SIST EN ISO 11816-1:2014

Milk and milk products - Determination of alkaline phosphatase activity - Part 1: Fluorimetric method for milk and milk-based drinks (ISO 11816-1:2013)

This part of ISO 11816|IDF 155 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 (7.1) so as to obtain a measurement not higher than 7 000 mU/l.

Milch und Milchprodukte - Bestimmung der Aktivität der alkalischen Phosphatase - Teil 1: Fluorimetrisches Verfahren für Milch und flüssige Milchprodukte (ISO 11816-1:2013)

Dieser Teil von ISO 11816 | IDF 155 legt ein fluorimetrisches Verfahren für die Bestimmung der Aktivität der alkalischen Phosphatase (ALP, EC 3.1.3.1) in Rohmilch und pasteurisierter Vollmilch, fettarmer Milch, Magermilch und aromatisierter Milch fest. Das Verfahren ist für Milch und flüssige Milchprodukte von Kühen, Schafen und Ziegen anwendbar. Es ist auch auf Milchpulver nach Rekonstituierung anwendbar.
Das Gerät kann Aktivitäten von mehr als 7 000 Milliunits je Liter (mU/l) auslesen. Wenn die Aktivität größer als 7 000 mU/l ist, dann ist mit Milch, die frei von alkalischer Phosphatase (7.1) ist, zu verdünnen, um ein Ergebnis kleiner als 7 000 mU/l zu erhalten.

Lait et produits laitiers - Détermination de l'activité de la phosphatase alcaline - Partie 1: Méthode fluorimétrique pour le lait et les boissons à base de lait (ISO 11816-1:2013)

L'ISO 11816-1|FIL 155-1:2013 spécifie une méthode fluorimétrique pour la détermination de l'activité de la phosphatase alcaline (ALP, EC 3.1.3.1) dans les laits crus et traités thermiquement entiers, demi-écrémés, écrémés et dans les laits aromatisés. La méthode est applicable au lait et aux boissons à base de lait de vache, de brebis et de chèvre. Elle est également applicable au lait en poudre après reconstitution.
L'appareillage peut lire des activités jusqu'à 7 000 milliunités par litre (mU/l). Si l'activité est supérieure à 7 000 mU/l, une dilution avec du lait exempt de phosphatase alcaline (7.1) est effectuée de façon à obtenir un mesurage ne dépassant pas 7 000 mU/l.

Mleko in mlečni proizvodi - Določevanje aktivnosti alkalne fosfataze - 1. del: Fluorimetrijska metoda za mleko in pijače na osnovi mleka (ISO 11816-1:2013)

Ta del standarda ISO 11816|IDF 155 določa fluorimetrijsko metodo za določevanje aktivnosti alkalne fosfataze (ALP, EC 3.1.3.1) v surovem in toplotno obdelanem polnomastnem mleku, delno posnetem mleku, posnetem mleku in aromatiziranem mleku. Ta metoda se uporablja za mleko in pijače na osnovi kravjega, ovčjega in kozjega mleka. Uporablja se tudi za mleko v prahu po pripravi. Instrument lahko beleži aktivnosti do 7000 milienot na liter (mU/l). Če je aktivnost višja od 7000 mU/l, se razredči z mlekom brez vsebnosti alkalne fosfataze (točka 7.1), da se pridobi meritev, ki ne presega 7000 mU/l.

General Information

• Status: Published
• Public Enquiry End Date: 19-Dec-2012
• Publication Date: 11-Dec-2013
• ICS: 67.100.10 - Milk and processed milk products
• Technical Committee: KŽP - Agricultural food products
• Current Stage: 6060 - National Implementation/Publication (Adopted Project)
• Start Date: 25-Nov-2013
• Due Date: 30-Jan-2014
• Completion Date: 12-Dec-2013
• Ref Project: EN ISO 11816-1:2013 - Milk and milk products - Determination of alkaline phosphatase activity - Part 1: Fluorimetric method for milk and milk-based drinks (ISO 11816-1:2013)

Relations

• Revises: oSIST prEN ISO 11816-1:2023 - Milk and milk products - Determination of alkaline phosphatase activity - Part 1: Fluorimetric method for milk and milk-based drinks (ISO/DIS 11816-1:2022)
• Effective Date: 18-Jan-2023
• Revises: oSIST prEN ISO 11816-1:2023 - Milk and milk products - Determination of alkaline phosphatase activity - Part 1: Fluorimetric method for milk and milk-based drinks (ISO/DIS 11816-1:2022)
• Effective Date: 12-Oct-2022
• Replaces: SIST EN ISO 11816-1:2006 - Milk and milk products - Determination of alkaline phosphatase activity - Part 1: Fluorimetric method for milk and milk-based drinks (ISO 11816-1:2006)
• Effective Date: 01-Jan-2014
• Replaces: SIST EN ISO 11816-1:2006 - Milk and milk products - Determination of alkaline phosphatase activity - Part 1: Fluorimetric method for milk and milk-based drinks (ISO 11816-1:2006)
• Effective Date: 08-Jun-2022

Standards Content (Sample)

SLOVENSKI STANDARD
SIST EN ISO 11816-1:2014
01-januar-2014
1DGRPHãþD
SIST EN ISO 11816-1:2006
0OHNRLQPOHþQLSURL]YRGL'RORþHYDQMHDNWLYQRVWLDONDOQHIRVIDWD]HGHO
)OXRULPHWULMVNDPHWRGD]DPOHNRLQSLMDþHQDRVQRYLPOHND,62

Milk and milk products - Determination of alkaline phosphatase activity - Part 1:

Fluorimetric method for milk and milk-based drinks (ISO 11816-1:2013)

Milch und Milchprodukte - Bestimmung der Aktivität der alkalischen Phosphatase - Teil 1:

Fluorimetrisches Verfahren für Milch und flüssige Milchprodukte (ISO 11816-1:2013)

Lait et produits laitiers - Détermination de l'activité de la phosphatase alcaline - Partie 1:

Méthode fluorimétrique pour le lait et les boissons à base de lait (ISO 11816-1:2013)

Ta slovenski standard je istoveten z: EN ISO 11816-1:2013
ICS:
67.100.10 0OHNRLQSUHGHODQLPOHþQL Milk and processed milk
SURL]YRGL products
SIST EN ISO 11816-1:2014 en

2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

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SIST EN ISO 11816-1:2014
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SIST EN ISO 11816-1:2014
EUROPEAN STANDARD
EN ISO 11816-1
NORME EUROPÉENNE
EUROPÄISCHE NORM
November 2013
ICS 67.100.10 Supersedes EN ISO 11816-1:2006
English Version
Milk and milk products - Determination of alkaline phosphatase
activity - Part 1: Fluorimetric method for milk and milk-based
drinks (ISO 11816-1:2013)

Lait et produits laitiers - Détermination de l'activité de la Milch und Milcherzeugnisse - Bestimmung der Aktivität der

phosphatase alcaline - Partie 1: Méthode fluorimétrique alkalischen Phosphatase - Teil 1: Fluorimetrisches

pour le lait et les boissons à base de lait (ISO 11816- Verfahren für Milch und flüssige Milchprodukte (ISO 11816-

1:2013) 1:2013)
This European Standard was approved by CEN on 14 September 2013.

