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The Development and Validation of a Multi-class Method for Analysis of Veterinary Drug Residues in Milk using Ultrahigh Performance Liquid Chromatography Electrospray Ionization Quadrupole Orbitrap Mass Spectrometry Jian Wang, Daniel Leung, Willis Chow, James S. Chang, and Jon W Wong J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.5b04096 • Publication Date (Web): 29 Sep 2015 Downloaded from http://pubs.acs.org on October 5, 2015

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Journal of Agricultural and Food Chemistry

The Development and Validation of a Multi-class Method for Analysis of

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Veterinary Drug Residues in Milk using Ultra-high Performance Liquid

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Chromatography Electrospray Ionization Quadrupole Orbitrap Mass Spectrometry

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Jian Wang*1, Daniel Leung1, Willis Chow1, James Chang2 and Jon W. Wong3

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* To whom correspondence should be addressed

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[phone: (403) 338-5273; fax: (403) 338-5299; e-mail: [email protected]]

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1

Canadian Food Inspection Agency, Calgary Laboratory, 3650-36th Street N.W.,

Calgary, Alberta, T2L 2L1, Canada

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2

ThermoFisher Scientific, 355 River Oaks Parkway, San Jose, California, 95134, USA

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US Food and Drug Administration, Center for Food Safety and Applied Nutrition, 5100

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Paint Branch Parkway, College Park, Maryland, 20740, USA

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1

ACS Paragon Plus Environment


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ABSTRACT

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This paper presents the development and validation of a multi-class method for

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the analysis of veterinary drug residues in milk using ultra-high performance liquid

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chromatography electrospray ionization quadrupole Orbitrap mass spectrometry

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(UHPLC/ESI Q-Orbitrap). The 12 classes of veterinary drugs (a total of 125) included in

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this study were endectocides, fluoroquinolones, ionophores, macrolides, nitroimidazole,

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NSAIDs, β-Lactams, penicillins, phenicols, sulfonamides, tetracyclines and

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aminoglycosides. Veterinary drug residues in milk were extracted using a modified

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salting-out supported liquid extraction (SOSLE) method, which entailed the precipitation

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of milk proteins using an extraction buffer (oxalic acid and EDTA, pH 3) and acetonitrile,

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a salting-out acetonitrile/water phase separation using ammonium sulfate, and solid-phase

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extraction (SPE) using polymeric reversed-phase sorbent cartridges. The final extracts

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were concentrated and reconstituted into a buffer solution and analyzed using

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UHPLC/ESI Q-Orbitrap mass spectrometry. The developed method was validated using a

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nested experimental design to evaluate the method performance characteristics such as

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overall recovery, intermediate precision and measurement uncertainty. The method was

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able to quantify or screen up to 105 veterinary drugs from 11 different classes except

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aminoglycosides. The limits of quantification were as low as 1.0 µg/kg with an analytical

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range from 1.0 to 100.0 µg/kg in milk.

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KEYWORDS: UHPLC/ESI Q-Orbitrap; High resolution mass spectrometry; Veterinary

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drugs; Milk; Quantification; Identification; Measurement uncertainty

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INTRODUCTION Veterinary drugs have been widely used in medical and veterinary practice to treat

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and prevent animal diseases and to enhance growth rate and feed efficiency.

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Consequently, incorrect administration of the drug or improper withdrawal time after

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treatment could lead to the presence of drug residues in foods of animal origin. The

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residues in turn may provoke allergic reactions in some hypersensitive individuals or

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encourage the spread of drug-resistant pathogenic bacterial strains.1-3 Furthermore,

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veterinary drugs present in milk can have negative implications on microbial processes

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(for example cheese production). Therefore, milk should be free of veterinary drugs or

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contain concentrations less than the relevant maximum tolerance levels. There is a need

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for analytical methods that are capable of detecting and monitoring an increasingly large

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number of veterinary drug residues that are potentially used for food production.4

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In general, veterinary drug residues in food are determined through biological

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screening methods such as microbial inhibition tests, immunochemical methods , etc, and

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quantitative and confirmatory methods such as liquid chromatography coupled to mass

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spectrometry (LC-MS). Veterinary drug residues, which were typically in a group of less

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than 20 compounds, were analyzed historically based on a single-class or related families.

