This document describes general workflows and protocols for the validation and the verification of qualitative screening tests for the detection of residues of veterinary drugs in liquid milk (raw, pasteurized, UHT and reconstituted milk powders and whey protein extracts) including biological methods. This guideline does not cover the validation of residue analysis by HPLC, UHPLC or LC-MS/MS.
This document is intended to be useful for manufacturers of screening test kits, laboratories validating screening methods or tests, competent authorities and dairies or end users of reagents or tests for the detection of veterinary drug residues in milk products. This document facilitates and improves the validation and verification of screening methods. The goals of this document are a harmonization in validation of methods or test kits in order for all stakeholders to have full trust in the result of residue screening and to limit the overlap and multiplication of validation work in different laboratories by sharing the validation results generated by an independent laboratory. Furthermore, a harmonized validation and verification procedure allows for comparison of the performance of different screening methods.
This document does not imply that all end users are bound to perform all verification work proposed.
The verification of the correct use of reagents/kits for the detection of antimicrobials is not part of the scope of this document.

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2013-05-15 LVO: Text arrived in ISO/CS (see notification of 2013-05-13 in dataservice).

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ISO - Taking over of an ISO Technical Corrigendum

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The International Standard describes a method for the determination of chloride content using a potentiometric titration method.

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ISO 14673-2|IDF 189-2:2004 specifies a routine method for the determination of the nitrate and nitrite contents of milk and milk products by segmented flow analysis. The method is applicable to milk, cheese, and liquid and dried milk products and infant foods.

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ISO 14673-1|IDF 189-1:2004 specifies a method for the determination of the nitrate and nitrite contents of milk and milk products by cadmium reduction and spectrometry. The method is applicable to: whole and partly skimmed and skimmed dried milk; hard, semi-hard and soft cheeses; processed cheese; whey cheese, caseins and caseinates and dried whey.
The method may be performed using automatic equipment, in particular by segmented flow analysis (SFA) or flow injection analysis (FIA), thus reducing cadmium contamination in laboratory work places and waste water.

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ISO 14673-3|IDF 189-3:2004 specifies a routine method for the determination of the nitrate and nitrite contents of milk and milk products by cadmium reduction and flow injection analysis (FIA). The method is applicable to hard, semi-hard and soft cheeses of various ages, and processed cheese. The detection limits of the method are 0,5 mg of nitrate ions per kilogram and 1,0 mg of nitrite ions per kilogram, respectively.
The method is also applicable to whey powder, milk powder and milk-based infant food.

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ISO 17189|IDF 194:2003 specifies a method for the determination of the fat content of butter, edible oil emulsions and spreadable fats (margarine, vegetable oil spreads, dairy spreads and blended spreads).

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ISO 18330|IDF 188:2003 gives guidelines for the standardized description of immunoassays or receptor assays for the detection of antimicrobial residues in milk and milk products.
It is intended to provide a framework and basis for the evaluation/validation of tests based on the binding of an antimicrobial compound to its specific antibody or to other types of detecting molecules.
In addition to immunoassays [e.g. enzyme-immunoassay (EIA) and radio-immunoassay (RIA)], there are several quantitative, semi-quantitative and qualitative test formats based on the binding of antimicrobial compounds to microbial receptors or to receptor proteins. Enzymatic assays and particle-based assays based on receptor proteins are referred to as receptor assays in ISO 18330|IDF 188:2003.

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ISO 3727-3|IDF 80-3:2003 specifies a method for the calculation of the fat content of butter.

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ISO 14675|IDF186:2002 give guidelines on the use of screening methods used for the determination of aflatoxin M1 content in milk and milk products, based upon competitive enzyme immuno-assays.
For legal purposes, positive enzyme immunoassay results require confirmation by an accepted reference method. However, depending on whether the test complies with the specifications given hereafter, enzyme immunoassays can be used for routine quality control, especially when the absence of aflatoxin M1 above the regulatory limit needs to be documented.

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ISO 14891|IDF 185:2002 specifies a routine method for the determination of the total nitrogen content of milk and milk products.

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TC forecasts updated : delay with the directives ( TC forecasts updating exercise.sj 00-11-09)++

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TC forecasts updated : delay with the directives ( TC forecasts updating exercise.sj 00-11-09)++

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ISO 11816-2:2016|IDF 155-2:2016 specifies a fluorimetric method for the determination of alkaline phosphatase (ALP, EC 3.1.3.1) activity in cheese.
This method is applicable to soft cheeses, semi-hard and hard cheeses provided that the mould is only on the surface of the cheese and not also in the inner part (e.g. blue veined cheeses). For large hard cheeses, specific conditions of sampling apply (see Clause 7).
The instrument can read activities in the supernatant up to 7 000 milliunits per litre (mU/l).