CEN members are bound to comply with the CEN/CENELEC Internal Regulations which stipulate the conditions for giving this European

Standard the status of a national standard without any alteration. Up-to-date lists and bibliographical references concerning such national

standards may be obtained on application to the CEN-CENELEC Management Centre or to any CEN member.

This European Standard exists in three official versions (English, French, German). A version in any other language made by translation

under the responsibility of a CEN member into its own language and notified to the CEN-CENELEC Management Centre has the same

status as the official versions.

CEN members are the national standards bodies of Austria, Belgium, Bulgaria, Croatia, Cyprus, Czech Republic, Denmark, Estonia,

Finland, Former Yugoslav Republic of Macedonia, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania,

Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and United

Kingdom.
EUROPEAN COMMITTEE FOR STANDARDIZATION
COMITÉ EUROPÉEN DE NORMALISATION
EUROPÄISCHES KOMITEE FÜR NORMUNG
CEN-CENELEC Management Centre: Avenue Marnix 17, B-1000 Brussels

© 2013 CEN All rights of exploitation in any form and by any means reserved Ref. No. EN ISO 11816-1:2013 E

worldwide for CEN national Members.
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SIST EN ISO 11816-1:2014
EN ISO 11816-1:2013 (E)
Contents Page

Foreword ..............................................................................................................................................................3

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SIST EN ISO 11816-1:2014
EN ISO 11816-1:2013 (E)
Foreword

This document (EN ISO 11816-1:2013) has been prepared by Technical Committee ISO/TC 34 “Food

products" in collaboration with Technical Committee CEN/TC 302 “Milk and milk products - Methods of

sampling and analysis” the secretariat of which is held by NEN.

This European Standard shall be given the status of a national standard, either by publication of an identical

text or by endorsement, at the latest by May 2014, and conflicting national standards shall be withdrawn at the

latest by May 2014.

Attention is drawn to the possibility that some of the elements of this document may be the subject of patent

rights. CEN [and/or CENELEC] shall not be held responsible for identifying any or all such patent rights.

This document supersedes EN ISO 11816-1:2006.

According to the CEN-CENELEC Internal Regulations, the national standards organizations of the following

countries are bound to implement this European Standard: Austria, Belgium, Bulgaria, Croatia, Cyprus, Czech

Republic, Denmark, Estonia, Finland, Former Yugoslav Republic of Macedonia, France, Germany, Greece,

Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal,

Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and the United Kingdom.

Endorsement notice

The text of ISO 11816-1:2013 has been approved by CEN as EN ISO 11816-1:2013 without any modification.

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SIST EN ISO 11816-1:2014
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SIST EN ISO 11816-1:2014
INTERNATIONAL ISO
STANDARD 11816-1
IDF
155-1
Third edition
2013-11-01
Milk and milk products —
Determination of alkaline
phosphatase activity —
Part 1:
Fluorimetric method for milk and
milk-based drinks
Lait et produits laitiers — Détermination de l’activité de la
phosphatase alcaline —
Partie 1: Méthode fluorimétrique pour le lait et les boissons à base de lait
Reference numbers
ISO 11816-1:2013(E)
IDF 155-1:2013(E)
ISO and IDF 2013
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SIST EN ISO 11816-1:2014
ISO 11816-1:2013(E)
IDF 155-1:2013(E)
COPYRIGHT PROTECTED DOCUMENT
© ISO/IDF 2013

All rights reserved. Unless otherwise specified, no part of this publication may be reproduced or utilized otherwise in any form or

by any means, electronic or mechanical, including photocopying, or posting on the internet or an intranet, without prior written

permission. Permission can be requested from either ISO or IDF at the respective address below or ISO’s member body in the

country of the requester.
ISO copyright office International Dairy Federation

Case postale 56 • CH-1211 Geneva 20 Silver Building • Bd Auguste Reyers 70/B • B-1030 Brussels

Tel. + 41 22 749 01 11 Tel. + 32 2 733 98 88
Fax + 41 22 749 09 47 Fax + 32 2 733 04 13
E-mail [e-mail] E-mail [e-mail]
Web www.iso.org Web www.fil-idf.org
Published in Switzerland
ii © ISO and IDF 2013 – All rights reserved
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SIST EN ISO 11816-1:2014
ISO 11816-1:2013(E)
IDF 155-1:2013(E)
Contents Page

Foreword ..........................................................................................................................................................................................................................................v

1 Scope ................................................................................................................................................................................................................................. 1

2 Terms and definitions ..................................................................................................................................................................................... 1

3 Principle ........................................................................................................................................................................................................................ 1

4 Reagents ........................................................................................................................................................................................................................ 1

5 Apparatus ..................................................................................................................................................................................................................... 2

6 Sampling ........................................................................................................................................................................................................................ 3

7 Preparations ............................................................................................................................................................................................................. 3

7.1 Alkaline phosphatase-free milk ................................................................................................................................................ 3

7.2 Preparation of test sample ............................................................................................................................................................ 4

8 Procedure..................................................................................................................................................................................................................... 4

8.1 Verification of instrument performance ........................................................................................................................... 4

8.2 Reagent controls to test the suitability of ready-to-use working substrate (4.3) ......................... 5

8.3 Calibration .................................................................................................................................................................................................. 5

8.4 Determination ......................................................................................................................................................................................... 6

8.5 Test sample-related controls ...................................................................................................................................................... 6

9 Calculation and expression of results ............................................................................................................................................. 7

9.1 Calibration ratio ..................................................................................................................................................................................... 7

9.2 Calculation .................................................................................................................................................................................................. 8

9.3 Expression of test results ............................................................................................................................................................... 8

10 Precision ....................................................................................................................................................................................................................... 8

10.1 Collaborative test .................................................................................................................................................................................. 8

10.2 Repeatability ............................................................................................................................................................................................. 8

10.3 Reproducibility ....................................................................................................................................................................................... 8

11 Test report ................................................................................................................................................................................................................... 9

Annex A
(informative)

Collaborative trials ..........................................................................................................................................................................................10

Bibliography .............................................................................................................................................................................................................................13

© ISO and IDF 2013 – All rights reserved iii
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SIST EN ISO 11816-1:2014
ISO 11816-1:2013(E)
IDF 155-1:2013(E)
Foreword

ISO (the International Organization for Standardization) is a worldwide federation of national

standards bodies (ISO member bodies). The work of preparing International Standards is normally

carried out through ISO technical committees. Each member body interested in a subject for which

a technical committee has been established has the right to be represented on that committee.

International organizations, governmental and non-governmental, in liaison with ISO, also take part

in the work. ISO collaborates closely with the International Electrotechnical Commission (IEC) on all

matters of electrotechnical standardization.

