Multiresidue Screening of Veterinary Drugs in Meat,Milk, Egg,and Fish Using Liquid Chromatography Coupled with Ion Trap Time-of-Flight Mass Spectrometry

New approaches to veterinary drug screening based on liquid chromatography-mass spectrometry (LC-MS/MS) and time-of-flight mass spectrometry (ToF/MS) are rapid and have high selectivity and sensitivity. In this study, we developed a multiresidue method for screening over 100 veterinary drug residues using ion trap (IT)-ToF/MS. The screened compounds comprised major drug classes used in veterinary practice, representing the following: amphenicols, anthelmintics, benzimidazoles, β-lactams, coccidiostats, ionophores, macrolides, non-steroidal anti-inflammatory drugs, quinolones, sulfonamides, tetracyclines, and tranquilizers. The method was developed based on chromatographic retention time, specific accurate mass, isotope distribution, and fragment data. Each compound was validated at three levels, and the mass accuracy, accuracy, and repeatability were calculated. All parameters showed acceptable values and conformed to the Commission Decision 2002/657/EC criteria. This screening method can simultaneously analyze over 100 veterinary drugs in meat, milk, eggs, and fish in a single analytical run.     

Feasibility of desorption electrospray ionization mass spectrometry for rapid screening of anabolic steroid esters in hair

Hormone and veterinary drug screening and forensics can benefit from the recent developments in desorption electrospray ionization (DESI) mass spectrometry (MS). In this work the feasibility of DESI application for the rapid screening of intact esters of anabolic steroids in bovine hair has been studied. Using a linear ion trap both full scan and data-dependent collision induced dissociation MS(n) spectra were acquired in minutes for testosterone cypionate, testosterone decanoate and estradiol benzoate standard solutions deposited on a glass or PTFE surface. However direct analysis of incurred hair failed due to inefficient desorption ionization and the minute quantities of steroid esters present. Therefore a simplified ultrasonic liquid extraction procedure was developed, allowing rapid DESI analysis of a few microliters of the concentrate and a total analysis time of 2-4h per batch instead of 3 days. The potential of this DESI approach is clearly demonstrated by MS(3) data from hair samples incurred with high levels (300-800 μg kg(-1)) of steroid esters, levels which do occur in samples from controlled- and illegally treated animals. For much lower levels state-of-the-art ultra high performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) screening methods remain the method of choice and might benefit from the proposed simplified extraction as well.     

Optimization and validation of a multiclass screening and confirmation method for drug residues in milk using high-performance liquid chromatography/tandem mass spectrometry

The further optimization and validation of a multiresidue veterinary drug screening method for milk is described. The drug residues of regulatory interest in milk include beta-lactams, sulfonamides, tetracyclines, fluoroquinolones, and macrolides. A previously published procedure has been modified to incorporate new compounds and to collect both screening and confirmatory ion transitions in one acquisition method. Milk samples were extracted with an equal volume of acetonitrile. The samples were then subjected to cleanup with a bonded SPE cartridge and a MW cutoff filter. The SPE protocol was modified to effectively recover a metabolite of flunixin. Established tolerance levels are set for most of these drugs in milk; thus, the screening procedure was semiquantitative, using positive controls for comparison. The positive controls, consisting of extracts from milk fortified with the drugs at their tolerance or safe level, were used to set statistically valid minimum response criteria for unknown samples. This updated method was validated with fortified milk, as well as with milk samples from animals administered veterinary drugs.     

Nontargeted screening of veterinary drugs and their metabolites in milk based on mass defect filtering using liquid chromatography–high-resolution mass spectrometry

The development of nontargeted screening strategy for veterinary drugs and their metabolites is very important for food safety. In this study, a nontargeted screening strategy was developed to find the potentially hazardous substances based on mass defect filtering (MDF) using liquid chromatography–high-resolution mass spectrometry. First, the drug metabolites of 112 veterinary drugs from seven classes of antimicrobials were predicted. Second, three MDF models were established, including the traditional rectangular MDF, the enhanced parallelogram MDF, and the polygonal MDF. Finally, the strategy was applied to nontargeted screening of veterinary drugs in 36 milk samples. The polygonal MDF model based on the distribution area of parent drugs and their metabolites showed a better filtering effect. After removing food components and performing MDF, about 10% of the substances remained, and four veterinary drugs and six drug metabolites were discovered and identified, showing the effectiveness of this strategy. The nontargeted screening strategy can rapidly remove interfering substances and find the suspected compounds. It can also be used for nontargeted screening of veterinary drugs and their metabolites in other food matrices.     

