AOAC Methods in Codex STAN 234 (Preliminary Methods Review)

~,ltCJ AOAC INTERNATIO Al.

AOAC Official Methods Board Working Group on Codex STAN 234 Methods            PRELIMINARY METHODS FOR REVIEW

~fl\:;J AOAC INTERNATIO AL

OFFICIAL METHODS BOARD WORKING GROUP ON CODEX STAN 234 METHODS  PRELIMINARY METHODS FOR REVIEW Table of Contents 

OMA Method:  AOAC 991.20 – Nitrogen (Total) in Milk Method 

3 

Manuscript for AOAC 991.20 

5 

OMB Method:  AOAC 982.23 – Cadmium and Lead in Food Method 

16 

Manuscript for AOAC 982.23  

18  38 

OMA Method:  AOAC 960.40 – Copper in Food 

39 

Method Manuscript for AOAC 960.40 

45 

OMA Method:  AOAC 920.116 – Moisture in Butter (no manuscript published)

D. Preparation of Test Solution Add 15.00 g K 2 SO 4 , 1 mLCuSO 4 O catalyst solution, and 8–10 boiling chips to digestion flask.Warmmilk to 38 ° ± 1°C. Mixmilk as in 925.21 ( see 33.2.02). Weigh warm sample (5 ± 0.1 mL) and immediately place in digestion flask. ( Note : Weights must be recorded to nearest 0.0001 g.) Add 25 mL H 2 SO 4 , rinsing any milk on neck of flask into bulb. Flask may be stoppered and held for digestion at later time. Digest and distill a blank (all reagents and no test product) each day. E. Determination ( a ) Digestion burner setting. —Conduct digestion over heating device that can be adjusted to bring 250 mL H 2 O at 25°C to rolling boil in ca 5–6 min. To determine maximum heater setting to be used during digestion, preheat 10 min (gas) or 30 min (electric) at burner setting to be evaluated. Add 3 or 4 boiling chips to 250 mL H 2 O at 25°C and place flask on preheated burner. Determine heater setting that brings water from 25°C to rolling boil in 5–6 min on each burner. This is maximumburner setting to be used during digestion. ( b ) Digestion. —Place flask in inclined position with fume ejection system on. Start on setting low enough so that test portion does not foam up neck of Kjeldahl flask. Digest at least 20 min or until white fumes appear in flask. Next, increase burner setting half way to maximum burner setting determined in ( a ) and heat for 15 min. Next, increase heat to maximum setting determined in ( a ). When digest clears (clear with light blue–green color), continue to boil 1–1.5 h at maximum setting (total time ca 1.8–2.25 h). To determine specific boil time needed for analysis conditions in laboratory, select a high protein, high fat milk test sample and determine protein content using different boil times (1–1.5 h) after clearing. Mean protein test increases with increasing (0–1.5 h) boil time, becomes constant, and then decreases when boil time is too long. Select boil time that yields maximum protein test. At end of digestion, digest should be clear and free of undigested material. Cool acid digest to room temperature (ca 25 min). Cooled digest should be liquid or liquid with few small crystals. (Large amount of crystallization before addition of water indicates too little residual H 2 SO 4 at end of digestion and can result in low test values.) After digest is cooled to room temperature, add 300 mL H 2 O to flask and swirl to mix (for 800 mL flasks add 400 mL H 2 O). When room temperature water is added some crystals may form and then go into solution; this is normal. Let mixture cool to room temperature before distillation. Flasks can be stoppered for distillation at later time. ( c ) Distillation. —Turn on condenser water. Add 50 mLH 3 BO 3 solution with indicator to graduated 500 mL Erlenmeyer titration flask and place flask under condenser tip so that tip is well below H 3 BO 3 solution surface. To room temperature diluted digest, carefully add 75 mL 50% NaOH down sidewall of Kjeldahl flask with no agitation. NaOH forms clear layer under the diluted digest. Immediately connect flask to distillation bulb on condenser. Vigorously swirl flask to mix contents thoroughly; heat until all NH 3 has been distilled ( ³ 150 mL distillate; ³ 200 mL total volume). Do not leave distillation unattended. Flasks (500 mL) may bump at this point. Lower receiving flask and let liquid drain from condenser tip. Turn off distillation heater. Titrate H 3 BO 3 receiving solution with standard 0.1000M HCl solution to first trace of pink. Lighted stir plate may aid visualization of end point. Record mL HCl to at least nearest 0.05 mL. × 5H 2

