EN 15055:2006

Non fatty foods - Determination of chlormequat and mepiquat - LC-MS/MS method

This draft European Standard specifies a method using high performance liquid chromatography/tandem mass spectrometry (LC-MS/MS) for the determination of the growth regulators chlormequat and mepiquat in non-fatty foods as chlormequat and mepiquat cation, respectively.
The method is applicable to all kinds of fruits, vegetables and cereal products. It has been collaboratively studied on mushrooms, pears, wheat flour, fruit puree and, additionally, on infant formula, see [1].

Fettarme Lebensmittel - Bestimmung von Chlormequat und Mepiquat - LC-MS/MS-Verfahren

Diese Europäische Norm legt ein hochleistungs-flüssigchromatographisches Verfahren mit Detektion durch
Tandem-Massenspektrometrie (LC-MS/MS) zur Bestimmung der Wachstumsregulatoren Chlormequat und
Mepiquat in fettarmen Lebensmitteln als Chlormequat- bzw. Mepiquat-Kation fest.
Das Verfahren ist anwendbar auf alle Arten von Obst, Gemüse und Getreideerzeugnissen. Es ist in einem
Ringversuch mit Pilzen, Birnen, Weizenmehl, Obstpüree und zusätzlich an einer Kindernahrung (Milchpulver)
geprüft worden, siehe [1].

Aliments non gras - Détermination du chlorméquate et du mépiquate - Méthode CL-SM/SM

La présente Norme européenne spécifie une méthode utilisant la Chromatographie liquide à haute performance/Spectrométrie de masse en tandem (CL-SM/SM) pour la détermination des substances ou régulateurs de croissance chlorméquat et mépiquat dans des aliments non gras respectivement sous la forme de cation chlorméquat et de cation mépiquat.
La méthode s'applique à toutes les sortes de fruits, légumes et produits céréaliers. Elle a fait l'objet d'études réalisées en collaboration sur des champignons, des poires, de la farine de blé, de la purée de fruit et, en outre, sur des préparations pour nourrissons (voir [1]).

Nemastna živila - Določevanje klormekvata in mepikvata - Metoda LC-MS/MS

General Information

• Status: Published
• Publication Date: 16-May-2006
• Withdrawal Date: 29-Nov-2006
• ICS: 65.060 - Agricultural machines, implements and equipment
• Technical Committee: CEN/TC 275 - Food analysis - Horizontal methods
• Drafting Committee: CEN/TC 275/WG 3 - Pesticides and PCB's in foods of animal origin
• Current Stage: 9093 - Decision to confirm - Review Enquiry
• Start Date: 05-Jun-2023
• Completion Date: 14-Apr-2025
• Ref Project: SIST EN 15055:2006 - Non fatty foods - Determination of chlormequat and mepiquat - LC-MS/MS method

Overview

EN 15055:2006 - Non fatty foods: Determination of chlormequat and mepiquat by LC‑MS/MS - is a CEN (European) standard that specifies an analytical method for measuring the quaternary ammonium growth regulators chlormequat and mepiquat (reported as their cations) in non‑fatty food matrices. The method uses high‑performance liquid chromatography coupled with tandem mass spectrometry (LC‑MS/MS, positive electrospray ionisation, multiple reaction monitoring) and was collaboratively validated on matrices including mushrooms, pears, wheat flour, fruit puree and infant formula. The standard is intended for routine residue analysis related to food safety and maximum residue level (MRL) monitoring.

Key Topics and Requirements

• Scope and matrices: Applicable to all kinds of fruits, vegetables and cereal products; collaborative study performed on selected matrices.
• Analytical principle: Homogenise sample, spike with deuterated internal standards (d4‑chlormequat and d3‑mepiquat), extract with water/methanol, centrifuge, filter and analyse by LC‑MS/MS using MRM transitions for selective detection.
• Calibration and quantification: Six calibration solutions covering typical residue range (equivalent to 0.01–3.00 mg/kg for a 20 g test portion) using internal standard calibration (response ratio).
• Instrumental conditions: LC‑MS/MS with electrospray interface operated in multi‑reaction mode; example LC conditions and MS/MS transitions are provided in Annex A as proven configurations.
• Quality control: Reagent blanks, spiked recovery tests and confirmatory procedures (e.g., alternative HPLC column or additional transitions) are required to verify selectivity and recovery.
• Performance data: Precision (repeatability and reproducibility) from inter‑laboratory studies is summarised in Annex B; users should consult Annex B for matrix‑specific performance characteristics.
• Reporting: Test report must identify the sample, reference the standard, list sampling/test dates, results with units and any deviations from the method.

