1. Introcuction

Veterinary drugs are substances used for the prevention, treatment and diagnosis of animal diseases or for the targeted regulation of physiological functions in animals  [1, 2], including but not limited to antibiotics, antiparasitic drugs, antifungal drugs and bronchodilators, and are essential for the prevention and treatment of animal disease [3]. Because of their many benefits, some breeders use veterinary drugs irrationally and even abuse prohibited drugs illegally in order to improve economic returns or due to lack of cognition of drug hazards or scientific guidance on their use [4, 5]. These veterinary drugs remain in animals and enter the human body through the food chain , further jeopardize human health [6-8]. Therefore, China, EU, USA, and others have set the maximum residue limits (MRL) [9-11] of veterinary drugs in animal derived food and they initiate legal procedures in any excessive cases; to meet the monitoring objectives and regulatory requirements, it is of great necessity to provide accurate test results and develop effective and reliable veterinary drug residue testing methods.

Veterinary drugs are mainly composed of involatile polar or low-polar compounds; gas chromatography (GC) or gas chromatography-tandem mass spectrometry (GC-MS/MS) is not suitable for testing such components [2], and the pure liquid chromatography (LC) performs limited detection of veterinary drugs and their metabolites and classified detection of some veterinary drugs due to its low sensitivity and poor selectivity, and has great limitations for simultaneous detection of f different veterinary drugs. In recent years, the method has been replaced by methods of chromatography tandem-mass spectrometry methods with better selectivity and higher testing efficiency [12-14]. The improvement of the test equipment has greatly increased the number of veterinary drugs that can be detected simultaneously: 80 drugs could be analysed by the method established by Zhao et al [15]; 210 drugs in pork could be analyzed by the method established by Yin et al [16]; 77 drugs in chicken could be analyzed by the method established by Alcantara-Duran et al [6]; however, these methods are mostly based on analysis on one and the same matrix in the pretreatment method of QuEChERS, and have limitations in analyzing multiple matrices. Li Xiaoqin et al established a method to screen 204 veterinary drugs [17]; Desmarchelier A established a qualitative LC-MS/MS method to detect 154 veterinary drugs [18], these methods could detect multiple matrices but cannot perform quantitative analysis. There are only several micrograms and even nanograms of veterinary drugs in food [12], and this is a major challenge for residue testing in animal derived food.

 The matrices of livestock and poultry products are complex due to the large amount of lipids, proteins and other macromolecular substances present in the products [19-21], resulting in false positives or false negatives test results. Therefore, sample preparation is still a challenging process for testing drug residues in animal derived foods. In this study, Clean-up LPAS columns made of novel high-polymer materials were used to rapidly filter and clean up multiple animal derived food, and by combining LC-MS/MS, a high-throughput test method was established that can rapidly, accurately and efficiently test 61 veterinary drugs in 13 categories simultaneously, including 22 types of sulfonamides, 10 types of quinolones, 9 types of ?-receptor agonists, 4 types of tetracyclines, 4 types of amphenicol, 3 types of macrolides, 2 types of sedatives, 2 types of lincosamide antibiotics, and 1 type of nitroimidazole, globigerina, corticosteroid, penicillin and antiviral drug respectively. The method was  verified with satisfactory results obtained and successfully applied in testing 731 real samples to investigate the safety conditions of veterinary drug residues in livestock and poultry products.

2 Experiments

2.1 Drugs and Reagents

1290-6470 LC-MS (Agilent, USA), TGL-16 High-speed Refrigerated Centrifuge (Sichuan Shuke Instrument Co., Ltd, China), KNS-2500 Multi-Tube Vortex Mixer (Krownus Scientific Experimental Instrument Co., Ltd., China), KH-500B Ultrasonic Cleaner (Kunshan Hechao Ultrasonic Instruments Co., Ltd., China), UPT-II-100L ULUPURE-series Ultrapure Water Machine (ULUPURE, China), Milli-Q Ultrapure Water Machine (Millipore, USA).

