Veterinary Drug Residues In Milk Research Articles

Determination of five veterinary drug residues in milk by ultra-high performance liquid chromatography-quadrupole/electrostatic field orbitrap mass spectrometry based on modified chitosan membrane purification

Milk is an important consumer product with high nutritional value. The presence of veterinary drug residues in milk owing to the indiscriminate use of veterinary drugs may affect consumer health. In the mass spectrometric analysis of trace compounds, chromatographic co-eluting components easily interfere with the mass spectral signals obtained, affecting the accuracy of qualitative and quantitative analyses. Matrix purification is a promising method to reduce the matrix effect. Chitosan is a natural biopolymer with numerous active functional groups such as amino, acetyl, and hydroxyl groups; these groups can adsorb lipids through hydrophobic and electrostatic interactions. Chitosan also has the advantages of low production cost, stable chemical properties, and convenient modification. Novel chitosan-based materials are promising candidates for lipid purification. In this study, a chitosan membrane was modified with trimethoxyoctadecylsilane (C18-CSM). C18-CSM was prepared through one-step hydrolysis and used as a dispersive solid phase extraction (DSPE) adsorbent to purify the matrix during milk pretreatment. We combined C18-CSM with ultra-high performance liquid chromatography-quadrupole/electrostatic field orbitrap mass spectrometry (UHPLC-Q/Exactive Orbitrap MS) to develop an effective method for the extraction and determination of ofloxacin, enrofloxacin, ciprofloxacin, diazepam, and metronidazole in milk. C18-CSM was characterized using scanning electron microscopy, Fourier transform infrared spectroscopy, and water contact angle testing. The results indicated that the material has a rough surface and uniformly dense cross-section. The water contact angle of C18-CSM was 104°, indicating its good hydrophobicity. The pretreatment conditions (extraction solvent, dosage of NaCl, extraction frequency, and dosage of C18-CSM) that influenced the recoveries of the five veterinary drugs were investigated in detail. The optimal conditions were established as follows: 5% formic acid in acetonitrile, 1 g NaCl, extraction 1 time, 20 mg C18-CSM. Separation was performed on a Hypersil GOLD VANQUISH column (100 mm×2.1 mm, 1.9 μm). The mobile phase consisted of 0.1% formic acid aqueous solution and 0.1% formic acid in acetonitrile, and was flowed at a rate of 0.3 mL/min. The sample injection volume was 1 μL, and the column temperature was maintained at 25 ℃. Mass spectrometric analysis was performed in positive electrospray ionization mode. To verify the necessity of the purification material, the matrix effect was investigated using the matrix-matched standard curve method. The use of C18-CSM reduced the matrix effects of the five necessity drugs from the range of -22%-8.8% to the range of -13%-3.6%, indicating that C18-CSM is a highly efficient DSPE material. Under optimal conditions, the developed method showed good linearities within the range of 0.5-100 μg/L, with correlation coefficients (r2)≥0.9970. The limits of detection(LODs) and quantification (LOQs) were 0.2 μg/L and 0.5 μg/L, respectively. To assess the accuracy and precision of the method, we prepared milk samples with three spiked levels (low, medium, and high). The recoveries of the five veterinary drugs were ranged from 79.5% to 115%, and the intra-day and inter-day relative standard deviations were 7.0%-13% (n=6) and 1.3%-11% (n=3), respectively. This study provides a simple, accurate, and reliable method for the rapid and simultaneous determination of the five veterinary drug residues in milk.

Advancements and greenification potential of magnetic molecularly imprinted polymers for chromatographic analysis of veterinary drug residues in milk.