Applications and Users

• Food control laboratories conducting pesticide/growth regulator residue analysis in fruits, vegetables, cereals and processed products.
• Regulatory agencies and monitoring programs enforcing MRLs for chlormequat and mepiquat.
• Contract testing and quality assurance labs in the food supply chain (producers, processors, import/export control).
• Method developers and instrument vendors requiring validated LC‑MS/MS conditions for low‑molecular‑weight cationic residues.

Related Standards

• EN 15054 - alternative method: Non fatty foods - Determination of chlormequat and mepiquat - LC‑MS method.
• ISO 5725 series - referenced for inter‑laboratory precision assessment.

Keywords: EN 15055:2006, chlormequat, mepiquat, LC‑MS/MS, non‑fatty foods, residue analysis, growth regulators, food safety, HPLC, tandem mass spectrometry.

Standards Content (Sample)

2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.Non fatty foods - Determination of chlormequat and mepiquat - LC-MS/MS method&0606Aliments non gras - Détermination du chlorméquate et du mépiquate - Méthode CL-SM/SMFettarme Lebensmittel - Bestimmung von Chlormequat und Mepiquat - LC-MS/MS-VerfahrenTa slovenski standard je istoveten z:EN 15055:2006SIST EN 15055:2006en67.050ICS:SLOVENSKI
STANDARDSIST EN 15055:200601-september-2006

EUROPEAN STANDARDNORME EUROPÉENNEEUROPÄISCHE NORMEN 15055May 2006ICS 65.060 English VersionNon fatty foods - Determination of chlormequat and mepiquat -LC-MS/MS methodAliments non gras - Détermination de la teneur enchlormequate et mepiquate - Méthode LC-MS/MSFettarme Lebensmittel - Bestimmung von Chlormequat undMepiquat - LC-MS/MS-VerfahrenThis European Standard was approved by CEN on 20 April 2006.CEN members are bound to comply with the CEN/CENELEC Internal Regulations which stipulate the conditions for giving this EuropeanStandard the status of a national standard without any alteration. Up-to-date lists and bibliographical references concerning such nationalstandards may be obtained on application to the Central Secretariat or to any CEN member.This European Standard exists in three official versions (English, French, German). A version in any other language made by translationunder the responsibility of a CEN member into its own language and notified to the Central Secretariat has the same status as the officialversions.CEN members are the national standards bodies of Austria, Belgium, Cyprus, Czech Republic, Denmark, Estonia, Finland, France,Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania,Slovakia, Slovenia, Spain, Sweden, Switzerland and United Kingdom.EUROPEAN COMMITTEE FOR STANDARDIZATIONCOMITÉ EUROPÉEN DE NORMALISATIONEUROPÄISCHES KOMITEE FÜR NORMUNGManagement Centre: rue de Stassart, 36
B-1050 Brussels© 2006 CENAll rights of exploitation in any form and by any means reservedworldwide for CEN national Members.Ref. No. EN 15055:2006: E

Examples for appropriate experimental conditions.10 Annex B (informative)
Precision data.18 Bibliography.19

LC-MS method" available. According to the CEN/CENELEC Internal Regulations, the national standards organizations of the following countries are bound to implement this European Standard: Austria, Belgium, Cyprus, Czech Republic, Denmark, Estonia, Finland, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland and United Kingdom.

WARNING — The use of this standard may involve hazardous materials, operations and equipment. This standard does not purport to address all the safety problems associated with its use. It is the responsibility of the user of this standard to establish appropriate safety and health practices and to determine the applicability of regulatory limitations prior to use. 3.2 Ammonium acetate
3.3 Ammonium formate
3.4 Filter aid, for example Celite® 5451)
3.5 Glacial acetic acid, mass fraction w at least 96 g/100 g 3.6 Acetonitrile, HPLC quality 3.7 Methanol, HPLC quality 3.8 Water, suitable for HPLC 3.9 Chlormequat chloride stock solution,mass concentration (C5H13NCl2) = 4,30 µg/ml in methanol
This stock solution contains 3,33 µg/ml chlormequat cation.
1) Celite 545 is a product supplied by Celite Corp. (World Minerals Inc., Santa Barbara, CA, USA). This information is given for the convenience of users of this European Standard and does not constitute an endorsement by CEN of the product named. Equivalent products may be used if they can be shown to lead to the same results.

