Animal feeding stuffs, cereals and milled cereal products - Guidelines for the application of near infrared spectrometry (ISO 12099:2017)

This document gives guidelines for the determination by near infrared spectroscopy of constituents
such as moisture, fat, protein, starch and crude fibre and parameters such as digestibility in animal
feeding stuffs, cereals and milled cereal products.
The determinations are based on spectrometric measurement in the near infrared spectral region.

Futtermittel, Getreide und gemahlene Getreideerzeugnisse - Anleitung für die Anwendung von Nahinfrarot-Spektrometrie (ISO 12099:2017)

Diese Internationale Norm stellt eine Anleitung für die Bestimmung von Bestandteilen, wie z. B. Feuchte, Fett, Protein, Stärke und Rohfaser, und von Parametern, wie z. B. Verdaubarkeit des Futtermittels, von Getreide und gemahlenen Getreideerzeugnissen, mit Nahinfrarot-Spektroskopie bereit.
Die Bestimmungen basieren auf einer spektrometrischen Messung im Nahinfrarotbereich.

Aliments des animaux, céréales et produits de mouture des céréales - Lignes directrices pour l'application de la spectrométrie dans le proche infrarouge (ISO 12099:2017)

ISO 12099:2017 fournit des lignes directrices pour la détermination par spectrométrie dans le proche infrarouge de constituants tels que l'eau, les matières grasses, les protéines, l'amidon et la cellulose brute, et des paramètres tels que la digestibilité des aliments pour animaux, des céréales et des produits de mouture des céréales.
Les déterminations sont basées sur des mesurages spectrométriques dans le domaine spectral du proche infrarouge.

Krma, žito in mlevski proizvodi - Smernice za uporabo bližnje infrardeče spektrometrije (ISO 12099:2017)

Ta dokument podaja smernice za določevanje lastnosti, kot so vlažnost, maščoba, beljakovine, škrob in surove vlaknine, ter parametrov, kot je prebavljivost, v krmi, žitu in mlevskih proizvodih z bližnjo infrardečo spektrometrijo.
Določitve temeljijo na spektrometričnih meritvah v bližnjem infrardečem spektralnem območju.

General Information

Status
Published
Public Enquiry End Date
24-Apr-2016
Publication Date
15-Oct-2017
Current Stage
6060 - National Implementation/Publication (Adopted Project)
Start Date
05-Oct-2017
Due Date
10-Dec-2017
Completion Date
16-Oct-2017

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Standards Content (Sample)

SLOVENSKI STANDARD
SIST EN ISO 12099:2017
01-november-2017
1DGRPHãþD
SIST EN ISO 12099:2010
.UPDåLWRLQPOHYVNLSURL]YRGL6PHUQLFH]DXSRUDEREOLåQMHLQIUDUGHþH
VSHNWURPHWULMH ,62
Animal feeding stuffs, cereals and milled cereal products - Guidelines for the application
of near infrared spectrometry (ISO 12099:2017)
Futtermittel, Getreide und gemahlene Getreideerzeugnisse - Anleitung für die
Anwendung von Nahinfrarot-Spektrometrie (ISO 12099:2017)
Aliments des animaux, céréales et produits de mouture des céréales - Lignes directrices
pour l'application de la spectrométrie dans le proche infrarouge (ISO 12099:2017)
Ta slovenski standard je istoveten z: EN ISO 12099:2017
ICS:
65.120 Krmila Animal feeding stuffs
67.060 äLWDVWURþQLFHLQSURL]YRGLL] Cereals, pulses and derived
QMLK products
SIST EN ISO 12099:2017 en
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

---------------------- Page: 1 ----------------------

SIST EN ISO 12099:2017

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SIST EN ISO 12099:2017


EN ISO 12099
EUROPEAN STANDARD

NORME EUROPÉENNE

September 2017
EUROPÄISCHE NORM
ICS 65.120 Supersedes EN ISO 12099:2010
English Version

Animal feeding stuffs, cereals and milled cereal products -
Guidelines for the application of near infrared
spectrometry (ISO 12099:2017)
Aliments des animaux, céréales et produits de mouture Futtermittel, Getreide und gemahlene
des céréales - Lignes directrices pour l'application de la Getreideerzeugnisse - Anleitung für die Anwendung
spectrométrie dans le proche infrarouge (ISO von Nahinfrarot-Spektrometrie (ISO 12099:2017)
12099:2017)
This European Standard was approved by CEN on 14 July 2017.

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, Serbia, 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
© 2017 CEN All rights of exploitation in any form and by any means reserved Ref. No. EN ISO 12099:2017 E
worldwide for CEN national Members.

