferrocyanide trihydrate, C ( d ), dissolve in an appropriate amount of water, and transfer to a 1000 mL volumetric flask. Dilute to volume using water. Mix well.

AOAC Official Method 2016.03 Chloride in Milk, Milk Powder, Whey Powder, Infant Formula, and Adult Nutritionals Potentiometric Titration Method First Action 2016 [Applicable to the determination of chloride in milk, milk powder, whey powder, infant formula, and adult nutritionals by potentiometry, with an analytical range of 0.35–1060 mg chloride/100 g reconstituted product or ready-to-feed (RTF) liquids.] Caution : Consult Material Safety Data Sheets for all substances A. Principle Reconstitute powder samples by dissolving 25 g powder sample in 200 g warm water (40°C); RTF products are ready to use as they are. Precipitate proteins by adding precipitation solutions I and II, and then centrifuge. Acidify the supernatant with nitric acid solution. Titrate chloride ions against standardized silver nitrate solution (0.1 M), potentiometrically using a silver electrode to detect the end point. B. Apparatus Common laboratory equipment and, in particular, the following: (a) Analytical balance .—Precision to 0.1 mg. (b) Centrifuge .—Tabletop with rotor for 50 mL conical tubes, capable of operating at ≥12000 × g . (c) Centrifuge tubes .—50 mL, conical, polypropylene. (d) Pipets .—1, 10, 20, 50, and 100 mL, Class Aglass volumetric or automatic (Eppendorf or equivalent). (e) One-mark volumetric flasks .—50, 100, 500, and 1000 mL, glass, Class A. (f) Graduated cylinders .—25, 100, and 500 mL, glass. (g) Autosampler beaker .—e.g., 120 mL, depending on the titrator used. (h) pH Meter/mV meter .—With a scale covering ±700 mV, and a 20 or 25 mL buret (Mettler-Toledo, Columbus, OH, USA), or equivalent. (i) Automatic titrator .—Autosampler (Mettler-Toledo Rondo Tower) and motorized piston buret (Mettler-Toledo T50) with remote-control dispensing and filling (Mettler-Toledo LabX 3.1 software) or the Metrohm 862 Compact Titrosampler with 800 Dosino and 10 mL Exchange Unit (Riverview, FL, USA), or equivalent. Alternatively, a semiautomated (e.g., Metrohm Titrado 905/907, with Metrohm tiamo TM software, or equivalent) or a manual titrator (using a buret with accuracy of 0.01 mL) may be used. (j) Combined ring silver electrode .—e.g., Mettler DM 141 or DMi145-SC, MetrohmAg Titrode No. 6.0430.100S, or equivalent. Alternatively, a silver electrode with reference electrode may be used. (k) Magnetic stirrer .—Heidolph MR 3000 (Schwabach, Germany) or Metrohm 804Ti Stand with 802 Rod Stirrer, or equivalent. (l) Water bath .—Capable of warming water to 40°C, or equivalent. (m) Ultrasonic cleaner .—Model AS2060B (Tianjin Automatic Science Instrument Co. Ltd, Nanyang, China), or equivalent. that are required and considered hazardous. Follow all laboratory safety precautions and wear proper personal protective equipment.

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(d) Precipitating solution (Carrez) II .—Weigh 220 g zinc acetate dihydrate, C ( e ), and transfer to a 1000 mL volumetric flask. Dissolve with an appropriate amount of water and add 30 mL glacial acetic acid, C ( i ). Dilute to volume using water. Mix well. (e) Nitric acid solution .—With care, add 100 mL concentrated nitric acid, C ( f ), to 300 mL water. Mix well. (f) Wash solution .—According to autosampler/titrator manufacturer’s instructions [e.g., acetone or nitric acid solution ( see e , above)], or other. (g) AgNO 3 solution, 0.025 M (optional) .—Into a 1000 mL volumetric flask, pipet 250 mL 0.1 M AgNO 3 solution, C ( g ), or D ( a ). Dilute to volume with water. Note : Prepare freshly before use, and then check the titer by titration of 25 mL against 0.025 M NaCl solution. (h) NaCl solution, 0.025 M (optional) .—Into a 100 mL volumetric flask, pipet 25 mL 0.1 M NaCl solution, D ( b ). Dilute to volume with water. Note : Prepare freshly before use. (i) KNO 3 solution, 1 M. —Weigh 10.11 g potassium nitrate, C ( j ), into a 100 mL volumetric flask. Add about 80 mLwater and place the flask in an ultrasonic cleaner, B ( m ), to dissolve with ultrasound and heating until dissolved thoroughly. Cool down to room temperature and dilute to volume with water. Filter using a 0.45 μm membrane disposable syringe before use. E. Sample Preparation (a) Milk product, infant formula, and adult/pediatric nutritional. —Mix well to ensure that sample is homogeneous. (b) Powder samples. —Reconstitute by dissolving 25 g powder sample in 200 g warm water (40°C). F. Extraction For high-protein samples requiring additional protein precipitation beyond that accomplished by addition of nitric acid solution, perform F ( a )–( c ), below. Otherwise, begin with F ( d ). (a)  Weigh an appropriate aliquot of RTF or reconstituted powder (e.g., 25 g, accurate to 0.1mg) into a 50mLcentrifuge tube. Note : For samples with a high chloride content, weigh a smaller test portion, e.g., 5 g reconstituted or RTF product. (b)  Transfer 2.5 mL precipitating solution I, D ( c ), and 2.5 mL precipitating solution II, D ( d ), into the tube. Dilute to 50 mL with water. Mix well. If foam impacts the constant volume, then one or two drops of defoaming agent, C ( l ), should be added. (c)  Centrifuge at 12 000 × g for 5 min at 4°C, and then equilibrate to room temperature. (d)  Accurately transfer either 10 mL supernatant from F ( a )–( c ) or weigh an appropriate aliquot of RTF or reconstituted powder (e.g., 25 g, accurate to 0.1 mg). Note : For samples with a high chloride content, weigh a smaller test portion, e.g., 5 g reconstituted or RTF product. Into a 120 mL sample beaker or autosampler cup, add 5 mL nitric acid solution, D ( e ), and 50 mL water before titration. Add a magnetic stirring rod (if the titrator does not have a built-in rod stirrer). Place the autosampler cup or beaker onto a magnetic stirrer and stir until dissolved or finely suspended. (e)  The pH of the test solution must be below 1.5. If in doubt, check pH by means of a pH meter and, if necessary, add a little more nitric acid solution, D ( e ). G. Instrument Operating Conditions (a)  Check and maintenance of the combined silver electrode .— Rinse electrode with deionized water and wipe before use.