Milk, as a widely consumed nutrient-rich food, is crucial for bone health, growth, and overall nutrition. The persistent application of veterinary drugs for controlling diseases and heightening milk yield has imparted substantial repercussions on human health and environmental ecosystems. Due to the high demand, fresh consumption, complex composition of milk, and the potential adverse impacts of drug residues, advanced greener analytical methods are necessitated. Among them, functional materials-based analytical methods attract wide concerns. The magnetic molecularly imprinted polymers (MMIPs), as a kind of typical functional material, possess excellent greenification characteristics and potencies, and they are easily integrated into various detection technologies, which have offered green approaches toward analytes such as veterinary drugs in milk. Despite their increasing applications and great potential, MMIPs' use in dairy matrices remains underexplored, especially regarding ecological sustainability. This work reviews recent advances in MMIPs' synthesis and application as efficient sorbents for veterinary drug extraction in milk followed by chromatographic analysis. The uniqueness and effectiveness of MMIPs in real milk samples are evaluated, current limitations are addressed, and greenification opportunities are proposed. MMIPs show promise in revolutionizing green analytical procedures for veterinary drug detection, aligning with the environmental goals of modern food production systems.

Extention and interlaboratory comparison of an LC-MS/MS multi-class method for the determination of 15 different classes of veterinary drug residues in milk and poultry feed

An HPLC-MS/MS multi-class method for quantitation of 15 different classes of veterinary drug residues (>140 analytes) in milk and poultry feed was developed and validated. Accuracy criteria for routine laboratories were met for the majority of analytes, > 83 % in milk and between 50 and 60 % in chicken feed, with an apparent recovery of 60–140 %. Extraction efficiency criteria were met for >95 % of the analytes for milk and > 80 % for chicken feed. Intermediate precision meets the SANTE criterion of RSD < 20 % for 80–90 % of the analytes in both matrices. For all analytes with an existing MRL in milk, the LOQ was below the related MRL. Twenty-nine samples of commercial milk and chicken feed were analyzed within the interlaboratory comparison. No residues of veterinary drugs were found in the milk samples. However, the feed samples exhibited high levels of nicarbazin, salinomycin, and decoquinate.

Improved matrix purification using a graphene oxide-coated melamine sponge for UPLC-MS/MS-based determination of 37 veterinary drugs in milks.

A rapid and highly sensitive method was established for the analysis of 37 veterinary drug residues in milk using a modified QuEChERS method based on a reduced graphene oxide-coated melamine sponge (rGO@MeS) coupled with UPLC-MS/MS. Under optimal chromatographic and mass spectrometric conditions, the effects of different dehydrated salts (MgSO4 and Na2SO4) and metal chelating agents (Na2EDTA) on extraction efficiency were first investigated. Next, the influence of a dynamic and static purification mode was evaluated in terms of drug recoveries. Calibration curves of 37 veterinary drugs were constructed in the range 0.6-500 μg kg-1, and good linearities were obtained with all determination coefficients (R2) ≥0.992. The limits of detection (LODs) and quantitation (LOQs) were in the range 0.3-1.1 μg kg-1 and 0.6-3.5 μg kg-1, respectively. The recoveries of all compounds were in the range 61.3-118.2% at three spiked levels (20, 100, and 200 μg kg-1) with RSDs ≤15.4% for both intra- and inter-day precisions. Compared to pristine melamine sponges and commercial adsorbents (C18, PSA, and GCB), rGO@MeS demonstrated an equal or even better purification performance in terms of recoveries, matrix effects, and matrix removal efficiency. This method is rapid, simple, efficient, and appropriate for the qualitative and quantitative analyses of 37 veterinary drug residues in milk, providing a new detection strategy and technical support for the routine analysis of animal-derived food.

Evaluation of Antibiotics Residues in Milk and Meat Using Different Analytical Methods.

Veterinary drugs are pharmacologically and biologically active chemical agents. At present, veterinary drugs are extensively used to prevent and treat animal diseases, to promote animal growth, and to improve the conversion rate of feed. However, the use of veterinary drugs in food-producing animals may leave residues of the parent compounds and/or their metabolites in food products resulting in harmful effects on humans. To ensure food safety, sensitive and effective analytical methods have been developing rapidly. This review describes sample extraction and cleanup methods, and different analytical techniques are used for the determination of veterinary drug residues in milk and meat. Sample extraction methods, such as solvent extraction, liquid-liquid extraction, and cleanup methods such as dispersive solid-phase extraction and immunoaffinity chromatography, were summarized. Different types of analytical methods such as microbial, immunological, biosensor, thin layer chromatography, high-performance liquid chromatography, and liquid chromatography-tandem mass spectrometry were discussed for the analysis of veterinary drug residues in animal-derived foods. Liquid chromatography-tandem mass spectrometry is the most widely used analytical technique for the determination of antibiotic drug residues. This is due to the powerful separation of LC and accurate identification of MS, and LC-MS/MS is more popular in the analysis of veterinary drug residues.

