Liquid Chromatography/ Mass Spectrometry APPLICATION NOTE Authors: Abir Khaled Janusz Pawliszyn Department of Chemistry, University of Waterloo 200 University Avenue West Waterloo, Ontario, N2L 3G1, Canada Jingcun Wu Feng Qin PerkinElmer Inc. Woodbridge, Ontario, Canada Introduction The use of veterinary drugs in modern day intensive farming of food-producing animals is essential to prevent and treat animal diseases. 1,2 However, improper dosage or failure to keep the specified withdrawal period of approved drugs may lead to excessive levels of drug residues in the edible tissues of treated animals, posing a potential risk to human health. Moreover, the overuse of antimicrobials in food-producing animals has been closely linked to the emergence of antibiotic-resistant bacteria. 3–9 Aiming to safeguard human health, many countries have established extensive regulatory measures to prevent veterinary drug exploitation in the food-animal industry. 10,11 In Canada, Health Canada establishes maximum residue limits (MRL) for veterinary drugs through the Veterinary Drugs Directorate (VDD). 12 Likewise, in the USA, the Food and Drug Administration (FDA) sets regulatory tolerances of registered veterinary drugs through the Center for Veterinary Medicine (CVM). 13 Accordingly, monitoring of veterinary drug residues in food products of animal origin is necessary to ensure the safety of meat products, and that maximum residue limits are not breached. Owing to the large number of samples to be analyzed and the wide array of residual drugs, there is increasing demand for high-throughput multi-class, multi-residue methods (MMM) that are capable of simultaneously detecting a wide variety of analytes in a single analytical run. 7,14 Automated High Throughput Multi-class Multi-residue Method for Analysis of Veterinary Drugs in Chicken by SPME-UHPLC/MS/MS
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Liquid Chromatography/ Mass Spectrometry
A P P L I C A T I O N N O T E
Authors:
Abir KhaledJanusz Pawliszyn
Department of Chemistry, University of Waterloo 200 University Avenue West Waterloo, Ontario, N2L 3G1, Canada
Jingcun WuFeng Qin
PerkinElmer Inc. Woodbridge, Ontario, Canada
IntroductionThe use of veterinary drugs in modern day intensive farming of food-producing animals is essential to prevent and treat animal diseases.1,2 However, improper dosage or failure to keep the
specified withdrawal period of approved drugs may lead to excessive levels of drug residues in the edible tissues of treated animals, posing a potential risk to human health. Moreover, the overuse of antimicrobials in food-producing animals has been closely linked to the emergence of antibiotic-resistant bacteria.3–9
Aiming to safeguard human health, many countries have established extensive regulatory measures to prevent veterinary drug exploitation in the food-animal industry.10,11 In Canada, Health Canada establishes maximum residue limits (MRL) for veterinary drugs through the Veterinary Drugs Directorate (VDD).12 Likewise, in the USA, the Food and Drug Administration (FDA) sets regulatory tolerances of registered veterinary drugs through the Center for Veterinary Medicine (CVM).13 Accordingly, monitoring of veterinary drug residues in food products of animal origin is necessary to ensure the safety of meat products, and that maximum residue limits are not breached. Owing to the large number of samples to be analyzed and the wide array of residual drugs, there is increasing demand for high-throughput multi-class, multi-residue methods (MMM) that are capable of simultaneously detecting a wide variety of analytes in a single analytical run.7,14
Automated High Throughput Multi-class Multi-residue Method for Analysis of Veterinary Drugs in Chicken by SPME-UHPLC/MS/MS
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Analysis of veterinary drug residues in animal tissue samples can be a challenging task owing to the high protein, fat and phospholipid content of biological samples.15 Improper sample preparation and clean-up can lead to the presence of matrix interferences, which might adversely affect the signal and compromise the analytical outcome.14,16,17 Conventional sample preparation procedures to reduce matrix effects often include solvent extraction (SE) followed by either solid phase extraction (SPE) or dispersive SPE (d-SPE) for additional extract clean-up.14,15,18,19 These sample preparation techniques often involve time-consuming procedures owing to the lack of automation and high-throughput capabilities. In addition, they often involve the use of large amounts of toxic organic solvents.
