ISO 23776:2021
(Main)Meat and meat products — Determination of total phosphorous content
Meat and meat products — Determination of total phosphorous content
This document specifies three methods for the determination of the total phosphorous content of all kinds of meat and meat products, including poultry and livestock: the inductively coupled plasma optical emission spectrometry (ICP-OES) method; the spectrometric method; the gravimetric method. For the ICP-OES method, the limit of detection (LOD) is 1,0 mg/kg and the limit of quantification (LOQ) is 3,0 mg/kg if the mass of the test portion is 0,5 g and the constant volume is 50 ml.
Viandes et produits à base de viande — Détermination de la teneur en phosphore
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INTERNATIONAL ISO
STANDARD 23776
First edition
2021-07
Corrected version
2022-02
Meat and meat products —
Determination of total phosphorous
content
Viandes et produits à base de viande — Détermination de la teneur en
phosphore
Reference number
ISO 23776:2021(E)
© ISO 2021
---------------------- Page: 1 ----------------------
ISO 23776:2021(E)
COPYRIGHT PROTECTED DOCUMENT
© ISO 2021
All rights reserved. Unless otherwise specified, or required in the context of its implementation, 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
CP 401 • Ch. de Blandonnet 8
CH-1214 Vernier, Geneva
Phone: +41 22 749 01 11
Email: copyright@iso.org
Website: www.iso.org
Published in Switzerland
ii
© ISO 2021 – All rights reserved
---------------------- Page: 2 ----------------------
ISO 23776:2021(E)
Contents Page
Foreword .v
1 Scope . 1
2 Normative references . 1
3 Terms and definitions . 1
4 Principle . 1
4.1 Inductively coupled plasma optical emission spectrometry (ICP-OES) method . 1
4.2 Spectrometric method . 2
4.3 Gravimetric method . . 2
5 Sampling . 2
6 Inductively coupled plasma optical emission spectrometry (ICP-OES) method.2
6.1 Reagents . 2
6.2 Apparatus . 2
6.3 Procedure . 3
6.3.1 Sample pre-treatment . 3
6.3.2 Sample digestion . 3
6.4 Determination . 4
6.4.1 Instrument reference conditions . 4
6.4.2 Standard curve drawing . . 4
6.4.3 Test portion . 4
6.5 Calculation and expression of results . 4
6.6 Limit of detection . 5
6.7 Precision . . . 5
6.8 Repeatability . 5
6.9 Reproducibility . 5
7 Spectrometric method . 5
7.1 Reagents . 5
7.2 Apparatus . 6
7.3 Preparation of test sample . 7
7.4 Procedure . 7
7.4.1 Test portion . 7
7.4.2 Determination . 7
7.5 Calibration graph photo-electric . 7
7.6 Calculation . 8
7.7 Precision . . . 8
7.8 Repeatability . 8
7.9 Reproducibility . 8
8 Gravimetric method .8
8.1 Reagents . 8
8.2 Apparatus . 9
8.3 Procedures . 9
8.3.1 Preparation of test sample . 9
8.3.2 Test portion . 9
8.3.3 Mineralization . 10
8.3.4 Determination . 10
8.3.5 Blank test . 10
8.3.6 Expression of results . 10
8.3.7 Repeatability . 11
8.4 Notes on procedure . 11
9 Test report .11
Annex A (informative) International laboratory ring test (ICP-OES method) .12
iii
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ISO 23776:2021(E)
Bibliography .18
iv
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ISO 23776:2021(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 of 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
www.iso.org/iso/foreword.html.
This document was prepared by Technical Committee ISO/TC 34, Food products, Subcommittee SC 6,
Meat, poultry, fish, eggs and their products.
This first edition cancels and replaces ISO 2294:1974 and ISO 13730:1996, which have been technically
revised. The main changes compared with ISO 2294:1974 and ISO 13730:1996 are as follows:
— a new test method, the inductively coupled plasma optical emission spectrometry (ICP-OES) method,
has been added;
— the structure of the document has been revised;
— the title of the document has been modified;
— the Scope has been modified.
This corrected version of ISO 23776:2021 incorporates the following corrections:
— the concentration of phosphate stock solution (7.1.6) has been increased from c(P) = 109 mg/l;
c(P O ) = 250 mg/l to c(P) = 218 mg/l; c(P O ) = 500 mg/l;
2 5 2 5
— the amount of phosphate stock solution (7.1.6) dissolved in water has been increased from 479,4 mg
to 958,8 mg.
Any feedback or questions on this document should be directed to the user’s national standards body. A
complete listing of these bodies can be found at www.iso.org/members.html.
v
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INTERNATIONAL STANDARD ISO 23776:2021(E)
Meat and meat products — Determination of total
phosphorous content
1 Scope
This document specifies three methods for the determination of the total phosphorous content of all
kinds of meat and meat products, including poultry and livestock:
— the inductively coupled plasma optical emission spectrometry (ICP-OES) method;
— the spectrometric method;
— the gravimetric method.
For the ICP-OES method, the limit of detection (LOD) is 1,0 mg/kg and the limit of quantification (LOQ)
is 3,0 mg/kg if the mass of the test portion is 0,5 g and the constant volume is 50 ml.
2 Normative references
The following documents are referred to in the text in such a way that some or all of their content
constitutes requirements of this document. For dated references, only the edition cited applies. For
undated references, the latest edition of the referenced document (including any amendments) applies.
ISO 936, Meat and meat products — Determination of total ash
ISO 3696, Water for analytical laboratory use — Specification and test methods
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:
— ISO Online browsing platform: available at https:// www .iso .org/ obp
— IEC Electropedia: available at http:// www .electropedia .org/
3.1
total phosphorous content of meat and meat products
mass of phosphorous pentoxide determined by the procedure specified in this document
Note 1 to entry: It is expressed as a percentage of the mass of the test portion.
4 Principle
4.1 Inductively coupled plasma optical emission spectrometry (ICP-OES) method
The test portion of the sample is microwave digested with nitric acid. The concentration of phosphorous
is determined by ICP-OES using external calibration. In a certain concentration range, the spectral line
signal intensity of phosphorous is proportional to its concentration, and is quantified by the standard
curve method.
1
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ISO 23776:2021(E)
4.2 Spectrometric method
Drying of the test portion and incineration of the residue. After cooling, hydrolysis of the ash with
nitric acid. Filtration and dilution followed by the formation of a yellow compound with a mixture of
ammonium monovanadate and ammonium heptamolybdate. Photometric measurement at a wavelength
of 430 nm.
4.3 Gravimetric method
Mineralization of a test portion with sulfuric and nitric acids. Precipitation of the phosphorous as
quinoline phosphomolybdate. Drying and weighing of the precipitate. An alternative method of
mineralization is described in 8.4.
5 Sampling
Sampling is not part of the method specified in this document. A recommended sampling method is
given in ISO 17604.
It is important that the laboratory receives a sample which is truly representative and has not been
damaged or changed during transport or storage.
Start from a representative sample of at least 200 g. Store the sample in such a way that deterioration
and change in composition are prevented.
6 Inductively coupled plasma optical emission spectrometry (ICP-OES) method
6.1 Reagents
Use only reagents of recognized analytical grade.
