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A rapid multiclass method for antibiotic residues in goat dairy products by UPLC-quadrupole/electrostatic field orbitrap high-resolution mass spectrometry



Sulfanilamides, quinolones, nitroimidazoles, tetracyclines, cephalosporins, macrolides, and β-lactam are common tools in agriculture and can be found in animal-based foods such as goat milk and goat dried milk. To evaluate the risk of these species, reliable analytical methods are needed for accurate concentration determination, especially in goat milk and goat dried milk.


We describe a method based on PRiME extraction coupled with UPLC-quadrupole/electrostatic field orbitrap high-resolution mass spectrometry to accomplish this task.


Under optimal conditions, the limit of quantification for all antibiotics was 0.5–100 μg/L in goat milk and goat dried milk samples. The recoveries were 60.6–110.0% for goat milk and 60.1–109.6% for goat dried milk with a coefficient of variation less than 15%. The detection limits were 0.5–1.0 μg/kg. The limits of quantification for the analytes were 5.0–10.0 μg/kg. Finally, the method was used to screen veterinary antibiotics in 50 local goat milk and goat dried milk samples; metronidazole and enrofloxacin were detected in goat milk.


This method offers good reliability and the capacity for simultaneous detection can be used to detect residual contents and evaluate health risks in goat milk and goat dried milk.


Veterinary antibiotics are widely used to prevent infections, increase reproduction, and improve animal husbandry (Han et al. 2015; Javorska et al. 2017; Li et al. 2016; Tran et al. 2016; Reinholds et al. 2016; Serra-Compte et al. 2017; Cámara et al. 2013). However, these drugs are often used in discriminately in cattle and goat feeding (Zorraquino et al. 2011), which can lead to adverse human health effects, especially for infants and children who consume large amounts of dairy products (Li et al. 2019; Li et al. 2017).

To ensure the safety of human food, several countries have established stringent food safety regulations for these antibiotics in animal-based foods such as eggs, milk, kidney, liver, fat, and muscle (Han et al. 2012). For example, the maximum residue limits (MRLs) of benzylpenicillin, chlortetracycline, and danofloxacinin bovine milk are 4 μg/kg, 100 μg/kg, and 30 μg/kg, respectively, via the European Commission (Directives2006/141/ECand2003/89/EC). China’s MRL are published (GB 31650-2019 National food safety standard-Maximum residue limits for veterinary drugs in foods 2020) and set the MRLs for benzylpenicillin, ampicillin, and moxicillin at 4 μg/kg. Other MRLs in bovine milk include 25 μg/kg sulfadimidine; 30 μg/kg oxacillin, danofloxacin, and cloxacillin; 40 μg/kg erythromycin; 50 μg/kg flumequine, trimethoprim, tilmicosin, and sulfonamides (parent drug); 100 μg/kg sulfonamides (expect sulfadimidine), tylosin, enrofloxacin, and ceftiofur; 150 μg/kg lincomycinas; and 200 μg/kg spiramycin. These MRLs are quite low; thus, a sensitive and selective analytical method is needed.

Liquid chromatography-tandem mass spectrometry (LC-MS/MS) is a common tool in the analysis of veterinary residues. Most studies of trace antibiotic levels are based on triple quadrupole (TQ) mass spectrometry (Heller et al. 2006; Zorraquino et al. 2011; De Almeida et al. 2015; Liu et al. 2016; Li et al. 2017; Zhang et al. 2016; Forgacsova et al. 2019; Oyedeji et al. 2019; Kazakova et al. 2018; Socas-Rodríguez et al. 2017; Li et al. 2018). Multiple reaction monitoring (MRM) and selected reaction monitoring (SRM) are usually the standard quantification method. However, matrix effects and ion interference in TQ-MS remain due to complicated food composites. For better confirmation at ion and higher throughout in analysis of multi-residue veterinaries, liquid-chromatography-high resolution mass spectrometry (LC-HRMS) has become increasingly popular, specifically time-of-flight mass spectrometry (Li et al. 2016; Li et al. 2016; Berendsen et al. 2017; Zhang et al. 2015; Liu et al. 2019; Fu et al. 2018; Emhofer et al. 2019; Weng et al. 2020; Moreno-González et al.  2017; Pan et al. 2016; Saito-Shida et al. 2018) and quadrupole/electrostatic field orbitrap mass spectrometry (Jia et al. 2014b; Hu et al. 2019; Zhao et al. 2017; Jia et al. 2014a; Casado et al. 2018; Paepe et al. 2019; Jia et al. 2017; Casado et al. 2018; Jia et al. 2018a, 2018b; Jia et al. 2018a, 2018b; Casado et al. 2019; Rusko et al. 2019; Abdallah et al. 2019; López-García et al. 2017; Paepe et al. 2018; Kim et al. 2018; Jia et al. 2017).

