Article pubs.acs.org/JAFC
Qualitative Multiresidue Screening Method for 143 Veterinary Drugs and Pharmaceuticals in Milk and Fish Tissue Using Liquid Chromatography Quadrupole-Time-of-Flight Mass Spectrometry Marilena E. Dasenaki, Anna A. Bletsou, George A. Koulis, and Nikolaos S. Thomaidis* Laboratory of Analytical Chemistry, Department of Chemistry, University of Athens, Panepistimiopolis Zographou, 15771 Athens, Greece S Supporting Information *
ABSTRACT: A wide-scope screening methodology has been developed for the identification of veterinary drugs and pharmaceuticals in fish tissue and milk using ultrahigh-performance liquid chromatography quadrupole time-of-flight mass spectrometry (UHPLC-QTOF MS). The method was validated using a qualitative approach at two concentration levels. The detection of the residues was accomplished by retention time, accurate mass, and the isotopic fit using an in-house database. Product-ion spectra were used for unequivocal identification of the compounds. Generic sample treatment was applied. The majority of the compounds were successfully detected and identified at concentration levels of 150 ng mL−1 in milk and 200 μg kg−1 in fish (>80% of the compounds in both matrices), whereas satisfactory results were also obtained at concentration levels of 15 ng mL−1 in milk and 20 μg kg−1 in fish (>60% of the compounds detected and identified). KEYWORDS: veterinary drugs, pharmaceuticals, milk, fish, UHPLC-QTOF MS, screening, qualitative validation
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INTRODUCTION
Liquid chromatography in combination with triple-quadrupole mass spectrometric detection (LC-QqQ MS) dominates in the field of multiresidue determination of veterinary drugs in complex matrices, due to its excellent sensitivity and selectivity.9−14 However, there is a limitation regarding the number of analytes that can be detected simultaneously, and a compromise must be made concerning the length of the dwell times and the number of data points across a chromatographic peak.15 The number of analyte peaks to be monitored can be increased if retention time window-based SRM methods are used, but potential retention time instability might lead to the constant need for the windows’ readjustment.15 Additionally, this approach focuses on a predefined list of compounds to be tested for, because the transitions must be preselected (target analysis only). The use of high-resolution (HR) MS analyzers, such as timeof-flight (TOF), hybrid quadrupole-time-of-flight (QTOF), or Orbitrap, is considered as a powerful alternative for screening analysis of contaminants.16 These analyzers provide high signal specificity through high-resolution and mass accuracy in full scan acquisition mode. Spectrometers working in full scan mode can register unlimited numbers of compounds, allowing the complete elimination of compound-specific MS method development and the simultaneous collection of mass spectral data on both targeted and nontargeted components. Data can also be evaluated retrospectively for possible contaminants, such as new drugs or metabolites.17,18
The use of veterinary drugs in food production has been an issue of increasing concern for consumers during recent years. The incorrect use of veterinary drugs administered to production animals as well as the disrespect of withdrawal time after treatment may lead to residues in milk, eggs, and edible tissues. These residues may include the nonaltered parent compound as well as metabolites and/or conjugates and may have direct toxic effects on consumers, for example, allergic reactions in hypersensitive individuals. Moreover, indirect problems in clinical treatment may be caused through induction of resistant strains of bacteria (development of bacterial resistance).1,2 Human pharmaceuticals (especially antibiotics) are considered as widespread emerging pollutants with potential to enter the food chain. They can be detected in surface water and drinking water due to incomplete removal in wastewater treatment plants (WWTPs)3,4 and can also be added to animal feed because of their commercial availability and low cost.5 To protect consumer health and ensure quality food products, many organizations, such as the Codex Alimentarius,6 European Union (EU),7 and U.S. Food and Drug Administration (FDA),8 have established maximum residue limits (MRLs) for veterinary medicinal products in foodstuffs from animal origin. Therefore, the need to develop rapid, sensitive, accurate, and high-throughput qualitative methods as well as confirmation and identification techniques for the determination of veterinary drugs and pharmaceuticals in food matrices has become imperative to meet with regulation requirements. Recently, a trend has been observed in residue analysis toward more generic methods for the detection, confirmation, and quantification of a broad range of compounds in a single run. © XXXX American Chemical Society