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ISO 11816-1|IDF 155-1:2013 specifies a fluorimetric method for the determination of alkaline phosphatase (ALP, EC 3.1.3.1) activity in raw and heat-treated whole milk, semi-skimmed milk, skimmed milk and flavoured milks. This method is applicable to milk and milk-based drinks from cows, sheep and goats. It is also applicable to milk powder after reconstitution.
The instrument can read activities up to 7 000 milliunits per litre (mU/l). If the activity is higher than 7 000 mU/l, it is diluted with alkaline phosphatase-free milk so as to obtain a measurement not higher than 7 000 mU/l.

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This deliverable specifies a method for the determination of the moisture content of all types of dried milk. The revised version will include the results of a recently conducted interlaboratory study in whey powders, dairy permeate powders, cream powder and powdered infant formula in ISO 5537|IDF 26 to further underpin the extent of its scope.

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This document specifies a method for the determination of the fat content of all types of cheese and processed cheese products containing lactose of below 5 % (mass fraction) of non-fat solids, and all types of caseins and caseinates.
The method is not applicable to fresh cheese types containing, for example, fruits, syrup or muesli. For such products, the Schmid-Bondzynski-Ratzlaff (SBR) principle is not applicable due to high concentrations of sugars. For these products, the method using the Weibull-Berntrop principle (see ISO 8262-3 | IDF 124-3) is appropriate.

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This document gives guidance on the sample preparation of fresh cheese, (semi)soft cheese, (semi)hard cheese, processed cheese and whey cheese for physical and chemical analysis, including analysis by applying instrumental methods.
This document describes the (sub)sampling, and sample preparation steps carried out after sampling according to ISO 707 | IDF 50 and prior to method-specific sample preparations, e.g. as with analytical methods listed in References [2] to [22].
NOTE            Analysis on volatile substances, minor components or allergens can require additional precautionary measures in sample preparation in order to avoid loss of or contamination with one or more target analytes.

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ISO 1735|IDF 5:2004 specifies the reference method for the determination of the fat content of all types of cheese and processed cheese products having lactose contents of below 5 % (mass fraction) of non-fat solids.

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This document specifies a method for the determination of aflatoxin M1 content in milk and milk powder. The lowest level of validation is 0,08 µg/kg for whole milk powder, i.e. 0,008 µg/l for reconstituted liquid milk. The limit of detection (LOD) is 0,05 μg/kg for milk powder and 0,005 μg/kg for liquid milk. The limit of quantification (LOQ) is 0,1 μg/kg for milk powder and 0,01 μg/kg for liquid milk.
The method is also applicable to low-fat milk, skimmed milk, low-fat milk powder and skimmed milk powder.

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This document gives guidelines for the establishment of a conversion relationship between the results of an alternative method and an anchor method, and its verification for the quantitative determination of the microbiological quality of milk.
NOTE     The conversion relationship can be used a) to convert results from an alternative method to the anchor basis or b) to convert results/limits, expressed on an anchor basis, to results in units of an alternative method.

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This document specifies a method for the determination of inulin-type fructans (including oligofructose, fructooligosaccharides) in infant formula and adult nutritionals (both powder and liquid) containing 0,03 g/100 g to 5,0 g/100 g of fructans in the product as prepared ready for consumption.
The method has been validated in a multi laboratory study[1] with reconstituted standard reference material (SRM), infant/adult nutritional formula at a level of 0,204 g/100 g, adult nutritionals ready-to-feed (RTF) at levels of 1,28 g/100 g and 2,67 g/100 g, infant formula RTF at a level of 0,300 g/100 g, reconstituted follow-up formula at levels of 0,209 g/100 g to 0,275 g/100 g, reconstituted infant formula at levels from 0,030 8 g/100 g to 0,264 g/100 g. During the single laboratory validation study[2], spike-recovery experiments were performed up to 5 g/100 g in reconstituted infant formula powders (milk-based, partially hydrolysed milk-based and soy-based), adult nutritional RTF and reconstituted adult nutritional powders.

