Abstract

BACKGROUND:

The objective of this study was to develop a comprehensive and simple method for the simultaneous determination of 59 veterinary drug residues in livestock products for safety management.

METHODS AND RESULTS:

For sample preparation, we used a modified liquid extraction method, according to which the sample was extracted with 80% acetonitrile followed by incubation at -20℃ for 30 min. After centrifugation, an aliquot of the extract was evaporated to dryness at 40℃ and analyzed using liquid chromatography combined with tandem mass spectrometry. The method was validated at three concentration levels for beef, pork, chicken, egg, and milk in accordance with the Codex Alimentarius Commission/Guidelines 71-2009. Quantitative analysis was performed using a matrix-matched calibration. As a results, at least 52 (77.6%) out of 66 compounds showed the proper method validation results in terms of both recovery of the target compound and coefficient of variation required by Codex guidelines in livestock products. The limit of quantitation of the method ranged from 0.2 to 1119.6 ng g-1 for all matrices.

CONCLUSION(S):

This method was accurate, effective, and comprehensive for 59 veterinary drugs determination in livestock products, and can be used to investigate veterinary drugs from different chemical families for safety management in livestock products.

Keyword

LC-MS/MS,Livestock products,Multi-class analysis,Veterinary drug

Introduction

Veterinary drugs have been widely used in medical and veterinary practice to treat and prevent diseases and enhance growth rate and feed efficiency. Currently, the most commonly used veterinary drugs are β-lactams, sulfonamides, macrolides, and quinolones [1-5]. If they are not used correctly, this practice could lead to the presence of veterinary drug residues in foods of animal origin. The possible adverse effects of these drugs on public health include allergic reactions in hypersensitive or sensitized individuals and the development of resistant strains of bacteria following the ingestion of sub-therapeutic doses of antimicrobials [6-12]. With the increase in public attention to food safety, regulation of drugs used in animal food production has been imposed in nearly every country [13, 14]. Analytical methods employed for the determination of veterinary drugs must meet the criteria established under regulatory guidance. To ensure food safety, strict regulations for analytical methods in terms of maximum residue limits (MRL) and minimum required performance limits (MRPL) have been stipulated by national and international agencies, such as the Codex Alimentarius Commission and the European Commission.

In Korea, varying degrees of drug MRLs are imposed on livestock products imported from different countries and these products are not inspected for drug residues outside the established MRLs or residue tolerances. To enhance food safety in Korea, the Ministry of Food and Drug Safety (MFDS) has been preparing to introduce a positive list system (PLS). The PLS program for veterinary drugs will be implemented by 2024 or after. Five major livestock products (beef, pork, chicken, eggs and milk) were first subjected to PLS. In the absence of established MRLs in Korea, Codex standards are used as a default policy; failing this, the lowest MRL set for similar products can be used. However, with the implementation of PLS, a default tolerance of 10 μg kg^-1 will be applied to drugs with no established Korean MRLs. Following the full implementation of the new system, residual drug substances without established MRLs or residual tolerances are subject to law enforcement [28]. Accordingly, the development of generic, fast, sensitive, and reliable analytical methods that can monitor unauthorized veterinary drugs is required to ensure successful implementation of PLS.

Methods for selecting classes of veterinary drugs are abundant. Multi-class, multi-residue methods are rare due to many analytical challenges which must be overcome in sample preparation. Challenges include the extraction of drugs with wide polarity ranges and characteristics, high concentrations of protein and fat coextractives in meat that complicate sample clean-up, and tissue enzymes (released during homogenization) that degrade some analytes during the extraction process. Foods of animal origin, such as muscle, liver, and eggs, are complex matrices that may contain 2-47% fat and 10-30% proteins [14]. Therefore, efficient sample extraction and selective matrix clean-up are usually challenging given the need to achieve the desired recovery of multiple veterinary drug compounds.

The analysis of multi-class, multi-residue veterinary drugs in livestock matrices has been reported in literature over the past few years. Several extraction approaches have been proposed for the analysis of feed stuffs and animal products, such as the quick, easy, cheap, effective, rugged, and safe (QuEChERS) approach [15-18], solid-phase extraction (SPE) [19, 26], pressurized liquid extraction (DVPLE) [20], liquid-liquid extraction (LLE) [21, 25], and matrix solid-phase dispersion [22-24].