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SIST EN ISO 12099:2017
EN ISO 12099:2017 (E)
Contents Page
European foreword . 3

2

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SIST EN ISO 12099:2017
EN ISO 12099:2017 (E)
European foreword
This document (EN ISO 12099:2017) has been prepared by Technical Committee ISO/TC 34 “Food
products" in collaboration with Technical Committee CEN/TC 327 “Animal feeding stuffs - 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 March 2018 and conflicting national standards shall be
withdrawn at the latest by March 2018.
Attention is drawn to the possibility that some of the elements of this document may be the subject of
patent rights. CEN shall not be held responsible for identifying any or all such patent rights.
This document supersedes EN ISO 12099:2010.
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, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland,
Turkey and the United Kingdom.
Endorsement notice
The text of ISO 12099:2017 has been approved by CEN as EN ISO 12099:2017 without any modification.
3

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SIST EN ISO 12099:2017

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SIST EN ISO 12099:2017
INTERNATIONAL ISO
STANDARD 12099
Second edition
2017-08
Animal feeding stuffs, cereals and
milled cereal products — Guidelines
for the application of near infrared
spectrometry
Aliments des animaux, céréales et produits de mouture des céréales —
Lignes directrices pour l’application de la spectrométrie dans le
proche infrarouge
Reference number
ISO 12099:2017(E)
©
ISO 2017

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SIST EN ISO 12099:2017
ISO 12099:2017(E)

COPYRIGHT PROTECTED DOCUMENT
© ISO 2017, Published in Switzerland
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 at the address below or ISO’s member body in the country of
the requester.
ISO copyright office
Ch. de Blandonnet 8 • CP 401
CH-1214 Vernier, Geneva, Switzerland
Tel. +41 22 749 01 11
Fax +41 22 749 09 47
copyright@iso.org
www.iso.org
ii © ISO 2017 – All rights reserved

---------------------- Page: 8 ----------------------

SIST EN ISO 12099:2017
ISO 12099:2017(E)

Contents Page
Foreword .iv
Introduction .v
1 Scope . 1
2 Normative references . 1
3 Terms and definitions . 1
4 Principle . 2
5 Apparatus . 2
6 Calibration and initial validation . 2
6.1 General . 2
6.2 Reference methods . 3
6.3 Outliers . 3
6.4 Validation of calibration models . 3
6.4.1 General. 3
6.4.2 Bias correction . 4
6.4.3 Slope adjustment . 4
6.4.4 Expansion of calibration set . 4
6.5 Changes in measuring and instrument conditions . 4
7 Statistics for performance measurement . 5
7.1 General . 5
7.2 Plot the results . 5
7.3 Bias . 6
7.4 Root mean square error of prediction (s ) . 8
RMSEP
7.5 Standard error of prediction (s ) . 8
SEP
7.6 Slope .10
8 Sampling .12
9 Procedure.12
9.1 Preparation of test sample .12
9.2 Measurement .12
9.3 Evaluation of result .12
10 Checking instrument stability .13
10.1 Control sample .13
10.2 Instrument diagnostics .13
10.3 Instruments in a network .13
11 Running performance check of calibration .13
11.1 General .13
11.2 Control charts using the difference between reference and NIR results .14
12 Precision and accuracy .15
12.1 Repeatability .15
12.2 Reproducibility .15
12.3 Accuracy .15
12.4 Uncertainty .15
13 Test report .15
Annex A (informative) Guidelines for specific NIR standards .16
Annex B (informative) Examples of outliers and control charts .17
Annex C (informative) Supplementary terms and definitions .23
Bibliography .28
© ISO 2017 – All rights reserved iii

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SIST EN ISO 12099:2017
ISO 12099:2017(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 (see 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 (see 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.
For an explanation on the voluntary nature of standards, the meaning of ISO specific terms and
expressions related to conformity assessment, as well as information about ISO’s adherence to the
World Trade Organization (WTO) principles in the Technical Barriers to Trade (TBT) see the following
URL: w w w . i s o .org/ iso/ foreword .html.
This document was prepared by Technical Committee ISO/TC 34, Food products, Subcommittee SC 10,
Animal feeding stuffs.
This second edition cancels and replaces the first edition (ISO 12099:2010), which has been technically
revised.
iv © ISO 2017 – All rights reserved

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SIST EN ISO 12099:2017
ISO 12099:2017(E)

Introduction
This document has been drafted using, as a basis, ISO 21543 | IDF 201, which was prepared by Technical
Committee ISO/TC 34, Food products, Subcommittee SC 5, Milk and milk products, and the International
Dairy Federation (IDF).
© ISO 2017 – All rights reserved v