Rapid determination of 103 common veterinary drug residues in milk and dairy products by ultra performance liquid chromatography tandem mass spectrometry.

A multi-residue method has been developed for the identification and quantification of 103 common veterinary drug residues in milk and dairy Products. This method was based on QuEChERS with dispersive solid-phase where C18 sorbent and anhydrous sodium sulfate were used to sample purification. After evaporation and reconstitution, the samples were analyzed by ultra-performance liquid chromatography-tandem mass spectrometry. The mean recovery results were all higher than 60% except ampicillin, pipemidic acid, enoxacin, and estriol, and the relative standard deviation was <20.0%. The limit of quantification ranged between 0.1 and 5 μg/kg for milk and between 0.5 and 25 μg/kg for milk powder. It was successfully used to detect residues of veterinary drug in real samples. This study proposes a simple and fast analytical method for monitoring multi-class veterinary drug residues to ensure food safety.

Fluorescent aptasensor based on DNA-AgNCs emitting in the visible red wavelength range for detection of kanamycin in milk

Fluorescence sensors detect veterinary drug residues in milk, which are usually interfered with by the complex milk matrix. The specific DNA template can produce DNA stabilized silver nanoclusters (DNA-AgNCs) emitting in the visible red wavelength range. This will reduce the fluorescence interference of milk by avoiding the fluorescence signal generated by the inherent component of milk in the relatively short wavelength range. Furthermore, the red fluorescence has relatively high penetration, which can also reduce the impact of interfering substances. Here, on the basis of fluorescence resonance energy transfer (FRET), we designed a fluorescent aptasensor using DNA-AgNCs and gold nanoparticles (AuNPs) to detect kanamycin (KAN) residue in milk. Due to electrostatic interaction, DNA adsorbs on the AuNPs. The decrease in the distance between DNA-AgNCs and AuNPs generates FRET, which causes the fluorescence of DNA-AgNCs to be quenched. When KAN is added, the aptamer and KAN can specifically bind with high affinity, resulting in a weakening of the electrostatic interaction between DNA-AgNCs and AuNPs. This results in a decrease in FRET between DNA-AgNCs and AuNPs, and recovery of the fluorescent signal intensity of DNA-AgNCs. The designed sensor has satisfactory specificity and sensitivity for the detection of KAN residues. The applied strategy showed the limit of KAN detection to be as low as 22.6 nM. Moreover, this work provides a way for reducing the interference of the milk background fluorescence signal in the fluorescence sensor and has broad application prospects.

Improving the QuEChERS Liquid/Liquid Extraction of Analytes With Widely Varying Physicochemical Properties: Example of 201 Veterinary Drugs in Milk.

QuEChERS is an extraction and sample processing technique widely used for multiresidue methods (e.g., pesticides or veterinary drugs). QuEChERS recovers a wide analyte polarity range, and yet low or even very low recoveries are observed for some polar analytes. This is especially relevant when analyzing veterinary drug residues in milk. Based on the Hoffmeister row, potassium hydrogenphosphate/dihydrogenphosphate were selected as salting-out agents. The methodology was termed p-QuEChERS. The avoidance of weak complexing agents (magnesium and probably sodium), as present in conventional QuEChERS salt mixtures, improved the partition of critical analytes into the organic phase. Significantly higher recoveries were obtained for analytes belonging to the families of tetracyclines, penicillins, and cephalosporins. The proposed p-QuEChERS concept, compared to conventional QuEChERS, improves the recovery of various polar analytes. Analytes benefiting from this methodology are drugs, which are particularly relevant in the specific field of multiresidue analysis in milk. On the other hand, none of the total 201 investigated analytes showed a reduced recovery. In addition, neither the sample handling nor the co-partition of involatile salts into the extracts was negatively affected. The p-QuEChERS based method has been fully validated according to European Community guidelines, using liquid chromatography coupled to high-resolution mass spectrometry instrumentation (time of flight (TOF) as well as Orbitrap). The developed multiresidue method quantifies 201 different veterinary drugs at relevant residue concentration in milk and is highly suitable for high throughput routine control.