Recently, new pre-treatment methods have been proposed for extraction and clean-up of each class of veterinary antibiotic residues in food samples. These include liquid-liquid extraction (LLE) for macrolides extraction from milk samples (Şanli et al. 2011) as well as a modified QuEChERS and solid-phase extraction (SPE) or dispersive SPE for clean-up of complex food samples (Junza et al. 2011; Jia et al. 2014a; Kaufmann and Widmer 2013; Dubreil-Chéneau et al. 2014; Heller et al. 2006; Chen et al. 2017). More recently, a novel phospholipids-removing SPE column–PriME HLB was developed based on the specific adsorption for phospholipids carrying fatty acid chains. In contrast to traditional SPE methods, this procedure removes interferences, fats, and phospholipids while simultaneously extracting multiple veterinary residues from milk and dried milk in one loading step; the method is convenient, fast, affordable, and green.

The objective of this study is to establish an effective method to simultaneously determine 60 selected veterinary antibiotic residues, including 17 sulfanilamides, 16 quinolones, 7 nitroimidazoles, 3 tetracyclines, 2 cephalosporins, 8 macrolides, and 7 β-lactams, in milk and dried milk samples by UPLC-quadrupole/electrostatic field orbitrap high-resolution mass spectrometry. The resulting method was then successfully used to screen veterinary antibiotic residues in local goat milk and goat dried milk samples.


Chemicals and reagents

We obtained the following from Dr. Ehrenstorfer GmbH (Augsburg, Germany): sulfamerazine (SMZ), sulfathiazole, trimethoprim (TMP), sulfamethizole, sulfisoxazole (SIZ), sulfadiazine (SD), sulfachlorpyridazine, sulfamethoxydiazine, sulfadimethoxypyrimidine, sulfaquinoxaline, sulfadimoxine (SDM), sulfamethoxypyridazine, sulfamethazine, sulfapyridine (SPD), sulfamethoxazole (SMX), sulfaguanidine, sulfaphenazole, lomefloxacin (LOM), ciprofloxacin (CIP), enrofloxacin (ENR), ofloxacin (OFX), norfloxacin (NOR), orbifloxacin (ORB), danofloxacin (DAN), sparfloxacin (SPA), sarafloxacin (SAR), marbofloxacin (MAR), enoxacin (ENO), flumequine (FLU), fleroxacin (FLE), difloxacin (DIF), pefloxacin (PEF), nalidixic acid, erythromycin, lincomycin (LIN), spiramycin, roxithromycin, tilmicosin (TIL), tylosin (TYL), clindamycin, kitasamycin, dimetridazole, hydroxymetronidazole, ipronidazole-OH, ipronidazole, ornidazole, metronidazole, 2-methyl-5-nitroimidazole, chlortetracycline (CLT), doxycycline (DOX), demeclocycline (DEM), ceftiofur (TIL), cefapirin, oxacillin (OXAC), dicloxacillin (DICL), cloxacillin (CLOX), nafcillin (NAFC), ampicillin (AMPI), penicillin G (PEG), and penicillin V (PEV).

Acetonitrile and methanol were HPLC gradient grade and purchased from Merck (Darmstadt, Germany). Formic acid and acetic acid were purchased from Anpu (Shanghai, China), and doubly deionized water was obtained from a Milli-Q gradient water system (Milipore, Bedford, MA).

Stock solutions of individual compounds were prepared in methanol (1000 mg L−1) and stored at − 20 °C in dark glass bottles during the three-month validity period. The working mixed standard solution was then diluted with 0.1% formic acid solution and kept at − 20 °C in dark glass bottles for one month. PRiME HLB solid phase extraction cartridges (60 mg, 3CC) were obtained from Waters (Milford, USA).