33.2.11

AOAC Official Method 991.20 Nitrogen (Total) in Milk

Kjeldahl Methods First Action 1991 Final Action 1994

IDF–ISO–AOAC Method Results of the interlaboratory study supporting acceptance of the method [expressed on a protein basis (N ´ 6.38)]: s r = 0.014; s R = 0.017; RSD r = 0.385%; RSD R = 0.504% A. Principle Milk is digested in H 2 SO 4 , using CuSO 4 × 5H 2 O as catalyst with K 2 SO 4 as boiling point elevator, to release nitrogen from protein and retain nitrogen as ammonium salt. Concentrated NaOH is added to release NH 3 , which is distilled, collected in H 3 BO 3 solution, and titrated. B. Apparatus ( a ) Digestion flasks. —Kjeldahl. Hard, moderately thick, well-annealed glass. Total capacity ca 500 or 800 mL. ( b ) Distillation flasks. —Same Kjeldahl flask as in ( a ), fitted with rubber stopper through which passes lower end of efficient rubber bulb or trap to prevent mechanical carryover of NaOH during distillation. Connect upper end of bulb to condenser tube by rubber tubing. Use graduated 500 mL Erlenmeyer titration flask to collect distillate. Trap outlet of condenser in manner to ensure complete absorption of NH 3 distilled into boric acid solution. ( c ) Digestion/distillation system. —Traditional apparatus with adjustable controls for individual flasks. ( d ) Titration buret. —50 mL. Class A or equivalent. C. Reagents ( a ) Sulfuric acid. —95–98% H 2 SO 4 . Nitrogen free. ( b ) Copper catalyst solution. —CuSO 4 × 5H 2 O. Nitrogen free. Prepare solution 0.05 g/mL H 2 O. ( c ) Potassium sulfate. —K 2 SO 4 . Nitrogen free. ( d ) Sodiumhydroxide solution. —50% (w/w) nitrate-free NaOH. ( e ) Boiling chips. —Mesh size 10 suggested. High purity, amphoteric alundum granules, plain. ( f ) Methyl red/bromocresol green indicator solution. —Dissolve 0.2 g methyl red and dilute to 100 mL in 95% ethanol. Dissolve 1.0 g bromocresol green and dilute to 500 mL in 95% ethanol. Mix 1 part methyl red solution with 5 parts bromocresol green solution (combine all of both solutions). ( g ) Boric acid solution. —4%, with indicator. Dissolve 40 g H 3 BO 3 and dilute to 1 L in water and add 3 mL methyl red/bromocresol green indicator solution, ( f ). Solution will be light orange color. ( h ) Hydrochloric acid standard solution. —0.1000M. Prepare as in 936.15 ( see A.1.06) or use premade solution of certified specification range 0.0995–0.1005M and use 0.1000M for calculation. ( i ) Ammonium sulfate. —99.9% (NH 4 ) 2 SO 4 . ( j ) Tryptophan or lysine hydrochloride. —99% C 11 H 12 N 2 O 2 or C 6 H 15 ClN 2 O 2 . ( k ) Sucrose. —Nitrogen free. Traditional Method

ã 2005 AOAC INTERNATIONAL

Codex Trial Method Review

F. Nitrogen Recovery Verification Run nitrogen recoveries to check accuracy of procedure and equipment. ( a ) Nitrogen loss. —Use 0.12 g ammonium sulfate and 0.85 g sucrose per flask. Add all other reagents as stated in D . Digest and distill under same conditions as for a milk test portion. Recoveries shall be at least 99%. ( b ) Digestion efficiency. —Use 0.16 g lysine hydrochloride or 0.18 g tryptophan, with 0.67 g sucrose per flask. Add all other reagents as stated in D . Digest and distill under same conditions as for a milk test portion. Recoveries shall be at least 98%. G. Calculations Calculate results as follows: = mL HCl titrant used for test portion and blank, respectively; M = molarity of HCl solution; and W = test portion weight, g. Multiply percent nitrogen by factor 6.38, to calculate percent “protein.” This is “protein” on a total nitrogen basis. Maximum recommended difference between duplicates is 0.03% “protein.” H. Repeatability and Reproducibility Values For results of interlaboratory study parameters obtained in collaborative study of this method, r value = 0.038 and R value = 0.049 (expressed on protein basis [N ´ 6.38]). I. Apparatus ( a ) Digestion block. —Aluminum alloy block or equivalent apparatus, with adjustable temperature control and device for measuring block temperature. ( b ) Digestion block tubes. —250 mL capacity. ( c ) Distillation unit. —For steam distillation. To accept 250 mL digestion tubes and 500 mL titration flasks. ( d ) Distillation titration flask. —500 mL graduated Erlenmeyer titration flask. ( e ) Titration buret. —50 mL. Class A or equivalent. J. Reagents See C ( a )–( k ). [ Note : 40% (w/w) NaOH may be used instead of 50% (w/w). Boiling chips should not be used if equipment manufacturer does not recommend such use.] K. Preparation of Test Solution Add 12 g K 2 SO 4 and 1mLCuSO 4 × 5H 2 O catalyst solution to digestion tube. Warm milk to 38 ° ± 1°C. Mix milk as in 925.21 ( see 33.2.02). Weighwarmtest portion (5 ± 0.1mL) and immediately place in digestion tube. ( Note : Weights must be recorded to nearest 0.0001 g.) Add 20 mL H 2 SO 4 . Tube may be stoppered and held for digestion at later time. Digest and distill a blank (all reagents and no test portion) each day. L. Determination ( a ) Digestion. —Set block at low initial temperature to control foaming (ca 180 ° –230°C). Place tubes with aspirator connected in block digestion; suction should be just enough to remove fumes. Nitrogen, % = 14007 . ( ) ´ - ´ V V M W s b where V s and V b Block Digestion/Steam Distillation Method