The procedures used to develop this document and those intended for its further maintenance are

described in the ISO/IEC Directives, Part 1. In particular the different approval criteria needed for the

different types of ISO documents should be noted. This document was drafted in accordance with the

editorial rules of the ISO/IEC Directives, Part 2. www.iso.org/directives

Attention is drawn to the possibility that some of the elements of this document may be the subject of

patent rights. ISO shall not be held responsible for identifying any or all such patent rights. Details of

any patent rights identified during the development of the document will be in the Introduction and/or

on the ISO list of patent declarations received. www.iso.org/patents

Any trade name used in this document is information given for the convenience of users and does not

constitute an endorsement.

The committee responsible for this document is ISO/TC 34, Food products, Subcommittee SC 5, Milk and

milk products, and the International Dairy Federation (IDF). It is being published jointly by ISO and IDF.

This third edition of ISO 11816-1|IDF 155-1 cancels and replaces the second edition (ISO 11816-1:2006),

which has been technically revised.

ISO 11816|IDF 155 consists of the following parts, under the general title Milk and milk products —

Determination of alkaline phosphatase activity:
— Part 1: Fluorimetric method for milk and milk-based drinks
— Part 2: Fluorimetric method for cheese
iv © ISO and IDF 2013 – All rights reserved
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SIST EN ISO 11816-1:2014
ISO 11816-1:2013(E)
IDF 155-1:2013(E)
Foreword

IDF (the International Dairy Federation) is a non-profit organization representing the dairy sector

worldwide. IDF membership comprises National Committees in every member country as well as

regional dairy associations having signed a formal agreement on cooperation with IDF. All members of

IDF have the right to be represented on the IDF Standing Committees carrying out the technical work.

IDF collaborates with ISO in the development of standard methods of analysis and sampling for milk and

milk products.

The main task of Standing Committees is to prepare International Standards. Draft International

Standards adopted by the Standing Committees are circulated to the National Committees for

endorsement prior to publication as an International Standard. Publication as an International Standard

requires approval by at least 50 % of IDF National Committees casting a vote.

Attention is drawn to the possibility that some of the elements of this document may be the subject of

patent rights. IDF shall not be held responsible for identifying any or all such patent rights.

Any trade name used in this document is information given for the convenience of users and does not

constitute an endorsement.

ISO 11816-1|IDF 155-1 was prepared by the International Dairy Federation (IDF) and Technical

Committee ISO/TC 34, Food products, Subcommittee SC 5, Milk and milk products. It is being published

jointly by IDF and ISO.

All work was carried out by the Joint ISO-IDF Project Group on Determination of alkaline phosphatase

activity – fluorimetric method, of the Standing Committee on Analytical Methods for Processing Aids and

Indicators, under the aegis of its project leader, Ms. Eileen Garry (USA).

This third edition of ISO 11816-1|IDF 155-1 cancels and replaces IDF 155-1:2006, which has been

technically revised.

ISO 11816|IDF 155 consists of the following parts, under the general title Milk and milk products —

Determination of alkaline phosphatase activity:
— Part 1: Fluorimetric method for milk and milk-based drinks
— Part 2: Fluorimetric method for cheese
© ISO and IDF 2013 – All rights reserved v
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SIST EN ISO 11816-1:2014
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SIST EN ISO 11816-1:2014
ISO 11816-1:2013(E)
INTERNATIONAL STANDARD
IDF 155-1:2013(E)
Milk and milk products — Determination of alkaline
phosphatase activity —
Part 1:
Fluorimetric method for milk and milk-based drinks
1 Scope

This part of ISO 11816|IDF 155 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 (7.1) so as to obtain a measurement not

higher than 7 000 mU/l.
2 Terms and definitions
For the purposes of this document, the following terms and definitions apply.
2.1
alkaline phosphatase (ALP) activity

activity of the alkaline phosphatase present in the product, determined by the specified procedure

Note 1 to entry: The alkaline phosphatase activity is expressed as milliunits of enzyme activity per litre of

sample (mU/l).
2.2
unit of alkaline phosphatase activity

amount of alkaline phosphatase enzyme that catalyses the transformation of 1 µmol of substrate per minute

3 Principle

The alkaline phosphatase activity of the sample is measured by a continuous fluorimetric direct

kinetic assay. A non-fluorescent aromatic monophosphoric ester substrate, 2’-[2-benzothiazolyl]-6’-

hydroxybenzothiazole phosphate, in the presence of any alkaline phosphatase derived from the sample,

undergoes hydrolysis of its phosphate radical, producing a highly fluorescent product. Fluorimetric

measurement of alkaline phosphatase (ALP) activity is measured at 38 °C over a 3-min period when

using Fluorophos®. This includes pre-incubation of substrate and sample, followed by multiple kinetic

readings of the reaction rate.

NOTE Although this is a 3-min test, the first minute is an equilibration period to ensure that the sample is at

38 °C. Measurements of activity are actually made from the beginning of the second minute to the end of the third

minute (i.e. over a 2-min period).
4 Reagents

Use only reagents of recognized analytical grade, unless otherwise specified, and distilled or

demineralized water, or water of equivalent purity.
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SIST EN ISO 11816-1:2014
ISO 11816-1:2013(E)
IDF 155-1:2013(E)

4.1 Fluorophos® substrate, in bottles, each containing 144 mg of Fluorophos® substrate powder,

molecular weight 580 grams per mole.

This is a non-fluorescent aromatic monophosphoric ester substrate, 2’-[2-benzothiazolyl]-6’-

hydroxybenzothiazole phosphate (Fluorophos®). The Fluorophos® substrate remains stable for

two years from the date of manufacture, provided it is stored in unopened bottles at between 2 °C and

8 °C; protect against light.

4.2 Substrate buffer solution, diethanolamine (DEA) buffer solution, c(DEA) = 2,4 mol/l, with pH

10,0, in bottles of 240 ml each. The substrate buffer solution remains stable for two years from the date

of manufacture, provided it is stored in unopened bottles at between 2 °C and 8 °C; protect against light.

4.3 Working substrate

Allow the Fluorophos® substrate (4.1) and the substrate buffer solution (4.2) to come to room

temperature. Add the content of one bottle of substrate buffer solution (240 ml) (4.2) to that of one bottle

of Fluorophos® substrate (144 mg) (4.1), and mix well by inversion for 3 min to create ~1,0 millimolar

(pH 10) solution. Use amber glass to protect against light.

Allow the obtained solution to stand at room temperature for at least 30 min prior to use.

Use the A/D (analogue-to-digital) test given in 8.2 to test the suitability of the ready-to-use working substrate.

Do not use the working substrate if a reading above 1 200 FLU (fluorescence units) is obtained (8.2).

The working substrate remains stable for 60 days when protected from light and stored at between 2 °C

and 8 °C, or for 8 h when stored at 38 °C.