Veterinary drug residues in food can be determined by liquid chromatography (LC) coupled with ultraviolet (UV), fluorescence detection (FLD), or mass spectrometry (MS). In recent years, multi-class multiresidue methods have been introduced to further increase monitoring efficiency [29, 33, 34]. Traditional analysis technologies, such as LC-UV [37, 38] and LCFLD [35, 36], cannot meet the requirements for simultaneous detection of multi-class drugs. LC-tandem mass spectrometry (LC-MS/MS) technologies have enabled the realization of multi-residue methodologies owing to their advantages of sensitivity, selectivity, and low interference. In addition, multi-detection methods for the analysis of veterinary drugs using LC coupled with time-of-flight MS (LC-ToF-MS) have been published [30-32]. One of the main advantages is the possibility of analyzing an unlimited number of analytes in a single run because the detection by ToFMS is not limited by the dwell time. Nevertheless, it can be applied for screening and quantification purposes, but cannot be used as a confirmatory method because of the requirements of regulatory and always requires confirmation of positive findings using an MS/MS detector [41].

This paper describes the development and validation of a simple and effective quantitative screening method using LC-MS/MS for the simultaneous detection of 66 compounds (59 veterinary drugs and 7 metabolites) in livestock products. The validation procedure followed the codex guidelines CAC/GL 71-2009 [27] to apply the method in routine analysis.

MaterialsandMethods

Chemicals and reagents

All 66 target compounds were of high-purity grade (>90%), and the majority of them were purchased from Dr. Ehrenstorfer (Augsburg, Germany). LC-MS-grade acetonitrile (ACN), methanol (MeOH), and methylene chloride (MC) were purchased from Merck (Darmstadt, Germany). Formic acid (>99%), dimethyl sulfoxide (DMSO), ethylenediaminetetraacetic acid disodium salt dihydrate (Na₂EDTA·2H₂O), sodium chloride (NaCl), and acetic acid (>99%) were purchased from Sigma-aldrich (St. Louis, MO, USA). Ammonium formate (>98%) was supplied by Alfa Aesar (Ward Hill, MA). Anhydrous magnesium sulfate (MgSO₄) from JUNSEI Chemical (Tokyo, Japan) and primary secondary amine (PSA) from Agilent Technologies (CA, USA) were supplied. C18 powder (particle size = 55–105 μm, pore size = 125 Å) was purchased from Waters (Milford, MA, USA). Distilled water (DW) was provided by a Milli-Q water system (Merck-Millipore, Zug, Switzerland). Polytetrafluoroethylene (PTFE), polyvinylidene (PVDF), nylon filter (diameter = 15 mm, pore size = 0.2 μm) were purchased from Teknokroma (Barcelona, Spain).

Standard solutions

Stock solutions of the individual veterinary drugs (standards) were prepared at a concentration of 1000 μg mL^-1 (calculated as a free base). Most of the analytes were dissolved in MeOH, and some of them were dissolved in ACN, DW, and DMSO. Stock solutions were stored at –20℃ in amber polypropylene tubes to avoid photodegradation and adsorption onto the glass. A working solution of the standard mixture was prepared and diluted step-by-step with a standard dilution solution to prepare different concentrations of standard solutions. An extra dilution of the analytes was prepared when a lower-fortification mixture was required.

Sample collection

Livestock samples (beef, pork, chicken, egg, milks) were obtained from local supermarkets in South Korea. Upon arrival at the laboratory, all animal tissue samples (over 500 g) were homogenized using a food blender and samples were placed in plastic bags in a freezer at -20℃ till analysis.

Comparison of sample preparation methods

The schemes of Testing method (T1) and testing method (T2) were shown in Fig. 1. ‘Testing method 1’ (T1), cited with minor modification from CLG- MRM 3.02 (FSIS, USDA), was a liquid extraction method with the precipitation under low temperature, and ‘testing method 2’ (T2), cited with minor modification Y.S, Choi (Dankook Univ., South Korea), was a modified QuEChERS method. EDTA was used in the extraction solution of the T2 method to prevent chelation, even though the target compounds did not appear to form chelates, because the scope of the method could be extended to chelating drugs [42, 43]. NaCl was added to induce phase separation between the aqueous and organic phases; as a result of this salting out effect, analytes in the aqueous phase were driven into the organic phase [42]. Then, the solution was left to stand at -20℃ for 30 min to promote the precipitation of lipids and proteins [39].

