Determination of 11 p-phenylenediamine antioxidants and their oxidation products in milk by ultra performance liquid chromatography-tandem mass spectrometry

ObjectiveTo establish the first method for the simultaneous determination of 11 p-phenylenediamine antioxidants （PPDs） and their oxidation products in milk by solid-phase extraction （SPE） coupled with ultra performance liquid chromatography tandem mass spectrometry （UPLC-MS/MS）.MethodsA 2.00 g milk sample was weighed， freeze-dried， and extracted with 10 mL of acetonitrile. The mixture was shaken， then centrifuged at low temperature. The supernatant was purified using an Oasis HLB solid-phase extraction column. The combined sample and purified liquid were evaporated under nitrogen and resuspended for injection and analysis. Chromatographic separation was performed using a gradient elution with a mobile phase of 0.1% formic acid aqueous solution and acetonitrile. The analysis was carried out on a WATERS ACQUITY BEH C18 column （2.1 mm×100 mm， 1.7 μm）. Detection was conducted in positive ion electrospray ionization （ESI+） multiple reaction monitoring （MRM） mode， and quantification was achieved using the internal standard method.ResultsThe correlation coefficients （R²） for all 11 PPDs and their oxidation products were greater than 0.990 within the concentration range of 0.1-50 μg/L. The method detection limits ranged from 0.004 to 0.152 μg/kg， and the quantification limits ranged from 0.013 to 0.507 μg/kg. Recovery rates for three different spiked concentrations ranged from 84.4% to 121.9%， with good intra-day and inter-day precision （RSD<14%）. When applied to 28 milk samples from different countries and regions， the detection rate for 6PPD was the highest， with positive concentrations ranging from 0.05 to 2.27 μg/kg. The detection rate for DTPD was 39%， with positive concentrations ranging from 0.07 to 0.79 μg/kg， and only two samples had 6PPD-Q concentrations above the limit of quantification.ConclusionThis method demonstrates excellent sensitivity， accuracy， and precision， making it suitable for detecting p-phenylenediamine-based antioxidants and their oxidation products in milk samples.