NOTE The volume of the working substrate (240 ml) obtained is sufficient for approximately 115 tests.

4.4 Calibrator solutions, Fluoroyellow®(FY) [2’-(2-benzothiazolyl)-6’-hydroxybenzothiazole] in

substrate buffer solution (4.2).

The calibrator solutions remain stable for 18 months from the date of manufacture, provided they are

stored in unopened bottles at between 2 °C and 8 °C. Mix gently prior to use to ensure optimal results.

4.4.1 Calibrator solution A, containing 0 μmol/l of Fluoroyellow®.
4.4.2 Calibrator solution B, containing 17,24 × 10 μmol/l of Fluoroyellow®.
4.4.3 Calibrator solution C, containing 34,48 × 10 μmol/l of Fluoroyellow®.

4.5 Daily instrument control solution, containing 34,48 x10 µmol/l of Fluoroyellow®.

The daily instrument control solution remains stable for 18 months from the date of manufacture,

provided it is stored in unopened bottles at between 2 °C and 8 °C. Mix gently prior to use to ensure

optimal results.
5 Apparatus
Usual laboratory equipment and, in particular, the following.

1) The reagents specified in 4.1 to 4.5 and the apparatus specified in 5.1 to 5.4 (except 5.3.3) comprise the Fluorophos

Test System, which is the trade name of a product supplied by Advanced Instruments, Inc., Two Technology Way,

Norwood, Massachusetts 02062, USA. The manufacturer may change the packaging configurations supplied with

Fluorophos Test system. The user should refer to the manufacturer’s instructions for preparing reagents if different

from those specified herein. Fluorophos and Fluoroyellow are trademarks of Advanced Instruments, Inc. This

information is given for the convenience of users of this document and does not constitute an endorsement by ISO

or IDF of the products named. Equivalent products may be used if they can be shown to lead to the same results.

2 © ISO and IDF 2013 – All rights reserved
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SIST EN ISO 11816-1:2014
ISO 11816-1:2013(E)
IDF 155-1:2013(E)

5.1 Filter fluorimeter, with thermostatically controlled cuvette holder, capable of operating at

38 °C ± 1 °C and right-angle optics, allowing excitation at a wavelength of 440 nm and emission at between

520 nm and 560 nm [e.g. Fluorophos® instrument ].
5.2 Cuvettes, disposable, of non-fluorescent glass.
5.3 Pipettes
5.3.1 Fixed-volume dispenser, capable of dispensing 2,0 ml.
5.3.2 Positive-displacement or air displacement pipette, of capacity 0,075 ml.

Follow strict instructions for pipetting technique as this is a critical step in generating accurate results.

Ensure that piston of pipette bore is tightly secured prior to use.
5.3.3 Pipettes, of capacity 2 ml and 3 ml.

5.4 Incubator block, capable of maintaining a temperature of 38 °C ± 1 °C, suitable for holding cuvettes.

5.5 Suitable laboratory-grade film.
5.6 Vortex mixer.

5.7 Water bath, capable of maintaining a temperature of 63 °C ± 1 °C and 95 °C ± 1 °C.

5.8 One-mark volumetric flasks, of capacity 100 ml.
6 Sampling

Sampling is not part of the method specified in this part of ISO 11816|IDF 155. A recommended sampling

method is given in ISO 707|IDF 50.

A representative sample should have been sent to the laboratory. It should not have been damaged or

changed during transport or storage.
7 Preparations
7.1 Alkaline phosphatase-free milk

Prepare phosphatase-free milk of the type to be tested by carefully dispensing the desired portion of

milk into a test tube or suitable container, ensuring that no milk touches the rim or sides of the container.

Place the tube or container with the milk portion in the water bath (5.7) set at 95 °C. Preheat the milk

portion to 95 °C, before starting its 5-min heating period at that temperature. Check the temperature by

using a thermometer or thermistor probe placed in the centre of the tube or container. When the milk

portion reaches 95 °C, immediately start its 5-min heating period. Cool the whole portion rapidly after

the heating period.

Test the thus-treated milk portion to ensure that its ALP activity is less than 10 mU/l.

© ISO and IDF 2013 – All rights reserved 3
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SIST EN ISO 11816-1:2014
ISO 11816-1:2013(E)
IDF 155-1:2013(E)
7.2 Preparation of test sample
7.2.1 General
Carefully mix all test samples prior to use.
NOTE It is usually not necessary to prewarm test samples.
7.2.2 Pasteurized test samples
Use pasteurized test samples as delivered, in amounts as required.
7.2.3 Dilution of test samples with high ALP values

Prepare dilutions of the test samples of milk using phosphatase-free milk (7.1) in order to bring their

ALP levels within the analytical range of assay (<7 000 mU/l). Mix the diluted solutions well.

8 Procedure
8.1 Verification of instrument performance
8.1.1 General

It is important to check instrument performance for drift, stray light and stability prior to analysing test

samples. Follow good laboratory practice principles when operating the filter fluorimeter (5.1).

Quality control tests include
a) the daily A/D test, used to check the proper functioning of the equipment,

b) the daily instrument control test, using the daily instrument control solution (4.5) to monitor any

electronic or optical drift in the fluorimeter, and

c) the use of external positive, negative and normal controls, described in 8.1.3, which are recommended

for monitoring daily instrument precision parameters.
8.1.2 A/D tests

8.1.2.1 When using the Fluorophos® instrument, perform the A/D tests daily before testing commences.

8.1.2.2 Access the A/D test through the “SETUP” menu. Press “SETUP” key, then select menu item

“A/D Test” by pressing < or > . With nothing in the cuvette holder, press “START”. Allow the figures

appearing on the display screen to stabilize. The display should read 302 ± 4. If the reading is outside that

range, clean the excitation and emission filters and repeat the A/D test.

8.1.2.3 Dispense 2,0 ml of daily instrument control solution (4.5) into a labelled cuvette. Place the

cuvette in the incubator block (5.4) set at 38°C for 20 min. Insert the prewarmed cuvette into the cuvette

holder. Close the lid. When the display is stable, record the displayed value, which should be 602 ± 12. If

outside that range, use the small screwdriver supplied to slowly turn the potentiometer screw on the left-

hand side of the instrument clockwise or anticlockwise, as necessary, until the display reads 602. Allow

the numbers to equilibrate for 15 min.
4 © ISO and IDF 2013 – All rights reserved
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SIST EN ISO 11816-1:2014
ISO 11816-1:2013(E)
IDF 155-1:2013(E)
8.1.3 Controls

Perform positive, negative and PhosphaCheck-N controls using a powdered milk base with phosphatase

and preservative.

The PhosphaCheck® pasteurization controls remain stable for 18 months from the date of manufacture,

provided they are stored in unopened and unreconstituted bottles between 2 °C and 8 °C. Once

reconstituted, the controls are stable for three days at between 2 °C and 8 °C. Do not freeze.