Digest 30 min or until white fumes develop. Increase temperature to 410 ° –430°C and digest until clear. It may be necessary to increase temperature gradually over ca 20 min to control foaming. Do not let foam within tube rise higher than ca 4–5 cm below surface of fume collection device inserted into top of tube. After digest clears (clear with light blue–green color), continue to boil (H 2 SO 4 must be boiling) for at least 1 h, total digestion time ca 1.75–2.5 h. To determine specific length of boil time needed for analysis conditions in your laboratory, select high protein, high fat milk test sample and determine protein content using different boil times (1–1.5 h) after clearing. Mean protein test increases with increasing (0–1.5 h) boil time, becomes constant, and then decreases when boil time is too long. Select boil time that yields maximum protein test. ( Note : Before removing hot tubes from block, make sure there is no condensate layer in aspirator manifold. If there is a liquid layer, increase aspiration to remove liquid.) At end of digestion, digest should be clear and free of undigested material. Cool digest to room temperature (ca 25 min). Cooled digest should be liquid or liquid with few small crystals at bottom of tube. (Excessive crystallization indicates too little residual H 2 SO 4 at end of digestion and may cause low results. To reduce acid loss during digestion, reduce fume aspiration rate.) After digest has cooled to room temperature, add 85mLH 2 O (blanksmay require 100mL) to each tube, swirl tomix, and let cool to room temperature.When room temperature water is added, some crystalsmay formand then go into solution; this is normal. Tubes can be stoppered for distillation at later time. ( b ) Distillation. —Place 50% (or 40%) NaOH in alkali tank of distillation unit. Adjust volume dispensed to 55 mL (65 mL for 40% NaOH). Attach digestion tube containing diluted digest to distillation unit. Place graduated 500 mL Erlenmeyer titration flask containing 50 mL H 3 BO 3 solution with indicator on receiving platform, with tube from condenser extending below surface of H 3 BO 3 solution. Steam-distill until ³ 150 mL distillate is collected ( ³ 200 mL total volume). Remove receiving flask. Titrate H 3 BO 3 receiving solution with standard 0.1000MHCl to first trace of pink. Lighted stir plate may aid visualization of end point. RecordmLHCl to at least nearest 0.05 mL. M. Nitrogen Recovery Verification Run nitrogen recoveries to check accuracy of procedure and equipment. ( a ) Nitrogen loss. —Use 0.12 g ammonium sulfate and 0.85 g sucrose per flask. Add all other reagents as stated in Preparation of Test Solution , K . Digest and distill under same conditions as for a milk test portion. Recoveries shall be at least 99%. ( b ) Digestion efficiency. —Use 0.16 g lysine hydrochloride or 0.18 g tryptophan, with 0.67 g sucrose per flask. Add all other reagents as stated in Preparation of Test Solution , K . Digest and distill under same conditions as for a milk test portion. Recoveries shall be at least 98%. N. Calculations See G . O. Repeatability and Reproducibility Values For results of interlaboratory study parameters obtained in collaborative study of thismethod, r value =0.038 andRvalue =0.049. Reference: JAOAC 73 , 849(1990). Revised: March 1996

ã 2005 AOAC INTERNATIONAL Codex Trial Method Review

849

BARBANO ET At.: J. ASSOC. OFF ANAi.. CHEM. (VOi., 73. NO. 6, 1990)

D. H. Kleyn and L. Lengyel, Rutgers University, New Brunswick, NJ R. K. Stuckey, T. Cronau, J. Siebodnik, and J. Scott, Indiana State Board of Health, Indianapolis, lN T. Way, C. Mayfield, and C. Strayer, Applied Microbiolo– gy Services, Inc., College Station, TX H. M. Wehr and B. Burwell, Oregon Dept of Agriculture, Salem, OR REFERENCES (I) Kay, H. D., & Graham, W. R. (1935)}. Dairy Res. 6, 191-203 (2) Official Methods ofAnalysis ( 1984) 14th Ed. ancl { 1990) 15th Ed., AOAC, Arlington, VA (3) Rocco,R.M. ( 1990)J.FoodProt 53, 588- 591

be conducted to assess the suitability ofthis metbod for other dairy products including cheese, whey, and cream.

Acknowledgment• The. cooperationof the following collaborators is gratefully acknowledged: R. L. Bradley, Jr, M. Lundberg, and J. Beyer, University llf Wisconsin, Madison, WI D. Elliott and B. Thornhill, Florida Dept of Agriculture, Winter Haven, FL L Hensel, Mid-America Dairymen, Inc., Winsted, MN J. Jaworski, R. Cyr, and L. Justis, Vermont Dept of Agri– culture, Montpelier. VT

Kjeldahl Method for Determination of Total Nitrogen Content of Milk: Collaborative Study

DAVfD M. BARBANO and JENNY L. CLARK Cornell University, Department ofFood Science, Ithaca, NY 14853 CHAPMAN E. DUNHAM and .J. RICHARD FLEMING' Texas Milk Markel, PO Box I 10939, Carrolflon, TX 75011

Collaborating Laboratories: Cornell University and Northeast Dairy Herd Improvement Cooperative; and laboratories operated by or under contract to the following Federal Milk Markets: Chicago Regional; Eastern Ohio/Western Pennsylvania; Greater Kansas City; New England; Texas; Upper Midwest

protein determination that accurately mellsures protein . ln· frared milk analyzers, now in commercial use by the dairy industry, can be calibrated to predict the protein content of milk [972.16, 15th Ed. (l)] based on infrared light absor– bance at 6.465 ,um wavelength by the N-H bonds within the protein. Data from an accurate reference method for milk protein determinlltion is necessary for proper calibration of infrared milk anaJyzers. The Kjeldahl method measures nitrogen and from the nitrogen content of a sample the protein content can be estimated. The Kjeldabl method has been widely studied (2- 14). Many researchers have attempted to substitute reagents (3- 7, IO, 11), vary reagent quantities (5, 8, 9, 12, 13), and optimize digestion parameters (8, 9, 12-14) to improve the test accuracy, decrease testing time, and eliminate hazardous chemicals (e.g., mercury) that have a detrimental impact on the environment. The Kjeldabl method uses an acid digestion to release bound organic nitrogen and retain it as ammonium sulfate Received for publication February 27, 1990. This report was prcsen1ed nt lhc 103rd AOAC Annual International Meet– ing, September 25-21!, 1989. a1 St. Loui~, MO. The report has been evaluated by the General Referee and the Cu111mit1ce Statistician and reviewed by the Committee on Foods I. The method was approved interim official first action by the Chairman of the Official Methods Board a nd was adopted official first action at the 104th AOAC Annual International Meeting, September 9-13. 1990, a1 New Orlcuns, LA. Assoc iation actions will be published in " Change, in Offi-.;ial Methods of Analysis'' ( 1991) J. Assoc. Off Anal. Chm,. 74, January/ February issue. 1 J. Richard Fleming ii, Chairman. Test Procedures Commi\tec of the Feder– al Milk Marketing Orders.

