Abstract：
Sodium nitrophenolate (SNP) is a widely used universal growth regulator consisting of 5-nitroguaiacol sodium (5NG), 4-nitrophenol sodium (PNP), and 2-nitrophenol sodium (ONP). SNP has a positive influence on plants and animals as a feed additive that accelerates growth but is potentially hazardous to humans. SNP has been reported to be cytotoxic and mutagenic, which may increase the risk of cancer and pose a great threat to food safety. There are neither mature detection nor standard methods for the trace analysis of SNP in animal food. Therefore, the development of an accurate and precise analytical method is imperative. This innovative method has theoretical and practical significance for the control of SNP residues, offering advantages such as cost-effectiveness and time efficiency. It will be beneficial for the establishment of detection standards and management measures in foodstuffs of animal origin.In this study, a reliable method for the simultaneous determination of SNP residues in animal food (porcine muscle, chicken tissue, fish, and liver) was developed. For realizing the perfect limit of quantification, the application of back extraction coupled with high performance liquid chromatography-atmospheric pressure chemical ionization-tandem mass spectrometry (HPLC-APCI-MS/MS) was proposed to combine high sensitivity and high selectivity. The optimal method was as follows. First, 2.0 g samples were extracted with 10 mL 0.5 mol/L sodium hydroxide solution, followed by adjustment of the pH to acidity with 3 mol/L hydrochloric acid and the addition of sodium chloride (5.0 g) to saturate the inorganic phase. After back-extraction twice with 16 mL acetonitrile, the solution was merged and again saturated with 5 mL of sodium chloride solution. Second, the merged organic phase was cleaned up with 10 mL of n-hexane for defatting. The middle acetonitrile layer was then concentrated to nearly 1.5 mL at 40 ℃ in a N2 stream before dilution with the mobile phase to a volume of 3.0 mL and filtered. Finally, the analytes were separated on a C18 column (100 mm×4.6 mm, 3 μm) and subjected to gradient elution with a mixed solution of methanol and water. Mass spectrometric analysis, which was quantified using the external standard method, was carried out with an atmospheric pressure chemical ionization negative ion source and based on multiple reaction monitoring (MRM) mode. The key parameters, such as the extraction solvent, extraction steps, and purification method, were optimized.The calibration curves were linear in the ranges of 0.5-10 (5NG), 1.0-20 (PNP), and 2.5-50 μg/L (ONP) with correlation coefficients greater than 0.9999. The limit of quantification (LOQ) for 5NG was 1.0 μg/kg, double for PNP, and five times for ONP. The recoveries of the three different concentration levels in all the four matrices were in the range of 81.5%-98.4%, 81.5%-102%, and 81.4%-95.1%. The repeatability, expressed as the relative standard deviations (RSDs) of the three compounds, ranged from 1.51% to 5.98%, 1.10% to 8.85% and 0.91% to 8.61% (n=6). The developed method is characterized by an excellent purification effect, sensitivity, and accuracy. This method is suitable for the simultaneous and quantitative determination of SNP residues in foodstuffs of animal origin.
摘要：
硝基苯酚钠（SNP）是一种广泛使用的通用生长调节剂，由5-硝基愈创木酚钠（5NG）组成，4-硝基苯酚钠（PNP），和2-硝基苯酚钠(ONP)。SNP作为饲料添加剂对植物和动物具有积极影响，可加速生长，但对人类具有潜在危险。据报道，SNP具有细胞毒性和诱变性，这可能会增加患癌症的风险，对食品安全构成巨大威胁。动物性食品中SNP的痕量分析既没有成熟的检测方法，也没有标准的方法。因此,发展准确、精确的分析方法势在必行。该创新方法对SNP残留的控制具有理论和现实意义,提供成本效益和时间效率等优势。这将有利于建立动物源性食品的检测标准和管理措施。在这项研究中,一种同时测定动物食品中SNP残留的可靠方法(猪肌肉，鸡组织,鱼,和肝脏）发育。为了实现量化的完美极限，提出了反萃取与高效液相色谱-常压化学电离串联质谱（HPLC-APCI-MS/MS）联用的高灵敏度和高选择性。最佳方法如下。首先,2.0g样品用10mL0.5mol/L氢氧化钠溶液提取，然后用3mol/L盐酸调节pH至酸性,并加入氯化钠(5.0g)以饱和无机相。用16mL乙腈反萃取两次后，合并该溶液并再次用5mL氯化钠溶液饱和。第二,合并的有机相用10mL正己烷净化,用于脱脂。然后将中间乙腈层在40°C下在N2流中浓缩至接近1.5mL,然后用流动相稀释至3.0mL的体积并过滤。最后,分析物在C18色谱柱上分离(100mm×4.6mm，3μm），并用甲醇和水的混合溶液进行梯度洗脱。质谱分析,使用外标法定量，采用常压化学电离负离子源并基于多反应监测(MRM)模式进行。关键参数，如提取溶剂，提取步骤,和纯化方法，进行了优化。校准曲线在0.5-10(5NG)范围内呈线性关系，1.0-20（PNP），和2.5-50μg/L（ONP），相关系数大于0.9999。5NG的定量限（LOQ）为1.0μg/kg，双PNP，和ONP的五次。三种不同浓度水平在四种基质中的回收率均在81.5%-98.4%之间,81.5%-102%，和81.4%-95.1%。可重复性,表示为三种化合物的相对标准偏差(RSD)，从1.51%到5.98%，1.10%至8.85%和0.91%至8.61%(n=6)。所开发的方法具有优异的纯化效果，灵敏度,和准确性。该方法适用于同时定量测定动物源性食品中的SNP残留。