Allow the controls to come to room temperature. Reconstitute the PhosphaCheck® pasteurization controls

before use. Remove the metal and rubber stopper. Add 3,0 ml of deionized water at room temperature.

Replace the stopper and mix gently by inversion for 1 min and then let stand for 15 min. Do not shake the

controls or allow them to foam. Mix gently before each use to ensure optimal results.

After calibrating an unused channel with the negative control, analyse the three control solutions (i.e.

positive, negative and PhophaCheck-N™) by adding 75 µl of each control solution to 2 ml of prewarmed

substrate. Perform the ALP test.

The reading for the negative control shall be < 10 mU/l, the Phosphacheck-N™ normal shall be between

10 and 40 mU/l, and the positive shall be 500 ± 100 mU/l. These controls can be used daily to monitor

the precision of the instrument.

8.2 Reagent controls to test the suitability of ready-to-use working substrate (4.3)

Dispense 2,0 ml of the working substrate (4.3) into a labelled cuvette. Place the cuvette in the heating

block (5.4) set at 38 °C for 20 min. Insert the prewarmed cuvette with the working substrate into the

cuvette holder. Close the lid. When the display is stable, record the displayed value.

Freshly made substrate alone in the A/D mode usually gives a display reading of about 650

...

SLOVENSKI STANDARD
oSIST prEN ISO 11816-1:2012
01-november-2012
0OHNRLQPOHþQLSURL]YRGL8JRWDYOMDQMHDNWLYQRVWLDONDOQHIRVIDWD]HV
IOXRURPHWULMVNRPHWRGRGHO)OXRURPHWULþQDPHWRGD]DPOHNRLQPOHþQHSLMDþH
,62',6

Milk and milk products - Determination of alkaline phosphatase activity - Part 1:

Fluorimetric method for milk and milk-based drinks (ISO/DIS 11816-1:2012)

Milch und Milchprodukte - Bestimmung der Aktivität der alkalischen Phosphatase - Teil 1:

Fluorimetrisches Verfahren für Milch und flüssige Milchprodukte (ISO/DIS 11816-1:2012)

Lait et produits laitiers - Détermination de l'activité de la phosphatase alcaline - Partie 1:

Méthode fluorimétrique pour le lait et les boissons à base de lait (ISO/DIS 11816-1:2012)

Ta slovenski standard je istoveten z: prEN ISO 11816-1 rev
ICS:
67.100.10 0OHNRLQSUHGHODQLPOHþQL Milk and processed milk
SURL]YRGL products
oSIST prEN ISO 11816-1:2012 en

2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

---------------------- Page: 1 ----------------------
oSIST prEN ISO 11816-1:2012
---------------------- Page: 2 ----------------------
oSIST prEN ISO 11816-1:2012
EUROPEAN STANDARD
DRAFT
prEN ISO 11816-1 rev
NORME EUROPÉENNE
EUROPÄISCHE NORM
September 2012
ICS 67.100.10 Will supersede EN ISO 11816-1:2006
English Version
Milk and milk products - Determination of alkaline phosphatase
activity - Part 1: Fluorimetric method for milk and milk-based
drinks (ISO/DIS 11816-1:2012)

Lait et produits laitiers - Détermination de l'activité de la Milch und Milchprodukte - Bestimmung der Aktivität der

phosphatase alcaline - Partie 1: Méthode fluorimétrique alkalischen Phosphatase - Teil 1: Fluorimetrisches

pour le lait et les boissons à base de lait (ISO/DIS 11816- Verfahren für Milch und flüssige Milchprodukte (ISO/DIS

1:2012) 11816-1:2012)

This draft European Standard is submitted to CEN members for parallel enquiry. It has been drawn up by the Technical Committee

CEN/TC 302.

If this draft becomes a European Standard, CEN members are bound to comply with the CEN/CENELEC Internal Regulations which

stipulate the conditions for giving this European Standard the status of a national standard without any alteration.

This draft European Standard was established by CEN in three official versions (English, French, German). A version in any other language

made by translation under the responsibility of a CEN member into its own language and notified to the CEN-CENELEC Management

Centre has the same status as the official versions.

CEN members are the national standards bodies of Austria, Belgium, Bulgaria, Croatia, Cyprus, Czech Republic, Denmark, Estonia,

Finland, Former Yugoslav Republic of Macedonia, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania,

Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and United

Kingdom.

Recipients of this draft are invited to submit, with their comments, notification of any relevant patent rights of which they are aware and to

provide supporting documentation.

Warning : This document is not a European Standard. It is distributed for review and comments. It is subject to change without notice and

shall not be referred to as a European Standard.
EUROPEAN COMMITTEE FOR STANDARDIZATION
COMITÉ EUROPÉEN DE NORMALISATION
EUROPÄISCHES KOMITEE FÜR NORMUNG
Management Centre: Avenue Marnix 17, B-1000 Brussels

© 2012 CEN All rights of exploitation in any form and by any means reserved Ref. No. prEN ISO 11816-1 rev:2012: E

worldwide for CEN national Members.
---------------------- Page: 3 ----------------------
oSIST prEN ISO 11816-1:2012
prEN ISO 11816-1:2012 (E)
Contents Page

Foreword ..............................................................................................................................................................3

---------------------- Page: 4 ----------------------
oSIST prEN ISO 11816-1:2012
prEN ISO 11816-1:2012 (E)
Foreword

This document (prEN ISO 11816-1:2012) has been prepared by Technical Committee ISO/TC 34 “Food

products" in collaboration with Technical Committee CEN/TC 302 “Milk and milk products - Methods of

sampling and analysis” the secretariat of which is held by NEN.
This document is currently submitted to the parallel Enquiry.
This document will supersede EN ISO 11816-1:2006.
Endorsement notice

The text of ISO/DIS 11816-1:2012 has been approved by CEN as a prEN ISO 11816-1:2012 without any

modification.
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oSIST prEN ISO 11816-1:2012
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oSIST prEN ISO 11816-1:2012
DRAFT INTERNATIONAL STANDARD ISO/DIS 11816-1
ISO/TC 34/SC 5 Secretariat: NEN
Voting begins on Voting terminates on
2012-09-06 2013-02-06

INTERNATIONAL ORGANIZATION FOR STANDARDIZATION  МЕЖДУНАРОДНАЯ ОРГАНИЗАЦИЯ ПО СТАНДАРТИЗАЦИИ  ORGANISATION INTERNATIONALE DE NORMALISATION

Milk and milk products — Determination of alkaline
phosphatase activity —
Part 1:
Fluorimetric method for milk and milk-based drinks

Lait et produits laitiers — Détermination de l'activité de la phosphatase alcaline —

Partie 1: Méthode fluorimétrique pour le lait et les boissons à base de lait
[Revision of second edition (ISO 11816-1:2006)]
ICS 67.100.10
ISO/CEN PARALLEL PROCESSING

This draft has been developed within the International Organization for Standardization (ISO), and

processed under the ISO-lead mode of collaboration as defined in the Vienna Agreement.