Controllable Preparation of Macroporous Molecularly Imprinted Monolithic Hplc Columns for On-Line Concentration, Purification, and Determination of Trace Fluoroquinolones Veterinary Drug Residues in Milk

Controllable Preparation of Macroporous Molecularly Imprinted Monolithic Hplc Columns for On-Line Concentration, Purification, and Determination of Trace Fluoroquinolones Veterinary Drug Residues in Milk

Target screening of 105 veterinary drug residues in milk using UHPLC/ESI Q-Orbitrap multiplexing data independent acquisition.

This paper presents a multi-class target screening method for the detection of 105 veterinary drug residues from 11 classes in milk using ultra-high performance liquid chromatography electrospray ionization quadrupole Orbitrap mass spectrometry (UHPLC/ESI Q-Orbitrap). The method is based on a non-target approach of full mass scan and multiplexing data-independent acquisition (Full MS/mDIA). The veterinary drugs include endectocides, fluoroquinolones, ionophores, macrolides, nitroimidazole, NSAIDs, β-lactams, penicillins, phenicols, sulfonamides, and tetracyclines. Veterinary drug residues were extracted from milk using a salting-out and solid-phase extraction (SOSPE) procedure, which entailed the precipitation of milk proteins by an extraction buffer (oxalic acid and EDTA, pH 3) and acetonitrile, a salting-out acetonitrile/water phase separation using ammonium sulfate, and solid-phase extraction for clean-up using polymeric reversed-phase sorbent cartridges. The Q-Orbitrap Full MS/dd-MS2 (data-dependent acquisition) was used to acquire product-ion spectra of individual veterinary drugs to build a compound database and a mass spectral library, whereas its Full MS/mDIA was utilized to acquire sample data from milk for target screening of veterinary drugs fortified at 1.0 or 10.0 μg/kg. The in-spectrum mass correction or solvent background lock-mass correction was used to minimize mass error when building the compound database from experimental dd-MS2 accurate mass data. Retention time alignment and response threshold adjustment were used to eliminate or reduce false negativesand/or false positive rates. The validated method was capable of screening 58% and 96% of 105 veterinary drugs at 1.0 and 10.0 μg/kg, respectively, without manually evaluating every compound during data processing, which will reduce the workload in routine practice.

Rapid determination of 88 veterinary drug residues in milk using automated TurborFlow online clean-up mode coupled to liquid chromatography-tandem mass spectrometry

A novel method based on TurborFlow online solid phase extraction (SPE) combined with liquid chromatography-tandem mass spectrometry (LC-MS/MS) has been established for simultaneous screening and confirmation of 88 wide-range veterinary drugs belonging to eight families (20 sulfonamides, 7 macrolides, 15 quinolones, 8 penicillins, 13 benzimidazoles, 4 tetracyclines, 2 sedatives, and 19 hormones) in milk. The preparation method consists of sample dilution and ultrasonic extraction, followed by an automated turbulent flow cyclone chromatography sample clean-up system. The detection was achieved in selected reaction monitoring mode (SRM). The total run time was within 39min, including automated extraction, analytical chromatography and re-equilibration of the turboflow system. The optimization of different experimental parameters including extraction, purification, separation, and detection were evaluated separately in this study. The developed method was validated and good performing characteristics were obtained. The linear regression coefficients (R2) of matrix-match calibration standard curves established for quantification were higher than 0.9930. The limits of detection (LOD) were in the range of 0.2–2.0μg/kg given by signal–noise ratio ≥3 (S/N) and the limits of quantification (LOQ, S/N≥10) ranged between 0.5μg/kg and 10μg/kg. Average recoveries of spiked target compounds with different levels were between 63.1% and 117.4%, with percentage relative standard deviations (RSD) in the range of 3.3–17.6%. The results indicated that the developed method has great potential for the routine laboratory analysis of large numbers of samples on measuring different classes of compounds. In comparison to traditional procedures, the automated sample clean-up ensures rapid, effective, sensitive analyses of veterinary drugs in milk.

