Analysis of chlormequat residue in milk by UPLC-MS/MS

Chlormequat is a plant growth regulator generally used to promote lateral branching and flowering of ornamental plants. It is also frequently used to improve yields for different types of cereals in Europe. These cereals are often processed into animal feed. Consequently chlormequat residues could potentially be found in matrices of animal origin. The MRL in milk were fixed at 0.05 mg/kg in annex I part B of the regulation 396/2005. Milk is a very complex matrix composed of different proteins and fat. Therefore, intensive clean-up is needed before injection to prevent interference or clogging during LC analysis. For the extraction, 4ml of water is added to 1g of milk just after the addition of chlormequat D4 as internal standard. The cleanup strategy selected was the use of dichloromethane andacetic acid to eliminate fat and precipitate protein, respectively. This step was followed by a solid phase extraction (SPE) of the aqueous phase on a Weak Cation Exchange cartridge. The chlormequat was eluted by using ammonium acetate, before injection on an UPLC-MS/MS system (Waters premier). Due to his ionic characteristic, no retention is observed on classical C18 column. The reverse phase polymethacrylate Shodex RSpak DE413 (150 × 4.6 mm) recommended in the CEN method prEN15054 has been selected. Isocratic condition (water/acetonitrile/ methanol/acetic acid - 71.25+17.5+7.5+3.75 V+V+V+V 50 mM ammonium acetate are used at a flow of 0.4 ml/min. The observed a retention time is 4.0 min. The analytical method has a limit of quantification of 0.025 mg/kg (limit of detection of 0.0125 mg/kg) and was validated following SANCO/12495/2011 criteria at the LOQ and at a higher level (10 times LOQ - 0.25 mg/kg). Results show a recovery of approximately 100% (between 100.6 and 103.0) with a repeatability and in-house reproducibility below 10%. These very good validation data are correlated to the robustness of the method and to the use of an isotopic surrogate.</p