6.1.1 Water, conforming to at least grade 1 in accordance with ISO 3696.
6.1.2 Nitric acid (HNO ), concentrated, not less than 65 % (mass fraction) or higher purity,
3
ρ = 1,42 g/ml.
20
6.1.3 Argon (Ar): argon (> 99,995 %, mass fraction) or liquid argon.
6.1.4 Nitric acid (5 + 95): take 50 ml nitric acid (6.1.2), slowly add 950 ml water and mix.
6.1.5 Phosphorous standard stock solution (1 000 mg/l), c(P) = 1 000 mg/l; c(P O ) = 2 294 mg/l.
2 5
This stock solution is stable for one month when stored in the dark at room temperature.
6.1.6 Standard series solution of phosphorous: accurate extract standard reserve liquid, dilute
standard series solution with nitric acid solution (5 + 95). The mass concentration is 0 mg/l, 20,0 mg/l,
40,0 mg/l, 60,0 mg/l, 80,0 mg/l and 100,0 mg/l.
According to the sensitivity of the instrument and the actual content of phosphorous in the sample, the
concentration range of the standard solution should be adjusted appropriately.
6.2 Apparatus
IMPORTANT — All glassware shall be thoroughly cleaned using a phosphate-free detergent and
then rinsed with water.
The usual laboratory apparatus and, in particular, the following shall be used.
2
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ISO 23776:2021(E)
6.2.1 Inductively coupled plasma optical emission spectrometer.
6.2.2 Analytical balance, capable of weighing to the nearest 0,000 1 g.
6.2.3 Microwave digestion instrument, with polytetrafluoroethylene digestion internal tank.
6.2.4 Electric hot plate with adjustable temperature control, or graphite digestion unit.
6.2.5 Ultrasonic water bath.
6.2.6 Homogenizer, high-speed pulverizer, capable of sample pulverizing and homogenizing.
6.2.7 One-mark volumetric flasks, of capacities 25 ml and 50 ml.
6.2.8 One-mark pipettes, of capacities 2 ml, 5 ml and 10 ml.
6.2.9 Graduated (automatic) pipettes, of capacities 2 ml, 5 ml and 10 ml.
6.2.10 Polytetrafluoroethylene digestion tube.
6.3 Procedure
6.3.1 Sample pre-treatment
Samples with low water content are mixed together after removing debris. The samples with high
water content are homogenized.
6.3.2 Sample digestion
Microwave digestion:
— weigh 0,2 g to 0,5 g of the test portion (accurate to 0,001 g, the sample with more moisture content
can be appropriately increased to 1 g to 2 g) in the microwave digestion internal tank (6.2.3) with
the polytetrafluoroethylene digestion tube (6.2.10);
— add 5 ml to 10 ml of nitric acid (6.1.2);
— stand for 1 h or overnight;
— screw the tank cap;
— follow the standard operation steps of the microwave digestion instrument (6.2.3) to digest;
— take out after cooling;
— slowly open the tank cap and vent;
— flush the inner cap with a little water;
— put the digestion tank (6.2.3) on the electric hot plate (with adjustable temperature control) (6.2.4)
or in the ultrasonic water bath (6.2.5);
— heat for 30 min at 100 °C or ultrasonic degassing (approximately 40 kHZ) for 2 min to 5 min;
— dilute with water to 25 ml or 50 ml and mix;
— do a blank test at the same time.
3
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ISO 23776:2021(E)
6.4 Determination
6.4.1 Instrument reference conditions
The process is as follows:
— optimize the operating conditions of the instrument;
— ensure the instrument sensitivity and other indicators meet the requirements of the analysis;
— the reference conditions for the instrument operation are observation mode:
— horizontal observation;
— power: 1 150 W;
— plasma gas flow: 15 l/min;
— auxiliary gas flow: 0,5 l/min;
— atomized gas flow: 0,65 l/min;
— measured line (select one): 213,6 nm, 214,9 nm, 178,3 nm, 177 nm or 177,4 nm.
6.4.2 Standard curve drawing
The standard series working solution is injected into the inductively coupled plasma optical emission
spectrometer (6.2.1) and the intensity signal response of the analytical spectral line is determined.
When the element concentration is abscissa, the spectral line intensity response value is y-axis and the
standard curve is drawn.
6.4.3 Test portion
For sample determination, the blank solution and sample solution are injected into the inductively
coupled plasma optical emission spectrometer (6.2.1). The signal response values of the spectral line
strength are measured, and the concentration of phosphorous in the solution is obtained according to
the standard curve.
6.5 Calculation and expression of results
Calculate the phosphorous content, X, in milligrams per kilogram, using Formula (1):
()ρρ− ××Vf
0
X = (1)
m
where
ρ is the phosphorous mass concentration of the test portion, in milligrams per litre;
ρ is the phosphorous mass concentration of the blank test, in milligrams per litre;
0
V is the constant volume of the sample digestion solution, in millilitre;
m is the numerical value of the mass, in grams of test portion;
f is the dilution factor.
Three-bit valid numbers are reserved for the results of the calculation.
4
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ISO 23776:2021(E)
6.6 Limit of detection
The LOD is 1,0 mg/kg and the LOQ is 3,0 mg/kg if the mass of the test portion is 0,5 g and the constant
volume is 50 ml.
6.7 Precision
The precision of the method was established by an international laboratory ring test, carried out in
accordance with ISO 5725-2.
6.8 Repeatability
The absolute difference between two independent single test results, obtained using the same method
on test material in the same laboratory by the same operator using the same equipment within a short
interval of time (see Annex A).
6.9 Reproducibility
The absolute difference between two independent single test results, obtained using the same method
on identical test material in different laboratories with different operators using different equipment
(see Annex A).
7 Spectrometric method
7.1 Reagents
Use only reagents of recognized analytical grade and distilled or demineralised water or water of at
least equivalent purity.
7.1.1 Nitric acid (HNO ), ρ = 1,42 g/ml, guarantee reagent or higher purity.
3 20
7.1.2 Nitric acid: water 1:2 (volume/volume).
Mix one volume of nitric acid [mass fraction of 65 %; ρ = 1, 42 g/ml (7.1.1)] with two volumes of water.
20
7.1.3 Ammonium metavanadate (ammonium monovanadate) solution (NH VO ), 2,5 g/l.
4 3
Dissolve 2,5 g of ammonium metavanadate in 500 ml of boiling water. Cool and add 20 ml of the nitric
acid (7.1.2), dilute to the mark 1 l with water and mix.
7.1.4 Ammonium heptamolybdate solution, [(NH ) Mo O ·4H O], 50 g/l (CAS 12027-67-7).
4 6 7 24 2
Dissolve 50 g of ammonium heptamolybdate tetrahydrate in about 800 ml of warm water (at
approximately 50 °C). Cool and transfer quantitatively to a 1 000 ml volumetric flask. Dilute to the
mark with water and mix.
7.1.5 Colour reagent.
Mix one volume of the nitric acid (7.1.2) with one volume of the ammonium metavanadate solution
(7.1.3). Subsequently add one volume of the ammonium heptamolybdate solution (7.1.4) and mix. Make
sure of the order of addition. It can be kept stable in the dark for one month.