Sample preparation

Goat milk sample

The target analytes were extracted from 1 g of milk sample with 4 mL of 0.2% formic acid/acetonitrile solution and vortexed for 30 s. The mixture was then shaken for 30 min and centrifuged at 10,000 r/min for 10 min at 4 °C. The total supernatant fraction was directly loaded on a PRiME HLB. All elutes were collected in a centrifugal tube and evaporated under nitrogen gas at 40 °C. The residue was added to 1 mL acetonitrile: 0.1% formic acid solution (1:9, v/v) and filtered with a 0.22-μm filter membrane. The final extract solution was transferred to vial and injected into UPLC-quadrupole/electrostatic field orbitrap mass spectrometer system under full ms/dd-ms2 optimized conditions for each compound.

Goat dried milk

The goat dried milk (0.45 g) was weighed in a centrifuge tube (50 mL) and dissolved with 3 mL water (40–50 °C). Next, 7 mL of acetonitrile with 0.2% formic acid was added as an extraction solvent, and the tube was vigorously mixed for 30 s. The tube was then immediately shaken for 30 min and then centrifuged for 20 min at 10,000 r/min at 4 °C. The upper layer was submitted to a PRiME HLB. All elutes were collected into a centrifugal tube and evaporated under nitrogen gas at 40 °C. The residue was added with 1 mL acetonitrile: 0.1% formic acid solution (1:9, v/v), and filtered with a 0.22-μm filter membrane. The final extract solution was analyzed like the goat milk samples.

UPLC-quadrupole /electrostatic field orbitrap mass analysis

The analytes were measured with an ultra-high performance liquid chromatography system (Ultimate 3000, USA) coupled with a quadrupole/electrostatic field orbitrap mass spectrometer (Thermo &Fisher Q Exactive, USA). A Thermo Hypersil GoldaQ (2.1 × 100 mm, 1.9 μm) column was used for separation. Mobile phase consisting of elute A (water, 0.1% formic acid) and elute B (acetonitrile) was used at a flow rate of 0.3 mL/min. All analytes were separated using gradient method: 0–1 min: 10% B; 1–6 min: 10% B to 80% B; 6–8 min: 80% B; 8.1–12 min: 10% B. The optimized sample injection volume was set at 10 μL. All 62 target analytes ware eluted over 0–6 min while the last 6 min were used for column cleaning and re-equilibration.

The quadrupole/electrostatic field orbitrap was equipped with a heated electrospray ionization (HESI) source. The temperature of the HESI was 350 °C, the capillary temperature was 320 °C, and the spray voltage was 3.8 kV for positive mode. All other quantitative data were acquired in full scan mode. Full MS/dd-MS2 was used for qualitative analysis. Precursor ions were selected by the quadrupole sent to the S-Lens in consideration of the detection of target analytes. The productions were then obtained from fragmented precursor ions via normalized collision energy (NCE).

The MS parameters of full MS/dd-MS2 were as follows: Full MS, inclusion on, resolution 70,000, maximum IT 200 ms, and AGC target 3.0e6. The dd-MS2 settings were as follows: inclusion on, resolution17,500, maximum IT 6 ms, AGC target 2.0e5, and isolation window 2.0 m/z. The accurate masses for the precursor ions and productions are shown in Table 1.

Table 1 UPLC-quadrupole/electrostatic field orbitrap parameters of the 60 veterinary antibiotic residues


