Simultaneous determination of three components of sodium nitrophenolate in foodstuffs of animal origin by high performance liquid chromatography-tandem mass spectrometry using atmospheric pressure chemical ionization

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.