7.1.6 Phosphate stock solution, c(P) = 218 mg/l; c(P O ) = 500 mg/l.
2 5
Potassium dihydrogen phosphate (KH PO ), (CAS: 7778-77-0, > 99,99 %).
2 4
5
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ISO 23776:2021(E)
Dissolve in water 958,8 mg of potassium dihydrogen phosphate (KH PO ), previously dried for 3 h at
2 4
(103 ± 2) °C and allowed to cool in desiccators.
Transfer quantitatively to a 1 000 ml volumetric flask. Dilute to the mark with water and mix.
7.1.7 Phosphate standard solutions, containing between 0,05 mg and 0,30 mg of P O per millilitre.
2 5
Transfer by pipette or burette to 100 ml volumetric flasks 10 ml, 20 ml, 30 ml, 40 ml, 50 ml and 60 ml of
the phosphate stock solution (7.1.6). Add 10 ml of the nitric acid (7.1.2). Dilute to the mark with water
and mix.
The range of standard curve can be adjusted according to the use of different concentrations of standard
substance. Use water to dilute.
7.1.8 Blank solution.
Pipette 2 ml of the nitric acid (7.1.2) and 30 ml of the colour reagent (7.1.5) into a 100 ml volumetric
flask. Dilute to the mark with water and mix.
7.2 Apparatus
IMPORTANT — All glassware shall be thoroughly cleaned using a phosphate-free detergent and
then rinsed with water.
The usual laboratory apparatus and, in particular, the following shall be used.
7.2.1 Mechanical or electrical equipment capable of homogenizing the laboratory sample,
including a high speed rotational cutter or a mincer fitted with a plate with holes not exceeding 4,5 mm
in diameter.
7.2.2 Water bath, capable of being maintained at 100 °C.
7.2.3 Fluted filter paper, of diameter 15 cm, phosphate free.
7.2.4 Spectrometer, capable of being used at a wavelength of (430 ± 2) nm, or a photo-electric
colorimeter with an interference filter with absorption maximum of (430 ± 2) nm.
7.2.5 Glass cells, of 10 mm optical path length.
7.2.6 Analytical balance, capable of weighing to an accuracy of ±0,001 g.
7.2.7 One-mark volumetric flasks, of capacities (100 ± 0,10) ml and (1 000 ± 0,40) ml.
7.2.8 Muffle furnace, with adjustable temperature control, temperature (550 ± 25) °С.
7.2.9 Electric hot plate, with adjustable temperature control.
7.2.10 Porcelain crucible, with 60 mm diameter, 25 mm high inclined wall.
7.2.11 Desiccator, provided with an effective desiccant.
For details of this and other apparatus needed for the incineration procedure, see ISO 936.
7.2.12 Mechanical meat mincer, laboratory size, fitted with a plate with holes of diameter not
exceeding 4 mm.
6
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ISO 23776:2021(E)
7.3 Preparation of test sample
Homogenize the laboratory sample with the appropriate equipment (7.2.1). Take care that the
temperature of the sample material does not rise above 25 °C. If a mincer is used, pass the sample at
least twice through the equipment.
Fill a suitable airtight container with the prepared test sample, close the container and store in such
a way that deterioration and change in composition are prevented. Analyse the test sample as soon as
practicable. The storage time should be not more than 24 h.
7.4 Procedure
7.4.1 Test portion
7.4.1.1 If it is necessary to check whether the repeatability requirement (see 7.8) is met, carry out
two single determinations in accordance with 7.4.1 to 7.5.
7.4.1.2 Weigh, to the nearest 0,001 g, about 5 g of the prepared test sample. Carry out the
mineralization of the test portion by using an incinerator (7.2.8) and the method described in ISO 936.
Take up the resulting ash in 10 ml of the nitric acid (7.1.2) using a stirring rod to aid dissolution. Cover
the dish with a watch glass and heat for 30 min on a boiling water bath (7.2.2). Allow to cool and transfer
the liquid quantitatively to a 100 ml volumetric flask (7.2.7). Dilute to the mark with water, mix and
filter through the filter paper (7.2.3), rejecting the first 5 ml to 10 ml of filtrate.
7.4.2 Determination
7.4.2.1 Pipette 20 ml of the clear and colourless filtrate (7.4.1.1) into a 100 ml volumetric flask (7.2.7)
and add 30 ml of the colour reagent (7.1.5) by pipette. Dilute to the mark with water and mix. Allow to
stand for at least 15 min.
7.4.2.2 Measure the absorbance at a wavelength of (430 ± 2) nm in a glass cell (7.2.5) against the
blank solution (7.1.8), using the spectrometer or the colorimeter equipped with an interference filter
(7.2.4).
7.4.2.3 Read the phosphorous concentration of the sample solution from the calibration graph
obtained as described in 7.5.
7.5 Calibration graph photo-electric
7.5.1 Pipette 20 ml of each phosphate standard solution (7.1.7) into 100 ml volumetric flasks. Add
to these solutions 30 ml of the colour reagent. Dilute to the mark with water to obtain concentrations
of 10 μg, 20 μg, 30 μg, 40 μg, 50 μg and 60 μg of P O per millilitre. Mix and allow to stand for at least
2 5
15 min.
7.5.2 C
...
INTERNATIONAL ISO
STANDARD 23776
First edition
2021-07
Meat and meat products —
Determination of total phosphorous
content
Viandes et produits à base de viande — Détermination de la teneur en
phosphore
Reference number
ISO 23776:2021(E)
©
ISO 2021
---------------------- Page: 1 ----------------------
ISO 23776:2021(E)
COPYRIGHT PROTECTED DOCUMENT
© ISO 2021
All rights reserved. Unless otherwise specified, or required in the context of its implementation, 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
CP 401 • Ch. de Blandonnet 8
CH-1214 Vernier, Geneva
Phone: +41 22 749 01 11
Email: copyright@iso.org
Website: www.iso.org
Published in Switzerland
ii © ISO 2021 – All rights reserved
---------------------- Page: 2 ----------------------
ISO 23776:2021(E)
Contents Page
Foreword .v
1 Scope . 1
2 Normative references . 1
3 Terms and definitions . 1
4 Principle . 1
4.1 Inductively coupled plasma optical emission spectrometry (ICP-OES) method . 1
4.2 Spectrometric method . 2
4.3 Gravimetric method . 2
5 Sampling . 2
6 Inductively coupled plasma optical emission spectrometry (ICP-OES) method .2
6.1 Reagents. 2
6.2 Apparatus . 2
6.3 Procedure . 3
6.3.1 Sample pre-treatment . 3
6.3.2 Sample digestion . 3
6.4 Determination . 4
6.4.1 Instrument reference conditions . 4
6.4.2 Standard curve drawing . 4
6.4.3 Test portion . 4
6.5 Calculation and expression of results. 4
6.6 Limit of detection . 5
6.7 Precision . 5
6.8 Repeatability . 5
6.9 Reproducibility . 5
7 Spectrometric method . 5
7.1 Reagents. 5
7.2 Apparatus . 6
7.3 Preparation of test sample . 7
7.4 Procedure . 7
7.4.1 Test portion . 7
7.4.2 Determination . 7
7.5 Calibration graph photo-electric . 7
7.6 Calculation . 8
7.7 Precision . 8
7.8 Repeatability . 8
7.9 Reproducibility . 8
8 Gravimetric method . 8
8.1 Reagents. 8
8.2 Apparatus . 9
8.3 Procedures . 9
8.3.1 Preparation of test sample . 9
8.3.2 Test portion . 9
8.3.3 Mineralization .10
8.3.4 Determination .10
8.3.5 Blank test .10
8.3.6 Expression of results .10
8.3.7 Repeatability .11
8.4 Notes on procedure .11
9 Test report .11
Annex A (informative) International laboratory ring test (ICP-OES method) .12
© ISO 2021 – All rights reserved iii
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ISO 23776:2021(E)
Bibliography .18
iv © ISO 2021 – All rights reserved
---------------------- Page: 4 ----------------------
ISO 23776:2021(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 of 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 www .iso .org/
iso/ foreword .html.