Carbinol (chromatographically pure, Fisher Chemical), acetonitrile (chromatographically pure, Fisher Chemical), formic acid (chromatographically pure, Fisher Chemical), ammonium acetate (chromatographically pure, Tianjin Kemiou Chemical Reagent Co., Ltd.), ammonium formate (GR (guarantee reagent)), clean-up column-LPAS (Beijing Knorth Technology Co., Ltd., China), and pure water used in this experiment were produced by the ultrapure water machines in the laboratory. All standard substances are standard solid substances with a purity of over 95% and were purchased from Dr. Ehrenstorfer GmbH. The names, CAS No. and groups of all standard substances are given in Table 1.

 2.2 Preparation of Standard Solutions

Standards  at 10 mg in each target group were accurately weighed and placed in 10 mL brown volumetric flasks, sufficiently dissolved with methanol to a constant volume, and then transferred to brown standard sample bottles after uniform mixing to prepare single standard stock solutions at a concentration of 1 mg/mL and stored at -20?. Amoxicillin was dissolved in acetonitrile: water (1:1) to a constant volume ; quinolones were dissolved in an appropriate amount of sodium hydroxide solution at a concentration of 0.03 mol/L and then dissolved in methanol to a constant volume. A mixed standard solution with a concentration of 10 ?g/mL containing all the diluted single standard methanol was prepared, and a series of working curves with seven concentrations of 0.5 ?g/L, 1.0 ?g/L, 2.0 ?g/L, 5.0 ?g/L, 10.0 ?g/L, 20.0?g/L, and 50.0 ?g/L were prepared after diluting the mixed standard solution with ultra-pure water or blank matrices.

2.3 Instruments

2.3.1 Instruments

1290-6470 LC-MS (Agilent, USA), TGL-16 High-speed Refrigerated Centrifuge (Sichuan Shuke Instrument Co., Ltd, China), KNS-2500 Multi-Tube Vortex Mixer (Krownus Scientific Experimental Instrument Co., Ltd., China), KH-500B Ultrasonic Cleaner (Kunshan Hechao Ultrasonic Instruments Co., Ltd., China), UPT-II-100L ULUPURE-series Ultrapure Water Machine (ULUPURE, China), Milli-Q Ultrapure Water Machine (Millipore, USA).

2.3.2 LC-MS/MS Instrument Conditions

In the test, detection was performed using Agilent 1290-6470 HPLC-MS/MS combined with Agilent Zorbax SB-C18 and 50 mm×0.30 mm×1.8 µm chromatographic columns, with the following specific conditions : sample volume 2.0 µL; mobile phase A is methanol and B is 5 mmol/L ammonium acetate solution containing 0.1% formic acid; gradient elution program: 0~2 min; 10% A; 2~7 min; 10%~80% A; 7~7.5 min,  80% A; 7.5~8 min, 80%~95% A; 8~9 min, 95% A; 9~9.5 min, 95%~10% A; 9.5~14 min, 10% A; column temperature: 35?; flow rate: 0.3 mL/min; electrospray ionisation (ESI) source, positive and negative ion modes; drying gas temperature: 325?; capillary drying gas flow: 7  L/min; nebulizer pressure : 35 psi; sheath gas flow: 12 L/min; sheath gas heater: 300?; the chromatographic conditions for dynamic multiple reaction monitoring (dMRM) detection, internal standard rations and each target veterinary drug  of interest are shown in Table 1.

2.4 Sample Preparation

    The collection, preparation and storage of pork, pig liver, chicken, egg and beef samples were carried out according to the methods of sample preparation and storage of animal and poultry products according to ^[22^]. The above samples, which were accurately weighed at 2.00 ± 0.05 g, were instilled with 100 ?L of internal standard mixture solution at a concentration of 0.2 ?g/mL, mixed by vortexing, and then placed in a 50 mL centrifuge tube and left in the dark for 20 min. Then the samples were instilled with 8mL acetonitrile/water solution (90:10) containing 0.2% formic acid, under vortex shaking for 2 min at the speed of 2000 r/min and ultrasonic extraction after 20 min of ice water bath, and then centrifuged at 4? for 5 min at the speed of 5000 r/min. 2 mL of supernatant was injected into the clean-up LPAS column at the rate of 1 drop per second, a 0.22?m organic filter head was installed at the lower end of the column to collect the filtrate into the sample bottle for analysis by LC-MS/MS, with the specific operation as shown in Figure 1.