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A single-class method was relatively easy to optimize for both extraction and instrument

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parameters due to the similar physical and chemical properties of veterinary drugs from

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the same group. However, in the last few years, there were an increasing number of

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publications on multi-class methods for analysis of veterinary drugs in food using LC-

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MS/MS, LC TOF-MS (time-of-flight) and LC Orbitrap MS instruments. Examples of

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procedural methods that have been reported for multi-veterinary drugs at various

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concentration levels include molecular weight cut-off filters (3 kD) for milk (150

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veterinary drugs);5 liquid/liquid extraction for honey (42 antibiotics);6 polymer-based

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sorbent solid-phase extraction for egg, milk, animal tissues (100 veterinary drugs);7,8 and

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salting-out acetonitrile/water extraction with or without dispersive solid-phase extraction

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cleanup (i.e. Quick Easy Cheap Effective Rugged Safe or QuEChERS) for animal tissues

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(41 veterinary drugs),9 milk (21 veterinary drugs),10 honey (54 veterinary drugs),11 and

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milk (59 veterinary drugs).11 Recently, Kaufmann et al. published a multi-residue method

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of over 100 veterinary drugs in milk, which introduced a novel extraction and cleanup

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technique, i.e. salting-out supported liquid extraction (SOSLE), to extract both polar and

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relatively non-polar veterinary drugs in a single sample preparation procedure.4

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In our current study, we further explored the applications of QuEChERS and

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SOSLE for a single analysis of 125 veterinary drugs from 12 different classes in milk,

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which were monitored under the Canadian National Chemical Residue Monitoring

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Program. This paper discusses the respective advantages of either QuEChERS or SOSLE

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in terms of method simplicity or inclusive coverage of veterinary drugs for quantification

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or screening in a single analysis. SOSLE was further modified and was adopted as the

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final extraction method for validation. Furthermore, we employed the same UHPLC/ESI

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Q-Orbitrap instrument parameters that we used for the determination of 451 pesticides in

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fruits and vegetables12 for analysis of the veterinary drugs in milk. The whole idea was to

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simplify the routine practice of instrument operations. The same Q-Orbitrap parameters,

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LC column, LC mobile phases and LC gradient profile could be used to analyze either

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pesticides or veterinary drugs in various matrices for different monitoring programs.

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Finally, this paper presents a multi-class method using UHPLC/ESI Q-Orbitrap

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MS along with SOSLE to quantify or screen up to 105 veterinary drug residues from 11

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different classes (except aminoglycosides) in milk at low parts-per-billion (ppb)

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concentration levels. Based on the method performance, the UHPLC/ESI Q-Orbitrap MS

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method proved to be robust and sensitive enough to determine over 100 veterinary drug

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residues of interest from 1.0 to 100.0 µg/kg in milk. The method allows for high

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throughput testing of routine samples, which greatly benefits monitoring programs, while

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satisfying regulatory purposes for analysis of a number large of veterinary drugs in a

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single analysis.

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MATERIALS and METHODS Materials and Reagents. Five batches of whole milk were collected from five

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different local farms. All milk samples, which were tested free of the veterinary drug

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residues using the developed UHPLC/ESI Q-Orbitrap method in current study, were kept

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at -20 °C. Pierce LTQ ESI positive ion calibration solution (10 mL) was purchased from

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ThermoFisher Scientific (Rockford, IL, USA). The calibration solution, which includes

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n-butylamine (m/z 74), caffeine (m/z 195 and its fragment m/z 138), Ultramark 1621 (m/z

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1022, 1122, 1222, 1322, 1422, 1522, 1622, 1722, 1822) and MRFA (m/z 524), was used

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to tune and calibrate the Q-Orbitrap. Ammonium acetate (reagent grade), ammonium

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sulfate (reagent plus, > 99.0%), formic acid (LC-MS grade, ~ 98%), ammonium formate

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(for mass spectrometry, > 99.0%), oxalic acid (reagent plus, > 99.0%), ammonium

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hydroxide (28-30%), and LC-MS acetonitrile (Chromasolv, 2.5 L) were purchased from

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Sigma-Aldrich Corp (MO, USA). Acetic acid (glacial acetic acid, reagent grade, 99.7%),

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acetonitrile (distilled in glass), methanol (distilled in glass), and EDTA disodium salt

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were obtained from Caledon Laboratories Ltd (Ont, Canada). Water (18.2 MΩ⋅cm) used

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for reagent and sample preparation was obtained from a Barnstead Nanopure system

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(Thermo Scientific, OH, USA). Veterinary drug standards (Table 1, column 1) were

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obtained from Sigma-Aldrich Corp or Toronto Research Chemicals (Ont, Canada).