Codex Trial Method Review Dairy farmers in some regions of the United States are paid on the basis of both the fat and protein contents of their milk or receive bonus payments for high milk protein content. Thus, it is very important to have a reference method for milk Amacro-KJeldahl ptocedure using a copper catalyst tor de– tentllnatlon of milk total nitrogen wa1 developed for both tradltlonal and block dlgeatot/steam dl1llller equipment, and the performance was evaluated by collaboratlve study. In the fll'1t trlal of the collaborative study, 9 pairs Of bllnd duplicate mllk 1amplee wet• analyzed for total nitrogen and total nHrogen was conver1,ed to "protein" by using a factor of 6.38. Prot•ln content of mllk samples ranged from 3.088 lo 3.810%. In the first trlal, It\ and R values for the block dlgeators were Influenced slgnlftcantty by protein concentra– tion; 1 11 and R values were not Influenced by protein concen· tratlon tot traditional equipment. H was hypothesized that total digestion time for some block dlgeafors In the first trlal waa not sufficient tor high ptoteln mllk samplee. Thut, a NCond trlal was undertaken wflh bolling time after clearing lnereased by 0.5 h. In the second trial, none of the parame- • lera for reproduclblllty with either type of equipment were · ll!fluenced by protern concentration. It waa concluded that laboratory-to-laboratory dltrerences In tine voltage mar re· quire different total digestion times In different laboratorlea, particularly those using bloekdJge1tor1. The i

BARBANO ET Al.: J. ASSOC. OFF. Al"AL. CHEM, (VOL. 73, NO. 6, 1990)

850

dissolved in sulfuric acid, Other reactions in the digestion involve destruction of organic compounds with the release of CO 2 and H 2 0. During the digestion, some of the sulfuric acid is c-0nvertcd to KHS04 (when H2S0 4 reacts with Ki$04). Other reaction products are H20, S02, S, and O when H 2 S0 4 reacts with sodium thiosulfate or salicylic acid ( 15). If the digest crystallizes during or after digestion, it is an indication of too little residual acid. Too little acid at the end of digestion often results in low test results because there is insufficient acid to completely retain all nitrogen. It has been reported that nitrogen loss occurs as the digest approaches the solid state (13). After complete digestion, alkali is added to release gaseous ammonia, which is volatilized by distilla– tion and trapped by absorption in an acid collection solution during distillation. The amount of ammonia collected is then quantitated by titration. A modification of the original Kjeldahl method by Wil– farth in 1885 ( 15) used a metal catalyst to speed digestion. Two popular catalysts arc mercury anti copp~r. Scbwab and Schwab-Agallidis (2) determined Kjeldahl reaction kinetics on aniline with different catalysts. They concluded that mer– cury was superior to copper in catalyzing the Kjeldahl reac– tion. Copper did not have any perceptible catalytic activity on release of nitrogen from aoiline. However, in their re· search, potassium sulfate was not used to elevate the boiling point of the digestion mixture. Mercury is thought to be the better of the 2 catalysts on hard-to-digest materials (3- 6). However, in many studies, researchers did not optimize di– gestion conditions for copper; researchers simply substituted copper for mercury. Other studies that have optimized the digestion parameters for copper catalyst have found copper to be as effective as mercury in catalyzing tbe Kjeldahl reactions (7-9). The International Dairy Federation (JDF) has published a provisional method (16) using copper cata– lyst for Kjeldahl determination of milk protein, which is based on the results of an interlaboratory study ( 17). The concentration of copper used as a catalyst is low, to avoid formation of ammonia-copper complexes that can cause un– derestimatton of nitrogen (I 7). AOAC has adopted the cop– per catalyst in Kjel

been investigated over the years by chemical analysis and theoretical calculations based on milk protein amino acid sequences. One of the more recent investigations proposed that a conversion factor of 6.355 would be more accurate for milk protein (20, 21). It is important for anaJysts to rccogniz" the sensitivity 01 test results to measurement errors within the method. Th innuence of errors in measuremeut of volume of titranl sample weight, and normality of titrant for a 3.3% protein sample are as follows: an error of ±0.05 mL m measuremcn1 of titrant volnme will cause a deviation of 0.0089%protein an error of 0.0005 g in measurement of sample weight will cause a deviation of ±0.0003% protein, and an error or 0.0005N in the normality of the titrant will cause a deviatioJ of :1:0. 0164% protein. Thus, the most critical factor is th~ titrant normality. Of course, all these examples assume tha1 the sampJe was completely digested and no nitrogen was l

Codex Trial Method Review

851

BARBANO ET AL.: J. ASSOC. Off. ANA\... CHEM. {VOL. 73, NO. 6, 1990)

