Veterinary drugs have played important roles in modern animal husbandry due to their prevention and treatment of diseases as well as growth promotion effects. In pursuit of higher profits, drug abuse phenomena were occasionally reported, which had caused serious residue problems and potential hazards to consumer health [1,2]. Despite improved awareness of medication regulations among livestock industry practitioners, routine monitoring of veterinary drugs served as the last line of defense to ensure drug safety. Therefore, different types of veterinary drug residue detection methods have been developed, mainly including enzyme-linked immunosorbent assay [3,4], colloidal gold immunochromatographic assay [5,6], biochip technique [7,8], fluorescence biosensor [[9], [10], [11]], surface-enhanced raman spectroscopy [12,13], and chromatographic methods as well as their coupling techniques with mass spectrometry [[14], [15], [16], [17], [18]]. Among the numerous detection techniques, liquid chromatography-tandem mass spectrometry coupled with an electrospray ionization source (ESI-LC-MS/MS) has shown its significant advantages in confirmatory analysis of veterinary drug residues due to its good selectivity, accuracy, and sensitivity as well as high throughput characteristics [15,19].

However, the co-extracted complex matrix in samples often played significantly impacts on the target signals during the electrospray ionization process, thus attenuating the accuracy, sensitivity, and reproducibility of the analysis results. Therefore, suitable sample pretreatment process is of an important prerequisite prior to LC-MS/MS analysis. Commonly used pretreatment techniques mainly include liquid-liquid extraction (LLE), solid phase extraction (SPE), and QuEChERS (quick, easy, cheap, effective, rugged, safe). Among them, LLE and SPE require either large consumptions of hazardous reagents or cumbersome sample processing processes. Comparatively, the emerging QuEChERS methods have already been widely applied in analysis of various foods due to their obvious advantages in simpleness, convenience, and effectiveness [16,17,[20], [21], [22]]. Considering the complex matrix interference, modified QuEChERS methods mainly focus on the preparation and synthesis of new matrix adsorbents tailored for different food origins. Currently, commercially available adsorbents commonly conclude octadecylsilane chemically bonded silica (C18), primary secondary amine (PSA), graphitized carbon black (GCB), carbon nanotubes (CNTs), and graphene as well as its oxides [21,[23], [24], [25], [26]]. Among them, graphene oxide (GO) has good hydrophilic and hydrophobic properties as well as large specific surface area, which should possess satisfactory matrix adsorption performance with a broad application prospect. However, the severe stacking and poor dispersion between GO layers significantly reduced its utilization and limited its widespread use. Decoration of GO on the three dimensional porous melamine sponge (MeS) serves as an intelligent solution to improve its utilization without worrying the above problems [27]. As an elastic and versatile material with high porosity of up to 99 %, commercially available MeS has good chemical stability and high mechanical strength in various extraction solvents, which could provide a good physical support for GO modification. In addition to good adsorption performance, the functionalized composite material could streamline the matrix adsorption and separation processes by simple and fast soaking and squeezing cycles.

According to the reported literatures [28,29], the high-throughput QuEChERS-based preparation method works efficiently for various classes of veterinary drug. However, the residual levels of multi-class veterinary drugs in animal-derived foods were extremely distinctive due to various reasons, such as different dosages, administration routes, and metabolic rates. Therefore, conventional QuEChERS method directly coupled with LC-MS techniques might still encounter the dilemma of insufficient sensitivity in some cases, especially for those drug residues below ∼µg/kg. Furthermore, the Chinese government, the US Food and Drug Administration, and European Union have increasingly strict requirements for both the permissible veterinary drugs with maximum residue limits as well as those prohibited drugs. As a result, there is an urgent need of developing convenient, efficient and appropriate drug enrichment methods prior to LC-MS analysis. However, the traditional offline enrichment method might suffer the drug degradation issues or transfer losses. By contrast, the online enrichment method not only improved the automation and convenience of the analytical process, but also enhances the accuracy and reproducibility of the detection results, which is of great significance for multi-class residue analysis of veterinary drugs [30,31]. As known, the online enrichment method could be directly achieved by large volume loading with different dilution strategies, such as fractionized sampling and stacking (FSS) [32,33], pre-column dilution [34], and bypass dilution [35]. Comparatively, the FSS interface could precisely control the dilution effects by adjusting the valve switching process without changing the flow rates of mobile phases or suffering the gradient variation of elution solvents. In FSS, tens of microliters of sample solutions could be fractionized into numerous micro-units (namely injection plugs) stored and transferred in stainless steel capillary, of which the elution strength was significantly weakened or eliminated by dilution with aqueous plugs. As a result, the monitored solute molecules could be efficiently stacked at the column head as expected to achieve large volumes of sample loading [32]. However, conventional FSS interface techniques typically require coupling at least two chromatography systems to complete the above enrichment process, which would increase the equipment cost and device adaptability as well as its operation complexity.

In this study, a simple and convenient FSS interface has been designed and constructed on one set of UPLC-MS/MS system for ultrasensitive analysis of multi-residue veterinary drugs in beef by integrated with a modified QuEChERS method. The fast and convenient matrix purification was achieved by soaking and squeezing within several seconds based on a prepared reduced GO coated melamine sponge (rGO@MeS). Large volumes of purified solution were efficiently loaded into the online UPLC-MS/MS system without observing any peak broadening or distortions by FSS interface, resulting in an enhanced sensitivity for simultaneously analyzing multi-residue veterinary drugs. Not only the embedded FSS interface does not change the original setup and sampling procedures of the commercial instruments, but also has little influence on the chromatographic separation gradient. The good compatibility and satisfactory results demonstrated its promising application and development prospects of the established technique in ultrasensitive determination of veterinary drug residues at trace levels in various foods.