3.13 Calibration solutions
Prepare six calibration solutions (no. 1 to no. 6) in 10 ml volumetric flasks as follows. Into the flasks, pipette 10 µl, 50 µl, 100 µl, 300 µl, 1 000 µl or 3 000 µl each of chlormequat chloride stock solution (3.9) and of mepiquat chloride stock solution (3.10). To all flasks add 10 µl of the internal standard solution 1 (3.11) and 10 µl of the internal standard solution 2 (3.12). Dilute the solutions no. 1 to no. 5 to the mark with a methanol/water mixture 1 + 1 (V+V) and solution no. 6 with water.
All six calibration solutions contain 16,7 ng/ml of d4-chlormequat cation and 16,7 ng/ml of d3-mepiquat cation. Solutions no. 1 to no. 6 contain 3,3 ng/ml, 16,7 ng/ml, 33,3 ng/ml, 100 ng/ml, 333 ng/ml and 1 000 ng/ml each of chlormequat cation and of mepiquat cation respectively. On the basis of a 20 g test portion, this corresponds to 0,01 mg/kg, 0,05 mg/kg, 0,10 mg/kg, 0,30 mg/kg, 1,00 mg/kg or 3,00 mg/kg each of chlormequat cation and mepiquat cation. 4 Apparatus Usual laboratory apparatus and, in particular, the following: 4.1 Homogenizer or high speed blender, fitted with jar
4.2 Centrifuge, capable of producing a centrifugal acceleration of at least 3 000 g at the bases of the centrifuge tubes
4.3 Syringe, capacity of at least 2 ml
4.4 Syringe filter, pore size 0,45 µm (polyamide or polytetrafluoroethylene)
4.5 Glass vials, 1,8 ml volume; applicable for automatic injection into an autosampler
4.6 LC-MS/MS system, equipped with electrospray interface
5 Procedure 5.1 Preparation of the samples Where possible, carry out the analysis of samples immediately upon their arrival in the laboratory. Do not analyse a laboratory sample which is wholly or extensively spoiled.

The reduction of the test sample shall be carried out in such a way that representative portions are obtained (e. g. by division into four and selection of opposite quadrants). When the samples are in small units (e. g. small fruits, legumes, cereals), the test sample shall be thoroughly mixed before weighing out the test portion. When the samples are in larger units, take wedge-shaped sections (e. g. large fruits and vegetables) or cross sections (e. g. cucumbers) which include the outer surface from each unit. From each test sample, remove those parts which would interfere with the analytical procedure. In the case of stone fruits, the stones should be removed. Care shall be taken that as little as possible of the remainder such as juice or flesh is lost. The basis for the calculation of the residue mass fraction is the mass of the original test sample (with stones).
Chop the test sample and weigh out test portions of masses of 20 g to an accuracy of ± 1 %. If samples have to be stored for more than 3 days, they should be deep-frozen at – 20 °C. To ensure that, even after thawing, representative samples can be taken, prepare portions of the product which are each sufficient for one analysis. 5.2 Extraction Transfer a representative test portion of mA = 20 g into the blender cup (4.1). For dry sample materials like cereal products, weigh a homogenised portion of 10 g (mA) into the cup. Add 60 µl of internal standard solution 1 (3.11) and 60 µl of internal standard solution 2 (3.12). Add sufficient water, that a total volume (added and natural) of 20 ml water is obtained. In the case of dry sample materials wait 10 min after addition of water. Add 40 ml of methanol (3.7) and blend for 2 min. The total volume of liquid extract (taking into account the natural water content of the sample) is 60 ml. Centrifuge an aliquot at approximately 3 000 g (4.2), transfer approximately 2 ml of the supernatant into the syringe (4.3) and filter the solution through the syringe filter (4.4) into a 1,8 ml glass vial (4.5). If the filter is likely to be blocked by suspended matter, place a 1 cm layer of filter aid (3.4) onto the bottom of the syringe before filling it with the extract. 5.3 Determination Inject equal volumes of the sample test solution derived from 5.2 and of the calibration solutions (3.13) into the LC-MS/MS system. The LC-MS/MS system shall be operated in the multi reaction mode with transitions selective for chlormequat, mepiquat and the internal standards (see Annex A). Make sure that the liquid chromatographic conditions (column length, stationary phase type, injection volume, column temperature, electrospray interface parameters, etc.) are such that the separation of chlormequat and mepiquat from possible interfering peaks originating from the samples is as complete as possible.
Typical LC-MS/MS conditions are given in Annex A. 5.4 Test for interference and recovery Prepare reagent blanks and carry out spiked recovery tests at levels appropriate to the maximum residue level. The chromatogram of the reagent blank should not show any significant peak at the retention time of chlormequat or mepiquat.

NOTE 1 Typically, a response ratio of about 1 s
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