This document was prepared by Technical Committee ISO/TC 34, Food products, Subcommittee SC 6,
Meat, poultry, fish, eggs and their products.
This first edition cancels and replaces ISO 2294:1974 and ISO 13730:1996, which have been technically
revised. The main changes compared with ISO 2294:1974 and ISO 13730:1996 are as follows:
— a new test method, the inductively coupled plasma optical emission spectrometry (ICP-OES) method,
has been added;
— the structure of the document has been revised;
— the title of the document has been modified;
— the Scope has been modified.
Any feedback or questions on this document should be directed to the user’s national standards body. A
complete listing of these bodies can be found at www .iso .org/ members .html.
© ISO 2021 – All rights reserved v
---------------------- Page: 5 ----------------------
INTERNATIONAL STANDARD ISO 23776:2021(E)
Meat and meat products — Determination of total
phosphorous content
1 Scope
This document specifies three methods for the determination of the total phosphorous content of all
kinds of meat and meat products, including poultry and livestock:
— the inductively coupled plasma optical emission spectrometry (ICP-OES) method;
— the spectrometric method;
— the gravimetric method.
For the ICP-OES method, the limit of detection (LOD) is 1,0 mg/kg and the limit of quantification (LOQ)
is 3,0 mg/kg if the mass of the test portion is 0,5 g and the constant volume is 50 ml.
2 Normative references
The following documents are referred to in the text in such a way that some or all of their content
constitutes requirements of this document. For dated references, only the edition cited applies. For
undated references, the latest edition of the referenced document (including any amendments) applies.
ISO 936, Meat and meat products — Determination of total ash
ISO 3696, Water for analytical laboratory use — Specification and test methods
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:
— ISO Online browsing platform: available at https:// www .iso .org/ obp
— IEC Electropedia: available at http:// www .electropedia .org/
3.1
total phosphorous content of meat and meat products
mass of phosphorous pentoxide determined by the procedure specified in this document
Note 1 to entry: It is expressed as a percentage of the mass of the test portion.
4 Principle
4.1 Inductively coupled plasma optical emission spectrometry (ICP-OES) method
The test portion of the sample is microwave digested with nitric acid. The concentration of phosphorous
is determined by ICP-OES using external calibration. In a certain concentration range, the spectral line
signal intensity of phosphorous is proportional to its concentration, and is quantified by the standard
curve method.
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ISO 23776:2021(E)
4.2 Spectrometric method
Drying of the test portion and incineration of the residue. After cooling, hydrolysis of the ash with
nitric acid. Filtration and dilution followed by the formation of a yellow compound with a mixture of
ammonium monovanadate and ammonium heptamolybdate. Photometric measurement at a wavelength
of 430 nm.
4.3 Gravimetric method
Mineralization of a test portion with sulfuric and nitric acids. Precipitation of the phosphorous as
quinoline phosphomolybdate. Drying and weighing of the precipitate. An alternative method of
mineralization is described in 8.4.
5 Sampling
Sampling is not part of the method specified in this document. A recommended sampling method is
given in ISO 17604.
It is important that the laboratory receives a sample which is truly representative and has not been
damaged or changed during transport or storage.
Start from a representative sample of at least 200 g. Store the sample in such a way that deterioration
and change in composition are prevented.
6 Inductively coupled plasma optical emission spectrometry (ICP-OES) method
6.1 Reagents
Use only reagents of recognized analytical grade.
6.1.1 Water, conforming to at least grade 1 in accordance with ISO 3696.
6.1.2 Nitric acid (HNO ), concentrated, not less than 65 % (mass fraction) or higher purity,
3
ρ = 1,42 g/ml.
20
6.1.3 Argon (Ar): argon (> 99,995 %, mass fraction) or liquid argon.
6.1.4 Nitric acid (5 + 95): take 50 ml nitric acid (6.1.2), slowly add 950 ml water and mix.
6.1.5 Phosphorous standard stock solution (1 000 mg/l), c(P) = 1 000 mg/l; c(P O ) = 2 294 mg/l.
2 5
This stock solution is stable for one month when stored in the dark at room temperature.
6.1.6 Standard series solution of phosphorous: accurate extract standard reserve liquid, dilute
standard series solution with nitric acid solution (5 + 95). The mass concentration is 0 mg/l, 20,0 mg/l,
40,0 mg/l, 60,0 mg/l, 80,0 mg/l and 100,0 mg/l.
According to the sensitivity of the instrument and the actual content of phosphorous in the sample, the
concentration range of the standard solution should be adjusted appropriately.
6.2 Apparatus
IMPORTANT — All glassware shall be thoroughly cleaned using a phosphate-free detergent and
then rinsed with water.
The usual laboratory apparatus and, in particular, the following shall be used.
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ISO 23776:2021(E)
6.2.1 Inductively coupled plasma optical emission spectrometer.
6.2.2 Analytical balance, capable of weighing to the nearest 0,000 1 g.
6.2.3 Microwave digestion instrument, with polytetrafluoroethylene digestion internal tank.
6.2.4 Electric hot plate with adjustable temperature control, or graphite digestion unit.
6.2.5 Ultrasonic water bath.
6.2.6 Homogenizer, high-speed pulverizer, capable of sample pulverizing and homogenizing.
6.2.7 One-mark volumetric flasks, of capacities 25 ml and 50 ml.
6.2.8 One-mark pipettes, of capacities 2 ml, 5 ml and 10 ml.
6.2.9 Graduated (automatic) pipettes, of capacities 2 ml, 5 ml and 10 ml.
6.2.10 Polytetrafluoroethylene digestion tube.
6.3 Procedure
6.3.1 Sample pre-treatment
Samples with low water content are mixed together after removing debris. The samples with high
water content are homogenized.
