Production of a specific monoclonal antibody and a sensitive immunoassay for the detection of diphacinone in biological samples.

Diphacinone (DPN) is an extensively used anticoagulant rodenticide that is also considered a hazardous chemical, which poses a threat to nontarget species. DPN poisoning cases in humans or other species frequently occur, while rapid and sensitive detection methods are rarely reported. Thus, it is meaningful to develop an immunoassay for DPN detection with high sensitivity and specificity. In this study, a hapten was synthesized and then conjugated with carrier proteins to prepare the immunogens with different conjugation ratios for the preparation of antibody. After evaluation of the antisera using an indirect competitive enzyme-linked immunosorbent assay (icELISA) and statistical analysis, we found that the immunogen prepared using the N,N-dicyclohexylcarbodiimide (DCC) method with a conjugation ratio of 28.5 could elicit mice to generate antibodies with high performance. Using hybridoma technology, we obtained the specific monoclonal antibody (mAb) 4G5 with a half maximal inhibitory concentration (IC50) of 0.82 ng/mL in buffer solution. We initially explored the recognition mechanism of DPN/CLDPN and mAb from both conformational and electronic aspects. Then, mAb 4G5 was applied to develop icELISA for biological samples. The limits of detection (LODs) of icELISA were 0.28 µg/L, 0.32 µg/L, and 0.55 µg/kg for swine plasma, urine, and liver samples, respectively, and the recoveries ranged from 72.3 to 103.3% with a coefficient of variation (CV) of less than 12.3% in spiked samples. In summary, we developed a sensitive, specific, and accurate icELISA for the detection of DPN in biological samples, which showed potential in food safety analysis and clinical diagnosis. Graphical abstract.

[Study on the enhancement diphacinone-europium fluorescence system and application for determination of the residue in urine].

OBJECTIVE: To establish an accurate, convenient and sensitive quantitative fluorescence method for the determination of diphacinone (DPN) in urine. METHODS: Diphacinone was complexed with europium ion (Eu3+) at the presence of DL-histidine, polyvinylpyrrolidone (PVP) and ammonia-ammonium chloride (NH3 -NH4 Cl) buffer solution to form a multiple complex of DPN-Eu(3+) -DL-histidine-NH3-PVP fluorescence system, and the fluorescence sensitivity was greatly enhanced by the rare earth element of yttrium ion (Y3+ ). The excitation and emission wavelengths were 330 nm and 612 nm, respectively. RESULTS: The optimum conditions were Eu3+ 10.0 mg/L,Y3+ 20.0 mg/L, DL-histidine 0.01%, PVP 0.05% at pH 9.0. Under the optimum conditions, fluorescence intensity of the system was a linear function of the concentration of diphacinone in the range of 0.2 - 10.0 mg/L, the limit of quantification was 0.2 mg/L. The spiked recoveries for DPN in urine were 87.0% - 97.3%, the intra-and inter-day RSDs were between 3.4% - 4.7% and 4.8% - 6.6%, respectively. CONCLUSION: The established method is simple, accurate and reliable, and it is satisfactory for the determination of DPN in urine and can be used for DPN poisoned patients for the rapid clinical diagnosis.

[Simultaneous determination of trace diphacinone and chlorophacinone in biological samples by high performance liquid chromatography coupled with ion trap mass spectrometry].

A rapid qualitative and quantitative method for the simultaneous determination of trace diphacinone and chlorophacinone in biological samples has been established. The method mainly serves for the emergent poisoning detection. The whole blood was treated with methanol-acetonitrile (50/50, v/v) and the urine was cleaned-up by Waters Oasis HLB SPE cartridges. The samples were separated on an Extend C18 column (150 mm x 4.6 mm, 5 microm) by using the mobile phase consisted of ammonium acetate-acetic acid (0.02 mol/L, pH 5.5) - methanol (15/85, v/v). The determination was performed by high performance liquid chromatography coupled with ion trap mass spectrometry (HPLC-IT-MS) using a negative electrospray ionization interface in the multiple reaction monitoring (MRM) mode. The transitions of m/z 339 --> 167 for diphacinone and m/z 373 --> 201 for chlorophacinone were selected for the quantificantions. For the whole blood samples, the calibration curves were linear within the ranges of 1.0 - 200.0 microg/L and 0.5 - 100.0 microg/L; the limits of quantification were 1.0 microg/L and 0.5 microg/L; the spike recoveries were 90.1% - 92.2% and 87.6% - 93.4%, the intra-day relative standard deviations (RSDs) were less than 6.8% and 7.4%, and the inter-day RSDs were less than 9.9% and 10.9% for diphacinone and chlorophacinone, respectively. For the urine samples, the calibration curves were linear within the ranges of 0.2 - 40.0 microg/L and 0.1 - 20.0 microg/L; the limits of quantification were 0.2 microg/L and 0.1 microg/L; the spike recoveries were 90.1% -94.5% and 90.0% -98.0%, the intra-day RSDs were less than 6.1% and 7.3%, and the inter-day RSDs were less than 8.9% and 11.2% for diphacinone and chlorophacinone, respectively. This method is simple and sensitive for the satisfactory determination of trace diphacinone and chlorophacinone residues in poisoned patients for the clinical diagnosis.

[Study of diphacinone in biological samples by high performance liquid chromatography/diode array detector].

An analytical approach has been developed for high performance liquid chromatographic determination of diphacinone extracted from liver, blood, urine and kidney of rabbit by solid phase extraction (SPE) cartridges (using SAX, CN or SILICA GEL) with coumarin as the internal standard. Diphacinone was separated by reversed-phase gradient chromatography with DAD detection at 286 nm. The Analytical column was Hypersil BDS C18(150 mm x 4.6 mm i.d., 5 microns) and the guard column was Phenomenex ODS(4 mm x 3.0 mm i.d.). The mobile phase was a gradient mixture of aqueous solution (A) and methanol solution (B) both containing 0.5% ion pair A. There was a good linear relationship between the concentration of diphacinone and the ratio of peak areas of diphacinone and coumarin (internal standard) (r = 0.9999). The linear range was 1 mg/L-100 mg/L, and the lower detection limit was 5 ng (S/N = 3). The average recoveries of diphacinone in urine, blood and liver were 88.4% (n = 3, RSD = 1.25%, SPE by CN column), 82.2% (n = 3, RSD = 1.67%, SPE by SAX column), 91.0% (n = 3, RSD = 2.77%, SPE by SILICA GEL column), respectively.