食品中99种兽药的一步式提取净化体系

建立了99种禁限用兽药的一步式提取净化体系,并通过高效液相色谱-四极杆/静电场轨道阱高分辨质谱（UHPLC-Q-Orbitrap HRMS）对其效果进行了评价。该提取净化体系基于载体辅助液液萃取技术,通过一次性前处理,完成常见的理化性质差异很大的8大类共计99种兽药残留的提取、净化工作,同时结合四极杆静电场轨道阱高分辨质谱实现了99种兽药残留的一步式多残留筛查。采用此体系对样品禁限用兽药进行测定分析,结果表明,该方法对液态乳、猪肉、鱼类等食品基质具有较强的适用性,检测的99种兽药线性范围为0.001~0.1 μg/mL,定量限为0.5~20.0 μg/kg,加标回收率为60%~120%,相对标准偏差小于15%。该方法的前处理和仪器分析过程对不同理化性质的化合物的兼容性强,检测效率高,可操作性强,检出限能满足所有受试的目标物,并且大大降低了检测成本。     

Use of biosensors for rapid drug residue analysis without sample deconjugation or clean-up: a possible way forward.

The drug salbutamol (SBL) is a beta-agonist that may be used illegally as an animal growth promoter. SBL is also a good example of a drug which is excreted in the form of glucuronides and sulfates. Such metabolites cause complexities in analysing for the presence of drug residues. In the majority of cases a process of deconjugation and sample clean-up is required prior to analysis. This is both time consuming and causes some loss of accuracy. In this study, the urine of calves treated with SBL orally for 3 d was collected during and after medication. Samples were assayed before and after hydrolysis by two different methods, radioimmunoassay (RIA) and a newly developed biosensor immunoassay (BIA). Some samples were also analysed by GC-MS. The results clearly showed that both screening assays (RIA and BIA) found high concentrations of SBL residues throughout the study. This was especially true in the BIA method. It was also demonstrated that urine sample analysis without the need for deconjugation or clean-up could be achieved. Results obtained by GC-MS tended to be an order of magnitude lower than the corresponding screening test results. This work showed that biosensor based veterinary drug residue testing procedures can be developed which can generate results in real time without the need for time consuming sample preparation.     

Review: Current Development of Immunoassay for Analyzing Veterinary Drug Residue in Foods and Food Products

Veterinary drug residues in foods and food products have been reported to be harmful for consumer health and must not be permitted in food intended for human consumption. These drugs include antibiotics, anthelmintics, beta-receptor agonists, and steroids. Immunoassay is recognized as the most powerful screening technique for a large number of different compounds. Because of the rapidity, sensitivity, and cost-effectiveness of this method, numerous immunoassays have already been developed for screening purposes to reduce sample loading for conventional analysis. In this review, the principles and an overview of advanced immunoassays for various veterinary drugs will be given.     

Ultra-performance liquid chromatography coupled to time of flight mass spectrometry (UPLC-TOF): a novel tool for multiresidue screening of veterinary drugs in urine

A multiresidue method for the screening of veterinary drugs in urine with ultra-performance liquid chromatography (UPLC) coupled to time of flight mass spectrometry (TOF) is proposed. The method covers currently more than 100 analytes belonging to different families of veterinary drugs. Urine samples are simply diluted and injected unfiltered into the UPLC-TOF. The short run time permit high-throughput of large series under a routine environment. The suggested approach includes the detection of some metabolites which are relevant to the urine matrix. The paper discusses the usefulness of metabolites as additional confirmation criteria for positive findings and suggests a revisiting of the identification point system in the light of additional resolution, as provided by the TOF and the UPLC technology. The proposed multiresidue approach was further broadened by monitoring drug group specific collision induced fragments (CID). Examples show the monitoring of generic CID fragments typical for sulfonamides and penicillins.     