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SIST EN ISO 12099:2017

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SIST EN ISO 12099:2017
INTERNATIONAL STANDARD ISO 12099:2017(E)
Animal feeding stuffs, cereals and milled cereal
products — Guidelines for the application of near infrared
spectrometry
1 Scope
This document gives guidelines for the determination by near infrared spectroscopy of constituents
such as moisture, fat, protein, starch and crude fibre and parameters such as digestibility in animal
feeding stuffs, cereals and milled cereal products.
The determinations are based on spectrometric measurement in the near infrared spectral region.
2 Normative references
There are no normative references in this document.
3 Terms and definitions
For the purposes of this document, the following terms and definitions apply.
ISO and IEC maintain terminological databases for use in standardization at the following addresses:
— IEC Electropedia: available at http:// www .electropedia .org/
— ISO Online browsing platform: available at http:// www .iso .org/ obp
3.1
near infrared instrument
NIR instrument
apparatus which, when used under the conditions defined in this document, predicts constituent
contents (3.3) and technological parameters (3.4) in animal feeding stuffs (3.2), cereals and milled cereal
products through relationships to absorptions in the near infrared range
3.2
animal feeding stuffs
substance or product, including additives, whether processed, partially processed or unprocessed,
intended to be used for oral feeding to animals
EXAMPLE Raw materials, fodder, meat and bone meal, mixed feed and other end products, pet food, etc.
3.3
constituent content
mass fraction of substances determined using the appropriate, standardized or validated chemical
method
Note 1 to entry: The mass fraction is often expressed as a percentage.
Note 2 to entry: For examples of appropriate methods, see References [1] to [12].
EXAMPLE Moisture, fat, protein, crude fibre, neutral detergent fibre and acid detergent fibre.
© ISO 2017 – All rights reserved 1

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SIST EN ISO 12099:2017
ISO 12099:2017(E)

3.4
technological parameter
property or functionality of animal feeding stuffs (3.2), cereals and milled cereal products that can be
determined using the appropriate, standardized or validated method(s)
Note 1 to entry: It is possible to develop and validate NIR methods for other parameters and sample types than
listed above, as long as the procedure from this document is observed. The measuring units of the parameters
determined follow the units used in the reference methods.
EXAMPLE Digestibility.
4 Principle
Spectral data in the near infrared region are collected and transformed to constituent or parameter
concentrations by calibration models developed on representative samples of the products concerned.
5 Apparatus
5.1 Near infrared instruments.
Instruments based on diffuse reflectance or transmittance measurement covering the near infrared
−1 −1
wavelength region of 770 nm to 2 500 nm (12 900 cm to 4 000 cm ) or segments of this or at selected
wavelengths or wavenumbers. The optical principle may be dispersive (e.g. grating monochromators),
interferometric or non-thermal (e.g. light emitting diodes, laser diodes and lasers). The instrument
should be provided with a diagnostic test system for testing photometric noise and reproducibility,
wavelength/wavenumber accuracy and wavelength/wavenumber precision (for scanning
spectrophotometers).
The instrument should measure a sufficiently large sample volume or surface to eliminate any
significant influence of inhomogeneity derived from chemical composition or physical properties of
the test sample. The sample path length (sample thickness) in transmittance measurements should
be optimized according to the manufacturer’s recommendation with respect to signal intensity for
obtaining linearity and maximum signal/noise ratio.
5.2 Appropriate milling or grinding device, for preparing the sample (if needed).
NOTE Changes in grinding or milling conditions can influence NIR measurements due, for example, to
heating which can drive off volatile components such as water.
6 Calibration and initial validation
6.1 General
The instrument shall be calibrated before use. Calibration involves the comparison with a reference
and adjustment processes to the instrument. Because a number of different calibration systems can be
applied with NIR instruments, no specific procedure can be given for calibration.
For an explanation of methods for calibration development, see Reference [16] and the respective
manufacturer’s manual. For the validation, it is important to have a sufficient number of representative
samples, covering variations such as the following:
a) combinations and composition ranges of major and minor sample components;
b) seasonal, geographic and genetic effects on forages, feed raw material and cereals;
c) processing techniques and conditions;
d) storage conditions;
2 © ISO 2017 – All rights reserved

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SIST EN ISO 12099:2017
ISO 12099:2017(E)