This draft is hereby submitted to the ISO member bodies and to the CEN member bodies for a parallel

five-month enquiry.

Should this draft be accepted, a final draft, established on the basis of comments received, will be

submitted to a parallel two-month approval vote in ISO and formal vote in CEN.

To expedite distribution, this document is circulated as received from the committee

secretariat. ISO Central Secretariat work of editing and text composition will be undertaken at

publication stage.

Pour accélérer la distribution, le présent document est distribué tel qu'il est parvenu du

secrétariat du comité. Le travail de rédaction et de composition de texte sera effectué au

Secrétariat central de l'ISO au stade de publication.

THIS DOCUMENT IS A DRAFT CIRCULATED FOR COMMENT AND APPROVAL. IT IS THEREFORE SUBJECT TO CHANGE AND MAY NOT BE

REFERRED TO AS AN INTERNATIONAL STANDARD UNTIL PUBLISHED AS SUCH.

IN ADDITION TO THEIR EVALUATION AS BEING ACCEPTABLE FOR INDUSTRIAL, TECHNOLOGICAL, COMMERCIAL AND USER PURPOSES,

DRAFT INTERNATIONAL STANDARDS MAY ON OCCASION HAVE TO BE CONSIDERED IN THE LIGHT OF THEIR POTENTIAL TO BECOME

STANDARDS TO WHICH REFERENCE MAY BE MADE IN NATIONAL REGULATIONS.

RECIPIENTS OF THIS DRAFT ARE INVITED TO SUBMIT, WITH THEIR COMMENTS, NOTIFICATION OF ANY RELEVANT PATENT RIGHTS OF WHICH

THEY ARE AWARE AND TO PROVIDE SUPPORTING DOCUMENTATION.
© International Organization for Standardization, 2012
---------------------- Page: 7 ----------------------
oSIST prEN ISO 11816-1:2012
ISO/DIS 11816-1
Copyright notice

This ISO document is a Draft International Standard and is copyright-protected by ISO. Except as permitted

under the applicable laws of the user’s country, neither this ISO draft nor any extract from it may be

reproduced, stored in a retrieval system or transmitted in any form or by any means, electronic,

photocopying, recording or otherwise, without prior written permission being secured.

Requests for permission to reproduce should be addressed to either ISO at the address below or ISO’s

member body in the country of the requester.
ISO copyright office
Case postale 56  CH-1211 Geneva 20
Tel. + 41 22 749 01 11
Fax + 41 22 749 09 47
E-mail [e-mail]
Web www.iso.org
Reproduction may be subject to royalty payments or a licensing agreement.
Violators may be prosecuted.
ii © ISO 2012 – All rights reserved
---------------------- Page: 8 ----------------------
oSIST prEN ISO 11816-1:2012
Contents Page

Foreword ............................................................................................................................................................. 4

Foreword ............................................................................................................................................................. 5

1  Scope ........................................................................................................................................................... 6

2  Terms and definitions ................................................................................................................................ 6

3  Principle ....................................................................................................................................................... 6

4  Reagents ...................................................................................................................................................... 6

5  Apparatus .................................................................................................................................................... 7

6  Sampling ...................................................................................................................................................... 8

7  Preparations ................................................................................................................................................ 8

8  Procedure .................................................................................................................................................... 8

9  Calculation and expression of results .................................................................................................... 11

10  Precision .................................................................................................................................................... 12

11  Test report ................................................................................................................................................. 13

Annex A (informative) Collaborative trials ..................................................................................................... 14

Bibliography ...................................................................................................................................................... 17

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oSIST prEN ISO 11816-1:2012
Foreword

ISO (the International Organization for Standardization) is a worldwide federation of national standards

bodies (ISO member bodies). The work of preparing International Standards is normally carried out through

ISO technical committees. Each member body interested in a subject for which a technical committee has

been established has the right to be represented on that committee. International organizations, governmental

and non-governmental, in liaison with ISO, also take part in the work. ISO collaborates closely with the

International Electrotechnical Commission (IEC) on all matters of electrotechnical standardization.

International Standards are drafted in accordance with the rules given in the ISO/IEC Directives, Part 2.

The main task of technical committees is to prepare International Standards. Draft International Standards

adopted by the technical committees are circulated to the member bodies for voting. Publication as an

International Standard requires approval by at least 75 % of the member bodies casting a vote.

Attention is drawn to the possibility that some of the elements of this document may be the subject of patent

rights. ISO shall not be held responsible for identifying any or all such patent rights.

ISO 11816-1 | IDF 155-1 was prepared by Technical Committee ISO/TC 34, Food products, Subcommittee

SC 5, Milk and milk products and the International Dairy Federation (IDF). It is being published jointly by ISO

and IDF.

This edition of ISO 11816-1 | IDF 155-1 cancels and replaces ISO 11816-1 Second edition 2006-04-15, which

has been technically revised.

ISO 11816 | IDF 155 consists of the following parts, under the general title Milk and milk products —

Determination of alkaline phosphatase activity:
Part 1: Fluorimetric method for milk and milk‐based drinks
Part 2: Fluorimetric method for cheese
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oSIST prEN ISO 11816-1:2012
Foreword

IDF (the International Dairy Federation) is a worldwide federation of the dairy sector with a National

Committee in every member country. Every National Committee has the right to be represented on the IDF

Standing Committees carrying out the technical work. IDF collaborates with ISO in the development of

standard methods of analysis and sampling for milk and milk products.

Draft International Standards adopted by the Action Teams and Standing Committees are circulated to the

National Committees for voting. Publication as an International Standard requires approval by at least 50% of

IDF National Committees casting a vote.

Attention is drawn to the possibility that some of the elements of this document may be the subject of patent

rights. IDF shall not be held responsible for identifying any or all such patent rights.

ISO 11816-1 | IDF 155-1 was prepared by the International Dairy Federation (IDF) and Technical Committee

ISO/TC 34, Food products, Subcommittee SC 5, Milk and milk products. It is being published jointly by IDF

and ISO.

All work was carried out by the Joint ISO-IDF Project Group on Determination of alkaline phosphatise activity

– fluorimetric method, of the Standing Committee on Analytical Methods for Processing Aids and Indicators,

under the aegis of its project leader, Ms. Eileen Garry (US). This edition of ISO 11816-1 | IDF 155-1 cancels

and replaces IDF 155-1 Second edition 2006-04-15, which has been technically revised.

ISO 11816 | IDF 155 consists of the following parts, under the general title Milk and milk products —

Determination of alkaline phosphatase activity:
Part 1: Fluorimetric method for milk and milk‐based drinks
Part 2: Fluorimetric method for cheese
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oSIST prEN ISO 11816-1:2012
1 Scope

This part of ISO 11816 | IDF 155 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. The method is applicable for milk and milk-based drinks from cows, sheep and goats. It is also

applicable to milk powder after reconstitution.