Assessment of raw milk quality and stakeholders' awareness on milk-borne health risks in Arusha City and Meru District, Tanzania.

A cross-sectional study was conducted to assess the quality of raw milk and stakeholders' awareness on milk-borne health risks and factors for poor milk hygiene in Arusha City and Meru District, Tanzania between October and December 2012. A total of 105 smallholder dairy farmers, milk vendors and milk retailers were interviewed, and milk samples were collected for physical, microbial and antibiotic residue analysis using standard procedures. Questionnaire results indicated high level of awareness (94%) that drinking raw milk can predispose consumers to milk-borne diseases; nevertheless, 65% of respondents consumed raw milk. Physicochemical analyses showed some of the milk had sediments (20%), bad smell (21%) and had clotted on alcohol test (27%). About 36% of milk samples had pH below 6.6, and 25% had specific gravity below 1.028g/ml. The mean total viable count (TVC) of milk from vendors is significantly (P < 0.05) higher than that from retailers and smallholder dairy farmers. Generally, 65% of milk samples assessed had a higher TVC than the level recommended (2.0 × 10(5)cfu/ml) by the East African Community (EAC) standards. Up to 91% of the milk samples had bacterial growth that included Eschericia coli (66%), Staphylococcus aureus (33%), Corynebacterium (11%) and Pseudomonas (10%). All smallholder dairy farmers were aware of drug residues, but majority (57%) were unaware of human health effects caused by veterinary drug residues in milk. Up to 97% of respondents reported to comply with drug withdrawal periods. This possibly led to all milk samples analysed to be negative from detectable levels of antibiotic residues. It is concluded that the level of awareness on milk quality is high, although practices associated with milking and post-harvest handling predispose milk to bacterial contamination which is a public health risk to milk consumers.

Determination of Tetracyclines Residues in Egg, Milk, and Milk Powder by Online Coupling of A Precolumn Packed with Molecular Imprinted Hybrid Composite Materials to RP-HPLC-UV

A rapid, specific, and sensitive method was developed for determination of tetracyclines (TCs) in egg, milk, and milk powder samples by online molecularly imprinted solid-phase extraction (MISPE) coupled to a reversed-phase high-performance liquid chromatography (HPLC). A molecularly imprinted polymer hybrid composite material (MIP-HCM) was prepared using TC as the template, methacrylic acid as the functional monomer, tetraethoxysilane as inorganic precursor, and methacryloxypropyltrimethoxysilane as the coupling agent. A new technique of online solid-phase extraction (SPE) coupled with HPLC was established. The conditions of online SPE were optimized. Under the optimal conditions, three TCs residues in egg, milk, and milk powder were separated and detected by the proposed method with better clean-up and recoveries. The recoveries of TCs ranged from 85.3% to 98.3%. The limits of detection and quantitation of the proposed method were in the range of 0.76–1.13 µg/kg and 2.53–3.77 µg/kg, respectively. The MIP-HCM was first used as online SPE packing and the hyphenated technique is promising as a general strategy for direct determination of veterinary drug residues in milk and other biological samples.

Challenges in Implementing a Screening Method for Veterinary Drugs in Milk Using Liquid Chromatography Quadrupole Time-of-Flight Mass Spectrometry

High resolution mass spectrometry (HRMS) is a valuable tool for the analysis of chemical contaminants in food. Our laboratory has successfully developed methods to screen for veterinary drug residues using liquid chromatography quadrupole time-of-flight (Q-TOF). There have been, however, significant challenges as methods are transferred from the development stage to routine regulatory analysis. Having experimental retention time and product ion information for analytes greatly facilitates the ability to determine if residues found by the HRMS searching software are false detects. These data were collected for over 200 veterinary drug residues using LC Q-TOF MS. The screening levels of detection for over 150 veterinary drug residues in milk were determined, and over half of those tested can be detected at concentrations of 10 ng/mL or less; 72% can be found in milk when present at 100 ng/mL. Tentative identification of the product ions from these analytes is also presented.

