关键词: 超高效液相色谱, 串联质谱, 饲料, 二氢吡啶

Abstract: 【Objective】As a new kind of feed additive, diludine has been widely used in livestock production because it can promote the growth of animals, increase efficiency of the feed, and improve the meat quality. However, diludine is not allowed to use as a medical feed additive. The residues of diludine in animal origin foods may cause severe hypotensive reaction in the sensitive population. In the past World Consumer Rights Day, also known as 3.15, the national broadcaster China Central Television (CCTV) exposed parts of illegal use of veterinary drugs (e.g. diludine) in some of the feed companies, which draw extensive concerns of the society and the government. It is, therefore, of great importance to develop sensitive and reliable analytical methods to monitor diludine in feedstuffs in order to protect consumer rights and to provide technical support for government regulation.【Method】Chromatographic separation was achieved using a BEH C₁₈ column, and different mobile phases consisting of methanol-water, acetonitrile-water, methanol-0.1% formic acid, and acetonitrile-0.1% formic acid at different concentrations were optimized. In order to achieve the maximum sensitivity of ESI-MS/MS, direct infusion of standard solution was carried out in positive ionization mode to optimize the ESI source parameters (e.g. capillary voltage, cone voltage, and desolvation gas flow rate). Then dissociation with argon was induced and different collision energies and dwell times were compared in order to find the daughter ions, and the most abundant product ion was used as the quantification ion. In order to find the best sample preparation method, different extraction solvent (methanol and acetonitrile) and solid phase extraction (SPE) cartridges (C₁₈, HLB, MCX and alumina B) were optimized. 【Result】The optimized sample preparation procedures were conditioned as follows: Feed samples were extracted using acetonitrile and the supernatants were evaporated to dryness under a gentle stream of nitrogen at 60℃. The residues were re-dissolved with acetonitrile/water (1:9), and then cleaned up on HLB SPE cartridges. The target compounds were identified and quantitatively determined by ultra-performance liquid chromatography coupled with electrospray ionization (ESI) tandem mass spectrometry (UPLC-MS/MS) operated in multiple reaction monitoring mode (MRM). The molecular ion [M+H]+ of diludine is m/z 254, and the loss of –OCH₂CH₃ (46 Da) is found to be common and leads to the major daughter ion m/z 208. The elimination of –CO (28 Da) from the ion m/z 208 gives the fragment at m/z 180, and then a subsequent loss of –COOCH₂CH₃ (73 Da) leads to the fragment at m/z 108. Therefore, in this study, the quantitative ion was m/z 254＞208, while m/z 254＞180 was used for qualitative ion. Under the current optimized chromatographic conditions, each LC run was completed in 10 min. Good linearity was obtained in the ranges of 0-500 μg·L^-1, with linear coefficients (R²) higher than 0.9992. The limits of detection (LODs) and quantification (LOQs) which defined as the concentration with a signal-to-noise ratio (S/N) of 3 and 10, were 10 μg·kg ^-1 and 50 μg·kg ^-1, respectively. Average recoveries from three fortification levels (10, 50 and 100 μg·kg ^-1) ranged between 82.6% and 101.0%, with relative standard deviations (RSD) lower than 9.2%. 【Conclusion】 The proposed method is fast, sensitive, and easy to perform, making it applicable for high-throughput daily monitoring.

Key words: ultra-performance liquid chromatography, tandem mass spectrometry, animal feeds, diludine