After that, evaporation and filtration steps were added to improve sensitivity with the testing method (T1). An aliquot of 5 mL of the upper layer (organic phase) was concentrated at 40℃ by evaporation with a nitrogen stream to dryness, and the dried samples was reconstituted with 50% ACN followed by filtration through a syringe filter of pore size 0.2 μm. Filtration with PTFE, PVDF, and nylon cartridges were compared to study filtration loss.

Final optimized Sample preparation method

A 2.0-g portion of the livestock samples (beef, pork, chicken, egg, and milks) was weighed into a 50 mL polypropylene centrifuge tube. After the addition of 10 mL 80% ACN (v/v) the samples were shaken for 10 min using a mechanical shaker. The sample tube was then incubated for 30 min at –20℃ in a freezer followed by centrifugation at 4,700 ×g at 4℃ for 10 min. The supernatant (5 mL) was evaporated to dryness under a stream of nitrogen at 40℃, 50% ACN (1 mL, v/v) was added to the residue. The final extracts were filtered through a 0.2-μm nylon syringe filter, and 5 μL of the filtered sample was injected for LCMS/MS analysis.

Instrumental conditions

Ultra-high-performance liquid chromatography UHPLCMS/MS analysis of veterinary drugs and metabolites was performed using a Shimadzu Nexera X2 system (Kyoto, Japan) coupled with a Shimadzu LCMS-8060. Chromatographic separation was achieved on an Xbridge C18 column (Waters, 150 mm × 2.1 mm, particle size = 3.5 μm), the column oven temperature was 40℃ and the injection volume was 5 μL. The analytes were eluted with a mobile phase composed of (A) 0.1% formic acid in DW and (B) 0.1% formic acid in ACN at a flow rate of 0.25 mL min^-1. The gradient profile was scheduled as follows: initial proportion (98% A and 2% B) for 1 min → linear increase to 70% (B) until 7 min → linear increase to 80% (B) until 7.3 min → linear increase to 98% (B) until 10.2 min → hold 98% (B) until 13.2 min, and then back to 2% (B) until 13.21 min → hold for 2% (B) until 16 min. The mass spectrometric instrument was operated with an electrospray ionization (ESI) interface in positive and negative ion modes. ESI parameters were as follows: positive interface voltage = 4.0 kV, negative interface voltage = -3.0 kV, interface temperature = 350℃, heat block temperature = 300℃, desolvation line (DL) temperature = 150℃, nebulizing gas flow = 3.0 L min^-1, and drying gas flow = 10 L min^-1. Considering the properties of the screening methods for trace residues, the MS/MS instrument was operated in multiple reactions monitoring (MRM) mode to achieve highly sensitive and selective analysis. MS/MS parameters of the MRM methods were optimized for each compound using automated flow injection analysis of individual standard solutions. Labsolutions (Ver. 5.99) software was used for instrument control and data processing.

Method validation

The performance characteristics of the optimized method were established by a validation procedure according to the Codex guidelines CAC/GL 71-2009. The analytical characteristics evaluated were linearity, accuracy, precision, limit of detection (LOD), and limit of quantitation (LOQ). Targeted testing levels were applied at the MRL level (if they existed) during the validation process. For compounds without MRLs, 10 μg kg^-1 was used as the target testing level for all five livestock matrices. Linearity was evaluated using matrix-matched calibration with six fortified levels of blank, 0.25×, 0.5×, 1×, 2×, and 4× MRL or 2.5, 5, 10, 20, and 40 μg kg^-1, while the blank, 1×, 2×, 5×, 10×, and 20× LOQ for prohibited compounds. Linear regression analysis was performed by plotting the peak area versus the analyte concentrations for compounds with no corresponding internal standard. The accuracy of this method was estimated using recovery studies. The precision of the method was evaluated by performing repeatability and reproducibility experiments. LOD and LOQ were determined as the amounts for which the signal-to-noise ratio (S/N) was higher than 3 and 10, respectively.

