SIST EN ISO 11816-1:2014
(Main)Milk and milk products - Determination of alkaline phosphatase activity - Part 1: Fluorimetric method for milk and milk-based drinks (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)
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
Relations
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 11816-1:2013ICS:
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
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.---------------------- Page: 3 ----------------------
SIST EN ISO 11816-1:2014
EN ISO 11816-1:2013 (E)
Contents Page
Foreword ..............................................................................................................................................................3
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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 noticeThe 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
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 88Fax + 41 22 749 09 47 Fax + 32 2 733 04 13
E-mail copyright@iso.org E-mail info@fil-idf.org
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---------------------- Page: 9 ----------------------
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/directivesAttention 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/patentsAny 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 PrincipleThe 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.© ISO and IDF 2013 – All rights reserved 1
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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 substrateAllow 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---------------------- Page: 14 ----------------------
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---------------------- Page: 15 ----------------------
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 Procedure8.1 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 includea) 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, andc) 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
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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 revICS:
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.
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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
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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.---------------------- Page: 5 ----------------------
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 alkalinephosphatase 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
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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
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Tel. + 41 22 749 01 11
Fax + 41 22 749 09 47
E-mail copyright@iso.org
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
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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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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 155NOTE The alkaline phosphatase activity is expressed as milliunits of enzyme activity per litre of sample (mU/l).
2.2 unit of alkaline phosphatase activityAmount of alkaline phosphatase enzyme that catalyses the transformation of 1 µmol of substrate per minute
3 PrincipleThe 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 ApparatusUsual 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
® 1560 nm [e.g. Fluorophos instrument ].
5.2 Cuvettes, disposable, non-fluorescent glass, of diameter 12 mm and of length 75 mm.
5.3 Pipettes5.3.1 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.
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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 sample7.2.1 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.
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b) the daily instrument control test, using the daily instrument control solution (4.5) to monitor any
electronic or optical drift in the fluorimeter; andc) 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.---------------------- Page: 15 ----------------------
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...
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