Special Issue: IUPAC - Analysis of Residues in Food Received: November 11, 2014 Revised: March 29, 2015 Accepted: March 31, 2015
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DOI: 10.1021/acs.jafc.5b00962 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
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Journal of Agricultural and Food Chemistry
extracted, and low sensitivity made the detection of some compounds and fragments at low concentrations unfeasible. Deng et al. presented a multiresidue/multiclass quantitative screening approach for 105 veterinary drugs in meat, milk, and egg with UHPLC-QTOF MS/MS,34 and Nácher-Mestre et al. described the development, qualitative validation, and application of a screening method for the detection and identification of about 70 organic compounds, including antibiotics, pesticides, and mycotoxins, in fish feed and fish fillets.27 Most recently, Turnipseed et al. presented an LC-QTOF MS/MS method for the screening of about 200 veterinary drugs in milk, providing experimental retention time and product ion information.35 The objective of this work is the development of a reliable, sensitive, and modern screening methodology for the detection and identification of 143 veterinary drugs and pharmaceuticals in milk and fish samples based on the use of an advanced UHPLC-QTOF MS technique. Generic sample extraction procedures were used, and their efficiency was evaluated. A qualitative validation of the screening method was performed, and levels of detection and identification were specified for all target analytes in both matrices. Subsequently, the method was applied to the analysis of different milk and fish samples to test its applicability. Experimental data including retention times, product ion spectra, and precursor and product ion mass accuracy as well as precursor−product ion ratios were obtained and presented for all target compounds.
Several methods using LC-TOF-MS that monitor veterinary drug and pharmaceutical residues in food products have been published.19−23 However, having MS data only for the precursor ions is not considered sufficient for residue identity confirmation, even with high mass accuracy. In some methods in-source fragmentation was performed (in-source CID) to generate fragment ions that could provide additional data for compound identification.22 However, the in-source CID fragmentation has significant drawbacks compared to tandem mass spectrometry. Matrix interferences could preclude its use, and large amounts of impurities can significantly complicate the analysis process.24 A hybrid quadrupole TOF (QTOF) has the additional ability to obtain MS/MS spectra that can be used to further characterize drug residues. QTOF MS allows working under broad-band collision-induced dissociation (bbCID) (or MSE) mode, switching alternately between low (LE) and high collision energy (HE) in the collision cell Q2, at the same run. The low-energy data set provides information for molecular (precursor) ions corresponding to a given retention time, whereas the second set contains their fragment ions.25 The information provided, along with the isotopic distribution observed in the spectra, allows the reliable identification of the compounds detected in the samples. In multiresidue analysis the LC part is responsible for some limitation as well, mainly due to the large number of target analytes that need to be separated simultaneously. Ultrahighperformance liquid chromatography (UHPLC) can eliminate these limitations, providing additional chromatographic resolution and significantly lowering analysis times using sub-2-μm particulate column packing material.21 With the demand for high-throughput analysis constantly increasing, the development and application of generic extraction procedures, able to extract a wide range of compounds with different physical/chemical properties, have become essential. A wide array of the proposed methodologies for analyzing veterinary drug residues in food products employs liquid−liquid extraction (LLE) or solid−liquid extraction (SE), with solid-phase extraction (SPE) being the most widely used technique for the cleanup of the extracts.26 The combination of solid−liquid extraction or liquid−liquid extraction followed by the SPE cleanup step has been satisfactorily applied to the analysis