Inter-laboratory validation from the two laboratories was conducted to evaluate the ruggedness of the method. Each sample was prepared at the same fortified levels, and analyzed following the same analytical procedure at each of the two participating laboratories, using an individual LC-MS/MS system. Linearity was demonstrated for all compounds by preparing a six-levels matrix-matched calibration curve in the range of target concentrations (blank, 0.25×, 0.5×, 1×, 2×, and 4× MRL or 2.5, 5, 10, 20, and 40 μg kg^-1, and blank, 1×, 2×, 5×, 10×, and 20× LOQ). Accuracy and precisions were determined by analyzing blank samples at three different concentration in five replicates for each concentration (0.5×, 1×, 2× MRL or 5, 10, 20 μg kg^-1, and 1×, 2×, 10× LOQ). The accuracy and precision of the five replicate measurements per laboratory were expressed as the recovery (%) and coefficient of variation (CV, %).

ResultsandDiscussion

LC-MS/MS analysis

The use of LC for the separation of veterinary drugs for simultaneous determination is particularly useful because veterinary drugs have highly variable chemical structures. Various parameters, such as the initial conditions, holding time, and gradient steps, were optimized for all compounds. All the target analytes were well separated and showed symmetric peaks in the total ion chromatogram, permitting their identification, confirmation, and quantitation. The retention time of the compounds ranged from 1.23 to 13.49 min within a 16-min run time. For quantitation, the characteristic ions of each compound were determined in the multiple reaction monitoring mode of electrospray ionization. Peak identification was achieved by comparing retention times and matching the area ratios of the characteristic ions. The LC-MS/MS parameters for each analyte are listed in Table 1.

Target compounds

To choose the target compounds, a list of candidate veterinary drugs was first made with the help of the MFDS. The candidate list included (1) drugs that established MRL, but were not included in 8.3.1 of the Korean Food Code [32], (2) prohibited drugs in foods by domestic or foreign regulations, and (3) drugs with foreign, but not domestic approval. Several drugs, such as nitrofurans and aminoglycosides, are not included in the candidate list because they are too hydrophilic and require a derivatization step in the sample preparation procedure. Finally, the list of 66 target compounds was completed with the addition of the metabolites of some drugs that should be monitored together.

Optimization of sample preparation

For sample preparation, several generic sample preparation methods were used and then applied to pork, representatively, and recoveries at 100 ng g^-1 were compared to find a more suitable method for the comprehensive and fast extraction of the 66 target compounds (Fig. 2).

The extraction and clean-up efficiencies were investigated based on the number of compounds, with a recovery criterion of 70-120% with coefficient of variation ≤20%. When using testing method 2 (T2), peaks were not observed for desacetyl cephapirin and desfuroyl ceftiofur in all five livestock matrices. Piperazine was only observed in the eggs. In contrast, acceptable recoveries for all target compounds were obtained using the T1 method. In addition, some highpolarity compounds, such as N,N-bis-(4-nitrophenyl) urea, iodo-hydroxyquinoline sulfonic acid, florfenicol amine, 2-hydroxy-4,5-dimethylpyrimidine, and amprolium, were not extracted using the T2 method. In beef and pork, the results for the number of analytes showed similar results with T1 and T2. However, there were significant differences between T1 and T2 in chicken, egg, and milk. Therefore, the T1 method was chosen for sample preparation in this study. The T1 method was beneficial with respect to time because it minimized the sample preparation steps. For different matrices, different types of sorbents, such as NH₄, PSA, and C₁₈, were selected to clean up the interference (pigments, organic acids, sugars, and lipids) from the matrix [18]. C₁₈ and PSA were used to effectively remove interfering substances and fat. C₁₈ is a hydrocarbon chain that eliminates fats and nonpolar interfering substances [14, 40]. PSA is a weak anion exchange sorbent that retains carboxylic acids and fatty acids in samples because it contains two amino groups [44]. However, some methods do not include a clean-up step in the QuEChERS procedure because of the poor recovery of analytes.