The method was validated according to the EU Commission 2002/657/EC. The blank milk matrix samples were carefully selected to account for the possible variation within a given matrix(e.g., fat content, protein content, and other organics). The method was evaluated for linearity, limit of detection (LOD), precision, and accuracy. In the experiment, matrix-matched instead of internal standard was used because of the level of matrix effects can by significantly reduced by matrix-matched calibration curve (Table 2). At the same time, internal standard can be found in a few antibiotics. A matrix-matched calibration curve was established for each target antibiotics separately. Six calibration levels were prepared by spiking the blank matrix with each antibiotic. The coefficients of determination (r2) were higher than 0.99 in all matrices. The veterinary antibiotics were divided into two groups according to the response value of each target analyte to mass spectrometry. Group1 included erythromycin, spiramycin, roxithromycin, TIL, TYL, clindamycin, CLT, DEM, ceftiofur, cefapirin, OXAC, DICL, CLOX, NAFC, AMPI, PEG, and PEV with the following spiking levels: 10, 20, and 50 μg/kg. Group 2 included SMZ, sulfathiazole, TMP, sulfamethizole, SIZ, SD, sulfachlorpyridazine, sulfamethoxydiazine, sulfamethazine, sulfaquinoxaline, SDM, sulfamethoxypyridazine, sulfadimethoxypyrimidine, SPD, SMX, sulfaguanidine, sulfaphenazole, LOM, CIP ENR, OFX, NOR, ORB, DAN, SPA, SAR, MAR, ENO, FLU, FLE, DIF, PEF, nalidixic acid, LIN, dimetridazole, metronidazole-OH, ipronidazole-OH, ipronidazole, ornidazole, metronidazole, 2-methyl-5-nitroimidazole, doxycycline, and josamycin with the following spiking levels: 5, 10, and 20 μg/kg. The accuracy was determined with recovery experiments using blank samples at LOQ spiking levels in triplicate. The repeat ability was evaluated via the relative standard deviation (RSD, %). The limits of detection (LOD) and quantification (LOQ) were defined as lowest concentrations with a signal-to-noise (S/N) ratio of 3 for LOD or 10 for LOQ.

Table 2 Matrix effect (ME) for 60 veterinary antibiotic residues spiked in blank milk and milk power sample for individual donors (n = 5)

The matrix effect (ME) was investigated by comparing the peak area of each antibiotic spiked in blank sample after extraction procedure at same concentration level, with peak area of each antibiotic in water (without matrix matched) at the same concentration. The peak area of each antibiotic in water was set at 100% (Javorska et al. 2017).

Results and discussion

Optimization of the UPLC-quadrupole/electrostatic field orbitrap conditions

Ultra-performance chromatography columns with sub-2-μm particles have outstanding separation capacity. They have facilitated the development of quantification methods for multi-residues within a short run time. Here, different types of chromatographic columns were investigated. Under the same determination conditions, DICL and PEV were weak retention on a Waters ACQUITY UPLC® BEH Shield RP 18 (100 mm × 2.1 mm, 1.7 μm), and CLOX and OXAC were unreserved on a Waters ACQUITY UPLC® HSS T3 column (100 mm × 2.1 mm,1.8 μm). However, the Thermo Hypersil GOLD aQ (100 mm × 2.1 mm, 1.9 μm) showed good performance in the separation of 60 veterinary antibiotics. The analysis process was completed within 12 min (Fig. 1).

Fig. 1
figure 1

Separation of 60 veterinary antibiotic residues in Thermo Hypersil GOLD aQ

Different solvents were tested to achieve better separation and retention of target analytes including cetonitrile, methanol, and 0.1% formic acid aqueous solutions. There needed to be some compromise between mobile phase composition and MS response for the 60 selected veterinary antibiotics. Consideration of the sensitivity (S/N) and the peak shape showed that the target analytes had better performance in acetonitrile than in methanol. When the aqueous solution was water, the peaks of quinolones, macrolides, and tetracyclines were asymmetrical and heavy-trailed. However, the shape of peak and the retention were well when formic acid was added into the aqueous solution. This is because the addition of formic acid improved the ionization efficiency. Therefore, acetonitrile and 0.1% formic acid were selected as the mobile phase.

The optimum mass spectrometric parameters for the identification and quantification of 60 veterinary antibiotics were obtained after analyzing the compounds by flow injection analysis. The sensitivity of target analytes was investigated via the chromatograms in full scan mode in positive ionization mode. Due to adduct formation with formic acid, all analytes showed strong formic/hydrogen adduct species ([M + H]+ ); these species appear to be the precursor ions in the mass spectrum. The target analytes could achieve better base separation with the interference peak. This was more efficient and lowered the matrix effects, thereby leading to a resolution of 70,000 versus 17,500.

The full MS/dd-MS2 mode led to a production spectrum with accurate mass measurement according to the inclusion list (a list of targeted accurate masses). This was defined as a data-dependent acquisition (dd-MS2). After full scan analysis, specific mass windows were extracted to screen the data for the presence of analytes. The effect of the isolation window on analyte selectivity was tested. The best results were achieved when an isolation window of 2.0 ppm was employed. Table 1 shows the optimal parameters of the UPLC-quadrupole/electrostatic field orbitrap.