Use of capillary electrophoresis with laser-induced fluorescence detection to screen and liquid chromatography–tandem mass spectrometry to confirm sulfonamide residues: Validation according to European Union 2002/657/EC

A multiresidue method is described for determining six sulfonamides (SAs) (sulfadiazine, sulfathiazole, sulfamethazine, sulfamethoxazole, sulfaquinoxaline and sulfadimethoxine) in liver by a capillary electrophoresis screening method and a liquid chromatography coupled to tandem mass spectrometry confirmatory assay. Samples were prepared by homogenizing the tissue, with sodium hydroxide and acetonitrile. After evaporation, extracts were injected in the capillary electrophoresis system or mass spectrometry system for confirmatory analysis. The detection of analytes was achieved by laser-induced fluorescence in capillary electrophoresis. Procedures were validated according to the European Union regulation 2002/657/EC determining specificity, selectivity and detection capability for screening method and decision limit, detection capability, specificity, selectivity, trueness and precision for confirmation method. The results of validation process demonstrate that the method is suitable for application in Brazilian statutory veterinary drug residue surveillance programs. Capillary electrophoresis was proved to be a fast, robust method with low time and reagents consumption.     

A generic fast solid-phase extraction high-performance liquid chromatography/mass spectrometry method for high-throughput drug discovery

High-performance liquid chromatography/mass spectrometry (HPLC/MS) is increasingly perceived to be an essential tool in drug discovery at many key steps, like drug screening, lead identification, ADME profiling, and drug metabolism and pharmacology studies. High-throughput screenings in the early phase for metabolic stability, protein binding, permeability (ADME) and bioavailability are widely used to weed out compounds that do not exhibit the necessary characteristics. For such high-throughput LC/MS studies, a generic LC/MS method that can be used for a variety of compounds is desired. In this study, we used a small set of compounds with a wide range of properties to guide method development, and achieved a sample throughput of 1.7 min/sample. Here, we present a generic fast method that achieves good peak separation and peak shape on conventional HPLC systems using a column-switching mechanism for on-line solid-phase extraction (SPE)-HPLC/MS analysis. The method has a linear response range from 1 to 500 nM for the tested compounds. When a larger set of 658 randomly picked small molecules were analyzed using this method, 612 were observed with good signal intensity and HPLC peak shapes. This generic fast SPE-LC/MS method has been used to screen more than 1.5 million compounds repetitively against over 200 protein targets for hit confirmation and semi-quantitation of binding constants from biological assays. Over 7000 different compounds for a variety of protein-binding assays have been studied using this method for quantitative analysis as well.     

Comprehensive screening and quantification of veterinary drugs in milk using UPLC–ToF-MS

Ultra-performance liquid chromatography combined with time-of-flight mass spectrometry (UPLC–ToF-MS) has been used for screening and quantification of more than 100 veterinary drugs in milk. The veterinary drugs represent different classes including benzimidazoles, macrolides, penicillins, quinolones, sulphonamides, pyrimidines, tetracylines, nitroimidazoles, tranquillizers, ionophores, amphenicols and non-steroidal anti-inflammatory agents (NSAIDs). After protein precipitation, centrifugation and solid-phase extraction (SPE), the extracts were analysed by UPLC–ToF-MS. From the acquired full scan data the drug-specific ions were extracted for construction of the chromatograms and evaluation of the results. The analytical method was validated according to the EU guidelines (2002/657/EC) for a quantitative screening method. At the concentration level of interest (MRL level) the results for repeatability (%RSD < 20% for 86% of the compounds), reproducibility (%RSD < 40% for 96% of the compounds) and the accuracy (80–120% for 88% of the compounds) were satisfactory. Evaluation of the CCβ values and the linearity results demonstrates that the developed method shows adequate sensitivity and linearity to provide quantitative results. Furthermore, the method is accurate enough to differentiate between suspected and negative samples or drug concentrations below or above the MRL. A set of 100 samples of raw milk were screened for residues. No suspected (positive) results were obtained except for the included blind reference sample containing sulphamethazine (88 μg/l) that tested positive for this compound. UPLC–ToF-MS combines high resolution for both LC and MS with high mass accuracy which is very powerful for the multi-compound analysis of veterinary drugs. The technique seems to be powerful enough for the analysis of not only veterinary drugs but also organic contaminants like pesticides, mycotoxins and plant toxins in one single method.     

QuEChERS-超高效液相色谱-串联质谱法测定鸡蛋中125种兽药残留

采用冷冻脂质过滤,结合分散固相萃取净化的QuEChERS方法,建立了超高效液相色谱-串联质谱法测定鸡蛋中125种兽药残留的检测方法。样品中的11类兽药（硝基咪唑类、苯并咪唑类、磺胺类、喹诺酮类、四环素类、大环内酯类、雄激素类、孕激素类、糖皮质醇类、雌激素类、氯霉素类）用70%（v/v）乙腈-水溶液（含0.1 mol/L EDTA）提取后,提取液在-20℃冷冻处理2 h,再经分散固相萃取法净化,净化液经稀释后,以超高效液相色谱-串联质谱法测定,外标法定量。结果显示125种兽药的线性相关系数R²≥0.99,定量限范围为2.0~60 μg/kg,回收率在60.4%~119.3%之间,相对标准偏差在0.3%~16.1%之间。该方法前处理简单、准确、成本较低,适用于鸡蛋中兽药残留的高通量快速检测分析。     