e) sample and instrument temperature;
f) instrument variations (i.e. differences between instruments).
NOTE For a solid validation, at least 20 samples are needed.
6.2 Reference methods
Internationally accepted reference methods for determination of moisture, fat, protein and other
constituents and parameters should be used. See References [1] to [12] for examples.
The reference method used for calibration should be in statistical control. It is essential to know the
precision of the reference method.
Where possible, references that provide measurement traceability to the SI (International system of
units), such as certified reference materials, should be used.
6.3 Outliers
In many situations, statistical outliers are observed during calibration and validation. Outliers may be
related to NIR data (spectral outliers, hereafter referred to as “x-outliers”) or errors in reference data
or samples with a different relationship between reference data and NIR data (hereafter referred to as
“y-outliers”); see Figures B.1 to B.5 for examples.
For the purpose of validation, samples are not to be regarded as outliers if they fulfil the following
conditions:
a) if they are within the working range of the constituents/parameters in the calibration(s);
b) if they are within the spectral variation of the calibration samples, as, for example, estimated by
Mahalanobis distance;
c) if the spectral residual is below a limit defined by the calibration process;
d) if the prediction residual is below a limit defined by the calibration process.
If a sample appears as an outlier, then it should be checked initially to see if it is an x-outlier. If it exceeds
the x-outlier limits defined for the calibration, it should be removed. If it is not an x-outlier, then both
the reference value and the NIR predicted value should be checked, e.g. by repeated measurements. If
these confirm the original values, then the sample should not be deleted and the validation statistics
should include this sample. If the repeat values show that either the original reference values or the NIR
predicted ones were in error, then the new values should be used.
6.4 Validation of calibration models
6.4.1 General
Before use, calibration equations shall be validated locally on an independent test set that is
representative of the sample population to be analysed. For the determination of bias, slope and for the
determination of standard error of prediction (SEP, see 7.5), at least 20 samples are needed. Validation
shall be carried out for each sample type, constituent/parameter, temperature and other factors known
to affect or expected to have an effect the measurement. The calibration is valid only for the variations,
i.e. sample types, range and temperature, used in the validation.
NOTE 1 Calibration models can only be used in the range they have been validated.
Results obtained on the independent test set are plotted, reference against NIR, and residuals against
reference results, to give a visual impression of the performance of the calibration. The SEP is calculated
(see 7.5) and the residual plot of data corrected for mean systematic error (bias) is examined for
outliers, i.e. samples with a residual exceeding ±3 s .
SEP
© ISO 2017 – All rights reserved 3

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SIST EN ISO 12099:2017
ISO 12099:2017(E)

If the validation process shows that the model cannot produce acceptable statistics, then it should not
be used.
NOTE 2 What will be acceptable will depend, for example, on the performance of the reference method, the
covered range, the purpose of the analysis, etc., and is up to the parties involved to decide.
Where available and suitable, reference materials or certified reference materials can be used as part of
validation of calibration models.
The next step is to fit NIR, y , and reference data, y , by linear regression (y = a + b × y ) to
NIRS ref ref NIRS
produce statistics that describe the validation results.
6.4.2 Bias correction
The data are also examined for a bias between the methods. If the difference between means of the
NIR predicted and reference values is significantly different from zero, then this indicates that the
calibration is biased. A bias may be removed by adjusting the constant term (see 7.3) in the calibration
equation.
6.4.3 Slope adjustment
If the slope, b, is significantly different from 1, the calibration is skewed.
Adjusting the slope/intercept of the calibration is generally not recommended unless the calibration
is applied to new types of samples or instruments. If a reinvestigation of the calibration does not
detect outliers, especially outliers with high leverage, it is preferable to expand the calibration set to
include more samples. However, if the slope is adjusted, the calibration should then be tested on a new
independent test set.
6.4.4 Expansion of calibration set
If the accuracy of the calibration does not meet expectations, the calibration set should be expanded
to include more samples or a new calibration should be made. In all cases when a new calibration
is developed on an expanded calibration set, the validation process should be repeated on a new
validation set. If necessary, expansion of the calibration set should be repeated until acceptable results
are obtained on a validation set.
6.5 Changes in measuring and instrument conditions
Unless additional calibration is performed, a local validation of a NIR method stating the accuracy of
the method can generally not be considered valid if the test conditions are changed.
For example, calibrations developed for a certain population of samples may not be valid for samples
outside this population, although the analyte concentration range is unchanged. A calibration developed
on grass silages from one area may not give the same accuracy on silages from another area if the
genetic, growing and processing parameters are different.
Changes in the sample presentation technique or the measuring conditions, e.g. temperature, not
included in the calibration set may also influence the analytical results.
Calibrations developed on a certain instrument cannot always be transferred directly to an identical
instrument operating under the same principle. It may be necessary to perform bias or slope /
intercept adjustments to calibration equations. In many cases, it will be nec
...

SLOVENSKI STANDARD
oSIST prEN ISO 12099:2016
01-april-2016
.UPDåLWRLQPOHYVNLSURL]YRGL6PHUQLFH]DXSRUDEREOLåQMHLQIUDUGHþH
VSHNWURPHWULMH ,62',6
Animal feeding stuffs, cereals and milled cereal products - Guidelines for the application
of near infrared spectrometry (ISO/DIS 12099:2016)
Futtermittel, Getreide und gemahlene Getreideerzeugnisse - Anleitung für die
Anwendung von Nahinfrarot-Spektrometrie (ISO/DIS 12099:2016)
Aliments des animaux, céréales et produits de mouture des céréales - Lignes directrices
pour l'application de la spectrométrie dans le proche infrarouge (ISO/DIS 12099:2016)
Ta slovenski standard je istoveten z: prEN ISO 12099
ICS:
65.120 Krmila Animal feeding stuffs
67.060 äLWDVWURþQLFHLQSURL]YRGLL] Cereals, pulses and derived
QMLK products
oSIST prEN ISO 12099:2016 en
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

