A quadruple-labeling luminescence strategy for multiplexed immunoassay of 51 drugs in milk with an automated pretreatment system

In this paper, a novel combination of quadruple-labeling luminescence strategy multiplexed immunoassay (QLL-MIA) and automated pretreatment system for simultaneous extraction and determination of 20 fluoroquinolones (FQs), 15 β-lactams, 15 sulfonamides (SAs), and chloramphenicol (CAP) in milk was developed. The strategy integrated 2 fluorescence probes, 2 chemiluminescence probes to achieve signal separation, and a new combination of nucleic acid extraction system and immunomagnetic beads (IMBs) as automated pretreatment system to achieve simultaneous determination of 51 drugs. Taking the advantage of automated pretreatment system for saving extraction time and concentration sample with 5 times, the limits of detection (LOD) for FQs, β-lactams, SAs, and CAP in QLL-MIA range from 29.1 ng/L for ciprofloxacin (CIP) to 8000.0 ng/L (8.0 μg/L) for trovafloxacin (TRO), 20.0 ng/L for ceftiofur (CEF) to 3409.1 ng/L (3.4 μg/L) for cephalexin (CEL), 8.6 ng/L for sulfadimethoxine (SDM) to 328.2 ng/L (0.3 μg/L) for sulfadiazine (SDZ), and 6.0 ng/L for CAP. Recoveries ranged from 80.6% to 105.5% at fortified concentration of LOD and 2LOD, with coefficient of variation <15%. Analysis of field milk samples by the combination of QLL-MIA and automated pretreatment system (the developed QLL-MIA) was in accordance with that of liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). The above results demonstrated that the developed QLL-MIA could simultaneously screen trace FQs, β-lactams, SAs, and CAP in filed samples, suitable for high-throughput screening of low-molecular weight contaminants.