ISO/FDIS 23822

ISO/TC 34/SC 6/WG 28
Secretariat: SAC
Date: 2026-03-0330
Eggs and egg products — Determination of nitroimidazole residues
— Liquid chromatography-tandem mass spectrometry method
FDIS stage
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Contents
Foreword . iv
1 Scope . 1
2 Normative references . 1
3 Terms and definitions . 1
4 Principle . 1
5 Sampling . 1
6 Preparation of test sample . 2
6.1 Preparation of sample . 2
6.2 Storage . 2
7 Reagents and materials . 2
7.1 Reagents . 2
7.2 Preparation of solution . 2
7.3 Standards . 3
7.4 Preparation of standard solution. 3
7.5 Materials . 4
8 Apparatus . 4
9 Procedure . 4
9.1 General. 4
9.2 Extraction . 4
9.3 Purification . 4
9.4 LC-MS/MS analysis . 4
9.5 Confirmation . 6
9.6 Quantification . 6
9.7 Blank test . 7
9.8 Calculation . 7
9.9 Limits of detection . 7
9.10 Limits of quantification . 7
9.11 Precision . 7
10 Test report . 8 ®
Annex A (informative) Compound name, CAS Registry Number , molecular formula, relative
molecular mass and structural formula . 9
Annex B (informative) LC-MS/MS chromatogramsof the quantification ion pairs for
nitroimidazoles from the mixed standard solution . 10
Annex C (informative) Collaborative interlaboratory test result. 20
Bibliography . 22

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Foreword
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poultry, fish, eggs and their products.
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Eggs and egg products — Determination of nitroimidazole residues —
Liquid chromatography-tandem mass spectrometry method
1 Scope
This document specifies a liquid chromatography tandem mass spectrometry method (LC-MS/MS) for the
determination of nitroimidazole residues in eggs and egg products.
This document is applicable to eggs and egg products, including whole eggs (fresh, frozen or pasteurized), egg
yolk (liquid, frozen, dried or pasteurized), egg white (liquid, frozen, dried or pasteurized), whole egg powder,
egg white powder, egg yolk powder, and liquid or dried albumin preparations.
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 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 terminology databases for use in standardization at the following addresses:
— ISO Online browsing platform: available at https://www.iso.org/obp
— IEC Electropedia: available at https://www.electropedia.org/
3.1
nitroimidazole residues
compounds including metronidazole, dimetridazole, ronidazole, ipronidazole, hydroxymetronidazole,
hydroxydimetridazole and hydroxyipronidazole
4 Principle
The test sample is extracted using acetonitrile. The extracted solution is purified through solid-phase
extraction. The nitroimidazole residues are determined and confirmed by LC-MS/MS, and quantified with an
internal standard method.
5 Sampling
Sampling is not part of the method specified in this document. A recommended sampling method is given in
CAC/GL 50-2004.
It is essential that the laboratory receivereceives a sample which is truly representative and remains
unaffected by damage or change during transport or storage.
Start from a representative sample of at least 1 kg.
Store the sample in such a way that deterioration and change in composition are prevented.
6 Preparation of test sample
6.1 Preparation of sample
6.1.1 Fresh eggs and egg products with shell
Take 16 (about 1 kg) fresh eggs or egg products and remove the shells. Homogenize the egg contents sample
with the appropriate equipment. Transfer the homogenized sample into a polyethylene bottle.
6.1.2 Other egg products
Homogenize the egg products samples thoroughly. Transfer the homogenized sample into a polyethylene
bottle.
For concentrated or dried egg products, weigh 5 g (to the nearest 0,01g01 g) of the homogenized sample into
a 50 ml centrifuge tube, add 20 ml of water for reconstitution, vortex for 10 min, and then sonicate for 10 min.
Ensure that the sample is evenly dispersed to obtain the reconsititutedreconstituted egg solutions.
NOTE This step is necessary to reconstitute dried (e.g.,. egg powder) or concentrated egg products to their ready-
to-use form by diluting them to the required concentration. Liquid and frozen products are already in a suitable state for
analysis and can bypass this procedure.
6.2 Storage
Store the test sample and standby sample at a temperature between −20 °C and −16 °C.
7 Reagents and materials
Reagents shall be of recognized analytical grade, unless otherwise specified.
7.1 Reagents
7.1.1 Water, grade 1 in accordance with ISO 3696.
7.1.2 Acetonitrile (CH CN), HPLC grade.
7.1.3 Methanol (CH OH), HPLC grade.
7.1.4 Formic acid (HCOOH), HPLC grade.
7.1.5 Sodium chloride (NaCl).
7.1.6 Dimethyl sulfoxide (DMSO).
7.1.7 n-Hexane (C H ).
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7.2 Preparation of solution
7.2.1 10 % Methanol aqueous solution (containing 0,1 % formic acid).
Pipette 10 ml of methanol and 0,1 ml of formic acid into a 100 ml one-mark volumetric flask. Dilute to the
mark with water and mix.
7.2.2 0,1 % Aqueous formic acid solution.
Pipette 1 ml of formic acid to a 1 000 ml one-mark volumetric flask. Dilute to the mark with water and mix
thoroughly.
7.2.3 Acetonitrile-saturated n-hexane.
Obtain the supernatant from the solution of acetonitrile and n-hexane (v/v = 18/82) after sufficient mixture.
7.3 Standards
7.3.1 Nitroimidazoles, purity ≥ 95 %, see Annex A.
7.3.2 Nitroimidazoles isotope internal standard.
Stock solutions of 100 mg/l for the following compounds: metronidazole-d , dimetridazole-d , ronidazole-d ,
3 3 3
ipronidazole-d , hydroxymetronidazole-d , hydroxydimetridazole-d and hydroxyipronidazole-d .
3 2 3 3
7.4 Preparation of standard solution
7.4.1 Standard stock solution, 1,00 mg/ml.
Weigh 10 mg (to the nearest 0,1 mg) of metronidazole, dimetridazole, ronidazole, ipronidazole,
hydroxymetronidazole, hydroxydimetridazole and hydroxyipronidazole. Transfer each solution to a separate
10 ml volumetric flask. Dilute to the mark with methanol and mix thoroughly.
The prepared stock solutions are stable for six months when stored in the dark at −18 °C.
7.4.2 Mixed standard intermediate solution I, 10,0 μg/ml.
Pipette 0,1 ml each of the standard stock solution (7.4.1) into a 10 ml one-mark volumetric flask. Dilute to the
mark with methanol and mix thoroughly.
This solution is stable for one month when stored in the dark at −18 °C.
7.4.3 Mixed standard intermediate solution II, 200 ng/ml.
Pipette 0,2 ml mixed standard intermediate solution I (7.4.2) into a 10 ml one-mark volumetric flask. Dilute
to the mark with methanol and mix.
The solution is for the current use.
7.4.4 Isotope internal standard intermediate solution, 1,00 μg/ml.
Pipette 0,1 ml of the isotope internal standard: stock solutions (7.3.2) into a 10 ml one-mark volumetric flask.
Dilute to the mark with methanol and mix thoroughly.
This solution is stable for one month when stored in the dark at −18 °C.
7.4.5 Mixed standard working solution.
Pipette a certain amount of mixed standard intermediate solution II (7.4.3) and isotope internal standard
intermediate solution (7.4.4) into seven volumetric flasks. Dilute with 10 % methanol aqueous solution (
...