Using final optimized sample preparation methods, the recoveries and CV at the MRL and LOQ were compared by filtration type. As shown in Fig. 3, the lowest numbers of analytes were observed in PTFE filtered beef and chicken samples, and PVDF filtered egg and milk samples. In particular, without filtration, the final egg extracts could not be analyzed because of the concerns about contamination of the analysis device, and also it could not be filtered with a PVDF filter. On the other hand, nylon retained the largest number of analytes during filtration.

Method validation

Table 2 and Table 3 summarizes the recovery and CV for each veterinary drug in the five matrices. A calibration curve was generated for all reference standards using matrix-matched calibrations at 6 target concentrations (blank, 0.25, 0.5, 1, 2, and 4× MRL; 2.5, 5, 10, 20, and 40 μg kg^-1; and blank, 1, 2, 5, 10, and 20×LOQ). Matrix effects are inevitable in LC-MS/MS analysis. Matrix-matched calibration curves were generated to offset this effect. The quantification of analytes in the samples was performed by spiking on blank samples before extraction with the target compounds at different concentrations. The calibration curves showed good linearity with determination coefficients (r²) above 0.98 in all cases. Accuracy and precision were examined at three different concentrations (0.5, 1, and 2× MRL, or 5, 10, and 20 μg kg^-1 and 1, 2, and 10× LOQ) for all matrices. The intra- and inter-laboratory validation results were in compliance with Codex guidelines (CAC/GL 71-2009). Recoveries of >88.1% in beef and milk and >83.6% in pork and eggs were achieved for the tested veterinary drugs with satisfactory CV. A total of 52 out of 67 compounds (77.6%) in chicken showed the proper method validation results. Table 4 shows the LOD and LOQ results for livestock products. The LOD and LOQ were determined based on analyte sensitivity (S/N ratio ≥3 or ≥10, respectively) in livestock products. The LODs were between 0.1 and 339.3 ng g^-1 for different compounds, while the LOQs ranged from 0.2 to 1119.6 ng g^-1.

Application to real samples

To evaluate the applicability of the proposed method for routine analysis, five different types of highly consumed livestock products were obtained from local supermarkets. Livestock product samples (65 beef, 112 pork, 56 chicken, 61 egg, and 73 milk) were transferred to our laboratory and analyzed according to the optimized procedure. Table 5 summarizes the quantified results of the sample. Three compounds (maduramycin, anthranilic acid, and fluralaner) were detected in livestock product samples. In our 367 samples, three samples (0.8%) were detected. The detected concentrations ranged from 10 to 560 ng g^-1. Maduramycin, anthranilic acid, and fluralaner have been approved for use in Korea. No compound was detected or quantitated, which would exceed the MRL established in Korea. Therefore, further investigation of the incurred samples is required to assess the extraction efficiency of the current analytical method.

Conclusions

A comprehensive and efficient multi-residue analytical method for the simultaneous determination of 66 compounds (59 veterinary drugs and 7 metabolites) in livestock products using LC-MS/MS was developed. The developed sample extraction method from different livestock matrices and clean-up steps were easy, rapid, and effective, and also minimized analyte loss during sample preparation. Furthermore, the use of UHPLC technology shortened the analysis times for all analytes. LC-MS/MS offers the best performance for screening purposes and can effectively provide concentration values. It is sufficiently accurate to differentiate between detection sample and blank samples or drug concentrations below or above the MRL.

Note

The authors declare no conflict of interest.

ACKNOWLEDGEMENT

This work was supported by the Ministry of Food and Drug safety of Republic of Korea (grant no. 21161 MFDS603) in 2021.

Tables & Figures

Fig. 1.

Schemes of the sample preparation protocol for analysis of veterinary drugs in livestock products.

Table 1.

Optimal MRM parameters for each target compound

Fig. 2.

Number of veterinary drugs satisfying the recovery rates of 70-120% and CV≤20% using the testing method 1 and 2.

Fig. 3.

Number of veterinary drugs satisfying the recovery rates and CV of Codex guidelines by filtration types.

Table 2.

Inter-laboratory validation results of analytical method for beef, pork, and chicken.

Table 3.

Inter-laboratory validation results of analytical method for egg and milk.