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oSIST prEN ISO 12099:2016

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oSIST prEN ISO 12099:2016
DRAFT INTERNATIONAL STANDARD
ISO/DIS 12099
ISO/TC 34/SC 10 Secretariat: ISIRI
Voting begins on: Voting terminates on:
2016-01-28 2016-04-28
Animal feeding stuffs, cereals and milled cereal products —
Guidelines for the application of near infrared spectrometry
Aliments des animaux, céréales et produits de mouture des céréales — Lignes directrices pour l’application
de la spectrométrie dans le proche infrarouge
ICS: 65.120
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
THIS DOCUMENT IS A DRAFT CIRCULATED
formal vote in CEN.
FOR COMMENT AND APPROVAL. IT IS
THEREFORE SUBJECT TO CHANGE AND MAY
NOT BE REFERRED TO AS AN INTERNATIONAL
STANDARD UNTIL PUBLISHED AS SUCH.
To expedite distribution, this document is circulated as received from the
IN ADDITION TO THEIR EVALUATION AS
committee secretariat. ISO Central Secretariat work of editing and text
BEING ACCEPTABLE FOR INDUSTRIAL,
composition will be undertaken at publication stage.
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
Reference number
NATIONAL REGULATIONS.
ISO/DIS 12099:2015(E)
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. ISO 2015

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oSIST prEN ISO 12099:2016
ISO/DIS 12099:2015(E)

COPYRIGHT PROTECTED DOCUMENT
© ISO 2015, Published in Switzerland
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 at the address below or ISO’s member body in the country of
the requester.
ISO copyright office
Ch. de Blandonnet 8 • CP 401
CH-1214 Vernier, Geneva, Switzerland
Tel. +41 22 749 01 11
Fax +41 22 749 09 47
copyright@iso.org
www.iso.org
ii © ISO 2015 – All rights reserved

---------------------- Page: 4 ----------------------
oSIST prEN ISO 12099:2016
Rev ISO/DIS 12099
Contents Page
Foreword .iv
Introduction.iv
1 Scope.1
2 Terms and definitions .1
3 Principle.2
4 Apparatus.2
5 Calibration and initial validation .2
6 Statistics for performance measurement .4
7 Sampling.10
8 Procedure.11
9 Checking instrument stability .11
10 Running performance check of calibration .12
11 Precision and accuracy .13
12 Test report.14
Annex A (informative) Guidelines for specific NIR standards.15
Annex B (informative) Example of figures.16
Annex C (informative) Supplementary terms and definitions .21
Bibliography.26
© ISO 2008 – All rights reserved iii

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oSIST prEN ISO 12099:2016
Rev ISO/DIS 12099
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 12099 was prepared by Technical Committee ISO/TC 34, Animal feeding stuffs, Subcommittee SC 10,
and by Technical Committee CEN/TC 327, Animal feeding stuffs in collaboration.
This second/third/. edition cancels and replaces the first/second/. edition (), [clause(s) / subclause(s) /
table(s) / figure(s) / annex(es)] of which [has / have] been technically revised.
iv © ISO 2008 – All rights reserved