Optimization of the extraction procedure

According to these reports, milk and dried milk contained a great deal of phospholipids. Two different solid-phase extraction (SPE) columns (PRiME HLB and Oasis-HLB) were compared to reduce the phospholipids of the milk samples. Twelve blank milk samples were prepared following the “Sample preparation” section; four of the samples were not treated with solid phase extraction columns, four were treated with HLB, and the last four were purified with PRiME HLB. All of these samples were injected into a UPLC-quadrupole/electrostatic field orbitrap analysis in full MS mode to acquire identifies phospholipids in milk.

Although the high-resolution quadrupole/electrostatic field orbitrap is selective, the complicated matrix can still affect target analyte ionization; this leads to ion suppression or enhancement. The recovery of veterinary antibiotics in the Oasis HLB column tailed off at 25% versus the PRiME HLB. Many components, such as phospholipids, aminoacids, and fat, in milk can lead to interference of mass response. As such, these components were not effectively removed by the PRiME HLB column.

Figure 2 shows that the peak intensities of phospholipids were significantly different among the three treatment modes. The peak intensities of these compounds were not influenced by HLB purification in milk samples versus untreated milk samples. The peak intensities of phospholipids significantly decreased, which confirmed that one step of pretreating milk samples by PRiME HLB led to effective removal of phospholipids for the high-throughput detection of multiple veterinary antibiotic residues.

Fig. 2
figure 2

Comparison the effect of different SPE on phospholipids

Previous studies showed that PRiME HLB removes phospholipids from milk via a single pretreatment step. There are no pre-equilibration and washing steps before eluting from the SPE. The effects of purifying the phospholipids including via absorption were compared for the SPE.

Here, different extraction solvents (pure acetonitrile, acetonitrile acidified with formic acid, or water) were evaluated, considering the acidic or basic character of these veterinary antibiotics. Commission Decision 2002/657/EC and GB/T 27404-2008 were used as guidelines to calculate recoveries and matrix effects (Fig. 3). Many target analytes had low recovery with 80% aqueous acetonitrile. Probably, the effect of precipitation of protein was weakened in acetonitrile aqueous solution, in which caused higher matrix effect and lower extraction recoveries. Acidic acetonitrile and pure acetonitrile both had good recoveries for most veterinary antibiotics. These results indicated that these solvents could prevent the interference of proteins and phospholipids. However, certain antibiotics (e.g., cefapirin, penicillin G, demethylchlortetracycline, chlorotetracycline, doxycycline, erythromycin, and penicillin V) had recoveries that were too low (below 50%) with pure acetonitrile. It was difficult to extract some highly polar components such as β-lactams when the concentration of acetonitrile in the solvent was too high. Therefore, acidic acetonitrile could be used for extraction. The 0.2% formic acid acetonitrile extracted more than 95% veterinary antibiotics spiked into blank milk samples and precipitated protein in milk sample; these results were better than extracted by pure acetonitrile.

Fig. 3
figure 3

Effect of different extraction solutions on the extraction properties (experiments were performed at spike levels of 10 g/kg for 60 veterinary antibiotic residues)

Method validation

PRiME HLB could eliminate the matrix effects, and matrix-matched calibration was also used to reduce the impact of matrix effects on precision and accuracy of the UPLC-quadrupole/electrostatic field orbitrap mass method.

The ME was calculated via the method in the “Validation” section. The result showed that the ME was established for each antibiotic spiked into milk and dried milk sample was not higher than 15%. Therefore, the matrix-matched calibration was applied for these matrices instead of internal standard. The results showed the matrix matched calibration can corrected the level of matrix effects (Table 2).

The resultant matrix-matched calibration curves using the instrument response were linear from 0.5 to 20 μg/L for sulfanilamides, quinolones, and nitroimidazoles. The range was 1–100 μg/L for tetracyclines, macrolides, and β-lactams. The response function was linear with a coefficient (r2) of 0.9906–0.9971 for milk samples and 0.9901–0.9998 for dried milk samples (Table 3).The sensitivity was evaluated via the limit of detection (LOD) and limit of quantification (LOQ). The LOQs were calculated at a signal-to-noise ratio (S/N) of 10; LODs used S/N of 3. These data are shown in Table 3.The LODs were 0.5 to 1.0 μg/kg and the LOQs ranged from5.0 to 10.0 μg/kg.