Table 4.

The LOD (ng g^-1) and LOQ (ng g^-1) in livestock products

Table 5.

The concentration (ng g^-1) of compounds in real sample analysis

References

1. Bretschneider, G., Elizalde, JC., & Pérez,FA. ((2008)). The effect of feeding antibiotic growth promoters on the performance of beef cattle consuming forage-based diets: A review.. Livestock Science 114. 135 - 149.

2. Kelly, L., Smith, DL., Snary, EL., Johnson, JA., Harris, AD., Wooldridge, M., & Morris,JG. ((2004)). Animal growth promoters: To ban or not to ban? A risk assessment approach.. International Journal of Antimicrobial Agents 24. 205 - 212.

3. Ternak,G. ((2005)). Antibiotic may cat as growth/obesity promoters in humans as an inadvertent result of antibiotic pollution?.. Medical Hypotheses 6. 14 - 16.

4. Wegener,HC. ((2003)). Antibiotics in animal feed and their role in resistance development.. Current Opinion in Microbiology 6. 439 - 445.

5. Lopes, RP., Reyes, RC., Romero-Gonzalez, R., Frenich, AG., & Vidal,JLM. ((2012)). Development and validation of a multiclass method for the determination of veterinary drug residues in chicken by ultrahigh performance liquid chromatography-tandem mass spectrometry.. Talanta 89. 201 - 20.

6. Albright, JL., Tuckey, SL., & Woods,GT. ((1961)). Antibiotics in milk-A Review.. Journal of Dairy Science 44. 779 - 807.

7. Herz, U., Lacy, P., Renz, H., & Erb,K. ((2000)). The influence of infections on the development and severity of allergic disorders.. Current Opinion in Immunology 12. 632 - 640.

8. Kemper,N. ((2008)). Veterinary antibiotics in the aquatic and terrestrial environment.. Ecological Indicators 8. 1 - 13.

9. Martinez,JL. ((2009)). Environmental pollution by antibiotics and by antibiotic resistance determinants.. Environmental Pollution 157. 2893 - 2902.

10. Gustafson,RH. ((1991)). Use of antibiotics in livestock and human health concerns.. Journal of Dairy Science 74. 1428 - 1432.

11. Sørum, H., & L’Abee-Lund,TM. ((2002)). Antibiotic resistance in food-related bacterial-a result of interfering with the global web of bacterial genetics.. International Journal of Food Microbiology 78. 43 - 56.

12. Wallmann,J. ((2006)). Monitoring of antimicrobial resistance in pathogenic bacteria from livestock animals.. International Journal of Medical Microbiology 296. 81 - 86.

13. Ellis,RL. ((2008)). Development of veterinary drug residue controls by the Codex Alimentarius Commission: a review.. Food Additives & Contaminants: Part A 25. 1432 - 1438.

14. Zhao, L., Lucas, D., Long, D., Richter, B., & Stevens,J. ((2018)). Multi-class multi-residue analysis of veterinary drugs in meat using enhanced matrix removal lipid cleanup and liquid chromatography-tandem mass spectrometry.. Journal of Chromatography A 1549. 14 - 24.

15. Gressler, V., Franzen, AR., de Lima, GJ., Tavernari, FC., Dalla Costa, OA., & Feddern,V. ((2016)). Development of a readily applied method to quantify ractopamine residue in meat and bone meal by QuEChERS-LC-MS /MS.. Journal of Chromatography B 1015-1016. 192 - 200.

16. Zhang, Y., Liu, X., Li, X., Zhang, J., Cao, Y., Su, M., Shi, Z., & Sun,ZH. ((2016)). Rapid screening and quantification of multi-class multi-residue veterinary drugs in royal jelly by ultra performance liquid chromatography coupled to quadrupole time-of-flight mass spectrometry.. Food Control 60. 667 - 676.

17. Pyo, MJ., Hah, DY., Choi, YJ., Jeong, KO., Han, CH., Park, YH., Kim, WG., Jung, JG., Kim, MK., & null,null. ((2015)). QuEChERS-based determination of tissue residues and acute toxicity of pyraclofos in rat.. Korean Journal of Veterinary Service 38. 173 - 180.