Solid phase microextraction (SPME) is a solvent-less, effective and versatile sample preparation technique, which has been widely used in environmental and other applications.20-22 Recently, with the development of new extraction phases and devices, SPME has found increased applicability to food analysis.22
In this work, we present a robust, high-throughput quantification method for the analysis of 73 veterinary drugs (belonging to more than 12 classes of drug compounds) in chicken tissue by using a fully automated solid phase micro-extraction (SPME) sample preparation technique and a QSight® UHPLC/MS/MS. The method offers an efficient and environmentally friendly alternative for the analysis of veterinary drug residues in animal tissue. In addition, it offers improved precision and accuracy as compared to traditional sampling procedures.
Experimental
Selection of Target Analytes and Target Levels in Chicken Tissue SamplesThe target analytes were selected from the list of priority veterinary drugs specified in the official United States Department of Agriculture method for the confirmation and screening of drug residues in animal tissues (CLG-MRM1.08).23 We aimed to include as many analytes from the list as possible to represent at least 12 drug classes, and to cover a wide range of polarities. The target levels were based on the Canadian MRLs and U.S. regulatory tolerances.24,25 In general, Canadian MRLs are either equal to or less than U.S. tolerances; however, in the case of ketoprofen and morantel, where Canadian MRLs were higher than U.S. tolerances, the U.S. regulatory tolerances were selected.
Materials and Supplies LC-MS grade acetonitrile (MeCN), methanol (MeOH), water, and formic acid (FA) were purchased from Fisher Scientific (Mississauga, ON, Canada). Polyacrylonitrile (PAN) and 1 mL and 2 mL Nunc U96 Deep Well plates made of polypropylene were purchased from VWR International (Mississauga, ON, Canada). Polypropylene inserts (600 mL) for the 1 mL plates were purchased from Analytical Sales and Services (NJ, USA). Desethylene ciprofloxacin HCl, hydroxy dimetridazole, and pirlimycin HCl were obtained from Toronto Research Chemicals (Toronto, ON, Canada). Haloxon was purchased from Cedarlane (Burlington, ON, Canada). All other veterinary drugs, internal standard (IS) flunixin-d3, and other reagents were purchased
from Sigma-Aldrich (Oakville, ON, Canada). SPME pins, shown in Figure 1, were manufactured using high-tolerance 304 stainless steel rods (1/16" diameter) purchased from McMaster-Carr (Aurora, OH, USA). The pins were cut to a length of 50 mm at the University of Waterloo's Science Technical Services (UW-STC), and coated with a slurry of HLB particles suspended in PAN as a binder at a thickness of 60 μm and a length of 2 cm, as previously described.26
Stock solutions of each veterinary drug and the deuterated internal standard were prepared separately by accurately weighing about 5 mg of each individual standard, and dissolving it in 5 mL of either acetonitrile (ACN), methanol (MeOH), water, or 10% dimethyl sulfoxide, depending on the solubility of each compound. An internal standard (IS) solution containing flunixin-d3, at 10 μg/mL, was prepared by appropriate dilution of IS stock solution in ACN. A composite solution containing β-Lactams/cephalosporins was prepared in water at a concentration of 200X (where X is the MRL). All other analytes were mixed in a composite solution and diluted to 100X with ACN. All stock and composite solutions were stored at -30 °C. Spiking solutions were prepared and used in the validation experiments within a week of preparation. Calibration standards were prepared by diluting the spiking solutions each day during validation and analysis. All standard solutions were stored in amber glass vials and closed with fitted PTFE, except for Lactams/cephalosporins solutions which were stored in plastic vials.
Organic chicken muscle tissue samples were purchased from three different local grocery stores in Waterloo, ON, Canada to serve as a pooled blank matrix. Five distinct samples of non-organic chicken were purchased from different local grocery stores for analysis of market samples.