mined that the block digestor does oot work well with the larger volumes of reagents that the traditional unit can ac– commodate. Thus, for the 250 mL tubes on block digestors, 5 g sample, l 2 g potassium sulfate, and 20 ml sulfuric acid (still a 0.6 ratio of salt to acid) worked well. The fourth phase of preliminary work involved an evalua– tion of titanium oxide plus copper sulfate (in the Associate Referee's laboratory) as an alternative catalyst to copper sulfate, as suggested by Kane (10). Milk was digested with and without titanium oxide (0.6 g/tlask) at 2 different ratios of potassium sulfate to acid (0.6 vs 0.835). The fifth phase of preliminary work was a collaborative study in July 1988 using recovery solutions (ammonium sul– fate and glycine) with the optimized Kjeldahl procedure fo.r milk. Copper catalyst was used. The procedure was the same as the method in the present. paper with the following excep– tions: 1 blanlc was run instead of 2; indicator was added dropwise to each distillate before tit ration; hydrogen perox– ide was used to suppress foaming during digestion for both traditional and block equipment ( 5 mL 30% hydrogen permc· ide was add-Cd immediately before digestion); both tbe block and traditional procedures used 12 g potassium sulfate, 20 ml. sulfuric acid, and 55 mL 50% sodium hydroxide; and boiling time of digest was I h after clearing. Three different levels of both ammonium sulfate and glycine were analyzed in blind duplicate by 9 laboratories (6 laboratories had block digestors and steam distillers; 3 laboratories had traditional macro-.Kjeldahl djgestors and distillers). Conclusions trom Preliminary Work The first phase of the preliminary worlc (May and June 1986), with each laboratory using its own KjeJde.hl method and current equipment, indicated that the micro-Kjeldahl methods did not perform as well as the macro-Kjeldahl meth· ods. The standard deviation of the difference between dupli– cates for percent nitrogen recovery for the micro-Kjeldahl methods was about 5 times that of the macro-Kjeldahl sys-– terns (1 .90 vs 0.39). There was no difference in average nitrogen recovery for copper vs mercury catalyst. The second phase of the preliminary work indiceted that equivalent nitrogen recoveries could be obtained with either copper. or mercury catalysts. We concluded that with optimi– zation of the digestion parameters instead of merely substi– tuting one catalyst for another, comparable results could be obtained with mercury or copper catalysts. The third phase of the preliminary work found that 0.05 g copper sulfate pentahydrate/nask was sufficient to yield good test resuhs. The optimum ratio of potassium sulfate to sulfuric acid was determined to be between 0.56 and 0.67. A digestion time of I to 2 h after clearing was determined to give best test results for both the iraditional and block equip– ment. Excessive foaming can be a problem on the block digestor (on the traditional system, when the sample foams, one need only rotate the nask to alleviate the problem). Both predrying the milk and adding hydrogen peroxide to the liquid sample immediately before digestion were found to help reduce foaming in block digestor systems. Neither of these 2 approaches were incorporated into the final procc· dure. The first approach would add a minimum of 4 to 5 h to the test, which is not acceptable. The second approach (use of hydrogen peroxide) is somewhat hazardous. Boiling rods and antifoam ta blets did not reduce the foeming problems on the block digestor. Programming the block. digestor temperature (starting at a low temperature and increasing gradually to

The primary objectives of the present research were to optimize the macro-Kjeldahl method for milk for both tradi– tional and block digestion equipment, to determine method performance (s, and sR), and to compare performance of the block digestor/steam distiller system to the traditional Kjel– dahl system. Pre-Collaborative Study Work 'The first phase of preliminary work involved an evaluation of equipment and methods that different laboratory person· nel used for determination of total protein by the Kjeldahl method. In May 1986, 8 laboratories participated in the first phase of the study. These laboratories were sent 6 recovery solutions (3 ammonium sulfate and 3 gtycine) in blind dupli· cate. The laboratories were asked to determine percent nitro– gen by the method with whieh they currently tested milk samples. Four laboratories used copper catalyst, 3 laborato– ries used mercury catalyst, and 1 laboratory used selenium catalyst. Amounts of potassium sulfate aod sulfuric acid used in digestions varied widely among laboratories. Six. of the laboratories used a macro-Kjeldahl system (both block diges– tor and traditional equipment) and 2 laboratories used a micro-Kjeldahl system. In June 1986, this first phase study was replicated. The se~ond phase of preliminary work was undertaken in August 1986 to determine if there was a difference in test results with mercury or copper catalysts. The same 8 labora· tories that participated in the first-phase study participated in the s~nd phase of the study. Analysts were requested to use their current method. They were supplied with the cata– lyst that I.hey did not normally use (i.e., if they normally used mercury, they were supplied with copper and vice versa). The laboratory that used selenium was supplied with both copper and mercury catalysts (along with instructions for both), Digestion conditions (ratio of potassium sulfate to sulfuric acid) were not changed; catalysts were merely substituted for what was currently being used in each laboratory. Analysts were ser:lt 3 ammonium sulfate nitrogen recovery solutions (io blind duplicate) and 3 milks (in blind duplicate). The third phase of preliminary work optimized the diges– tion/distillation parameters for each equipment type with copper catalyst and established a well documented, clearly defined method for milk analysis. Optimum chemical qua nti– ties for digestion (catalyst, potassium sulfate. sulfuric acid, and sample) were determined, AOAC and (DF have re· quired a 5 g sample size, 15 g potassium sulfate, and 25 mL sulfuric acid for Kjeldahl nitrogen determination for milk. Although AOAC currently requires mercury catalyst for milk analysis, IDF has switched to the use of copper sulfate (1 ml of a 0.05 g/mL copper sulfate pentahydrat_e solution}. Optimization of the digestion procedure involved varying the amount of catalyst (0 to 0.84 g CuSO4-5H2O) and potassium sulfate/sulfuric acid ratio (0.3 to 0.7). Digestion time needed to be established (AOAC digests for ~30 min after clearing with mercury catalyst; IDF digests for 1.5 h after clearing with copper catalyst). Digestion times of 0.5 to 2.0 .h after clearing were evaluated. Approaches to decrease the amount of sample foaming on the block digestors were also evaluated. These methods in– volved programming the block temperature during digestion, drying the milk in a 1 OQ°C oven before adding reagents, addition of 5 mL 30% hydrogen peroxide to the samples immediately before digestion, addition of antifoam tablets, and addition of boiling rods during digestion. lt was deter·

Codex Trial Method Review

BARBANO ET AL.: J. ASSOC. OFF. ANi\l. CHEM (VOL. 73, NO. 6. 1990)