Renew the electrolyte with 1 M KNO 3 manufacturer’s recommendations. If fat sticks to the electrodes during a series of analyses, then eliminate it by briefly immersing the electrode in acetone. The silver electrode must be stored in 1 M KNO 3 , D ( i ), after appropriate cleaning. Note : Instead of the combined silver electrode, separate silver and reference electrodes may also be used. (b)  Titration .—Connect the combined silver electrode to the titration apparatus according to the manufacturer’s indications. Ensure that the titration vessels are correctly placed on the autosampler and that there are enough reagents: both nitric acid solution [if added automatically, D ( e )] and 0.1 M AgNO 3 , C ( g ) or D ( a ). If no autosampler is available, then place the sample solutions manually under the titration equipment. Put the wash solution, D ( f ), in the washing position if an autosampler used. Ensure that the volume of wash solution is adequate. Under continuous stirring and without touching the electrode, titrate the sample solution automatically with 0.1 M standardized silver nitrate solution, C ( g ) or D ( a ), up to the end potential. The consumption of 0.1 M of silver nitrate solution, C ( g ) or D ( a ), should be recorded automatically and can be read from the titrator software or documented in the titrator operating records. For manual titration, using a buret, add 0.1 M standardized silver nitrate solution, C ( g ) or D ( a ), until the end potential has nearly been met. Continue to titrate slowly until the end point is met, as observed by the two small additions (about 0.05 mL) of silver nitrate solution. See Figures 2016.03A and 2016.03B . (c)  Special case: determination of very low amounts of chloride. —When determining low chloride concentrations such as found in desalted whey powder, for greater precision, it is preferable to use a standardized 0.025 mol/LAgNO 3 , D ( g ), solution for the titration. Figure 2016.03A. Automatic titration end point recognition using the dynamic titration mode on a Methohm Titrodo 905 titrator. U[mV], voltage of Ag electrode detected during titration; V[mV], volume of consumption of the standardized AgNO 3 titrant during titration; ERC, first derivative of the titration curve drawn by voltage of electrode versus volume of titrant consumption. , D ( i ), periodically per the

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(d)  Blank test: determination of reagent background content of chloride .—Perform a blank test using reagents, substituting water, C ( a ), for the sample portion. The titrant consumption of the blank test obtained at the end point should be less than 0.05 mL when using the 0.1 M standardized silver nitrate, and less than 0.2 mL when using the 0.025 M standardized silver nitrate. Otherwise, check the reagents and water involved into the procedures and then perform the blank test again until the criterion is achieved. H. System Suitability Test Perform a system suitability test prior to use. (a)  Transfer 5 mL NaCl solution, C ( h ) or D ( b ), into a 120 mL sample beaker. If 0.025 MAgNO 3 titrant is required, then use 1 mL NaCl solution. (b)  Add 5 mL nitric acid solution, D ( e ), and 50 mL water. (c)  Place the washing solution, D ( f ), in the washing position of the auto sampler. (d)  Titrate with 0.1 M standardized silver nitrate solution, C ( g ) or D ( a ), up to the end potential using an automatic, semiautomatic, or manual titrator. (e)  Repeat in quadruplicate. (f)  Calculate concentration of the silver nitrate solution according to I ( a ). The difference between the calculated concentration and the certified value should be within 0.5%. If it is outside the acceptance value, check the experimental procedures and titration system. If the issue is not resolved, then use fresh standardized

silver nitrate. If fresh standardized silver nitrate does not provide an acceptable result, replace the electrolyte of the electrode and check the operating condition of the dosing unit. I. Calculations (a)  Calculate silver nitrate concentration for system suitability verification, and report to four decimal places .—If using in-house made standardized AgNO 3 solution [ D ( a )], = × × V ( / ) 5.844 10 1 1 SNC mol L m where SNC is the silver nitrate concentration (mol/L), m 1 is the weight (mg) of sodium chloride in 5 mL or 1 mL standard solution, D ( b ), V 1 is the volume (mL) of 0.1 M or 0.02 MAgNO 3 consumed at titration end point, 5.844 is the sodium chloride weight (μg) corresponding to 1 mL of 0.1 mol/L AgNO 3 , and 10 is the mass conversion from titer to the concentration of titrant. Or, if using purchased standard grade 0.10 M NaCl, C ( g ), = × V ( / ) 0.1 3 1 SNC mol L V where SNC is the silver nitrate concentration (mol/L), V 3 is the volume (mL) of purchased standard grade 0.10 M sodium chloride added, and V 1 is the volume (mL) of 0.1 M or 0.025 M AgNO 3 consumed at titration end point.

Figure 2016.03B. Example of titration curve from a Mettler autotitrator.

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