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Internal standards dimetridazole-d3, HMMNI-d3, ipronidazole-d3, ipronidazole-OH-d3,

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and ronidazole-d3 were purchased from Sigma-Aldrich Corp. A LC vial was a 0.45 µm

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PVDF Syringeless Filter Device Mini-UniPrep with polypropylene housing (GE

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Healthcare UK Limited, UK). ENVIRO-CLEAN QuEChERS Mylar pouches that contain

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6 g MgSO4 and 1.5 g sodium acetate were purchased from UCT Inc (PA, USA). OASIS

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HLB Plus 225 mg cartridges were purchased from Waters Corp (MA, USA).

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Insert Table 1 here.

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Preparation of Standard Solutions. Individual veterinary drug standard stock

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solutions were generally prepared at a concentration of 1000 or 2000 µg/mL in methanol,

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acetonitrile or water. Intermediate veterinary drug working solutions were prepared at 5.0

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µg/mL in methanol from stock solutions. Stock and intermediate solutions were stored at

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-20 °C. A six-level veterinary drug standard mix working solution was prepared by

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transferring 0.1, 0.5, 2.0, 4.0, 6.0 and 10.0 mL of 5.0 µg/mL into six separate 50 mL

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volumetric flasks for their respective concentration levels, and then making up to volume

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with acetonitrile. The resulting concentrations were 0.010, 0.050, 0.200, 0.400, 0.600 and

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1.000 µg/mL, which were used to construct the matrix-matched standard calibration

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curves. Four-level sample spike veterinary drug standard working solutions were

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prepared by transferring 4.0, 10.0, 20.0 and 32.0 mL of 5.0 µg/mL into four separate 100

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mL volumetric flasks and making up to volume with acetonitrile for their respective

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concentration levels. The resulting concentrations were 0.200, 0.500, 1.000 and 1.600

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µg/mL, which were used for sample fortification. Internal standard working solutions (5.0

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µg/mL and 0.5 µg/mL) including dimetridazole-d3, HMMNI-d3, ipronidazole-d3,

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ipronidazole-OH-d3, and ronidazole-d3 were prepared in acetonitrile. All working

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solutions were stored at 4 °C.

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UHPLC/ESI Q-Orbitrap Parameters UHPLC/ESI Q-Orbitrap system consisted of an Accela 1250 LC pump and an

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Accela open autosampler coupled with a Q-Exactive mass spectrometer (ThermoFisher

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Scientific, Germany). The system was controlled by Xcalibur 2.2 software. The

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UHPLC/ESI Q-Orbitrap instrument parameters were the same as those we used for the

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determination of 451 pesticides in fruits and vegetables.12

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(a) Ultra-high Pressure Liquid Chromatography.

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UHPLC mobile phases A and B consisted of 4 mM ammonium formate and

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0.10% formic acid in water and methanol, respectively. The UHPLC column utilized was

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a Hypersil Gold, 100 mm × 2.1 mm, 1.9 µm column (Thermo Scientific, USA). UHPLC

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guard column was an Accucore aQ 10 × 2.1 mm, 2.6 µm Defender cartridge (Thermo

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Scientific, USA). The UHPLC gradient profile and flow rate are shown in Figure 1.

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Column oven temperature was set at 45 °C and auto-sampler temperature was set at 5 °C.

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Injection volume was 5 µL and the total run-time was 14 min.

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Insert Figure 1 here

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(b) Q-Orbitrap Parameters The Q-Exactive ion source was equipped with a heated electrospray ionization

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(HESI) probe and the Q-Orbitrap was tuned and calibrated using positive LTQ calibration

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solution once a week. The Q-Exactive was operated in either Full MS-SIM or Full

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MS/dd-MS2 in positive mode. In Full MS-SIM, the Q-Orbitrap performed full MS scan

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without HCD (high energy collision dissociation) fragmentation for quantification. The

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full MS scan range was set from m/z 80 to 1100 (0 to 12.0 min). The mass resolution was

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set to 70,000 FWHM at m/z 200 and the instrument was tuned for maximum ion

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throughput. AGC (automatic gain control) target or the number of ions to fill C-Trap was

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set at 1.0E6 with a maximum injection time (IT) of 250 milliseconds. All quantitative

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data in this study were acquired using Full MS-SIM mode. Q-Orbitrap MS means the Q-

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Exactive that was operated in Full MS-SIM mode for quantification throughout the text.

