BACKGROUND Nitrophenol compounds are important organic chemical raw materials, which have high toxicity and carcinogenicity. In recent years, with the increasing use of pesticide and dyestuff in modern society, nitrophenol compounds have attracted widespread attention from scholars at home and abroad through different pathways into soil media such as atmosphere, water, causing harm to human health and environment pollution. The U.S. Environmental Protection Agency has included 4-nitrophenol and 2,4-dinitrophenol in the “Priority Control Pollutants List”. China has included 4-nitrophenol in the “Priority Control Pollutants Blacklist”. The value of 2,4-dinitrophenol for soil pollution risk control was specified in GB 36600—2018. Therefore, the simultaneous detection method of three nitrophenol compounds in soil is necessary. In the past, the common methods for the analysis of nitrophenol compounds in soil samples include gas chromatography and gas chromatography-mass spectrometry. However, there are challenges such as low sensitivity and poor peak shape. In order to improve the sensitivity, it usually was derivatized. However, the method of derivatization is time-consuming and cumbersome.

OBJECTIVES To establish a method for the determination of three nitrophenol compounds in soil by high performance liquid chromatography-mass spectrometry (HPLC-MS).

METHODS A method for the determination of three nitrophenol compounds in soil by ultrasonic extraction high performance liquid chromatography-mass spectrometry was established. Firstly, the chromatographic conditions were selected by examining the separation and sensitivity of three nitrophenol compounds. Then, high performance liquid chromatography using Kinetex C18 column with packing particle size of 2.6μm was used to directly separate all target compounds at 30℃, with gradient elution. The mobile phase was composed of 0.01mol/L ammonium formate-formic acid aqueous solution and methanol, and the flow rate was set to 0.2mL/min. Electrospray ionization (in negative) time of mass spectrometry was applied to detect target compounds in full scan mode, and quantification was performed using an external standard determination. Secondly, the sample pretreatment conditions were optimized, including the selection of extraction reagents, the injection solvents, and replace solvent. Three extraction reagents (dichloromethane, dichloromethane n-hexane (V/V=2∶1) and dichloromethane ethyl acetate (V/V=2∶1) were selected for optimization. The mixture of methylene chloride and n-hexane (V/V=2∶1) was used as solvent to extract nitrophenol compounds in soil by ultrasonic. The extracting solution was purified by adding alkaline aqueous solution (pH＞12), and the nitrophenolic compounds were dissolved in aqueous solution. Separating the organic phase, the aqueous solution was adjusted to acid (pH＜2). Three nitrophenolic compounds were extracted with dichloromethane-ethyl acetate (V/V=4∶1) mixed solvent. The extract was concentrated and diluted to 10.0mL with 10% acetonitrile aqueous solution. Finally, it was determined by high performance liquid chromatography-mass spectrometry external standard method．

RESULTS The Kinetex C18 separation system was used in this study because of the short analysis time and high sensitivity of three nitrophenol compounds. After optimization and selection, the final analysis method was determined as follows: dichloromethane n-hexane (V/V=2∶1) was the most effective extraction reagent for three nitrophenol compounds; therefore dichloromethane n-hexane (V/V=2∶1) was chosen as the extraction reagent. The aqueous solution was adjusted to acid (pH＜2), dichloromethane-ethyl acetate (V/V=4∶1) was chosen as the extraction reagent. HPLC-MS can be used to effectively determine nitrophenol compounds in soil samples. Under the optimum experimental conditions, three nitrophenol compounds in soil were well separated within 20 minutes. Through the quality control of the experimental process by blank samples and replicate samples, the analytical results of this method were accurate and reliable.The proposed method had good precision (RSD was less than 10.0%), the correlation coefficient (R²) was not less than 0.9992 in the linear range, The detection limits of three nitrophenol compounds in soil were 0.1-0.2μg/kg, and the recovery rate of three nitrophenol compounds in blank soil was in the range of 61.7%-90.8%, thereby indicating that the developed method would be suitable to determine the target three nitrophenol compounds in soil. The detection limits of three nitrophenol compounds in soil.

CONCLUSIONS A method for the determination of three nitrophenol compounds in soil by high performance liquid chromatography-mass spectrometry is established by selecting and optimizing the sample pretreatment and analytical conditions. The method has the advantages of simple pretreatment, high sensitivity and can be effectively applied to the determination and evaluation of three nitrophenol compounds in soil.