Determination of Tripamide in human urine by high-performance liquid chromatography and high-performance liquid chromatography/electrospray ionization tandem mass spectrometry

Tripamide is a drug widely used in clinical practice for the treatment of hypertension and edema. This work evaluated a screening method for Tripamide and its urinary metabolites in human urine, using high-performance liquid chromatography diode-array detection (HPLC/DAD). Identification of these metabolites was investigated by high-performance liquid chromatography/electrospray ionization tandem mass spectrometry (HPLC/ESI-MS/MS) after dosing with 15 mg Tripamide. Acid hydrolysis showed that Tripamide is conjugated in the body. Two suspected metabolites were detected by HPLC/DAD. HPLC/ESI-MS/MS analysis suggested that these metabolites were probably hydroxylated together with loss of the -NH(2) group and dehydrogenation. These results will be useful in confirmation methods for Tripamide in doping control.     

Simultaneous Screening for Five Veterinary Drug Residues by a Developed Multiplexed Luminex Assay

Abstract

The development of sensitive and rapid methods for simultaneous determination of multiple residual veterinary drugs in foods is increasingly important. In this study we reported a new method to simultaneously detect five veterinary drug residues, including ractopamine, clenbuterol, furazolidone, chlortetracycline and qlaquindox. The experimental results indicated that the developed multiplex assay enable to detect all the five drugs at the concentration of 0.1 ng/mL, with desirable specificity, suggesting that it could be applicable for rapid screening of five residual veterinary drugs in a faster turnaround time and higher throughput.     

High-performance liquid chromatography of seized drugs at elevated pressure with 1.7 microm hybrid C18 stationary phase columns

High-performance liquid chromatography (HPLC) separation of drugs at elevated pressure with 1.7 microm hybrid C18 stationary phase columns was investigated. This technique, which uses instrumentation engineered to handle the narrow peaks and high back pressures generated by 1.7 microm particle columns, provided significantly better resolution and/or faster analysis than conventional HPLC and capillary electrophoresis (CE). The use of 2mm internal diameter (i.d.) columns of 3-10 cm length has been evaluated for the separation of basic and neutral drugs, drug profiling, and general screening (including acidic drugs). For these applications, compared to conventional HPLC and CE, it provided up to 12x and 3x faster analyses, respectively. Precision was excellent for both isocratic and gradient analyses. For retention time and peak area, RSDs of < or =0.1% were obtainable. Fifteen anabolic steroids and esters were well separated in a 2.5 min gradient. For drug profiling, compared to HPLC and CE, approximately twice as many peaks were resolved. HPLC at elevated pressure is also well suited as a general screening technique. Twenty-four solutes of varying drug classes including narcotic analgesics, stimulants, depressants, hallucinogens, and anabolic steroids were fully separated in a 13.5 min gradient.     

Multiclass and multiresidue screening of veterinary drugs and pesticides in infant formula using Quadrupole‐Orbitrap MS with PRM scan mode

A multiclass and multiresidue method for screening veterinary drugs and pesticides in infant formula was developed and validated using ultrahigh‐performance liquid chromatography coupled to Quadrupole‐Orbitrap high‐resolution mass spectrometry (UHPLC‐HRMS). A total of 49 veterinary drugs and pesticides investigated belong to 11 classes including antivirals, anticoccidials, macrolides, pyrethroids, insecticides, sulfonamides, beta‐agonists, sedatives, thyreostats, nonsteroidal anti‐inflammatory drugs, and other pharmacologically active substances. A generic sample preparation and highly selective acquisition mode of parallel reaction monitoring (PRM) were deliberately incorporated to perform efficient screening analysis. As a result, the screening target concentrations of the analytes varied from 1 to 500 μg/kg with ≤5% of false compliant rate as specified in Decision 2002/657/EC for screening analysis. The average recoveries ranged from 40.7 to 124.9% as well as the relative standard deviations from 4.2 to 26.6%, respectively. The matrix effects and interferences were effectively controlled by integrated application of dispersive solid phase extraction, PRM scan mode, and matrix‐matched standard calibration. The proposed method will be helpful to provide applicable strategy for screening residues in infant formula with surveillance purpose.     