Table 3 Validation parameters for 60 veterinary antibiotic residues at three concentration levels in blank milk samples and milk powder samples.

The intra-day and inter-day relative standard deviations (RSDs) were adopted for precision validation. The intra-day precision was evaluated via three repeated analyses at different concentrations on three sequential runs with six replicates. The inter-day precision was performed by analyzing spiked samples over five days. The RSDs were 0.4% to 10.5% for intra-day and 2.0% to 11.5% for inter-day experiments; these values were all less than 15%. It indicated that the developed method was reliable and reproducible within its analytical range. The recoveries were assessed by spiking blank dairy samples at three concentration levels (LOQs, 2×LOQs, 4×LOQs) with six replicate sat each level. The average recoveries were 60.6–110.0% for milk samples in all fortification levels; the values were 60.1–109.6% for dried milk samples.

Figure 4 shows the typical chromatograms from a full MS/dd-MS2 experiment of analytes detected in positive samples. With the UPLC-quadrupole/electrostatic field orbitrap high-resolution mass spectrometry method, not only accuracy was enhanced but also the low concentration antibiotic residues; this suggests that the UPLC-quadrupole/electrostatic field orbitrap high-resolution mass spectrometry method was appropriate for the screening of antibiotic residues in milk and dried milk samples.

Fig. 4
figure 4

Example of typical chromatography and spectra from a full MS/dd-MS2 experiment: (A1) extracted ion chromatogram of enrofloxacin [M + H]+ m/z 360.17081 in sample N0. 17; (A2) dd-MS2 total ion chromatogram of enrofloxacin of [M + H]+ m/z 245.10895 in sample No. 17

Method applications

Next, 25 goat milk and 35 dried milk samples were collected from local dairy farms in Shaanxi province, China. Traces of three veterinary antibiotic residues over allowable levels were detected in six samples: 2.45 μg/kg, 5.02 μg/kg of metronidazole in sample No. 3 (goat milk) and No. 15 (goat milk), and 112.4 μg/kg of enrofloxacin (goat milk) in sample No.17. These results suggest that one-step extraction by PRiME HLB combined with UPLC-quadrupole/electrostatic field orbitrap high-resolution mass spectrometry for milk products is a simple and effective method for analyses in goat milk and goat dried milk samples.


A methodology for the analysis of veterinary antibiotic residues in goat milk products based on PRiME HLB extraction combined with UPLC-quadrupole/electrostatic field orbitrap high-resolution mass spectrometry. The method can achieved the simultaneous analysis of sixty-two veterinary antibiotics belong to six different classification. The method showed good performance on recoveries, precision, accuracy, MDL, and MQL, proving the effectiveness of the methodology for analysis these compounds. Compared with traditional methods, the sensitivity was enhanced, and the accuracy was improved, leading to effective method for screening antibiotic residues in milk products.

Availability of data and materials

The data sets supporting the results of this article are included within the article and its additional files.







































































Penicillin G


Penicillin V


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This study was supported by the Technology Program of Shaanxi Entry and Exit Inspection and Quarantine Bureau (Shaan K-201606), the Science and Technology Resource Opening Sharing Platform of Shaanxi Province (2017KTPT-06), Science and Technology Project of General Administration of Customs, P. R. China (Grant No. 2018IK057), and Shaanxi Key Research & Development Program (2017NY-165).


Technology Program of Shaanxi Entry and Exit Inspection and Quarantine Bureau (ShaanK-201606), Science and Technology Resource Opening Sharing Platform of Shaanxi Province (2017KTPT-06), Science and Technology Project of General Administration of Customs, P. R. China (Grant No. 2018IK057), Shaanxi Key Research & Development Program (2017NY-165).

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LZ contributed to the conception of the study and data analyses and wrote the manuscript. LS performed the experiment. QH contributed significantly to analysis and manuscript preparation. YL helped perform the analysis with constructive discussions. The authors read and approved the final manuscript.

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Correspondence to Lu Zhang.

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Supplementary Information

Additional file 1.

National food safety standard-Maximum residue limits for veterinary drugs in foods.

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Zhang, L., Shi, L., He, Q. et al. A rapid multiclass method for antibiotic residues in goat dairy products by UPLC-quadrupole/electrostatic field orbitrap high-resolution mass spectrometry. J Anal Sci Technol 12, 14 (2021).

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