of several antibacterials in milk,19 meat,20,23 and eggs and fish.23 An effort was made to omit cleanup steps by performing a single-solvent extraction in fish and feed matrices,27 but matrix effects proved to be problematic leading to reduced sensitivity in some cases. To conclude, for an accurate and efficient multiresidue screening method, both a sensitive full scan technique and a comprehensive sample preparation are required. So far, the number of applications of LC-QTOF MS determination of veterinary drug residues in food and feed matrices is quite limited.5,27−35 Some of these studies cover a single class of drugs such as macrolides and quinolones in fish29,30 or fewer than 30 compounds in milk28,31 and aquaculture species.32 Two of them cope with feed matrices: Boix et al. presented a qualitative screening of 116 veterinary drugs, validated on the basis of a previously reported validation protocol,5 and Aguilera-Luiz et al. developed a wide-scope multiresidue method able to cover the analysis of >200 pesticide and veterinary residues.33 As Aguilera-Luiz et al. stated in the conclusions of their study,33 the developed method showed some limitations because some families of drugs, such as tetracyclines, quinolones, and penicillins, were not properly
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MATERIALS AND METHODS
Reagents and Chemicals. All veterinary drug and pharmaceutical standards were of high-purity grade (>90%). The majority of them were purchased from Sigma-Aldrich (Steinheim, Germany). Sulfadoxine (SDX) and sulfaclozine (SClZ) were donated by the National Laboratory of Residue Analysis of Food of Animal Origin of the Hellenic Ministry of Rural Development and Food. Bacitracin, halofuginone, arprinocid, salinomycin, semduramicin, manduramycin, nigericin, narasin, albendazole sulfone, carprofen, diclofenac, flunixin, mefenamic acid, meloxicam, niflumic acid, and tolfenamic acid were donated by the Veterinary Drug Residues Laboratory of the State General Laboratory of Cyprus. Acetonitrile (ACN) and methanol (MeOH) of LC-MS grade were purchased from Merck (Darmstadt, Germany), whereas 2-propanol of LC-MS grade was from Fisher Scientific (Geel, Belgium). Sodium hydroxide monohydrate (NaOH) for trace analysis ≥99.9995% and formic acid 99% were purchased from Fluka (Buchs, Switzerland). Hexane (pesticide analysis grade, 95%) was purchased from Carlo Erba (Milan, Italy), and distilled water was provided by a Milli-Q purification apparatus (Millipore Direct-Q UV, Bedford, MA, USA). The ethylenediaminetetraacetic acid disodium salt (EDTA) and the trichloroacetic acid (TCA) were of analytical grade and were purchased from Panreac (Barcelona, Spain) and Fisher Scientific (Loughborough, UK), respectively. Regenerated cellulose (RC) syringe filters (15 mm diameter, 0.22 μm pore size) were provided from Phenomenex (Torrance, CA, USA). Solid phase extraction cartridges were Oasis HLB 3 cc (60 mg) from Waters (Milford, MA, USA). Stock standard solutions of individual compounds (1000 μg mL−1) were prepared in methanol and stored at −20 °C in brown glass to prevent photodegradation. Penicillins, cefalosporines, and metformin were dissolved in Milli-Q water and stored at 4 °C. In quinolone standard solutions, 100 μL of formic acid was added to enhance solubility. Four intermediate standard solutions containing the analytes were prepared by dilution of the stock solutions with methanol. The final concentration of these multicomponent solutions was 10 μg mL−1, and they were also stored at −20 °C. New intermediate standard solutions were prepared every month. Working solutions and B
DOI: 10.1021/acs.jafc.5b00962 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
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Journal of Agricultural and Food Chemistry calibration standards, containing all of the target analytes, were obtained by gradient dilution of the intermediate solutions, in concentrations varying from 1 μg mL−1 to 1 ng mL−1. The working solutions were kept at −20 °C and renewed weekly. Instrumentation. An ultrahigh-performance liquid chromatography (UHPLC) system (Dionex UltiMate 3000 RSLC, Thermo Fisher Scientific, Germany) interfaced to a QTOF mass spectrometer (Maxis Impact, Bruker Daltonics, Bremen, Germany) was used for the screening analysis. The chromatographic separation was performed on an ACQUITY UHPLC BEH C18 column (2.1 × 50 mm, 1.7 μm) from Waters (Ireland), thermostated at 30 °C. The mobile phases were (A) MeOH and (B) an aqueous solution with 0.01% formic acid. The flow rate was 0.1 mL min−1 despite the fact that UHPLC allows higher flow rates (