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This document specifies the quantitative liquid chromatographic determination of specific sugars (galactose, glucose, fructose, sucrose, lactose and maltose) in various milk and milk products, applying arabinose as an internal standard.
The method is applicable to the following dairy matrices: milk, sweetened condensed milk, milk powder, cheese, whey powder, infant formula, milk dessert and yoghurt.
The method does not apply to dairy products containing soy or to the determination of the lactose content in low-lactose milk products at levels below 1 mg/g.
A high performance anion exchange chromatography method in combination with pulsed amperometric detection (HPAEC-PAD) method is applied[5][3][4]. With this method, thirteen different monosaccharides, disaccharides and trisaccharides can be separated: fucose, arabinose, galactose, glucose, fructose, sucrose, lactose, lactulose, maltose, melibiose, trehalose, isomaltulose and maltotriose.
The method is applicable to labelling for the six most important sugars that can be present by nature or by addition in milk and milk products. The method does not apply to sugar contents less than 0,1 %.

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ISO 14501│IDF 171:2007 specifies a method for the determination of aflatoxin M1 content in milk and milk powder. The limit of detection is 0,08 µg/kg for whole milk powder, i.e. 0,008 µg/l for reconstituted liquid milk.
The method is also applicable to low fat milk, skimmed milk, low fat milk powder, and skimmed milk powder.

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ISO 21187|IDF 196:2004 gives guidelines for the establishment of a conversion relationship between the results of a routine method and an anchor method, and its verification for the quantitative determination of the bacteriological quality of milk.

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ISO 20647:2015 specifies a method for the quantitative determination of total iodine in infant formula and adult nutritional formula.[1] The method is applicable to the measurement of total iodine in infant formula and adult nutritional formula from 0,5 µg/100g to 1 500 µg/100g reconstituted final product and for ready-to-feed products from 2,5 µg/100 g to 1 000 µg/100 g using ICP-MS.
Using various infant formula and adult nutritional products, the method was subjected to an interlaboratory study. Levels obtained ranged from 3,47 µg/100 g to 124 µg/100 g. For all precision data related to the interlaboratory study, see Table A.1 located in Annex A.

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ISO 16958:2015 specifies a method for the quantification of individual and/or all fatty acids in the profile of milk, milk products, infant formula and adult nutritional formula, containing milk fat and/or vegetable oils, supplemented or not supplemented with oils rich in long chain polyunsaturated fatty acids (LC-PUFA). This also includes groups of fatty acids often labelled [i.e. trans fatty acids (TFA), saturated fatty acids (SFA), monounsaturated fatty acids (MUFA), polyunsaturated fatty acids (PUFA), omega-3, omega-6 and omega-9 fatty acids] and/or individual fatty acids [i.e. linoleic acid (LA), α-linolenic acid (ALA), arachidonic acid (ARA), eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA)].
The determination is performed by direct transesterification in food matrices, without prior fat extraction, and consequently it is applicable to liquid samples or reconstituted powder samples with water having total fat ≥ 1,5 % m/m.
The fat extracted from products containing less than 1,5 % m/m fat can be analysed with the same method after a preliminary fat extraction using methods referenced in Clause 2. Dairy products, like soft or hard cheeses with acidity level ≤ 1 mmol/100 g of fat, can be analysed after a preliminary fat extraction using methods referenced in Clause 2. For products supplemented or enriched with PUFA with fish oil or algae origins, the evaporation of solvents should be performed at the lowest possible temperature (e.g. max. 40 °C) to recover these sensitive fatty acids.

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Le présent document spécifie une méthode de détermination quantitative de la teneur en calcium (Ca), cuivre (Cu), fer (Fe), magnésium (Mg), manganèse (Mn), phosphore (P), potassium (K), sodium (Na) et zinc (Zn) par spectrométrie d'émission atomique avec plasma induit par haute fréquence (ICP-AES). La méthode est applicable au lait, au lait en poudre, au beurre, au fromage, au lactosérum, au lactosérum en poudre, aux formules infantiles et aux produits nutritionnels pour adultes dans les plages données dans le Tableau 1.

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This document specifies a method for the quantitative determination of calcium (Ca), copper (Cu), iron (Fe), magnesium (Mg), manganese (Mn), phosphorus (P), potassium (K), sodium (Na), zinc (Zn), chromium (Cr), molybdenum (Mo) and selenium (Se) using inductively coupled plasma and mass spectrometry (ICP-MS).
The method is applicable for the determination of all 12 elements in infant formula and adult nutritional products. The method is also applicable for milk, milk powder, whey powder, butter and cheese excluding the determination of Cr, because all Cr results were below the quantification limit and reproducibility could not be determined in these matrices[1]. The present method is an extension of ISO 20649 | IDF 235 (AOAC 2011.19[2]) which was validated only for Cr, Mo and Se in infant formula and adult nutritional products.