Efficient hydrophilic interaction liquid chromatography-tandem mass spectrometry for the multiclass analysis of veterinary drugs in chicken muscle

A simple and sensitive method has been developed for multiresidue analysis of 24 important veterinary drugs (including 3 aminoglycosides, 3 β-lactams, 2 lincosamides, 4 macrolides, 4 quinolones, 4 sulfonamides, 3 tetracyclines, and amprolium) in chicken muscle. The method involved a simple extraction using (1:1, v/v) of 2% trichloroacetic acid in water-acetonitrile, followed by removing fat with hexane, dilution of sample extract, and filtration prior to liquid chromatography-tandem mass spectrometric (LC-MS/MS) analysis. Hydrophilic interaction liquid chromatography (HILIC) proved to be very effective for separation of a wide range of polar and hydrophilic compounds (providing high sensitivity and good peak shape) compared to reversed phase and ion-pair separation. The method was successfully validated according to the European Decision 2002/657/EC. Average recoveries were 53-99% at 0.5-MRL, MRL, and 1.5-MRL spiking levels, with satisfactory precision ≤15% RSD. The limit of detection of the method was 0.1-10 μgkg(-1) for 22 analytes and 20 μgkg(-1) for aminoglycosides. These values were lower than the maximum residue limits (MRLs) established by the European Union. The evaluated method provides reliable screening, quantification, and identification of 24 veterinary drug residues in foods of animal origin. It has been successfully tested in real samples (such as chicken muscle, shrimp, and egg).     

Simultaneous determination of dyes of different classes in aquaculture products and spices using HPLC–high-resolution quadrupole time-of-flight mass spectrometry

A simple method of sample preparation, rapid screening, and determination of dyes—veterinary drugs and illegally used dyes (coloring product counterfeiters)—in spices by HPLC–high-resolution quadrupole time-of-flight mass spectrometry is proposed. Sample preparation involves the extraction of analytes from a solid matrix with acetonitrile containing 0.1% of formic acid, twofold dilution of the extract with deionized water, filtration, and chromatographic separation. The limits of detection were 0.01–0.4 ng/g. The procedure of screening and determination of dyes in foods includes the identification of dyes by an accurate monoisotopic mass of the ion (m/z), retention time (t _R), and coincidence of the isotopic distribution (mSigma), and, if dyes are detected, their determination by the standard addition method. The relative standard deviation of the results does not exceed 15%. The duration of the analysis is 1–2 h.     

Screening of cardioprotective diseases bioactive components from Danshen extracts and LC–MS analysis

A rapid and efficient analysis and screening method is adopted for cell affinity capture coupled with HPLC–MS (CAC–HPLC–MS) analysis of bioactive components that have possible efficiency against cardiovascular diseases. This method involves affinity capture, concentration, and separation of bioactive components from Danshen library using oxidatively damaged endothelial cells induced by H₂O₂, as well as analysis and identification of targeted compounds with HPLC and MS. It combines the specific interaction between cell membrane receptors and bioactive components with the powerful analysis and identification function of HPLC–MS. The CAC–HPLC–MS method was also used for analysis and screening of bioactive components from crude extracts of Danshen. A total of 19 components were found to be bound to oxidatively damaged endothelial cells with seven of these identified. Existing literature confirms that these seven components have many activities related to cardioprotective diseases. Therefore, the combination of biological affinity capture with HPLC–MS should be regarded as an attractive method with great potential for rapid and efficient screening of bioactive components related to anti-cardiovascular diseases from natural product libraries.     

Determination of Pharmaceuticals by Dynamic Cell Membrane Chromatography Coupled with High-Performance Liquid Chromatography

As a biological affinity chromatographic method, cell membrane chromatography (CMC) using a silica stationary phase covered with specific cell membrane has been used in screening active components. The innovation of this work is that the bioactive cell membrane and the chromatographic packing are mixed and absorbed for the first time to form the pre-column. The pre-column was placed in front of a C₁₈ column to create dynamic CMC online high-performance liquid chromatography (HPLC) system. The retention behavior and dynamic changes of pharmaceuticals were studied for this system. The results indicate that the retention time of the drug was increased and the symmetry factor reached the analytical level after the addition of the dynamic cell membrane pre-column. Therefore, the dynamic CMC coupled with HPLC system may be a potentially rapid and efficient drug analysis approach for the interaction of drug molecule and receptor on red blood cell membranes.