Glufosinate (GLUF) is a broad-spectrum, non-selective herbicide and widely used for weed control in agriculture, forestry, and urban areas [1,2]. Similar in chemical structure to glyphosate (GLY), GLUF is a polar chemical containing phosphine and amino acid groups. As the use of GLY has become more widespread, it has also been shown to be ineffective in some crops, and weed resistance increases as the dose is increased [3,4], leading to a vicious cycle of increasing the dose again. The structure of the herbicide market has changed in recent years. Although GLY is still the number one product, GLUF continues to gain market share and has moved into second place. According to statistics, the market sales of GLUF in China reached US$1.05 billion in 2020.

The widespread use of GLUF has led to its ubiquitous presence in food and environmental matrices [5,6]. Many cases of accidental and suicidal poisoning have been attributed to the ingestion of GLUF [7]. GLUF poisoning is characterized by a variety of neurological symptoms, including altered consciousness, convulsions and apnoea, occurring several hours after ingestion [8]. The nomogram reported by Koyama et al [9] has been shown to be useful in predicting the severity of delayed effects of GLUF as a function of blood GLUF concentration. As there is a possibility of severe effects if a patient's blood GLUF level is above the line connecting the log of 70 g/mL (2 h after ingestion) and the log of 5 g/mL (8 h after ingestion), airway intubation and mechanical ventilation are required to prevent worsening of symptoms. A simple and rapid method for predicting the severity of GLUF poisoning is therefore needed. Unfortunately, such a method has not yet been demonstrated in practice [10]. In addition, the confirmation and determination of metabolites of GLUF in plasma can also be used to help predict intoxication in patients and to determine the stage of intoxication. The main metabolite of GLUF in humans reported in the literature is MPPA [7], but there are few reports on the metabolite of GLUF, N-acetyl GLUF, in human biological samples.

Liquid chromatography-tandem mass spectrometry (LC-MS/MS) has become the preferred method for the determination of GLUF and its metabolites due to its advantages of high sensitivity and high selectivity [1,4,7,[10], [11], [12], [13], [14], [15], [16], [17], [18]]. With the rapid development of hydrophilic interaction liquid chromatography (HILIC) technology, it is expected that more types and brands of commercial HILIC columns will become more accessible, which is expected to achieve a non-derivatization method of LC-MS/MS for the detection of GLUF and its metabolites. Obelisic N column has been used to analyze polar herbicides including GLY, GLUF and their metabolites [19], but column degradation and poor retention time reproducibility were observed. Recently, Pan et al [20] used a polymer-based Dikma Polyamino HILIC column to achieve the chromatographic separation of GLY and its four metabolites in plasma, which has the advantages of peak shape symmetry and strong chromatographic retention ability. It is expected to provide highly sensitive detection of GLUF and its metabolites in plasma under strong alkaline conditions.

However, due to the large amounts of impurities such as phospholipids, proteins, fatty acids and inorganic salts in plasma [21], the matrix interference effect on the detection of GLUF and its metabolites by LC-MS/MS is not negligible, and a suitable sample pretreatment method should be selected to eliminate or reduce it. Some researchers have used the water dilution method with water for the determination of GLY and GLUF in human urine [22]. It is simple and efficient, but it is difficult to effectively remove impurities such as inorganic salts and proteins in biological samples, and it is more damaging to the detection equipment. More researchers preferred to use solid phase extraction (SPE) as a pre-treatment of biological samples, and commercial SPE columns such as SAX [3], MCX/MAX [3], SCX [23], C18 [1], etc. have been used for the purification of GLUF and its metabolites in biological samples. Although the SPE method has the advantages of simple operation and good reproducibility, the limited types of commercial SPE columns and the poor self-modification of SPE materials make it difficult to meet practical detection requirements. Magnetic solid phase extraction (MSPE), as a new sample pretreatment technique, has been widely used for the purification and detection of biological samples due to its advantages of rapidity, simplicity and modifiability [24,25]. The core of the MSPE method lies in the design and synthesis of functionalized magnetic adsorbents with high selective adsorption and purification capabilities according to the structural characteristics of the target compounds and the sample matrix [25]. However, due to the complex matrix of plasma samples, it is difficult to simultaneously and effectively remove impurities such as phospholipids, fatty acids and inorganic salts from plasma by relying on a single functionalized magnetic material. Due to the high modifiability of functionalized magnetic materials, multi-functionalized modifications on the surface of magnetic polymers can be considered to enhance their ability to remove impurities in plasma samples.

The main objective of this study was to develop a rapid, simple, accurate and sensitive LC-MS/MS analytical method for the determination of GLUF and its metabolites, N-acetyl GLUF and MPP, in plasma samples (the chemical structures of the three target analytes were in Fig. S1 (Supplementary material). In this study, the Dikma Polyamino HILIC column was chosen as the separation column for the efficient retention and separation of GLUF and its metabolites in plasma samples. The innovative addition of a low concentration of ammonium fluoride solution to the mobile phase effectively increased the ionization efficiency of GLUF and its metabolites on the MS/MS detector, thereby significantly improving the sensitivity of the method. In addition, based on the chemical structure characteristics of GLUF and its metabolites in plasma, this study was the first to design and synthesize novel core-shell mixed-mode WCX/C18 bifunctional magnetic polymers (Fe3O4@WCX/C18) as an MSPE purification adsorbents. The focus was on the effect of the molar ratio of the bifunctional monomers 4-VBA and 1-octadecene on the purification performance during the adsorbent synthesis process. The experimental results show that the bifunctional components WCX/C18 have a synergistic effect on the determination of GLUF and its metabolites in plasma samples, and the optimal molar ratio of 4-VBA and 1-octadecene is 3:1. Meanwhile, compared with the commercial Oasis® WCX SPE cartridge, the Fe3O4@WCX/C18-based MSPE method synthesized in the present work has a better ability to reduce the substrate concentration than the commercial Oasis® WCX SPE cartridge. The MSPE-LC-MS/MS method established in this study is based on the detection of not only the prototype of GLUF but also its two metabolites in the confirmation of GLUF poisoning, providing a strong technical support for accurate clinical management.