Instruments and Conditions Chromatographic separation of veterinary drugs was conducted utilizing a PerkinElmer UHPLC system, with analyte determination achieved using a PerkinElmer QSight 220 triple quadrupole mass detector with dual ionization source. As the mass detector is capable of fast polarity switching, both positive and negative ionization modes were used. All instrument control, data acquisition, and data processing were performed using Simplicity 3Q™ software.
The optimized LC method and MS source parameters are shown in Table 1. The multiple reaction monitoring mode (MRM) transitions of the veterinary drugs under study are shown in Table 2. Two MRM transitions were monitored for each analyte for improved selectivity,27 whereas only one transition was needed for the IS. MS/MS analyte transitions and conditions were optimized via the software by direct infusion from methanolic and acetonitrile standards. Source conditions were optimized by flow injection (FIA) method. Based on the optimized conditions, the acquisition MS method is generated automatically by selecting the veterinary drugs of interest from the built-in compound library in the time-managed-MRM module of the Simplicity software, including both positive and negative analytes.
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Sample Preparation ProcedureChicken samples were homogenized with dry ice using a Vitamix blender to obtain a uniform powder. Organic chicken samples were then combined in equal weights and mixed to produce a pooled blank matrix. The homogenized chicken samples were first kept at -30 °C overnight to allow sublimation of residual dry ice, and then stored at -80 °C until analysis. Spiked chicken samples were prepared by adding 100 µL of the working solution, containing all analytes under study at their respective designated
concentrations, and 30 µL of the internal standard working solution to 2.0 g of homogenized pooled chicken blank matrix. Samples were vortexed manually for one minute, then placed on a benchtop agitator for one hour. Samples were then stored at 4 °C overnight, so as to allow for binding equilibria between matrix and analytes to be established. Following overnight refrigeration, the chicken samples were placed on the benchtop agitator for one hour prior to extraction to allow samples to reach room temperature. Next, 6 mL of water was added to each individual spiked chicken sample. Samples were then vortexed for one minute to attain a homogeneous consistency suitable for volumetric dispensing of 1500 μL of the diluted chicken samples into 96 well-plates.
Automated SPME ProcedureThe Concept 96 robotic sample preparation station (Professional Analytical System (PAS) Technology, Magdala, Germany) used in this work for SPME sample preparation, as shown in Figure 1, is a fully automated, software-operated, off-line bench-top station that executes all steps of the SPME protocol.28 The robotic arm of the Concept 96 is fitted with an SPME brush made of 96 SPME pins which are compatible with standard commercial 96-well-plates, thus allowing high throughput extraction of 96 samples simultaneously. The automated SPME workflow used in this method consisted of the following steps: (1) conditioning of the SPME coating for 30 minutes with MeOH:H2O (50:50, v/v); (2) static extraction for 60 minutes from 1.5 mL of diluted chicken tissue samples spiked with the target compounds; (3) rinsing of coating in water for 10 seconds so as to remove any loosely attached matrix constituents; and (4) solvent desorption in 400 μL of MeOH:MeCN:H2O (37.5:37.5:25, v/v/v) for 20 minutes. The final extracts from Step 4 were then transferred to the LC/MS/MS autosampler for injection and further analysis.
Table 1. LC Method and MS Source Conditions.
LC Conditions
LC Column Acquity UPLC HSS T3, 100 X 2.1 mm, 1.8 µm
Mobile Phase A 0.1% Formic Acid in Water
Mobile Phase B0.1% Formic Acid and 10 % Methanol in Acetonitrile
Mobile Phase Gradient
Start at 3% Mobile Phase B and Hold it for 1 Min, Then Increase B to 100% in 11 Min, and Keep at 100% B For 2 Mins. Finally Equilibrate The Column at Initial Condition for 3 Min.
Column Oven Temperature 40 °C
Auto Sampler Temperature 4 °C
Injection Volume 3.0 µl
MS Source ConditionsESI Voltage (Positive) 5000 V
ESI Voltage (Negative) -4000V
Drying Gas 120
Nebulizer Gas 200
Source Temperature 400 °C
HSID Temperature 320 °C
Detection Mode Time-managed MRM™
Figure 1. Concept 96-SPME device equipped with 96 SPME pins. Optimized conditions (time, agitation, solvent, and volume) for conditioning, extraction, rinsing, and desorption steps are shown, respectively, under each agitator-station.