852

there were 11 complete sets of results from 10 different laboratories. A )-digit sample coding system was used. A computer program was prepared to collect data and to translate the codes to match blind duplicate test results, as well as to calculate AOAC statistical parameters and to conduct outli– er tests (25, 26). Special data forms were sent to each labora– tory to minimize the probability ofsample mix-up. There was 1 data form for each set of 18 milk samples. The form had all the sample numbers (3-digit code) printed on it in the order that the analyst was to test the samples. There was a form for the analyst to write comments about each individual sample, and a questionnaire (to be completed by the analyst) for each testing method. Information such as sample arrival time and actual testing conditions during the analysis was collected to help ensure that the analysts followed all the details of the procedures. Six: oz Whirl·Pak bags were coded with sample identifica– tion numbers. Raw milks (S L) were collected from 9 differ– ent farms on a Monday morning (day I) and transported on ice to the central laboratory, On Tuesday (day 2), milk samples were cold-split in the central laboratory: the milk from each farm was mixed, poured into 1 large plastic con– tainer, and agitated continuously with a motor driven stirrer while the milk was drawn from a spout (at the bottom of the container) directly into coded sample bags. Forty-six. or more 6 oz Whirl-Pak bags of milk were prepared (80 mL/bag). Samples were refrigerated immediately after splitting. Sam– ple splitting uniformity was verified immediately after split· ting with a Dairylab 2 infrared milk analyzer [16.083, 14th Ed.( I) J by checking the fat and protei.n test of the (irsl, middle, and last sample bag. On Tuesday afternoon, when milk from all 9 farms had been split, the samples were put into appropriate groupings, packed in ice, placed in insulated shipping containers, and sent by overnight air delivery to participating laboratories. Also, a clean, dry, 250 mL wide-moutb, screw-cap plastic container was sent to each laboratory so that the analyst could ship 250 ml of 0, lOOON HCI titrant (a representative sample from the acid used to titrate the distillates) to the central laboratory for analysis. On Wednesday (day 3), sam– ples arrived at the laboratories and testing was initiated as soon as possible. Arrival temperature of the milks was always ~4°C. All testing was completed by Monday (day 8). Test results and completed questionnaires arrived at the data analysis laboratory by Wednesday (day 10). AU data were summariz.ed and returned to individual testing laboratories by day 16. Collaborators verified that the data compiled for their laboratory were correct. V•rlllcaflon of MIik Sampl• Quality A set ofmilk samples was tested for somatic cell count with a Foss-o-Matic electronic somatic cell counter (27), which was calibrated by direct microscopic somatic cell count (28). This set ofsamples had been exposed to all the same handling and shipping conditions as the samples that were tested by the Kjeldabl method, Collaborat/11e Study Trllll• For the first collaborative study (11 sets of samples, 9 milks in blind duplicate in each) conducted in Septembel' 1988, the methods used were as stated in the methods section except analysts were requested to digest samples for I h after clearing. Based on the results of the first trial, a second

the final digestion temperature) was effective in controlling sample foaming during digestion. Laboratories participating in the study preferred this approach to control foaming. The fourth phase of preliminary work found that titanium oxide did not improve test results or substantially decrease digestion time. Kane ( I0) found that titanium oxide im– proved test results and decreased testing time on animal feeds. In this work, he also used a 0.835 ratio of potassium sulfate to sulfuric acid (instead of 0.6), which may explain the shorter testing time required for complete digestion. Higher ratios of K 2 S04 to H 1 S0 4 will give a faster digestion, but can result in excessive acid loss during digestion, crystal– li1.ation of tJ10 digest on cooling, and low test results ( 15). At a salt-acid ratio of 0.6, the mean test results for milk were 3.08% protein without titanium and 3.08% protein with tita– nium using the same time from start of digest.ion to clear and 60-min boil after clearing. With a 45-min boil after clear, lhc results were 3.08% protein without titanium and 3.07% pro– tci11 with added titanium. At a salt-acid ratio of 0.85, the mean test results for milk were 3.09% protein without titani– um and 3.09% protein with titanium using the same time from start of digestion to clear and 60-min boil after clear. With a 45-min boil after clear the results were J.09% without titanium and 3.08% with titanium. The digests with the high– er salt- acid ratio did not crystallize after cooling, but were extremely viscous and hard to mix with the water prior to distillation. With the higher salt- acid ratio, peak nitrogen recovery may be reached slightly sooner during the boiling period after clearing. There were no significant advantages to the use of titanium dioxide or a higher sail-acid ratio for determination of total nitrogen content of milk samples by Kjelda.hl analysis. The fifth phase of the preliminary work with 9 laboratories in July 1988 indicated that nitrogen ree-0veries in all labora– tories were acceptable. In a meeting attended by the Asso– c.1ate Referee and laboratory representatives in August I 988, it was suggested that hydrogen peroxide be eliminated from the digestion and that sample foaming be controlled in the block digestors by starting at a lower initial digestion tem– perature. Also, it was requested that 2 blanks instead of I blank be run by each l~boratory in the collaborative study, because l erroneous blank value would bias all the results from a laboratory. Finally, it was suggested that indicator be added to the boric acid receiving solution when the boric acid is first prepared. This "batch" addition of indicator would eliminate variation in amount of indicator between different samples and make the titration end color more consistent among samples. After completion of prelimfoary work and prior to the beginning of the collaborative study, complete and detailed descriptions of the final testing methods were developed and sent to all laboratories. Collaboratlve Study For the collaborative study, each of 10 laboratories re– ceived a set of 18 raw milk samples (9 pairs of blind dupli– cates) . Each pair of blind duplicates represented milk from I farm. Sample coding was designed such that all samples had different identification numbers in all laboratories. Sample testing order was randomized between laboratories. Ten lab– oratories participated and one of these laboratories conduct– ed 2 sets of analyses, one with traditional equipment and one with block digc.stor equipment (2 different sets of milk with different sample coding were sent to this laboratory). Thus,

Codex Trial Method Review

853

BARllANO ET AL.; J, ASSOC. OFF. ANAi, CHEM , {VOL. 73, NO, 6, 19901

~ollaborative study (10 sets of samples, 9 milks in blind Juplicate in each) was conducted in October 1988. The nethods were the same as in the present paper except ana– lysts were instructed to digest samples for l.5 h after clear.. ing.

cation range 0.0995-0.IO0SN and use 0. lO0ON for calcula– tion. (i) Ammonium su/fate.- 99.9% (NH4)iSQ4. (j) Tryptophan or lysine hydrochloride.-99%

C11H12N2O2 or C6H1sCIN2O2. (k) Sucrose.-Nitrogen free.