6.3.2 Sample digestion
Microwave digestion:
— weigh 0,2 g to 0,5 g of the test portion (accurate to 0,001 g, the sample with more moisture content
can be appropriately increased to 1 g to 2 g) in the microwave digestion internal tank (6.2.3) with
the polytetrafluoroethylene digestion tube (6.2.10);
— add 5 ml to 10 ml of nitric acid (6.1.2);
— stand for 1 h or overnight;
— screw the tank cap;
— follow the standard operation steps of the microwave digestion instrument (6.2.3) to digest;
— take out after cooling;
— slowly open the tank cap and vent;
— flush the inner cap with a little water;
— put the digestion tank (6.2.3) on the electric hot plate (with adjustable temperature control) (6.2.4)
or in the ultrasonic water bath (6.2.5);
— heat for 30 min at 100 °C or ultrasonic degassing (approximately 40 kHZ) for 2 min to 5 min;
— dilute with water to 25 ml or 50 ml and mix;
— do a blank test at the same time.
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ISO 23776:2021(E)
6.4 Determination
6.4.1 Instrument reference conditions
The process is as follows:
— optimize the operating conditions of the instrument;
— ensure the instrument sensitivity and other indicators meet the requirements of the analysis;
— the reference conditions for the instrument operation are observation mode:
— horizontal observation;
— power: 1 150 W;
— plasma gas flow: 15 l/min;
— auxiliary gas flow: 0,5 l/min;
— atomized gas flow: 0,65 l/min;
— measured line (select one): 213,6 nm, 214,9 nm, 178,3 nm, 177 nm or 177,4 nm.
6.4.2 Standard curve drawing
The standard series working solution is injected into the inductively coupled plasma optical emission
spectrometer (6.2.1) and the intensity signal response of the analytical spectral line is determined.
When the element concentration is abscissa, the spectral line intensity response value is y-axis and the
standard curve is drawn.
6.4.3 Test portion
For sample determination, the blank solution and sample solution are injected into the inductively
coupled plasma optical emission spectrometer (6.2.1). The signal response values of the spectral line
strength are measured, and the concentration of phosphorous in the solution is obtained according to
the standard curve.
6.5 Calculation and expression of results
Calculate the phosphorous content, X, in milligrams per kilogram, using Formula (1):
()ρρ− ××Vf
0
X = (1)
m
where
ρ is the phosphorous mass concentration of the test portion, in milligrams per litre;
ρ is the phosphorous mass concentration of the blank test, in milligrams per litre;
0
V is the constant volume of the sample digestion solution, in millilitre;
m is the numerical value of the mass, in grams of test portion;
f is the dilution factor.
Three-bit valid numbers are reserved for the results of the calculation.
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ISO 23776:2021(E)
6.6 Limit of detection
The LOD is 1,0 mg/kg and the LOQ is 3,0 mg/kg if the mass of the test portion is 0,5 g and the constant
volume is 50 ml.
6.7 Precision
The precision of the method was established by an international laboratory ring test, carried out in
accordance with ISO 5725-2.
6.8 Repeatability
The absolute difference between two independent single test results, obtained using the same method
on test material in the same laboratory by the same operator using the same equipment within a short
interval of time (see Annex A).
6.9 Reproducibility
The absolute difference between two independent single test results, obtained using the same method
on identical test material in different laboratories with different operators using different equipment
(see Annex A).
7 Spectrometric method
7.1 Reagents
Use only reagents of recognized analytical grade and distilled or demineralised water or water of at
least equivalent purity.
7.1.1 Nitric acid (HNO ), ρ = 1,42 g/ml, guarantee reagent or higher purity.
3 20
7.1.2 Nitric acid: water 1:2 (volume/volume).
Mix one volume of nitric acid [mass fraction of 65 %; ρ = 1, 42 g/ml (7.1.1)] with two volumes of water.
20
7.1.3 Ammonium metavanadate (ammonium monovanadate) solution (NH VO ), 2,5 g/l.
4 3
Dissolve 2,5 g of ammonium metavanadate in 500 ml of boiling water. Cool and add 20 ml of the nitric
acid (7.1.2), dilute to the mark 1 l with water and mix.
7.1.4 Ammonium heptamolybdate solution, [(NH ) Mo O ·4H O], 50 g/l (CAS 12027-67-7).
4 6 7 24 2
Dissolve 50 g of ammonium heptamolybdate tetrahydrate in about 800 ml of warm water (at
approximately 50 °C). Cool and transfer quantitatively to a 1 000 ml volumetric flask. Dilute to the
mark with water and mix.
7.1.5 Colour reagent.
Mix one volume of the nitric acid (7.1.2) with one volume of the ammonium metavanadate solution
(7.1.3). Subsequently add one volume of the ammonium heptamolybdate solution (7.1.4) and mix. Make
sure of the order of addition. It can be kept stable in the dark for one month.
7.1.6 Phosphate stock solution, c(P) = 109 mg/l; c(P O ) = 250 mg/l.
2 5
Potassium dihydrogen phosphate (KH PO ), (CAS: 7778-77-0, > 99,99 %).
2 4
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ISO 23776:2021(E)
Dissolve in water 479,4 mg of potassium dihydrogen phosphate (KH PO ), previously dried for 3 h at
2 4
(103 ± 2) °C and allowed to cool in desiccators.
Transfer quantitatively to a 1 000 ml volumetric flask. Dilute to the mark with water and mix.
7.1.7 Phosphate standard solutions, containing between 0,05 mg and 0,30 mg of P O per millilitre.
2 5
Transfer by pipette or burette to 100 ml volumetric flasks 10 ml, 20 ml, 30 ml, 40 ml, 50 ml and 60 ml of
the phosphate stock solution (7.1.6). Add 10 ml of the nitric acid (7.1.2). Dilute to the mark with water
and mix.
The range of standard curve can be adjusted according to the use of different concentrations of standard
substance. Use water to dilute.
7.1.8 Blank solution.
Pipette 2 ml of the nitric acid (7.1.2) and 30 ml of the colour reagent (7.1.5) into a 100 ml volumetric
flask. Dilute to the mark with water and mix.
7.2 Apparatus
IMPORTANT — All glassware shall be thoroughly cleaned using a phosphate-free detergent and
then rinsed with water.
The usual laboratory apparatus and, in particular, the following shall be used.
7.2.1 Mechanical or electrical equipment capable of homogenizing the laboratory sample,
including a high speed rotational cutter or a mincer fitted with a plate with holes not exceeding 4,5 mm
in diameter.
7.2.2 Water bath, capable of being maintained at 100 °C.
7.2.3 Fluted filter paper, of diameter 15 cm, phosphate free.
7.2.4 Spectrometer, capable of being used at a wavelength of (430 ± 2) nm, or a photo-electric
colorimeter with an interference filter with absorption maximum of (430 ± 2) nm.
7.2.5 Glass cells, of 10 mm optical path length.
7.2.6 Analytical balance, capable of weighing to an accuracy of ±0,001 g.
7.2.7 One-mark volumetric flasks, of capacities (100 ± 0,10) ml and (1 000 ± 0,40) ml.
7.2.8 Muffle furnace, with adjustable temperature control, temperature (550 ± 25) °С.
7.2.9 Electric hot plate, with adjustable temperature control.
7.2.10 Porcelain crucible, with 60 mm diameter, 25 mm high inclined wall.
7.2.11 Desiccator, provided with an effective desiccant.
For details of this and other apparatus needed for the incineration procedure, see ISO 936.