2.5 Method Validation

Method validation includes accuracy, precision, LOD, LOQ, linear range and matrix effect. Linearity was evaluated using the correlation coefficient (R²) of the matrix-matched calibration curve of each veterinary drug. Accuracy and precision were obtained in the recovery tests at the three addition levels (low, medium and high: 5 ?g/kg, 10 ?g/kg, and 50 ?g/kg) (n=3). In accordance with the standards set up in (EU) 2021/808 [23], the LOQ is defined so that the accuracy and precision of the quantitative results are at the lowest peak levels in the acceptable range. Therefore, in the experiment, 0.25 times MRL standard solutions were added to blank matrix samples (n=10) (reference value 0.05mg/kg for MRL-free pesticides). The LOD of each matrix was calculated using the following formula [4].

LOD = 3.3 × (SD_MRL*0.25/S)

LOQ = 10 × (SD_MRL*0.25/S)

ME were calculated from the ratios of the slopes of the matrix-matched calibration curves to the solvent calibration curves [23, 24].

3 Results and Discussions

3.1 Instrument Condition Optimization

To optimise the LC conditions, mobile phases, gradient elution programmes and other key LC parameters were repeatedly adjusted. The test focused the emphases were put on comparisons between methyl alcohol and acetonitrile for organic phases, and between 0.1% formic acid water and 5mmol/L ammonium acetate solution containing 0.1% formic acid for water phases, and the mobile phase gradients were constantly adjusted to ensure that each parameter could peak normally. When the organic phase was acetonitrile, tetracyclines and some quinolones tended to have leading or tailing peaks and low response values. When the water phase was 0.1% formic acid water, drugs such as chloramphenicol, thiamphenicol, florfenicol, nicarbazin, etc., had no response or extremely low response when they were scanned in the negative ion monitoring mode. After several experiments, it was found that the response of the target objects improved significantly after the addition of 5 mmol/L ammonium acetate solution. Through the above experiments and considering the peak patterns, test sensitivity and isomeric separation of each target compound, it was finally confirmed that the optimum mobile phases for the test were methyl alcohol-5mmol/L ammonium acetate solution containing 0.1% formic acid. After adjusting the mobile phase gradient elution programme several times during the test, the gradient elution conditions in 2.3.1 were finally confirmed to test 61 veterinary drugs simultaneously and to achieve satisfactory separation effects.

In the well-optimised chromatographic conditions, parent ions were under full scan (MS₂Scan) with a single standard solution (2mg/L), and several groups of different fragment voltages (Fragment/V) were set, a mass-to-charge ratio identical to a relative molecular weight was considered as the parent ion of the latter after comparing the relative molecular weight of each standard sample, and a fragment voltage below the maximum response value was selected as the subsequent optimal fragment voltage. Then the product ion scan was performed, the collision energy (CE) optimised, the characteristic daughter ion and its optimal CE were discovered simultaneously to ensure that each parameter, except for the internal standards, possessed at least two characteristic ion pairs. Finally, the well-optimised conditions were adopted to perform dynamic multiple reaction monitoring (dMRM) analysis, and the retention time of 61 veterinary drug components, MRM ion pairs, monitoring mode and optimal CE areprovided in Table 1, and the total MRM ion diagrams in Figure 2(A,B).

3.2 Optimization of Extraction Reagents

The common extraction agents used in the pretreatment of veterinary drug residue tests include solutions of acetonitrile, acidified acetonitrile, ethyl acetate, phosphate buffer ^[25^], among others. There are many lipids, proteins and so on in animal derived food; when used as an extraction agent, acetonitrile could reduce lipid extraction and facilitate albumin precipitation ^[26^], and has a certain dehydration; samples with low water content are rapidly dehydrated and caked, which works against extraction. Therefore, the extraction efficiency was compared between acetonitrile and acetonitril:water solutions with water contents of 10%, 20%, 30% and 40% respectively. The results of the experiment is shown in Figure 3 and shows that there was the best extraction effect when the acetonitrile/water solution with water content of 10% was the extraction agent as there were 47 target objects whose recoveries were in the range of 60-120%.

To improve the extraction efficiency of each target object, comparisons were made between acetonitrile, 0.1% FA, 0.5% and 1% FA in acetonitrile in the experiment. The result of experiment is shown in Figure 4. As shown in the figure, when the extraction agent was 0.2% FA in acetonitrile, there were 54 veterinary drugs whose recovery rate was in the range of 60%-120%, which means that the recovery effects were satisfactory.