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Table 2. MRM Transitions.
Compound Name Polarity Precursor Ion Product Ions CE EV CCL2
UHPLC/MS/MS Method OptimizationIn this study, different columns and mobile phase conditions were evaluated to achieve better separation and sensitivity for the target compounds. It was found that three columns produced similar results with slightly different retention time profiles, and can thus be used for the separation of the compounds. The columns are: Waters Acquity C18 UPLC HSS T3 (1.8 µm, 2.1 x 100 mm), PerkinElmer Brownlee SPP Phenyl-Hexyl (2.7 µm, 2.1 x 100 mm), and Restek Raptor Biphenyl (2.7 µm, 2.1 x 100 mm). It was also found that utilizing 10% methanol in acetonitrile as the organic mobile phase provided the best overall performance in terms of analyte peak shape, retention and mass signal intensity. In particular, signal intensity was improved for the late-eluting compounds when 10% methanol was present in the organic mobile phase.
Traditional MRM method development is not suitable for the analysis of a large number of analytes, such as 73 drug residues, in a single run. It is both time-consuming and labor intensive to input all the mass transitions into a method manually. More importantly, the advanced time-managed-MRM acquisition method can intelligently utilize the retention time of a chromatographic peak, and automatically optimize the residence time of the MRM to achieve the best quantitative data. Therefore, a time-managed-MRM is applied for method development in this study to improve efficiency, data quality and method performance. The fast polarity switching capability of the mass spectrometer also provided high detectability for both positive and negative ions in a single analytical run.
Matrix EffectsMatrix effects (MEs) are a major concern in bioanalytical method development and validation using LC/MS/MS. As a result of the complexity of animal tissue composition, interferences from
co-eluting matrix components can alter the ionization efficiency of target analytes, leading to either suppression or enhancement of signals.29,30 These matrix effects can dramatically influence the qualitative and quantitative performance of the method. To overcome MEs, several approaches can be used, such as sample dilution, intensive sample clean-up, using alternative ionization sources, as well as the use of matrix matched calibration curves and isotopically labeled internal standards. While, the ideal internal standard for each analyte is a stable isotope labeled form of the target analyte, using stable isotopically labeled internal standards (IS) for each analyte is not practical in multiresidue methods owing to aspects of availability, cost, and convenience. Therefore, one of the best approaches to reduce MEs is to employ a sample preparation technique that allows isolation and enrichment of a wide range of analytes, while at the same time, delivering a high degree of sample clean-up. In this regard, SPME has consistently demonstrated its ability to isolate and enrich a wide variety of analytes with minimal coextraction of matrix macromolecules.31–33
Figure 2. Matrix effects (%) for target analytes in chicken tissue plotted vs. retention time.
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In this study, MEs were evaluated by comparing the slopes of calibration curves obtained from reagent-only (RO) samples, with slopes obtained from matrix matched standards. MEs (%) for each analyte were calculated by the equation:
ME% = (slope of matrix-matched calibration curve − slope of RO calibration curve) × 100%/slope of RO calibration curve.
When the percentage difference is positive, there is a signal enhancement effect, whereas a negative value indicates signal suppression effect. As shown in Figure 2 and Table 3, matrix effect values for the majority of compounds were less than 20%. Only florfenicol amine and closantel displayed significant signal suppression (23% and 22% respectively), while desethylene ciprofloxacin and betamethasone displayed significant ion enhancement (23% and 30% respectively). The minimal matrix effects achieved in this study confirm that SPME provides an excellent sample clean-up. These results can be mainly attributed to the biocompatible PAN layer, which prevents interfering matrix components, such as proteins and lipids, from adsorbing to the SPME coating while still allowing target analytes to adsorb on the HLB coating.