The instrument can read activities of more than 7 000 milliunits per litre (mU/l). If the activity is higher than

7 000 mU/l, then dilute with alkaline phosphatase free milk (7.1) so as to obtain a measurement not higher

than 7 000 mU/l.
2 Terms and definitions
For the purposes of this document, the following terms and definitions apply.
2.1 alkaline phosphatase (ALP) activity

Activity of the alkaline phosphatase present in the product, determined by the procedure specified in this part

of ISO 11816 | IDF 155

NOTE The alkaline phosphatase activity is expressed as milliunits of enzyme activity per litre of sample (mU/l).

2.2 unit of alkaline phosphatase activity

Amount of alkaline phosphatase enzyme that catalyses the transformation of 1 µmol of substrate per minute

3 Principle

The alkaline phosphatase activity of the sample is measured by a continuous fluorimetric direct kinetic assay.

A non-fluorescent aromatic monophosphoric ester substrate, 2’-[2-benzothiazolyl]-6’-hydroxybenzothiazole

phosphate, in the presence of any alkaline phosphatase derived from the sample, undergoes hydrolysis of its

phosphate radical, producing a highly fluorescent product. Fluorimetric measurement of alkaline phosphatase

(ALP) activity is measured at 38 °C over a 3-min period when using Fluorophos . This includes pre-incubation

of substrate and sample, followed by multiple kinetic readings of the reaction rate.

NOTE Although this is a 3 min test, the first min is an equilibration period to ensure that the sample is at 38 C.

Measurements of activity are actually made from the beginning of the second minute to the end of the third minute (i.e.

over a 2 min period).
4 Reagents

Use only reagents of recognized analytical grade, unless otherwise specified, and distilled or demineralized

water, or water of equivalent purity.
® 

4.1 Fluorophos substrate , in bottles, each containing 144 mg of Fluorophos substrate powder.

This is a non-fluorescent aromatic monophosphoric ester substrate, 2’-[2-benzothiazolyl]-6’-

 ®

hydroxybenzothiazole phosphate (Fluorophos ). The Fluorophos substrate remains stable for 2 years from

manufacturing date when stored in unopened bottles at between 2 °C and 8 °C.

4.2 Substrate buffer solution, diethanolamine (DEA) buffer solution, c(DEA) = 2,4 mol/l, with pH 10,0, in

1The reagents specified in 4.1 to 4.5 and the apparatus specified in 5.1 to 5.4 (except 5.3.3) are available as Fluorophos Test System from Advanced

Instruments, Inc., Two Technology Way, Norwood, Massachusetts 02062, USA.  The manufacturer may change packaging configurations supplied with

Fluorophos Test system.  The user should refer to the manufacturer’s instructions for preparing reagents if different from those specified herein.

Fluorophos and Fluoroyellow are registered trademarks of Advanced Instruments, Inc., and are examples of suitable products available commercially.

The information is given for the convenience of users of this document and does not constitute an endorsement by either ISO of IDF of these

products.
---------------------- Page: 12 ----------------------
oSIST prEN ISO 11816-1:2012

bottles of 240 ml each. The substrate buffer solution remains stable for 2 years from manufacturing date when

stored in unopened bottles at between 2 °C and 8 °C.
4.3 Working substrate

Allow the Fluorophos substrate (4.1) and the substrate buffer solution (4.2) to come to room temperature.

Add the content of one bottle substrate buffer solution (240 ml) (4.2) to that of one bottle Fluorophos

substrate (144 mg) (4.1) and mix well by inversion for 3 min. Use amber glass to protect against light.

Allow the obtained solution to stand at room temperature for at least 30 min prior to use.

Use the A/D (analog-to-digital) test given in 8.2 to test the suitability of the ready-to-use working substrate.

Do not use the working substrate if a reading above 1200 is obtained (8.2).

The working substrate remains stable for 60 days when protected from light and stored at between 2 °C and

8 °C, or for 8 h when stored at 38 °C.

NOTE The obtained volume of the working substrate (240 ml) is sufficient for approximately 115 tests.

4.4 Working calibrator solutions, Fluoroyellow (FY) [2'-(2-benzothiazolyl)-6'-hydroxybenzothiazole] in

substrate buffer solution (4.2).

The working calibrator solutions remain stable for 18 months from manufacturing date when stored in

unopened bottles at between 2 °C and 8 °C. Mix gently prior to use to assure optimal results.

4.4.1 Calibrator solution A, containing 0 μmol/l of Fluoroyellow .
-3 ®
4.4.2 Calibrator solution B, containing 17,24 x 10 μmol/l of Fluoroyellow .
-3 ®
4.4.3 Calibrator solution C, containing 34,48 x 10 μmol/l of Fluoroyellow .

4.5 Daily instrument control solution, containing 34,48 x10-³ µmol/l of Fluoroyellow .

The daily instrument control solution remains stable for 18 months from manufacturing date when stored in

unopened bottles at between 2 °C and 8 °C. Mix gently prior to use to assure optimal results.

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

5.1 Filter fluorimeter, with thermostatically controlled cuvette holder, capable of operating at 38 °C ± 1 °C

and right-angle optics, allowing excitation at a wavelength of 440 nm and emission at between 520 nm and

® 1
560 nm [e.g. Fluorophos instrument ].

5.2 Cuvettes, disposable, non-fluorescent glass, of diameter 12 mm and of length 75 mm.

5.3 Pipettes
5.3.1 Fixed-volume dispenser, capable of dispensing 2,0 ml.
5.3.2 Positive-displacement or air displacement pipette, of capacity 0,075 ml.

NOTE Follow strict instructions for pipetting technique as this is a critical step in generating accurate results. Ensure

that piston of pipette bore is tightly secured prior to use.
5.3.3 Pipette, of capacity 2 ml or 3 ml.

5.4 Incubator block, capable of maintaining a temperature of 38 °C ± 1 °C, suitable for holding cuvettes.

---------------------- Page: 13 ----------------------
oSIST prEN ISO 11816-1:2012
5.5 Parafilm or other suitable laboratory-grade film.
5.6 Vortex mixer.

5.7 Water bath, capable of maintaining a temperature of 63 °C ± 1 °C and 95 °C ± 1 °C.

5.8 One-mark volumetric flasks, of capacity 100 ml.
6 Sampling

A representative sample should have been sent to the laboratory. It should not have been damaged or

changed during transport or storage.

Sampling is not part of the method specified in this part of ISO 11816 І IDF 155. A recommended sampling

method is given in ISO 707 | IDF 50.
7 Preparations
7.1 Alkaline phosphatase-free milk

Prepare phosphatase-free milk of the type to be tested by carefully dispensing the desired portion of milk into

a test tube or suitable container, ensuring that no milk touches the rim or sides of the container.

Place the tube or container with the milk portion in the water bath (5.7) set at 95 °C. Pre-heat the milk portion

to 95 °C, before starting its 5-min heating period at that temperature. Check the temperature by using a

thermometer or thermistor probe placed in the centre of the tube or container. When the milk portion reaches

95 °C, immediately start its 5-min heating period. Cool the whole portion rapidly after the heating period.