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This document specifies a protocol for the evaluation of instrumental alternative methods for total bacterial count in raw milk from animals of different species.
NOTE    The document is complementary to ISO 16140-2 and ISO 8196 | IDF 128 (all parts).

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ISO 16297|IDF161:2013 gives guidelines for the evaluation of instrumental alternative methods for total bacterial count in raw milk from animals of different species.

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This document specifies a reference method for the determination of milk fat purity using gas chromatographic analysis of triglycerides. The method utilizes the differences in triglyceride fingerprint of milk fat from the individual triglyceride fingerprints of other fats and oils to determine samples which are outside the range normally observed for milk fat. This is achieved by using the defined triglyceride formulae based on the normalized weighted sum of individual triglyceride peaks which are sensitive to the integrity of the milk[6][7]. The integrity of the milk fat can be determined by comparing the result of these formulae with those previously observed for a range of pure milk fat samples[12]. Both vegetable fats and animal fats such as beef tallow and lard can be detected.
The method is applicable to bulk milk, or products made thereof, irrespective of the variation in common feeding practices, breed or lactation conditions. In particular, the method is applicable to fat extracted from milk products purporting to contain pure milk fat with unchanged composition, such as butter, cream, milk and milk powder.
Because a false-positive result can occur, the method does not apply to milk fat related to these circumstances:
a)    obtained from bovine milk other than cow's milk;
b)    obtained from single cows;
c)    obtained from cows whose diet contained a particularly high proportion of vegetable oils such as rapeseed, cotton or palm oil, etc.;
d)    obtained from cows suffering from serious underfeeding (strong energy deficit);
e)    obtained from colostrum;
f)     subjected to technological treatment such as removal of cholesterol or fractionation;
g)    obtained from skim milk, buttermilk or whey;
h)    obtained from cheeses showing increased lipolysis;
i)     extracted using the Gerber, Weibull?Berntrop or Schmid?Bondzynski?Ratzlaff methods, or that has been isolated using detergents (e.g. the Bureau of Dairy Industries method).
With the extraction methods specified in i), substantial quantities of partial glycerides or phospholipids can pass into the fat phase.
NOTE 1    In nature, butyric (n-butanoic) acid (C4) occurs exclusively in milk fat and enables quantitative estimations of low to moderate amounts of milk fat in vegetable and animal fats to be made. Due to the large variation of C4, for which the approximate content ranges from 3,1 % fat mass fraction to 3,8 % fat mass fraction, it is difficult to provide qualitative and quantitative information for foreign fat to pure milk fat ratios of up to 20 % mass fraction[11].
NOTE 2    In practice, quantitative results cannot be derived from the sterol content of vegetable fats, because they depend on production and processing conditions. Furthermore, the qualitative determination of foreign fat using sterols is ambiguous.
NOTE 3    Due to special feeding practices such as those related to c) and d), false-positive results have sometimes been reported for milk from certain Asian regions[15]. Moreover, grass-only diets such as mountain and, in particular, highland pasture feeding sometimes cause false-positive results, which can be substantiated by a content of conjugated linoleic acid (C18:2 c9t11) of ≥ 1,3 % fatty acid mass fraction[16][17]. Nevertheless, results conforming to the criteria of milk fat purity specified in this document are accepted, even if samples were undoubtedly produced under conditions reported in this note, including those described in h).
NOTE 4    In ca

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ISO 17678|IDF 202:2010 specifies a reference method for the determination of milk fat purity using gas chromatographic analysis of triglycerides. Both vegetable fats and animal fats such as beef tallow and lard can be detected. By using defined triglyceride equations, the integrity of milk fat is determined.
Basically, the method applies to bulk milk, or products made thereof, irrespective of feeding, breed or lactation conditions. In particular, the method is applicable to fat extracted from milk products purporting to contain pure milk fat with unchanged composition, such as butter, cream, milk, and milk powder.
However, under the circumstances listed hereafter, a false positive result can be obtained. Hence, the method is not applicable to milk fat: a) obtained from bovine milk other than cow's milk; b) obtained from single cows; c) obtained from cows which received an exceptionally high feeding of pure vegetable oils such as rapeseed oil; d) obtained from colostrum; e) subjected to technological treatment such as removal of cholesterol or fractionation; f) obtained from skim milk or buttermilk; g) extracted by using the Gerber, Weibull–Berntrop or Schmid–Bondzynski–Ratzlaff methods, or that has been isolated using detergents (e.g. the Bureau of Dairy Industries method).
With the extraction methods specified in g), substantial quantities of partial glycerides or phospholipids can pass into the fat phase. Consequently, the scope of ISO 17678|IDF 202:2010 excludes certain products and particularly cheese, whose ripening process can also affect the fat composition to such a degree that a false positive result is obtained.