3.3 Optimization of Extraction Volumes

Having optimised the best extraction solvent, the dosage of extraction solvent has also been optimized. Typically, the smaller the extraction volume is, the higher the matrix concentration and the more difficult the subsequent clean-up, but too large an extraction volume wastes reagents, increases economic costs and is not environmentally or human friendly. Therefore, half (35 types) of the target objects were randomly selected to be extracted in extraction volumes of 4 mL, 6 mL, 8 mL and 10 mL and comparisons were made, with the experimental result shown in figure 5.

As shown in the figure, as the extraction volume increased, the matrix effect was gradually reduced and the overall recovery rate decreased. When the extraction volume was 10 mL, the recoveries of 26 target objects were reduced to the range of 60%~80%, and that of metronidazole was below 60%; when it was 8mL, the recoveries of 30 target objects were between 70% and 120%, showing a satisfactory recovery effect; when it was 6mL and 4mL respectively, 8 and 25 target objects showed recoveries of over 120%. Therefore, 8 mL of 0.2% FA in acetonitrile was used to extract the samples in the test.

3.4 Optimization of Clean-up Processes

Solid phase extraction (SPE) is widely used in the detection of veterinary drug residues in animal derived foods due to its high purification efficiency and reliable recovery rates. Despite its effectiveness, SPE has obvious drawbacks. Different adsorbent columns are required depending on the veterinary drugs being detected. Adsorbents such as MAX, MCX, HLB, C18, PSA, and GCB are selected based on their adsorption properties. They operate through silica - based or ion - exchange mechanisms to separate target compounds from complex biological matrices. The standard SPE procedure includes column activation, sample loading, equilibration, washing, elution, and subsequent nitrogen evaporation and re - dissolution of the analyte. This results in a multi - step process that increases both time and cost. In addition, repeated extractions with various organic solvents not only complicate the operation but also require high - level professional skills. As a result, SPE is labor - intensive and costly, limiting its application in high - throughput detection.

Subsequently, QuEChERS has been developed as an alternative method. It is a simpler and more efficient solution for simultaneously detecting multiple veterinary drugs [4, 12]. This method combines liquid - liquid extraction (LLE) with dispersive solid - phase extraction (d-SPE), and then, through centrifugation, rapidly separates the target compounds from interfering substances. Commonly used adsorbents include GCB, C18, PSA, and silica gel [27, 28]. Although compared with solid - phase extraction, it has significant advantages in terms of ease of use, speed, and cost effectiveness, the matrix effect after purification is significantly increased, the recovery rate of some target drugs is relatively low, and the purification tubes are expensive. Therefore, d-SPE is mainly used for the screening (qualitative detection) of a large number of samples. Thus, in addition to effective extraction, effectively removing interfering substances is another crucial sample preparation step, as the complex matrix effect poses a major challenge to the rapid and accurate detection of veterinary drugs.

 Based on d-SPE and SPE, needle-cylinder clean-up columns were adopted in the test. The needle-cylinder clean-up columns are pre-filled with LPAS (lipid and protein adsorbent) prepared by the chemical bond modification technique, which can absorb interfering substances of lipid and protein better than C₁₈, PSA and GCB, and is more suitable determination of multiple veterinary drug residues in animal derived food. The clean-up procedures of stripping, vortex clean-up, secondary centrifugation and secondary transfer of the supernatant have been eliminated compared with the traditional QuEChERS method, and the clean-up is completed by extracting the supernatant into clean-up tubes, gently pressing the tubes and transferring it to LPAS cartridges at the rate of 1 drop per second, it is easier and faster (specific procedures are shown in figure 1).

The experiment compared the reagent consumption, time consumption, waste liquid generation, etc. of two commonly used pretreatment methods with the LPAS syringe type purification, as listed in Table 2. As can be seen from Table 2, the LPAS syringe - type purification reduces the consumption of solution reagents, vessels, and reagents compared with SPE and QuEChERS. The amount of waste liquid generated is 6.0 mL, only one - fourth of that of SPE, showing a significant advantage in reducing pollution emissions. The operation is simpler. The single sample detection time of the LPAS syringe - type purification does not exceed 30 minutes, only 1/4 of that of SPE and1/2 of that of QuEChERS. Therefore, compared with SPE, LPAS has obvious advantages in the purification steps. It can process larger batches of samples in a short time and is friendly to both the health of testers and the environment.