Nitrogen (Total) In MIik

D. Sample Preparation Add 15.00 g K2SO4, 1 mL CuSO 4 O catalyst solution, and 8- 10 boiling chips to digestion flask. Warm milk to 38 ± 1°. Mix milk as in 92,.lt. Weigh warm sample (5 ± 0.1 mL) and immediately place in digestion flask. (Note: Weights must be recorded to nearest 0.0001 g.) Add 25 mL H 2 SO 4 , rinsing any milk on neck of flask down into bulb. Flask may be stoppered and held for digestion at later time. Digest and distill a blank (all reagents and no sa:mple) each day, E. Determination (a) Digestion burner selting.-Conduct digestion over heating device that can be adjusted to bring 250 mL H 2 O at 25° to rolling boil in ca 5-6 min. To determine maximum heater setting to be used during digestion, preheat IO min (gas) or 30 min (electric) at burner setting to be evaluated, Add 3 or 4 boiling chips to 250 mL H 2 O at 25° and place flask on preheated burner. Determine heater setting that brings water from 25° to rolling boil in 5-6 min on each burner. This is maximum burner setting to be used during digestion. (b) Digestion.-Place nask in inclined position with fume ejection system on. Start on setting low enough so that sam– pledoes not foam up neck of Kjeldahl flask. Digest at least 20 min or untiJ white fumes appear in flask. Next, increase burner setting half way to maximum burner setting deter· mined in (a) and heat for IS min. At the end of l S min• increase heat to maximum setting determined in (a). When digest clears (clear with light blue-green color), continue to boil 1-1.5 hat maximum setting (total time ca 1.8-2.25 h). To determine specific boil time needed for analysis condi– tions in your laboratory, select a 'high protein, high fat milk sample and determine protein content using different boil times (1-1.5 h) after clearing. Mean protein test increases with increasing (0-1.5 h) boil time, becomes constant. and then decreases when boil time is too long. Select boil time that yields maximum protein test. At end of digestion, digest should be clear and free of undigested material. Cool acid digest to room temperature (ca 25 min). Cooled digest should be fiquid or liquid with few small crystals. (Large amount of crystallization before addi– tion of water indicates too little residual H 2SO 4 at end of digestion and can result in low test values.) After digest is cooled to room temperature, add 300 ml H 2 O to flask and swirl to mix (for 800 mL flasks add 400 ml H 2 O). When room temperature water is added some crystals may form and then go into solution; this is normal. Let mixture cool to room temperature before distillation. Flasks can be stop– pered for distillation at later time. (c) Distil/ation.-Turn on condenser water. Add 50 mL H38O3 solution with indicator to graduated 500 ml Erlen– meyer titration flask and place fiask under condenser tip so that tip is well below HJBOJ solution surface. To room tem– perature diluted digest, carefully add 75 mL 50% NaOH down sidewall of Kjeldahl flask with no agitation. NaOH forms clear layer under the diluted digest. Immediately con– nect flask to distillation bulb on condenser. Vigorously swirl •5H 2

KJeldahl Methods

Codex Trial Method Review (h) Hydrochloric acid standard solution.-0. lODON, Pre– ire as in 936.15 or use premade solution of certified specifi- Flrat Action (Caution: See safety notes on H2SO 4 , HCI, fuming acids, NaOH, ethanol, and use of electrical equipment.) \fethcd Performance: s, = 0.014; SR= 0.Q17; RSD, = 0.385%; RSDR = 0.504% SO4, using CuSO4, · SH2O as catalyst ~1th K2SO4 as boiling point elevator, to release nitrogen from protein and retain nitrogen as ammonium salt. Concentrated ~aOH is added to release Nfh, which is distilled, collected in H38O3 solution, and titrated. Traditions/ Method !I, A.pparatus (a) Digestionflasks.- Kjeldahl. Hard, moderately thick, 1~enannealed glass. Total capacity ca 500 or 800 mL. (b) Distillation flasks. - Same Kjeldahl flask as in (a), r1tted with rubber stopper through which pa.sses lower end of ~fficient rubber bulb or trap to prevent mechanical carryover 1f NaOH during distillation. Connect upper end of bulb to ~ondenser tube by rubber tubing. Use graduated 500 ml [•rlenmeyer titration flask to collect distillate. Trap outlet of ~ ndenser in manner to ensure complete absorption of NH 3 llstilled into boric acid solution. (c) Digestion/distillation system.- Traditional apparatus with adjustable controls for individual flasks. (d) Titration buret.- 50 mL Class A or equivalent. SO4. Nitrogen free. (b) Copper catalyst solution.-CuSO4·5H2O. Nitrogen ~e. Prepare solution 0.05 g/mL H 2 O. (c) Potassium suifate. - K 2 SO4. Nitrogen free. (d) Sodium hydroxide solution.-50% w /w nitrate-free ,aOH. (e) Boiling chips.-Mes.h size 10 suggested. High purity, niphoteric alundum granules, plain. (f) Me1hyl red/bromocresol green indicator solution.– lissolve 0.2 g methyl red and dilute to 100 mL in 95% 1hanol. Dissolve 1.0 g bromocresol green and dilute to 500 1L in 95% ethanol. Mix 1 part methyl red solution with 5 irts bromocresol green solution (combine all of both solu– ~ns). \g) Boric acid solution.- 4%, with indicator, Dissolve 40 g 1803 and dilute to 1 Lin water and add 3 mL methyl red/ omocresol green indicator solution, (O. Solution will be {ht orange color. ~- Prine/pie Milk is digested in H 2 1 c. Reagflnls (a) Sulfuric acid.-95-98% H 2

h h

g

y

" ff n s

d

BARIIANO ET AL,: J. ASSOC. OFP. ANAL CHEM, (VOL. 7.3, NO. 6, 1990)

