关键词: 高效液相色谱-串联质谱, 磁性纳米粒子, 黄曲霉毒素B₁, 谷物, 抗体

Abstract:

In this study, a magnetic nanomaterial antibody (Ab)-SiO₂@Fe₃O₄ was synthesized, which was employed to absorb aflatoxin B₁ (AFB₁) in complicated grain matrices. The Ab-SiO₂@Fe₃O₄ material was then paired with high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) for subsequent accurate detection. The Ab-SiO₂@Fe₃O₄ material has a specific adsorption capacity for AFB₁ because of the stable and specific biological binding between antigen and antibody. This process can achieve the identification between the material and food matrix quickly, thereby completing the separation and enrichment process. Then, high sensitivity and high accuracy HPLC-MS/MS were employed for signal readout and actual quantification, which can significantly increase the detection efficiency and enable high-throughput detection of numerous samples. In the pretreatment process, Fe₃O₄ was first synthesized by microwave-assisted hydrothermal synthesis within 1 h, and Ab-SiO₂@Fe₃O₄ was then produced using the enhanced Stober’s approach. This material with high adsorption performance was synthesized under relatively mild conditions and short time. To obtain Ab-SiO₂@Fe₃O₄ materials with uniform particle size, magnetic properties, and dispersibility that met the requirements, synthesis conditions of Ab-SiO₂@Fe₃O₄ and conditions for capturing the AFB₁ target were analyzed. The findings demonstrated that the best effect was obtained when the dosage of FeCl₃·6H₂O was 10.0 mmol, the heating time was 40 min, and 100 μL tetraethoxysilane was employed for SiO₂ coating. The AFB₁ antibody was then combined with the surface of SiO₂@Fe₃O₄ under several conditions. The findings revealed that the best coupling efficiency of Ab could be obtained when the concentration of 2-morpholinoethanesulfonic acid monohydrate (MES) was 10 mmol/L, pH was 6.5, and the molar ratio of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC)∶N-hydroxysuccinimide substances (NHS) was 2∶1. The coupling buffer was then selected as phosphate buffer (PBS) with pH=7.4, and 8 mg Ab-SiO₂@Fe₃O₄ was employed to separate and enrich AFB₁ at 37 ℃ for 10 min. In the actual detection, acetonitrile-water-formic acid (85∶10∶5, v/v/v) was employed as the extraction solution. After ultrasonic extraction for 10 min, Ab-SiO₂@Fe₃O₄ was employed to separate and enrich AFB₁ in the extract. The supernatant was dried with nitrogen and reconstituted with 1-mL acetonitrile. The solution was then filtered through a 0.22 μm filter and detected using HPLC-MS/MS, thereby realizing the quick and quantitative detection of AFB₁. AFB₁ had an excellent linear relationship in the range of 2-50 μg/L under the optimal analytical conditions, and the correlation coefficient was less than 0.99. The LOD was 0.04 μg/kg, and the LOQ was 0.13 μg/kg. The spiked recoveries of AFB₁ in three grain matrices ranged from 76.21% to 92.85% with RSD≤5.29% at four different spiked levels. The approach was applied to the determination and analysis of AFB₁ in 30 real grain samples of rice, corn, and wheat. The findings demonstrated that AFB₁ was detected in one wheat sample and two corn samples, and its content was 0.38, 0.13, and 0.47 μg/kg, respectively, and no toxins were found in other samples. The approach combined Ab-SiO₂@Fe₃O₄ magnetic nanomaterials with HPLC-MS/MS, which could obtain high-efficiency separation and enrichment of AFB₁. Furthermore, the low-cost Ab-SiO₂@Fe₃O₄ could be stored for more than a week and complete the pretreatment process within 30 min. This effective pretreatment process combined with HPLC-MS/MS could realize the analysis of several samples within a short time, and had a promising application prospect in the detection of AFB₁ in grains.

Key words: high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS), magnetic nanoparticles, aflatoxin B₁ (AFB₁), grain, antibody (Ab)