Test the thus-treated milk portion to ensure that its ALP activity is less than 10 mU/l.

7.2 Preparation of test sample
7.2.1 General
Carefully mix all test samples prior to use.
NOTE It is usually not necessary to prewarm test samples.
7.2.2 Pasteurized test samples
Use pasteurized test samples as delivered, in amounts as required.
7.2.3 Dilution of test samples with high ALP values

Prepare dilutions of the milks using phosphatase-free milk (7.1) in order to bring their ALP levels within the

analytical range of assay (< 7 000 mU/l). Mix the diluted solutions well.
8 Procedure
8.1 Verification of Instrument performance

It is important to check instrument performance for drift, stray light and stability prior to analysing test samples.

Follow Good Laboratory Practice standards when operating the filter fluorimeter (5.1).

Quality control tests include:
a) the daily A/D test, used to check the proper functioning of the equipment;

2 Parafilm is an example of a suitable product available commercially.  This information is given for the convenience of users of this part of ISO 11816

| IDF 155 and does not constitute an endorsement by either ISO or IDF of this product.

---------------------- Page: 14 ----------------------
oSIST prEN ISO 11816-1:2012

b) the daily instrument control test, using the daily instrument control solution (4.5) to monitor any

electronic or optical drift in the fluorimeter; and

c) the use of external positive, negative and normal controls, described in 8.1.2, are recommended for

monitoring daily instrument precision parameters.
8.1.1 A/D Tests

When using the Fluorophos instrument, perform the A/D tests daily before testing commences.

8.1.1.1 Access the A/D test through the “SETUP” menu. Press “SETUP” key, then select menu item “A/D

Test” by pressing < or >. With nothing in the cuvette holder, press “START”. Allow the figures appearing on

the display screen to stabilize. The display should read 302 ± 4. If the reading is outside that range, clean the

excitation and emission filters and repeat the A/D test.

8.1.1.2 Dispense 2,0 ml of daily instrument control solution (4.5) into a labelled cuvette. Place the

cuvette in the incubator block (5.4) set at 38°C for 20 min. Insert the prewarmed cuvette into the cuvette

holder. Close the lid. When the display is stable, record the displayed value, which should be 602 ±12.

If outside that range, use the small screwdriver supplied to slowly turn the potentiometer screw on the left-

hand side of the instrument clockwise or anticlockwise, as necessary, until the display reads 602. Allow the

numbers to equilibrate for 15 minutes.

8.1.2 Positive, negative and PhosphaCheck-N controls using a powdered milk base with

phosphatase and preservative.

The PhosphaCheck pasteurization controls remain stable for 18 months from manufacturing date when

stored in unopened and unreconstituted bottles between 2 °C and 8 °C. Once reconstituted, the controls are

stable for 3 days at between 2 °C and 8 °C. Do not freeze.

Allow the controls to come to room temperature. Reconstitute the PhosphaCheck pasteurization controls

before use. Remove the metal and rubber stopper. Add 3,0 ml of deionised water at room temperature.

Replace the stopper and mix gently by inversion for 1 min and then let stand for 15 min. Do not shake the

controls or allow them to foam. Mix gently before each use to assure optimal results.

After calibrating an unused channel with the negative control, analyse the three control solutions (i.e. positive,

negative and PhophaCheck-N™) by adding 75 µl of each control solution to 2 ml of prewarmed substrate.

Perform the ALP test.

The reading for the negative control shall be < 10 mU/l, the Phosphacheck N™ normal shall be between 10

and 40 mU/l, and the positive shall be 500 ± 100 mU/l. These controls can be used daily to monitor the

precision of the instrument.

8.2 Reagent controls to test the suitability of ready-to-use working substrate (4.3)

Dispense 2,0 ml of the working substrate (4.3) into a labelled cuvette. Place the cuvette in the heating block

(5.4) set at 38 ºC for 20 min. Insert the prewarmed cuvette with the working substrate into the cuvette holder.

Close the lid. When the display is stable, record the displayed value.

Freshly made substrate alone in the A/D mode usually gives a display reading of about 650 FLU which

increases over time. Do not use the working substrate when a display reading of above 1 200 FLU is

obtained.

 The controls and instrument performance check instructions are available from Advanced Instruments, Inc., Two Technology Way, Norwood,

Massachusetts 02062, USA.

This information is given for the convenience of users of this document and does not constitute an endorsement by either ISO or IDF of these

products.
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oSIST prEN ISO 11816-1:2012
8.3 Calibration

Calibration curves are usually stable. However, recalibrate the instrument, which has already been calibrated,

when the fluorimeter is initially installed, whenever servicing procedures (i.e. lamp or filter replacement) are

likely to affect the stored calibration, or when assayed control values show unacceptable results.

If there are changes in the calibration curve, recalibrate the instrument using a new set of calibrator solutions

A, B and C (4.4.1, 4.4.2 and 4.4.3). Establish a calibration curve for each type of product to be tested.

Mix calibrator solutions A, B and C by gentle inversion prior to use. Transfer, using the pipette (5.3.3), 2,0 ml

of calibrator solution A, of calibrator solution B and of calibrator solution C (4.4.1, 4.4.2 and 4.4.3) respectively,

each in duplicate, to six prelabelled cuvettes (5.2). Place the cuvettes in the incubator block (5.4) set at 38 °C

and preheat for 20 min.

Add using the positive displacement or air displacement pipette (5.3.2), 0,075 ml of alkaline phosphatase-free

milk (7.1) to all six cuvettes. Cover the cuvettes with parafilm (5.5). Mix their contents using the vortex mixer

(5.6) for 5 s or by gently inverting the cuvettes. Return the cuvettes to the incubator block (5.4). Complete the

calibration within 10 min after the addition of the test sample to the calibrator.

Starting with calibrator solution A, perform the following calibration routine. Wipe the outside of each cuvette

with soft tissue before placing the cuvette in the filter fluorimeter (5.1). When using the Fluorophos

instrument, press “CALIB” and select the “ALP Dairy” menu. Scroll through the menu and press “ENTER”

when the product to be calibrated is displayed. Beginning with calibrator solution A (4.4.1), insert this solution

in the fluorimeter and press “START”. When the measurement is finished, measure the second A calibrator

solution.

Follow the same procedure for the B (4.4.2) and C (4.4.3) calibrator solutions until the procedure is completed.

The Fluorophos instrument automatically calculates the amount of fluorescence obtained with calibrator

solution B and C against calibrator solution A to set the calibration ratio within the instrument.

Once calibration is completed, proceed to analyse the test samples.
8.4 Determination

Dispense, using the fixed volume dispenser (5.3.1) or pipette, 2,0 ml of working substrate (4.3) into a labelled

cuvette. Place the cuvette in the incubator block (5.4) set at 38 °C and hea
...