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oSIST prEN ISO 12099:2016
Rev ISO/DIS 12099
Introduction
This document has been drafted using as a basis the ISO 21543 / IDF 201 standard [15], which was prepared by
Technical Committee ISO/TC 34, Food products, Subcommittee SC 5, Milk and milk products, and the International
Dairy Federation (IDF).
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DRAFT INTERNATIONAL STANDARD ISO/DIS 12099
Animal feeding stuff, cereals and milled cereal products —
Guidance for the application of near infrared spectrometry
1 Scope
This International Standard gives guide lines for the determination by near infrared spectroscopy of
constituents such as moisture, fat, protein, starch and crude fibre and parameters such as digestibility in
animal feeding stuffs, cereals and milled cereal products.
The determinations are based on spectrometric measurement in the near infrared spectral region.
2 Terms and definitions
For the purposes of this International Standard, the following terms and definitions apply.
2.1 near infrared (NIR) instrument
proprietary apparatus which, when used under the conditions defined in this International Standard, predicts
the mass fractions of constituents and technological parameters as described below in animal feeding stuffs,
cereals and milled cereal products through relationships to absorptions in the near infrared range.
2.2 animal feeding stuffs
any substance or product, including additives, whether processed, partially processed or unprocessed,
intended to be used for oral feeding to animals.
Examples: Raw materials, fodder, animal flour, mixed feed and other end products, pet food etc.
2.3 constituent content
mass fraction of substances determined using the appropriate, standardized or validated chemical method .
NOTE 1 The mass fraction is often expressed as a percentage.
NOTE 2 Examples of constituents determined include moisture, fat, protein, crude fibre, neutral detergent fibre, and acid
detergent fibre. For appropriate methods see e.g. [1-12].
2.4 technological parameters
Property or functionality of animal feeding stuff, cereals and milled cereal products that can be determined
using the appropriate, standardized or validated method(s).
Example of such a parameter is the digestibility.
NOTE It is possible to develop and validate NIR methods for other parameters and sample types than listed above,
as long as the procedure from this standard is observed. The measuring units of the parameters determined have to follow
the units used in the reference methods.
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3 Principle
Spectral data in the near infrared region are collected and transformed to constituent or parameter
concentrations by calibration models developed on representative samples of the concerned products.
4 Apparatus
4.1 Near-infrared instruments
Instruments based on diffuse reflectance or transmittance measurement covering the near infrared
-1 -1
wavelength region of 770–2500 nm (12 900 cm – 4 000 cm ) or segments of this or at selected wavelengths
or wavenumbers. The optical principle may be dispersive (e.g. grating monochromators), interferometric or
non-thermal (e.g. light emitting diodes, laser diodes and lasers). The instrument should be provided with a
diagnostic test system for testing photometric noise and reproducibility, wavelength/wavenumber accuracy
and wavelength/wavenumber precision (for scanning spectrophotometers).
The instrument should measure a sufficiently large sample volume or surface to eliminate any significant
influence of inhomogeneity derived from chemical composition or physical properties of the test sample. The
sample path length (sample thickness) in transmittance measurements should be optimized according to the
manufacturer’s recommendation with respect to signal intensity for obtaining linearity and maximum
signal/noise ratio.
4.2 Appropriate milling or grinding device, for preparing the sample (if needed).
NOTE Changes in grinding or milling conditions may influence NIR measurements.
5 Calibration and initial validation
5.1 General
The instrument has to be calibrated before use. Because a number of different calibration systems can be
applied with NIR instruments, no specific procedure can be given for calibration.
For an explanation of methods for calibration development see a current textbook [16] and respective
manufacturers manuals. For the validation it is important to have a sufficient number of representative
samples, covering variations such as
a) Combinations and composition ranges of major and minor sample components
b) Seasonal, geographic and genetic effects on forages, feed raw material and cereals
c) Processing techniques and conditions
d) Storage conditions
e) Sample and instrument temperature
f) Instrument variations (differences between instruments)
NOTE : For a solid validation at least 20 samples are needed.
5.2 Reference methods
Internationally accepted reference methods for determination of moisture, fat, protein and other constituents
and parameters should be used. See bibliography [1-12] for examples.
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5.4.2 Slope adjustment
If the slope (b) is significantly different from 1, the calibration is skewed.
Adjusting the slope/intercept of the calibration is generally not recommended unless the calibration is applied
to new types of samples or instruments. If a reinvestigation of the calibration does not detect outliers,
especially outliers with high leverage, it is preferable to expand the calibration set to include more samples.
However, if the slope is adjusted, the calibration should then be tested on a new independent test set.
5.4.3 Expansion of calibration set
If the accuracy of the calibration is not meeting the expectations the calibration set should be expanded to
include more samples or a new calibration be made. In all cases when a new calibration is developed on an
expanded calibration set, the validation process should be repeated on a new validation set. If necessary,
expansion of the calibration set should be repeated until acceptable results are obtained on a validation set.
5.5 Changes in measuring and instrument conditions
Unless additional calibration is performed, a local validation of a NIR method stating the accuracy of the
method can generally not be considered valid if the test conditions are changed.
For example, calibrations developed for a certain population of samples may not be valid for samples outside
this population, although the analyte concentration range is unchanged. A calibration developed on grass
silages from one area may not give the same accuracy on silages from another area if the genetic, growing
and processing parameters are different.
Changes in the sample presentation technique or the measuring conditions (e.g. temperature) not included in
the calibration set may also influence the analytical results.
Calibrations developed on a certain instrument cannot always be transferred directly to an identical instrument
operating under the same principle. It may be necessary to perform bias or slope /intercept adjustments to
calibration equations. In many cases it will be necessary to standardize the two instruments against each
other before calibration equations can be transferred [16]. Standardization procedures can be used to transfer
calibrations between instruments of different types provided that samples are measured in the same way
(reflectance, transmittance) and that the spectral region is common.
If the conditions are changed, a supplementary validation should be performed.
The calibrations should be checked whenever any major part of the instrument (optical system, detector) has
been changed or repaired.
6 Statistics for performance measurement
6.1 General
The performances of a prediction model must be determined by a set of validation samples. This set consists
of samples which are independent of the calibration set. In a plant, it will be new batches; in agriculture, it will
be a new crop or a new experiment location.
This set of samples must be carefully analyzed following the reference methods. The care to analyze
validation samples must be emphasized and the precision of these results is more important for the validation
set than for the samples used at the calibration phase.
The number of validation samples must be at least 20 to compute the statistics with some confidence.
The NIR protocol used for the determination of the performances of the prediction model must be the same as
that used in routine (one measurement or two measurements).
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9 Checking instrument stability
9.1 Control sample
At least one control sample should be measured at least once per day to check instrument hardware stability
and to detect any malfunction. Knowledge of the true concentration of the analyte in the control sample is not
necessary. The sample material should be stable and, as far as possible, resemble the samples to be
analyzed. The parameter(s) measured should be stable and, as far as possible identical to or at least
biochemically close to the sample analyte. A sample is prepared as in 8.1 and stored in such a way as to
maximize the storage life. These samples are normally stable for lengthy periods but the stability should be
tested in the actual cases. Control samples should be overlapped to secure uninterrupted control.