18. Kang, J., Fan, CL., Chang, QY., Bu, MN., Zhao, ZY., Wang, W., & Pang,GF. ((2014)). Simultaneous determination of multi-class veterinary drug residues in different muscle tissues by modification QuEChERS combined with HPLC-MS/MS.. Analytical Methods 6. 6285 - 6293.

19. Zhu, WX., Yang, JZ., Wang, ZX., Wang, CJ., Liu, YF., & Zhang,L. ((2016)). Rapid determination of 88 veterinary drug residues in milk using automated TurborFlow online clean-up mode coupled to liquid chromatographytandem mass spectrometry.. Talanta 148. 401 - 411.

20. Boscher, A., Guignar, C., Pellet, T., Hoffmann, L., & Bohn,T. ((2010)). Development of a multi-class method for the quantification of veterinary drug reisudes in feedingstuffs by liquid chromatography-tandem mass spectrometry.. Journal of Chromatography A 1217. 6394 - 6404.

21. De Ruyck, H., Daeseleire, E., Ridder, H., & van Renterghem,R. ((2002)). Development and validation of a liquid chromatographic-electrospray tandem mass spectrometric multiresidue method for anthelmintics in milk.. Journal of Chromatography A 976. 181 - 194.

22. Lu, Y., Shen, Q., Dai, Z., Zhang, H., & Wang,H. ((2011)). Development of an on-line matrix solid-phase dispersion/fast liquid chromatography/tandem mass spectrometry system for the rapid and simultaneous determination of 13 sulfonamides in gas carp tissues.. Journal of Chromatography A 1218. 929 - 937.

23. Maldaner, L., Santana, CC., & Jardim,IC. ((2008)). HPLC determination of pesticides in soybeans using matrix solid phase dispersion.. Journal of Liquid Chromatography & Related Technologies 31. 972 - 983.

24. Martínez Vidal, JL., Frenich, AG., Aguilera-Luiz, MM., & Romero-González,R. ((2010)). Development of fast screening methods for the analysis of veterinary drug residues in milk by liquid chromatography-triple quadrupole mass spectrometry.. Analytical and Bioanalytical Chemistry 397. 2777 - 2790.

25. Yamada, R., Kozono, M., Ohmori, T., Morimatsu, F., & Kitayama,M. ((2006)). Simultaneous determination of residual veterinary drugs in bovine, porcine, and chicken muscle using liquid chromatography coupled with electrospray ionization tandem mass spectrometry.. Bioscience, Biotechnology, and Biochemistry 70. 54 - 65.

26. Kaufmann, A., Butcher, P., Maden, K., & Widmer,M. ((2008)). Quantitative multiresidue method for about 100 veterinary drugs in different meat matrices by sub 2-μm particulate high-performance liquid chromatography coupled to time of flight mass spectrometry.. Journal of Chromatography A 1194. 66 - 79.

27. ((2012)). Guidelines for the design and implementation of national regulatory food safety assurance programme associated with the use of veterinary drugs in food producing animals (CAC/GL 71-2009). 1 - 30.

28. Kim, EJ., Park, HJ., Park, SH., Choi, JD., Yun, HJ., & Kim,JH. ((2021)). Simultaneous determination of multi-class veterinary drugs in fishery products with liquid chromatography-tandem mass spectrometry.. Applied Biological Chemistry 64. 40.

29. Dasenaki, ME., & Thomaidis,NS. ((2015)). Multi-residue determination of 115 veterinary drugs and pharmaceutical residues in milk powder, butter, fish tissue and eggs using liquid chromatography-tandem mass spectrometry.. Analytical Chimica Acta 880. 103 - 121.

30. Villar-Pulido, M., Gilbert-López, B., García-Reyes, JF., Martos, NR., & Molina-Díaz,A. ((2011)). Multiclass detection and quantitation of antibiotics and veterinary drugs in shrimps by fast liquid chromatography time of flight mass spectrometry.. Talanta 85. 1419 - 1427.