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ISO 9233-2 | IDF 140-2:2018 specifies a method for the determination of natamycin mass fraction in cheese, cheese rind and processed cheese of above 0,5 mg/kg and of the surface-area-related natamycin mass in cheese rind of above 0,03 mg/dm2.

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ISO 9233-1 | IDF 140-1:2018 specifies a method for the determination in cheese rind of natamycin mass fraction of above 0,5 mg/kg and surface-area-related natamycin mass of above 0,03 mg/dm2.
NOTE       It is possible that the method is suitable for detecting migration of natamycin into the cheese.

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ISO 9233-2|IDF 140-2:2007 specifies a method for the determination of natamycin mass fraction in cheese, cheese rind and processed cheese of above 0,5 mg/kg and of the surface-area-related natamycin mass in cheese rind of above 0,03 mg/dm2.

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ISO 9233-1|IDF 140-1:2007 specifies a method for the determination in cheese rind of natamycin mass fraction of above 0,5 mg/kg and surface-area-related natamycin mass of above 0,03 mg/dm2.

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ISO 6785|IDF 93:2001 specifies a method for the detection of Salmonella spp. in milk and milk products.

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ISO 11816-2:2016|IDF 155-2:2016 specifies a fluorimetric method for the determination of alkaline phosphatase (ALP, EC 3.1.3.1) activity in cheese.
This method is applicable to soft cheeses, semi-hard and hard cheeses provided that the mould is only on the surface of the cheese and not also in the inner part (e.g. blue veined cheeses). For large hard cheeses, specific conditions of sampling apply (see Clause 7).
The instrument can read activities in the supernatant up to 7 000 milliunits per litre (mU/l).

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ISO 8968-4|IDF 20-4:2016 specifies a method for the direct and indirect determination of the protein nitrogen content of liquid, whole or skimmed milk.

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ISO 11816-2|IDF 155-2:2003 specifies a fluorometric method for the determination of alkaline phosphatase activity in cheese.
This method is also applicable to soft cheeses and semi-hard cheeses for distinguishing raw milk cheeses from cheese produced with pasteurized milk, provided that the mould is only on the surface and not also in the inner part (e.g. blue-veined cheeses). This method can also be used to check the proper pasteurization of cheese or its raw material.
In large hard cheeses where the whey curd mixture is scalded at temperatures above 50 degrees Celsius, high temperatures remain for a relatively long time. This is especially the case in the centre of these cheeses, thus promoting phosphatase inactivation. To distinguish, therefore, between hard cheeses from raw milk and hard cheeses from pasteurized milk is difficult and often even impossible by this method.

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ISO 8968-5|IDF 20-5:2001 specifies a method for the direct determination of the protein-nitrogen content of liquid milk, whole or skimmed.
An alternative indirect method using calculations is also described.

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ISO 8968-4|IDF 20-4:2001 specifies a method for the determination of the non-protein nitrogen content of liquid milk, whole or skimmed.

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ISO 8968-1|IDF 20-1:2014 specifies a method for the determination of the nitrogen content and crude protein calculation of milk and milk products by the Kjeldahl principle, using traditional and block digestion methods. The methods are applicable to: liquid cow's (whole, partially skimmed or skimmed milk), goat's and sheep's whole milk; hard, semi-hard and processed cheese; dried milk and dried milk products (including milk-based infant formulae, milk protein concentrate, whey protein concentrate, casein and caseinate). The methods are not applicable to samples containing ammonium caseinate.

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ISO 11816-1|IDF 155-1:2013 specifies a fluorimetric method for the determination of alkaline phosphatase (ALP, EC 3.1.3.1) activity in raw and heat-treated whole milk, semi-skimmed milk, skimmed milk and flavoured milks. This method is applicable to milk and milk-based drinks from cows, sheep and goats. It is also applicable to milk powder after reconstitution.
The instrument can read activities up to 7 000 milliunits per litre (mU/l). If the activity is higher than 7 000 mU/l, it is diluted with alkaline phosphatase-free milk so as to obtain a measurement not higher than 7 000 mU/l.

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ISO 8968-2|IDF 20-2:2001 specifies a method for the determination of the nitrogen content of liquid milk, whole or skimmed, by the block-digestion principle.

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