7.2.12 Mechanical meat mincer, laboratory size, fitted with a plate with holes of diameter not
exceeding 4 mm.
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ISO 23776:2021(E)
7.3 Preparation of test sample
Homogenize the laboratory sample with the appropriate equipment (7.2.1). Take care that the
temperature of the sample material does not rise above 25 °C. If a mincer is used, pass the sample at
least twice through the equipment.
Fill a suitable airtight container with the prepared test sample, close the container and store in such
a way that deterioration and change in composition are prevented. Analyse the test sample as soon as
practicable. The storage time should be not more than 24 h.
7.4 Procedure
7.4.1 Test portion
7.4.1.1 If it is necessary to check whether the repeatability requirement (see 7.8) is met, carry out two
single determinations in accordance with 7.4.1 to 7.5.
7.4.1.2 Weigh, to the nearest 0,001 g, about 5 g of the prepared test sample. Carry out the mineralization
of the test portion by using an incinerator (7.2.8) and the method described in ISO 936. Take up the
resulting ash in 10 ml of the nitric acid (7.1.2) using a stirring rod to aid dissolution. Cover the dish with
a watch glass and heat for 30 min on a boiling water bath (7.2.2). Allow to cool and transfer the liquid
quantitatively to a 100 ml volumetric flask (7.2.7). Dilute to the mark with water, mix and filter through
the filter paper (7.2.3), rejecting the first 5 ml to 10 ml of filtrate.
7.4.2 Determination
7.4.2.1 Pipette 20 ml of the clear and colourless filtrate (7.4.1.1) into a 100 ml volumetric flask (7.2.7)
and add 30 ml of the colour reagent (7.1.5) by pipette. Dilute to the mark with water and mix. Allow to
stand for at least 15 min.
7.4.2.2 Measure the absorbance at a wavelength of (430 ± 2) nm in a glass cell (7.2.5) against the blank
solution (7.1.8), using the spectrometer or the colorimeter equipped with an interference filter (7.2.4).
7.4.2.3 Read the phosphorous concentration of the sample solution from the calibration graph
obtained as described in 7.5.
7.5 Calibration graph photo-electric
7.5.1 Pipette 20 ml of each phosphate standard solution (7.1.7) into 100 ml volumetric flasks. Add
to these solutions 30 ml of the colour reagent. Dilute to the mark with water to obtain concentrations
of 10 μg, 20 μg, 30 μg, 40 μg, 50 μg and 60 μg of P O per millilitre. Mix and allow to stand for at least
2 5
15 min.
7.5.2 Carry out the procedure described in 7.4.2.1.
7.5.3 Plot the measured absorbance values, corrected for the blank value, against the concentrations
of the diluted phosphate standard solutions (7.5.1). Construct the best-fitting straight line through the
2
plotted points and the origin. The specified minimum determination coefficient R ≥ 0,95.
It is necessary to prepare a new c
...
INTERNATIONAL ISO
STANDARD 23776
First edition
Meat and meat products —
Determination of total phosphorous
content
Viandes et produits à base de viande — Détermination de la teneur en
phosphore
PROOF/ÉPREUVE
Reference number
ISO 23776:2021(E)
©
ISO 2021
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ISO 23776:2021(E)
COPYRIGHT PROTECTED DOCUMENT
© ISO 2021
All rights reserved. Unless otherwise specified, or required in the context of its implementation, 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
CP 401 • Ch. de Blandonnet 8
CH-1214 Vernier, Geneva
Phone: +41 22 749 01 11
Email: copyright@iso.org
Website: www.iso.org
Published in Switzerland
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ISO 23776:2021(E)
Contents Page
Foreword .v
1 Scope . 1
2 Normative references . 1
3 Terms and definitions . 1
4 Principle . 1
4.1 Inductively coupled plasma optical emission spectrometry (ICP-OES) method . 1
4.2 Spectrometric method . 2
4.3 Gravimetric method . 2
5 Sampling . 2
6 Inductively coupled plasma optical emission spectrometry (ICP-OES) method .2
6.1 Reagents. 2
6.2 Apparatus . 2
6.3 Procedure . 3
6.3.1 Sample pre-treatment . 3
6.3.2 Sample digestion . 3
6.4 Determination . 4
6.4.1 Instrument reference conditions . 4
6.4.2 Standard curve drawing . 4
6.4.3 Test portion . 4
6.5 Calculation and expression of results. 4
6.6 Limit of detection . 5
6.7 Precision . 5
6.8 Repeatability . 5
6.9 Reproducibility . 5
7 Spectrometric method . 5
7.1 Reagents. 5
7.2 Apparatus . 6
7.3 Preparation of test sample . 7
7.4 Procedure . 7
7.4.1 Test portion . 7
7.4.2 Determination . 7
7.5 Calibration graph photo-electric . 7
7.6 Calculation . 8
7.7 Precision . 8
7.8 Repeatability . 8
7.9 Reproducibility . 8
8 Gravimetric method . 8
8.1 Reagents. 8
8.2 Apparatus . 9
8.3 Procedures . 9
8.3.1 Preparation of test sample . 9
8.3.2 Test portion . 9
8.3.3 Mineralization .10
8.3.4 Determination .10
8.3.5 Blank test .10
8.3.6 Expression of results .10
8.3.7 Repeatability .11
8.4 Notes on procedure .11
9 Test report .11
Annex A (informative) International laboratory ring test (ICP-OES method) .12
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ISO 23776:2021(E)
Bibliography .18
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ISO 23776:2021(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 of 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 www .iso .org/
iso/ foreword .html.
This document was prepared by Technical Committee ISO/TC 34, Food products, Subcommittee SC 6,
Meat, poultry, fish, eggs and their products.
This first edition cancels and replaces ISO 2294:1974 and ISO 13730:1996, which have been technically
revised. The main changes compared with ISO 2294:1974 and ISO 13730:1996 are as follows:
— a new test method, the inductively coupled plasma optical emission spectrometry (ICP-OES) method,
has been added;
— the structure of the document has been revised;
— the title of the document has been modified;
— the Scope has been modified.
Any feedback or questions on this document should be directed to the user’s national standards body. A
complete listing of these bodies can be found at www .iso .org/ members .html.
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INTERNATIONAL STANDARD ISO 23776:2021(E)
Meat and meat products — Determination of total
phosphorous content
1 Scope
This document specifies three methods for the determination of the total phosphorous content of all
kinds of meat and meat products, including poultry and livestock:
— the inductively coupled plasma optical emission spectrometry (ICP-OES) method;
— the spectrometric method;
— the gravimetric method.
For the ICP-OES method, the limit of detection (LOD) is 1,0 mg/kg and the limit of quantification (LOQ)
is 3,0 mg/kg if the mass of the test portion is 0,5 g and the constant volume is 50 ml.
2 Normative references
The following documents are referred to in the text in such a way that some or all of their content
constitutes requirements of this document. For dated references, only the edition cited applies. For
undated references, the latest edition of the referenced document (including any amendments) applies.