854

925.21. Weigh warm sample (5 ± 0.1 mL) and immediately place in digestion tube. (Note: Weights must be recorded to nearest 0.0001 g.) Add 20 mL H2S04. Tube may be stop– pered and held for digestion at later time. Digest and distill a blank (all reagents and no sample) each day. L Determination (a) Digestion.- Set block at low initial temperature to control foaming (ca 180- 230°) . Place tubes with aspirator connected in block digestor; suctjoo should be just enough to remove fumes. Digest 30 min or until white fumes develop. Increase temperature to 410-430° and digest until clear, It may be necessary to increase temperature ·gradually over ca 20 min to control foaming. Do not let foam within tube rise higher than ca 4-5 cm below surface of fume collection device inserted into top of tube. After digest clears (clear with light blue-green color), continue to boil (H2S04 must be boiling) for at least l b, total digestion time ca I.75- 2.5 h. To determine specific length of boil time needed for analy– sis conditions in your laboratory, select high protein, high fat milk sample and determine protein content using different boil times (l-1.5 h) after clearing. Mean protein test in· creases with increasing (0-1.5 h) boil time, becomes con– stant, and then decreases when boil time is too long. Selee1 boil time that yields maximum protein test. (Note: Before removing hot tubes from block, make sure there is no conden– sate layer in aspirator manifold. lf there is a liquid layer. increase aspiration to remove liquid.) At end of digestion, digest should be clear and free of undigested material. Cool digest to room temperature (ca 25 min). Cooled digest should be liquid or liquid with few small crystals at bottom of tube. (Excessive crystallization indi– cates too little residual H2S04 at end of digestion and may cause low results. To reduce acid loss during digestion, re– duce fume aspiration rate.) After digest has cooled to room temperature, add 85 mL H20 (blanks may require 100 ml} to each tube, swirl to mix, and let cool to room temperatu.re. When room temperature water is added some crystals may form and then go into solution; this is normal. Tubes can be stoppered for distillation at later time. (b) Disti/latlon.-Plo.ce 50% (or 40%) NaOH in alkali tank of distillation unit. Adjust volume dispensed to 55 ml (65 mL for 40% NaOH). Attach digestion tube containing diluted digest to distillation unit. Place graduated 500 ml Erlenmeyer titration flask containing 50 mL H3B03 solutio1 with indicator on receiving platform, with tube, from cor, denser extending below surface of H3B03 solution. Stean1 distill until 2: 150 mL distillate is collected (2:200 mL tota volume). Remove receiving flask. Titrate H3803 receivlnf solution with standard 0.1000N HCI to first trace of pin~ Lighted stir plate may aid visualization of end point. Record ml HCI to at least nearei1t 0.05 mL. M. N/trogt,n Recovery Verification Run nitrogen recoveries to check accuracy of procedur and equipment. (a) Nitrogen /oss.-Use 0.12 g ammonium sulfate an 0.85 g sucrose per flask. Add all other reagents as stakl under Sample Preparation, K. Digest and distill under sam, conditions as for a milk sample. Recoveries shall be at leas~ 99%. (b) Digestion efftciency.-Use OJ 6 g lysine hydrochlori~ or 0.18 g tryptophan, with 0.67 g sucrose per flaslc. Add a other reagents as stated under Sample Preparation, K. Dige-.

llask to mix contents thoroughly; heat until all NH 3 has b-een distilled (:2:.150 mL distillate; 2:200 mL total volume). Do not leave distillation unattended. Flasks (500 ml) may bump at this point (ca 150 ml distillate; 200 mL total volume). Lower receiving flask and let liquid drain from condenser tip. Turn off distillation heater. Titrate H3B03 receiving solution with standard O. IOOON HCI solution to first trace of pink:. Lighted stir plate may aid visu11.liz.ation of end point. Record mL HCI to at least nearest 0.05 mL. F. Nllrogen Recovery Ver/I/cation Run nitrogen recoveries to check accuracy of procedure and equipment. (a) Nitrogen loss.-Use 0.12 g ammonium sulfate and 0.85 g sucrose per llask. Add aU other reagents as stated under Sample Preparation, D. Digest and distill under same conditions as for a milk sample. Recoveries shall be at least 99%. (b) Digestion efficiency,-Use 0.16 g lysine hydrochloride or 0.18 g tryptopban, with 0.67 g sucrose per flask. Add all other reagents as stated under Sample Preparation, D. Digest and distill under same conditions as for a milk sample. Re· coveries shall be at least 98%. G. Calculatlons Calculate results as follows: where v. and V 0 -= mL HCl titrani used for sample and blank, respectively; N = normality ofHCl solution; and W = sample weight, g. Multiply percent nitrogen by factor 6.38, to calculate per· cent "protein." This is "protein" on a total nitrogen basis. Maximum recommended difference between dupUcates is 0.03% ..protein." H. Repeatability and Reproduclblllty Values For method performance parameters obtained in collabo– rative study of this method, r value = 0.038 and R value = 0.049. Block Dlgestor/Steem DIBllllatlon Method I. Apparatus (a) Digestion b/ock.-Aluminum alloy block or equivalent apparatus, with adjustable temperature control and device for measuring block temperature. (b) Digestion block tubes.- 250 mL capacity. (c) Distillation unit.-For steam distillation. To accept 250 mL digestion tubes and 500 mL titration flasks. (d) Disti/Jation titration flask.-500 mL graduated Er– lenmeyer titration tlaslc. (e) Titration buret.-50 mL. Class A or equivalent. J. Reagents See C(a)-(k). Note: 40% w/ w NaOH may be used instead of 50% w /w. Boiling chips should not be used if equipment manufacturer does not recommend such use. K. Sample Preparellon Add 12 g K 2 S0 4 and l mL CuS04·5H20 catalyst solution to digestion tube. Warm milk to 38 ± l O • Mix milk as in Nitrogen,%,.. (J.4007 X (Vs - Vb) X N]/W

Codex Trial Method Review