The recorded day-to-day variation should be plotted in control charts and investigated for significant patterns
or trends.
9.2 Instrument diagnostics
For scanning spectrophotometers the wavelength/wavenumber (see above 4.1) accuracy and precision
should be checked at least once a week, or more frequently if recommended by the instrument manufacturer,
and the results should be compared to specifications and requirements (4.1).
A similar check of the instrument noise shall be carried out weekly, or at intervals recommended by the
manufacturer.
9.3 Instruments in a network
If several instruments are used in a network, special attention has to be given to standardization of the
instruments according to the manufacturers recommendations.
10 Running performance check of calibration
10.1 General
The suitability of the calibration for the measurement of individual samples should be checked. The outlier
measures used in the calibration development and validation can be applied e.g. Mahalanobis distance and
spectral residuals. In most instruments this is done automatically.
If the sample does not pass the test, i.e. the samples does not fit into the population of the samples used for
calibration and/or validation, it cannot be determined by the prediction model, unless the model is changed.
Thus the outlier measures can be used to decide which samples should be selected for reference analysis
and included in a calibration model update.
If the calibration model is found to be suitable for the measured sample, the spectrum is evaluated according
to the validated calibration model.
NIR methods should be validated continuously against reference methods to secure steady optimal
performance of calibrations and observance of accuracy. The frequency of checking the NIR method should
be sufficient to ensure that the method is operating under steady control with respect to systematic and
random deviations from the reference method. The frequency depends inter alia on the number of samples
analyzed per day and the rate of changes in sample population.
The running validation should be performed on samples selected randomly from the pool of analyzed samples.
It may be necessary to resort to some sampling strategy to ensure a balanced sample distribution over the
entire calibration range, e.g. segmentation of concentration range and random selection of test samples within
each segment or to ensure that samples with a commercially important range are covered.
The number of samples for the running validation should be sufficient for the statistics used to check the
performance. For a solid validation at least 20 samples are needed (to expect a normal distribution of
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variance). One can fill in the results of the independent validation set for starting the running validation. To
continue about 5 to 10 samples every week is quite sufficient to monitor the performance properly. Using less
samples it is hard to take the right decision in case one of the results is outside the control limits.
10.2 Control charts using the difference between reference and NIR results
Results should be assessed by control charts, plotting running sample numbers on the abscissa and the
difference between results obtained by reference and NIR methods on the ordinate; ± 2 s (95% probability)
SEP
and ± 3 s (99,8 % probability) may be used as warning and action limits where the S has been obtained
SEP SEP
on a test set collected independently of calibration samples.
If the calibration and the reference laboratories are performing as they should, then only 1 point in 20 points
should plot outside the warning limits and 2 point in 1 000 points outside the action limits.
Control charts should be checked for systematic bias drifts from zero, systematic patterns, and excessive
variation of results. General rules applied for Shewart control charts may be used in the assessment. However,
too many rules applied simultaneously may result in too many false alarms.
The following rules used in combination have proved to be useful in detection of problems.
1) One point outside either action limit.
2) Two out of three points in a row outside a warning limit.
3) Nine points in a row on the same side of the zero line.
Additional control charts plotting other features of the running control (e.g. mean difference between NIR and
reference results) and additional rules may be applied to strengthen decisions.
In assessment of results it should be remembered that s and measured differences between NIR and
SEP
reference results also include the imprecision of reference results. This contribution can be reduced to a
negligible part if the imprecision of reference results is reduced to less than one third of the s [17].
SEP
To reduce the risk of false alarms, the control samples should be analyzed independently (in different series)
by both NIR spectrometry and reference methods to avoid the influence of day-to-day systematic differences
in e.g. reference analyses.
If the warning limits are often exceeded and the control chart only shows random fluctuations (as opposed to
trends or systematic bias), the control limits may have been based on a too optimistic s value. An attempt
SEP
to force the results within the limits by frequent adjustments of the calibration will not improve the situation in
practice. The s should instead be re-evaluated using the latest results.
SEP
If the calibration equations after a period of stability begin to move out of control, the calibration should be
updated. Before this is done, an evaluation should be made of whether the changes could be due to changes
in reference analyses, unintended changes in measuring conditions (e.g. caused by a new operator),
instrument drift or malfunction etc. In some cases a simple adjustment of the constant term in the calibration
equation may be sufficient (an example is shown in Figure B.6). In other cases it may be necessary to run a
complete re-calibration procedure, where the complete or a part of the basic calibration set is expanded to
include samples from the running validation, and perhaps additional samples selected for this purpose (an
example is shown in Figure B.7).
Considering that the reference analyses are in statistical control and the measuring conditions and instrument
performance are unchanged, significant biases or increased s values can be due to changes in the
SEP
chemical, biological or physical properties of the samples compared to the underlying calibration set.
Other control charts, e.g. using z-scores may be used.
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11 Precision and accuracy
11.1 Repeatability
The repeatability, i.e. the difference between two individual single test results, obtained with the same method
on identical test material in the same laboratory by the same operator using the same equipment within a
short interval of time, which should not be exceeded in more than 5% of cases, depends on the sample
material, the analyte, sample and analyte variation ranges, method of sample presentation, instrument type,
and the calibration strategy used. The repeatability should be determined in each case.
11.2 Reproducibility
The reproducibility, i.e. the difference between two individual single test results, obtained on identical test
material by different laboratories and by different operators at different times, which should not be exceeded in
more than 5% of cases, depends on the sample material, the analyte, sample and analyte variation ranges,
method of sample presentation, instrument type, and the calibration strategy used. The reproducibility should
be determined in each case.
11.3 Accuracy
The accuracy, which includes uncertainty from systematic deviation from the true value on the individual
sample (trueness) and uncertainty from random variation (precision), depends inter alia on the sample
material, the analyte, sample and analyte variation ranges, method of sample presentation, instrument type,
and the calibration strategy used. The accuracy should be determined in each case. The reported S and
SEP
S values also include uncertainty of reference results which may vary from case to case.
RMSEP
11.4 Uncertainty
Uncertainty, Ue, is a parameter characterizing the dispersion of values that can reasonably be attributed to the
result. For NIR predicted results the uncertainty is usually expressed as
Ue = ±2 s
RMSEP
Note:The s has to be determined locally.
RMSEP
12 Test report
The test report shall specify:
a) all information necessary for complete identification of the sample;
b) the test method used, with reference to the relevant International Standard;
c) all operating conditions not specified in this International Standard, or regarded as optional,
d) and any circumstances which may have influenced the results;
e) the test result(s) obtained;
f) the current s and bias (if statistically significant), estimated from running a performance test on at
SEP
least 20 test samples (clause 10).
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Annex B
(informative)
Example of figures
275
225
175
125
75
75 125 175 225 275
NIR
Figure B.1 — Example: No outliers.
Determination of crude protein in forages: Results obtained on an independent test set (95 samples) using the
developed calibration equation: s = 4,02; s = 6,05; Slope b = 1,04.
SEP RMSEP
= ± 3 s limits, where s is standard deviation
= 45 degree line (ideal line with slope = 1 and bias = 0)
= regression line
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Reference