ISO 936, Meat and meat products — Determination of total ash
ISO 3696, Water for analytical laboratory use — Specification and test methods
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:
— ISO Online browsing platform: available at https:// www .iso .org/ obp
— IEC Electropedia: available at http:// www .electropedia .org/
3.1
total phosphorous content of meat and meat products
mass of phosphorous pentoxide determined by the procedure specified in this document
Note 1 to entry: It is expressed as a percentage of the mass of the test portion.
4 Principle
4.1 Inductively coupled plasma optical emission spectrometry (ICP-OES) method
The test portion of the sample is microwave digested with nitric acid. The concentration of phosphorous
is determined by ICP-OES using external calibration. In a certain concentration range, the spectral line
signal intensity of phosphorous is proportional to its concentration, and is quantified by the standard
curve method.
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ISO 23776:2021(E)
4.2 Spectrometric method
Drying of the test portion and incineration of the residue. After cooling, hydrolysis of the ash with
nitric acid. Filtration and dilution followed by the formation of a yellow compound with a mixture of
ammonium monovanadate and ammonium heptamolybdate. Photometric measurement at a wavelength
of 430 nm.
4.3 Gravimetric method
Mineralization of a test portion with sulfuric and nitric acids. Precipitation of the phosphorous as
quinoline phosphomolybdate. Drying and weighing of the precipitate. An alternative method of
mineralization is described in 8.4.
5 Sampling
Sampling is not part of the method specified in this document. A recommended sampling method is
given in ISO 17604.
It is important that the laboratory receives a sample which is truly representative and has not been
damaged or changed during transport or storage.
Start from a representative sample of at least 200 g. Store the sample in such a way that deterioration
and change in composition are prevented.
6 Inductively coupled plasma optical emission spectrometry (ICP-OES) method
6.1 Reagents
Use only reagents of recognized analytical grade.
6.1.1 Water, conforming to at least grade 1 in accordance with ISO 3696.
6.1.2 Nitric acid (HNO ), concentrated, not less than 65 % (mass fraction) or higher purity,
3
ρ = 1,42 g/ml.
20
6.1.3 Argon (Ar): argon (> 99,995 %, mass fraction) or liquid argon.
6.1.4 Nitric acid (5 + 95): take 50 ml nitric acid (6.1.2), slowly add 950 ml water and mix.
6.1.5 Phosphorous standard stock solution (1 000 mg/l), c(P) = 1 000 mg/l; c(P O ) = 2 294 mg/l.
2 5
This stock solution is stable for one month when stored in the dark at room temperature.
6.1.6 Standard series solution of phosphorous: accurate extract standard reserve liquid, dilute
standard series solution with nitric acid solution (5 + 95). The mass concentration is 0 mg/l, 20,0 mg/l,
40,0 mg/l, 60,0 mg/l, 80,0 mg/l and 100,0 mg/l.
According to the sensitivity of the instrument and the actual content of phosphorous in the sample, the
concentration range of the standard solution should be adjusted appropriately.
6.2 Apparatus
IMPORTANT — All glassware shall be thoroughly cleaned using a phosphate-free detergent and
then rinsed with water.
The usual laboratory apparatus and, in particular, the following shall be used.
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ISO 23776:2021(E)
6.2.1 Inductively coupled plasma optical emission spectrometer.
6.2.2 Analytical balance, capable of weighing to the nearest 0,000 1 g.
6.2.3 Microwave digestion instrument, with polytetrafluoroethylene digestion internal tank.
6.2.4 Electric hot plate with adjustable temperature control, or graphite digestion unit.
6.2.5 Ultrasonic water bath.
6.2.6 Homogenizer, high-speed pulverizer, capable of sample pulverizing and homogenizing.
6.2.7 One-mark volumetric flasks, of capacities 25 ml and 50 ml.
6.2.8 One-mark pipettes, of capacities 2 ml, 5 ml and 10 ml.
6.2.9 Graduated (automatic) pipettes, of capacities 2 ml, 5 ml and 10 ml.
6.2.10 Polytetrafluoroethylene digestion tube.
6.3 Procedure
6.3.1 Sample pre-treatment
Samples with low water content are mixed together after removing debris. The samples with high
water content are homogenized.
6.3.2 Sample digestion
Microwave digestion:
— weigh 0,2 g to 0,5 g of the test portion (accurate to 0,001 g, the sample with more moisture content
can be appropriately increased to 1 g to 2 g) in the microwave digestion internal tank (6.2.3) with
the polytetrafluoroethylene digestion tube (6.2.10);
— add 5 ml to 10 ml of nitric acid (6.1.2);
— stand for 1 h or overnight;
— screw the tank cap;
— follow the standard operation steps of the microwave digestion instrument (6.2.3) to digest;
— take out after cooling;
— slowly open the tank cap and vent;
— flush the inner cap with a little water;
— put the digestion tank (6.2.3) on the electric hot plate (with adjustable temperature control) (6.2.4)
or in the ultrasonic water bath (6.2.5);
— heat for 30 min at 100 °C or ultrasonic degassing (approximately 40 kHZ) for 2 min to 5 min;
— dilute with water to 25 ml or 50 ml and mix;
— do a blank test at the same time.
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ISO 23776:2021(E)
6.4 Determination
6.4.1 Instrument reference conditions
The process is as follows:
— optimize the operating conditions of the instrument;
— ensure the instrument sensitivity and other indicators meet the requirements of the analysis;
— the reference conditions for the instrument operation are observation mode:
— horizontal observation;
— power: 1 150 W;
— plasma gas flow: 15 l/min;
— auxiliary gas flow: 0,5 l/min;
— atomized gas flow: 0,65 l/min;
— measured line (select one): 213,6 nm, 214,9 nm, 178,3 nm, 177 nm or 177,4 nm.
6.4.2 Standard curve drawing
The standard series working solution is injected into the inductively coupled plasma optical emission
spectrometer (6.2.1) and the intensity signal response of the analytical spectral line is determined.
When the element concentration is abscissa, the spectral line intensity response value is y-axis and the
standard curve is drawn.
6.4.3 Test portion
For sample determination, the blank solution and sample solution are injected into the inductively
coupled plasma optical emission spectrometer (6.2.1). The signal response values of the spectral line
strength are measured, and the concentration of phosphorous in the solution is obtained according to
the standard curve.
6.5 Calculation and expression of results
Calculate the phosphorous content, X, in milligrams per kilogram, using Formula (1):
()ρρ− ××Vf
0
X = (1)
m
where
ρ is the phosphorous mass concentration of the test portion, in milligrams per litre;
ρ is the phosphorous mass concentration of the blank test, in milligrams per litre;
0
V is the constant volume of the sample digestion solution, in millilitre;
m is the numerical value of the mass, in grams of test portion;
f is the dilution factor.
Three-bit valid numbers are reserved for the results of the calculation.
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ISO 23776:2021(E)
6.6 Limit of detection
The LOD is 1,0 mg/kg and the LOQ is 3,0 mg/kg if the mass of the test portion is 0,5 g and the constant
volume is 50 ml.
6.7 Precision
The precision of the method was established by an international laboratory ring test, carried out in
accordance with ISO 5725-2.