Development of a LC-IT-TOF MS Procedure to Quantify Veterinary Drug Residues in Milk Employing a QuEChERS Approach

A sensitive liquid chromatography coupled to high-resolution tandem mass spectrometry (LC-IT-TOF MS) with electrospray ionization method for the identification and quantification of five antibiotic residues in milk has been developed. For sample preparation, a simple modification of the QuEChERS method was used with further sample cleanup using dispersive solid phase extraction according to AOAC official standards 2007.01. The method yielded acceptable accuracy values for each drug at each level, with mean recoveries (n = 3) ranging from 83 to 92 %. The recovery of antibiotics of the intra-assay ranged from 85 to 95 % (RSD < 9 %) and the inter-assay from 84 to 95 % (RSD < 11 %). A total of 31 milk samples, either pasteurized or fresh whole milk, were analyzed following the method. Amoxicillin and oxytetracycline (two of the pasteurized milk samples), ampicillin and amoxicillin (3 of 25 fresh milk samples), and tetracycline and oxytetracycline (two other fresh milk samples out of 25) were detected at levels below the current Brazilian maximum residue levels.

The challenges of developing a generic extraction procedure to analyze multi‐class veterinary drug residues in milk and honey using ultra‐high pressure liquid chromatography quadrupole time‐of‐flight mass spectrometry

This paper discusses the analytical challenges to develop a generic extraction procedure to analyze or screen multi-class veterinary drugs in milk and honey using ultra-high pressure liquid chromatography quadrupole time-of-flight mass spectrometry (UHPLC QqTOF MS). The veterinary drugs in this study included aminoglycosides, endectocides, fluoroquinolones, ionophores, β-lactams or penicillins, macrolides, NSAIDs, phenicols, sulfonamides and tetracyclines. Veterinary drugs were extracted using a QuEChERS (quick, easy, cheap, effective, rugged, and safe) method, which entailed the use of acetonitrile containing 1% acetic acid, sodium acetate, ethylenediaminetetra acetic acid disodium (EDTA) and magnesium sulfate, and no clean-up was performed. Chromatographic separation was achieved on a reversed-phase Acquity UPLC BEH C(18) , 100 × 2.1 mm, 1.7 µm column with 0.1% formic acid and 10 mM ammonium formate in water, and acetonitrile as mobile phases. Due to poor chromatographic retention, aminoglycosides were first dropped from the list, and because of poor extractability, β-lactams and tetracyclines were also excluded from the method. The method was able to quantify 31 or screen up to 54 drugs (unbound) in honey, and to quantify 34 or screen up to 59 drugs in milk. UHPLC QqTOF data were acquired in TOF MS full-scan mode that allowed both quantification and confirmation of veterinary drugs and identification of their degradation products in samples. The method could achieve detection limits as low as 1 µg/kg with analytical range from 1 to 100 µg/kg. The developed method was intended to be used for screening of as many analytes as possible in one single analysis, or unequivocal confirmation of positive findings and degradation product identification based on accurate mass measurement and isotopic patterns.

Multiclass analysis of 23 veterinary drugs in milk by ultraperformance liquid chromatography–electrospray tandem mass spectrometry

An ultraperformance liquid chromatography–electrospray tandem mass spectrometry (UPLC–MS/MS) method for the simultaneous detection and confirmation of 23 veterinary (multiclass) drugs in milk was developed and validated. The analytes were extracted by acetonitrile, evaporated and injected into the UPLC–MS/MS system on a Waters UPLC HSS T3 column in gradient mode. Data acquisition under MS/MS was achieved by applying multiple reaction monitoring (MRM) of two ion transitions per compound to provide a high degree of specificity. Results showed good repeatability, and recoveries for the 12 macrolide, 7 β-lactam and 2 lincosamide antibiotics and 2 other veterinary drugs (morantel, orbifloxacin) used in milk averaged 51.8–139.0%, 51.5–100.6%, 82.4–102.5% and 87.5–99.4%, respectively. The coefficients of variation (C.V.) of the recoveries were less than 15% for intraday and interday precisions. The limits of quantification (LOQs) were all lower than 5 ng/ml. This method was applied to 17 fresh milk samples and only lincomycin was found in milk samples under allowable levels. Overall, this method is a suitable and rapid tool to confirm the presence of 23 veterinary drug residues in milk.