In the test, commercially available clean-up columns were selected because their fillers are fixed and therefore the clean-up effect is dependent on the volume of clean-up liquid added. In the test, 1mL, 1.5mL, 2mL and 2.5mL of pork supernatant (the addition amount was 10?g/kg) samples were extracted and filter liquid was collected through clean-up columns; it was found that when the volume of liquid to be cleaned up was 2mL, there was the best clean-up effect with less interference from blank matrix impurities and the best recoveries of the 61 veterinary drugs, therefore 2mL of sample liquid was selected for filtration clean-up in the method. At the same time, a comparison of speeds through the cartridges was made and there was the best clean-up effect when the clean-up speed was 1 drop per second.

 3.5 Method Validation

3.5.1 Accuracy and Precision

Different animal derived foods have different substances and different interferences with veterinary drug residue testing; to eliminate the interfering factors, the 5 types of animal derived food: pork, pig liver, chicken, egg and beef, which are widely consumed by the residents in daily life and tend to be positive, were selected as samples, adding recovery experiments were conducted by adding low, medium and high (5 ?g/kg, 10 ?g/kg and 50 ?g/kg) levels to their blank samples (n=3), their average recovery and RSD were calculated to verify the applicability, accuracy and precision of the method. The results have shown (Table 3) that at the low, medium and high addition levels, the total recoveries of the 61 veterinary drugs were in the range of 70.9%~119.0%, and their RSDs were in the range of 0.1%~10.9%; with satisfactory accuracy and precision, the method could satisfy the demand for testing 61 veterinary drug residues in multiple animal derived food.

3.5.2 LOD, LOQ and Linear Range

Calibration working curves were drawn with the response value of the target compounds as the vertical coordinate and quality concentration as the horizontal coordinate. The results showed that the 61 veterinary drugs had strong linearity in the range of 0.5~50 ?g/L with correlation coefficients (R²) ? 0.995. The total LODs of the 61 veterinary drugs in 5 animal derived food were in the range of 0.03~1.5 ?g/kg, and their LOQs were in the range of 0.1~5.0 ?g/kg, which wre much lower than the MRLs set by the a majority of countries; therefore, it could meet the demand for the detection of multiple veterinary drugs in multiple livestock and poultry products. As ?-receptor agonists are hardly used in livestock and poultry breeding, and many countries have not yet set the MRLs of such drugs in livestock and poultry products, no LOD or LOQ was investigated in chicken and eggs in the study.

3.5.3 Matrix Effects

Matrix effects (ME) refer to phenomena where other interfering substances present in matrices lead to varying degrees of signal enhancement or attenuation of analytes  ^[24^]. Matrix effects are widespread in instrumental analysis, such as GC, GC-MS/MS, LC-MS/MS, etc. [31], and affect the accuracy and precision of the determination results. When LC-MS/MS was used to analyse complex samples, such as pig liver and egg, especially in the ESI mode, matrix effects were particularly evident and directly affected quantitative accuracy, unless such matrix effects were minimised or compensated ^[20].

Matrix effects were obtained by comparing the slope ratio of the calibration curve prepared by matrix blank solutions (k₁) and that of the reagent calibration curve (k₂) (ME= k₁/k₂). Assuming that matrix enhancement occurs when ME>1, that matrix attenuation occurs when ME<1, and that no matrix effect occurs when ME=1. Comparisons were made between the matrix effects of the 61 veterinary drugs in the 5 types of animal derived food of pork, pig liver, chicken, egg and beef in the test with the results as shown in table 4.

It is known from Table 4 that the ME of 55 veterinary drugs in pork matrix, 38 types in chicken matrix and 37 types in beef matrix were in the range of 0.8~1.2, those of 26 veterinary drugs in pig liver were less than 0.8 and those of 4 types were greater than 1.2, those of 28 veterinary drugs in eggs were below 0.8 and those of 2 types were greater than 1.2. This might be caused by the fact that pig liver and eggs contain a huge amount of protein, lipid etc., more complex components resulted in more severe matrix interferences. In order to effectively compensate for matrix effects, the test was calibrated with blank matrices.