31. Deng, XJ., Yang, HQ., Li, JZ., Song, Y., Guo, DH., Luo, Y., Du, XN., & Bo,T. ((2011)). Multiclass residues screening of 105 veterinary drugs in meat, milk, and egg using ultra high performance liquid chromatography tandem quadrupole time-of-flight mass spectrometry.. Journal of Liquid Chromatography & Related Technologies 34. 2286 - 2303.

32. Dasenaki, ME., Bletsou, AA., Koulis, GA., & Thomaidis,NS. ((2015)). Qualitative multiresidue screening method for 143 veterinary drugs and pharmaceuticals in milk and fish tissue using liquid chromatography quadrupole-time-of-flight mass spectrometry.. Journal of Agricultural and Food Chemistry 63. 4493 - 4508.

33. Biselli, S., Schwalb, U., Meyer, A., & Hartig,L. ((2012)). A multi-class, multi-analyte method for routine analysis of 84 veterinary drugs in chicken muscle using simple extraction and LC-MS/MS.. Food Additives & Contaminants: Part A 30. 921 - 939.

34. Stubbings, G., & Bigwood,T. ((2009)). The development and validation of a multiclass liquid chromatography tandem mass spectrometry (LC-MS/MS) procedure for the determination of veterinary drug residues in animal tissue using QuEChERS (QUick, Easy, CHeap, Effective, Rugged and Safe) approach.. Analytica Chimica Acta 637. 68 - 78.

35. Zhao, S., Jian, H., Li, X., Mi, T., Li, C., & Shen,J. ((2007)). Simultaneous determination of trace levels of 10 quinolones in swine, chicken, and shrimp muscle tissues using HPLC with programmable fluorescence detection.. Journal of Agricultural and Food Chemistry 55. 3829 - 3834.

36. Gajda, A., Posyniak, A., Zmudzki, J., Gbylik, M., & Bladek,T. ((2012)). Determination of (fluoro)quinolones in eggs by liquid chromatography with fluorescence detection and confirmation by liquid chromatography-tandem mass spectrometry.. Food Chemistry 135. 430 - 439.

37. Kumar, P., & Companyó,R. ((2011)). Development and validation of an LC-UV method for the determination of sulfonamides in animal feeds.. Drug Testing and Analysis 4. 368 - 375.

38. Hermo, MP., Barrón, D., & Barbosa,J. ((2006)). Development of analytical methods for multiresidue determination of quinolones in pig muscle samples by liquid chromatography with ultraviolet detection, liquid chromatography-mass spectrometry and liquid chromatography-tandem mass spectrometry.. Journal of Chromatography A 1104. 132 - 139.

39. Kim, YR., & Kang,HS. ((2021)). Multi-residue determination of twenty aminoglycoside antibiotics in various food matrices by dispersive solid phase extraction and liquid chromatography-tandem mass spectrometry.. Food Control 130. 108374.

40. Hou, X., Lei, S., Qiu, S., Guo, L., Yi, S., & Liu,W. ((2014)). A multi-residue method for the determination of pesticides in tea using multi-walled carbon nanotubes as a dispersive solid phase extraction absorbent.. Food Chemistry 153. 121 - 129.

41. Freitas, A., Barbosa, J., & Ramos,F. ((2014)). Multi-residue and multi-class method for the determination of antibiotics in bovine muscle by ultra-high-performance liquid chromatography tandem mass spectrometry.. Meat Science 98. 58 - 64.

42. Kim, J., Suh, JH., Cho, HD., Kang, W., Choi, YS., & Han,SB. ((2016)). Analytical method for fast screening and confirmation of multi-class veterinary drug residues in fish and shrimp by LC-M/SMS.. Food Additives & Contaminants: Part A 33. 420 - 432.

43. Valese, AC., Molognoni, L., de Souza, NC., de Sá Ploêncio, LA., Costa, ACO., Barreto, F., & Daguer,H. ((2017)). Development, validation and different approaches for the measurement uncertainty of a multi-class veterinary drugs residues LC-MS method for feeds.. Journal of Chromatography B 1053. 48 - 59.

44. Park, H., Kim, J., Kang, HS., Cho, BH., & Oh,JH. ((2020)). Multiresidue analysis of 18 dye residues in animal products by liquid chromatography-tandem mass spectrometry.. Journal of Food Hygiene and Safety 35. 109 - 117.