Development of a multi-class method for the identification and quantification of residues of antibiotics, coccidiostats and corticosteroids in milk by liquid chromatography–tandem mass spectrometry

Even if coccidiostats are not generally employed in dairy cows, maximum levels for these substances in milk samples have been set up in Commission Regulation 124/2009. Due to public health concerns regarding the presence of veterinary drug residues in milk, the aim of the present work is to report a multi-class high performance liquid chromatography–tandem mass spectrometry method for the simultaneous extraction, identification and quantification of seven coccidiostats, seven antimicrobial agents, three corticosteroids and an antifungal agent. Validation was conducted following the guidelines established in the European Commission Decision 2002/657/CE at the maximum concentration permitted in milk by the European Union for each drug. The method was successfully applied in 100 raw milk samples collected from ten local dairy farms and 15 milk samples bought in supermarkets. This method could be applied in routine analysis of milk samples by the dairy industry, replacing the current, non-differentiating, screening method.

Analysis of Veterinary Drugs and Metabolites in Milk Using Quadrupole Time-of-Flight Liquid Chromatography−Mass Spectrometry

A quadrupole time-of-flight (Q-TOF) liquid chromatography-mass spectrometry (LC-MS) method was developed to analyze veterinary drug residues in milk. Milk samples were extracted with acetonitrile. A molecular weight cutoff filter was the only cleanup step in the procedure. Initially, a set of target compounds (including representative sulfonamides, tetracyclines, β-lactams, and macrolides) was used for validation. Screening of residues was accomplished by collecting TOF (MS(1)) data and comparing the accurate mass and retention times of found compounds to a database containing information for veterinary drugs. The residues included in the study could be detected in samples fortified at the levels of concern with this procedure 97% of the time. Although the method was intended to be qualitative, an evaluation of the MS data indicated a linear response and acceptable recoveries for a majority of target compounds. In addition, MS/MS data were also generated for the [M + H](+) ions. Product ions for each compound were identified, and their mass accuracy was compared to theoretical values. Finally, incurred milk samples from cows dosed with veterinary drugs, including sulfamethazine, flunixin, cephapirin, or enrofloxacin, were analyzed with Q-TOF LC-MS. In addition to monitoring for the parent residues, several metabolites were detected in these samples by TOF. Proposed identification of these residues could be made by evaluating the MS and MS/MS data. For example, several plausible metabolites of enrofloxacin, some not previously observed in milk, are reported in this study.

Development of fast screening methods for the analysis of veterinary drug residues in milk by liquid chromatography-triple quadrupole mass spectrometry

Two rapid multi-residue screening methods for the determination of 21 veterinary drugs in milk by ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) have been developed and compared. For both methods, veterinary drugs were extracted from milk samples using a rapid extraction procedure based on the modification of the well-known QuEChERS (Quick, Easy, Cheap, Effective, Rugged, and Safe) method, and no further clean-up steps were necessary. One screening method was based on the selection of a characteristic neutral loss or product ion of the various families of compounds, whereas another one was based on the choice of a selected reaction monitoring (SRM) for each compound. These methods were compared with regards to false negatives, cut-off values and the unreliability region. The total run time for both methods was 3 min, allowing quick selection of samples that contained veterinary drugs. Non-negative samples were re-analyzed by the UHPLC-MS/MS confirmation/quantification method, which consisted in the monitoring of two SRM for each compound. The methods were validated according to international guides. The proposed analytical methods allow for the identification and confirmation of the target veterinary drugs at trace levels employing quick analysis time.