Selectivity, Linearity, and Limit of QuantificationThe selectivity of the proposed method was evaluated by analyzing 10 blank chicken samples. No background peaks above a signal-to-
noise ratio of three were present at the same elution time as the target analytes, showing that the method is free of interferences. Analyte confirmations were evaluated by comparing the retention time and MRM chromatograms of each compound in the chicken samples with those of reference standards. At least two MRM ion pairs were used in the method to meet the regulatory requirements. The example MRM chromatograms of some veterinary drugs in a pooled chicken sample are illustrated in Figure 3.
Linearity was assessed for each analyte using matrix-matched calibration curves at seven concentration levels in the range of 0.1–3X, with three replicates at each level. Despite the use of only one internal standard, determination coefficient (R2) were higher than 0.99 for 95% of the analytes. Figure 4 shows typical examples of calibration curves obtained. The estimated limit of quantification (LOQ) for the method was established as the lowest point of the matrix-matched calibration curve that produced a response that is both accurate when compared to the expected value (calculated via linear regression), within a 70−120% range of the true concentrations, as well as precise (≤25% RSD). All analytes under study achieved LOQs that are below the ½ X, which is low enough to meet both U.S. regulatory tolerances and Canadian MRLs. These results demonstrated that the developed method can be applied for fast screening and quantification of multi-class veterinary drug residues in chicken samples at the regulatory levels.
Figure 3. Example MRM chromatograms of veterinary drugs in the pooled chicken sample spiked at 0.3X.
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Figure 4. Calibration curves for representative compounds, 5-hydroxy-thiabendazole, orbifloxacin, sulfaquinoxaline and tetracycline obtained from matrix-matched samples (analyte concentrations range from 0.1X to 3X).
Accuracy and PrecisionThe accuracy and precision of the method, with respect to intraday repeatability, were evaluated by analyzing pooled matrix blanks that were spiked at three validation levels- low, mid, and high (0.3X, 0.9X and 2.5X)- using six replicates per concentration. Accuracy was calculated based on estimated concentration values obtained from the linear regression equation of the matrix-matched calibration curve. As shown in Table 3, all analytes fell within the 70–120% range of the true concentrations of compounds, with RSD values ≤ 20% at the 0.3X, 0.9X and 2.5X validation levels, indicating that these
compounds can be quantitatively determined by the proposed method except for 6-Phenyl-2-thiouracil and haloxon. Despite the use of only one internal standard, these excellent accuracy and precision results demonstrate the effective sample clean-up offered by the method, as evidenced by the minimal matrix effects. The overlapped total ion chromatograms (TIC) shown in Figure 5 were generated from the six replicate chicken samples prepared by SPME at a low 0.3X spiking level, demonstrating good detectability and precision of the method.
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Table 3. Results of retention time, linearity, accuracy, intraday repeatability (%RSD), and % matrix effects (%ME) for target analytes in chicken tissue.
Market Sample AnalysisThe developed method was applied for the analysis of veterinary drugs in five chicken samples fortified with internal standards. None of the studied drugs were detected in the samples based on the retention time and mass spectra information (two MRM transitions) in comparison with the corresponding reference standards.
Figure 5. The overlapped TIC of the six replicate chicken samples prepared by SPME at 0.3X spiking level.
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Conclusions
Analytical method development for multiresidue veterinary drugs in animal tissues is highly challenging owing to the complexity of the matrix, the large number of analytes to be monitored, the wide variation in their physico-chemical properties, and the often very low concentration levels that the method must be able to detect. Therefore, investigations related to the safety of food products of animal origin requires the use of powerful qualitative and quantitative analytical instrumentation that allows trace quantification and identification of drug residues. In addition to powerful instrumentation, sample preparation and clean-up are critical in relation to sample turnaround and the reliability of the analytical outcome. With the growing public concern regarding the safety of food products of animal origin, the demand for reliable automated and high-throughput analytical procedures has become an unmet need around the globe.
In this study, a high-throughput MMM for veterinary drug analysis in chicken was developed by using a fully automated SPME method, and coupling a UHPLC system to a QSight 220 triple-quad mass spectrometer. The validation results, and the minimal matrix effects achieved in this work, demonstrate that the proposed method can be successfully applied for the fast screening as well as quantitative analysis of more than 70 veterinary drugs in chicken tissue, with LOQs well below regulatory levels.
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