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4,5
4
Series1
Series2
3,5
Series3
Series4
3
Series5
Series6
2,5
2
Figure B.2 — Absorbance spectra with X-outlier.
The series 1 (upper) spectrum is indicating a spectral outlier.
Figure B.3 — PCA score plot with X-outlier (o).
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850
866
882
898
914
930
946
962
978
994
1010
1026
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Figure B.4 — Scatter plot with Y-outlier. (o).
The plot of reference vs predicted values (or vice versa) is showing one sample (o) that is strongly deviating from the other
samples. If the reason for this deviation is not related to NIR data (X-outlier) this sample will be a Y-outlier, due to
erroneous reference data or a different relationship between reference data and spectral data.
Figure B.5 — Example Determination of ADF in forages with Y-outlier (o)
= +/- 3 s limits
= 45 degree line
= regression line
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Figure B.6 — Example: Control chart for determination of percent fat in cereals.
No points are outside the upper action limit (UAL) or the lower action limit (LAL). However, 9 points in row (e.g.
14 to 22) are on the same side of the zero line. That indicates a bias problem. Two points (27 and 28) out of 3
points are outside the lower warning limit (LWL) but none are outside the upper warning limit (UWL). This also
indicates a bias problem. No increase in random variation is observed. The spread is still less than 3 s .
SEP
In conclusion, the calibration should be bias adjusted.
(Difference Reference – NIR is plotted. UAL/LAL = 3 s and UWL/LWL = 2 s )
SEP SEP
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