6.8 Repeatability
The absolute difference between two independent single test results, obtained using the same method
on test material in the same laboratory by the same operator using the same equipment within a short
interval of time (see Annex A).
6.9 Reproducibility
The absolute difference between two independent single test results, obtained using the same method
on identical test material in different laboratories with different operators using different equipment
(see Annex A).
7 Spectrometric method
7.1 Reagents
Use only reagents of recognized analytical grade and distilled or demineralised water or water of at
least equivalent purity.
7.1.1 Nitric acid (HNO ), ρ = 1,42 g/ml, guarantee reagent or higher purity.
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7.1.2 Nitric acid: water 1:2 (volume/volume).
Mix one volume of nitric acid [mass fraction of 65 %; ρ = 1, 42 g/ml (7.1.1)] with two volumes of water.
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7.1.3 Ammonium metavanadate (ammonium monovanadate) solution (NH VO ), 2,5 g/l.
4 3
Dissolve 2,5 g of ammonium metavanadate in 500 ml of boiling water. Cool and add 20 ml of the nitric
acid (7.1.2), dilute to the mark 1 l with water and mix.
7.1.4 Ammonium heptamolybdate solution, [(NH ) Mo O ·4H O], 50 g/l (CAS 12027-67-7).
4 6 7 24 2
Dissolve 50 g of ammonium heptamolybdate tetrahydrate in about 800 ml of warm water (at
approximately 50 °C). Cool and transfer quantitatively to a 1 000 ml volumetric flask. Dilute to the
mark with water and mix.
7.1.5 Colour reagent.
Mix one volume of the nitric acid (7.1.2) with one volume of the ammonium metavanadate solution
(7.1.3). Subsequently add one volume of the ammonium heptamolybdate solution (7.1.4) and mix. Make
sure of the order of addition. It can be kept stable in the dark for one month.
7.1.6 Phosphate stock solution, c(P) = 109 mg/l; c(P O ) = 250 mg/l.
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Potassium dihydrogen phosphate (KH PO ), (CAS: 7778-77-0, > 99,99 %).
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ISO 23776:2021(E)
Dissolve in water 479,4 mg of potassium dihydrogen phosphate (KH PO ), previously dried for 3 h at
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(103 ± 2) °C and allowed to cool in desiccators.
Transfer quantitatively to a 1 000 ml volumetric flask. Dilute to the mark with water and mix.
7.1.7 Phosphate standard solutions, containing between 0,05 mg and 0,30 mg of P O per millilitre.
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Transfer by pipette or burette to 100 ml volumetric flasks 10 ml, 20 ml, 30 ml, 40 ml, 50 ml and 60 ml of
the phosphate stock solution (7.1.6). Add 10 ml of the nitric acid (7.1.2). Dilute to the mark with water
and mix.
The range of standard curve can be adjusted according to the use of different concentrations of standard
substance. Use water to dilute.
7.1.8 Blank solution.
Pipette 2 ml of the nitric acid (7.1.2) and 30 ml of the colour reagent (7.1.5) into a 100 ml volumetric
flask. Dilute to the mark with water and mix.
7.2 Apparatus
IMPORTANT — All glassware shall be thoroughly cleaned using a phosphate-free detergent and
then rinsed with water.
The usual laboratory apparatus and, in particular, the following shall be used.
7.2.1 Mechanical or electrical equipment capable of homogenizing the laboratory sample,
including a high speed rotational cutter or a mincer fitted with a plate with holes not exceeding 4,5 mm
in diameter.
7.2.2 Water bath, capable of being maintained at 100 °C.
7.2.3 Fluted filter paper, of diameter 15 cm, phosphate free.
7.2.4 Spectrometer, capable of being used at a wavelength of (430 ± 2) nm, or a photo-electric
colorimeter with an interference filter with absorption maximum of (430 ± 2) nm.
7.2.5 Glass cells, of 10 mm optical path length.
7.2.6 Analytical balance, capable of weighing to an accuracy of ±0,001 g.
7.2.7 One-mark volumetric flasks, of capacities (100 ± 0,10) ml and (1 000 ± 0,40) ml.
7.2.8 Muffle furnace, with adjustable temperature control, temperature (550 ± 25) °С.
7.2.9 Electric hot plate, with adjustable temperature control.
7.2.10 Porcelain crucible, with 60 mm diameter, 25 mm high inclined wall.
7.2.11 Desiccator, provided with an effective desiccant.
For details of this and other apparatus needed for the incineration procedure, see ISO 936.
7.2.12 Mechanical meat mincer, laboratory size, fitted with a plate with holes of diameter not
exceeding 4 mm.
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ISO 23776:2021(E)
7.3 Preparation of test sample
Homogenize the laboratory sample with the appropriate equipment (7.2.1). Take care that the
temperature of the sample material does not rise above 25 °C. If a mincer is used, pass the sample at
least twice through the equipment.
Fill a suitable airtight container with the prepared test sample, close the container and store in such
a way that deterioration and change in composition are prevented. Analyse the test sample as soon as
practicable. The storage time should be not more than 24 h.
7.4 Procedure
7.4.1 Test portion
7.4.1.1 If it is necessary to check whether the repeatability requirement (see 7.8) is met, carry out two
single determinations in accordance with 7.4.1 to 7.5.
7.4.1.2 Weigh, to the nearest 0,001 g, about 5 g of the prepared test sample. Carry out the mineralization
of the test portion by using an incinerator (7.2.8) and the method described in ISO 936. Take up the
resulting ash in 10 ml of the nitric acid (7.1.2) using a stirring rod to aid dissolution. Cover the dish with
a watch glass and heat for 30 min on a boiling water bath (7.2.2). Allow to cool and transfer the liquid
quantitatively to a 100 ml volumetric flask (7.2.7). Dilute to the mark with water, mix and filter through
the filter paper (7.2.3), rejecting the first 5 ml to 10 ml of filtrate.
7.4.2 Determination
7.4.2.1 Pipette 20 ml of the clear and colourless filtrate (7.4.1.1) into a 100 ml volumetric flask (7.2.7)
and add 30 ml of the colour reagent (7.1.5) by pipette. Dilute to the mark with water and mix. Allow to
stand for at least 15 min.
7.4.2.2 Measure the absorbance at a wavelength of (430 ± 2) nm in a glass cell (7.2.5) against the blank
solution (7.1.8), using the spectrometer or the colorimeter equipped with an interference filter (7.2.4).
7.4.2.3 Read the phosphorous concentration of the sample solution from the calibration graph
obtained as described in 7.5.
7.5 Calibration graph photo-electric
7.5.1 Pipette 20 ml of each phosphate standard solution (7.1.7) into 100 ml volumetric flasks. Add
to these solutions 30 ml of the colour reagent. Dilute to the mark with water to obtain concentrations
of 10 μg, 20 μg, 30 μg, 40 μg, 50 μg and 60 μg of P O per millilitre. Mix and allow to stand for at least
2 5
15 min.
7.5.2 Carry out the procedure described in 7.4.2.1.
7.5.3 Plot the measured absorbance values, corrected for the blank value, against the concentrations
of the diluted phosphate standard solutions (7.5.1). Construct the best-fitting straight line t
...
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