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Targeted identification was achieved by full scan MS and if a targeted veterinary

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drug was present, its precursor ion scan, provided by an inclusion list, triggered a data-

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dependent MS2 (dd-MS2) scan. During full MS scan, the mass resolution was set at

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70,000 FWHM, AGC target at 1.0E6, maximum IT 250 ms, and scan range from m/z 80

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to 1100. If the targeted compound was detected within the 10 ppm mass error window

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and achieved by a designated intensity threshold (i.e., setting of 1.7E5), the precursor

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ions in the inclusion list were then isolated by the quadrupole, and sent to the HCD

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collision cell for fragmentation via the C-trap. The inclusion list consists of precursor

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ions that are of interest for targeted identification, and is provided in Table 1 (columns 6-

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9). The precursor ions were fragmented with stepped normalized collision energy (NCE)

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to generate the resulting dd-MS2 product-ion spectra. At this stage, the mass resolution of

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the Orbitrap analyzer was set at 35,000 FWHM, AGC target at 2E5, maximum IT 120

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ms, isolation window 1.0 m/z, NCE 40 ±50%, underfill ratio 10%, intensity threshold

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1.7E5, apex trigger 2 to 4 s, and dynamic exclusion 10.0 s.

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Other Q-Exactive generic parameters were sheath gas flow rate set at 60, Aux gas

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flow rate 30, Sweep gas flow rate 2, Spray voltage 3.50 kV, Capillary temp 350 °C, S-

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lens level 55.0 and Heater temp 350 °C.

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During the initial method development stage, 125 veterinary drugs and 5

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isotopically labeled standards (Table 1, column 1) were injected individually onto the

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UHPLC column (Hypersil Gold, 100 mm × 2.1 mm, 1.9 µm) and the Q-Orbitrap was

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operated in Full MS/dd-MS2 positive mode to determine the retention time and identity of

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each veterinary drug.

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Method Development on Sample Extraction.

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Method A: This is the conventional QuEChERS method after slight modification

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of a published method for the determination of 59 veterinary drug residues in milk11 with

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references to other methods.13,14 QuEChERS performed in this study means a procedure

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that consists of two steps throughout the text, i.e. Step 1 salting-out acetonitrile/water

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extraction, and Step 2 dispersive solid-phase extraction (d-SPE) clean-up.

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Step 1 Extraction. Triplicate milk samples (10 g/sample) were weighed into 50

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mL centrifuge tubes (VWR International, Canada). Five hundred µL of 1 ppm working

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solution was spiked to each sample, which was 50.0 µg/kg of veterinary drugs equivalent

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in sample. After 15 minutes, 10 mL of a mixture of acetonitrile and acetic acid (99+1,

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v/v) was added to the sample. The centrifuge tube was capped and vortexed to mix for 45

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s, and then 6 g MgSO4 and 1.5 g sodium acetate (ENVIRO-CLEAN QuEChERS Mylar

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pouch) were added to the sample. The sample mixture was capped and shaken at 1500

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rpm for 1 min using a 2010 Geno/Grinder (SPEX SamplePrep, Metuchen, NJ, USA) and 11



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then centrifuged at 3000 rpm (~2100 × g) for 3 min using a centrifuge (Allegra X-15R

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Centrifuge, Beckman Coulter Inc., CA, USA).

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Step 2 d-SPE Clean-up. Seven mL of supernatant from Step 1 was transferred to a

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15 mL centrifuge tube that contained 500 mg of end-capped C18 (obtained from the Sep-

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Pak tC18, Waters Corp.). Five mL of hexane (pre-saturated with acetonitrile) was added

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to the supernatant, which was shaken for 30 second by hand. The mixture was

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centrifuged at 3000 rpm (~2100 × g) for 3 min. The top hexane layer was aspirated to

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waste. Three mL of sample extracts were transferred into individual 5 mL PYREX brand

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centrifuge tubes, which was pre-calibrated with 1 mL volume accuracy (VWR

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International, Edmonton, AB, Canada). The sample extracts were evaporated to 0.1-0.2

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mL, which took approximately 20 min, using an N-EVAP nitrogen evaporator

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(Organomation Associates Inc., Berlin, MA, USA) at 50 °C under a stream of nitrogen.

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The extracts were reconstituted by making up to 0.5 mL with acetonitrile and then to 1.0

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mL with 0.1 M ammonium acetate. The extracts were vortexed for 30 s. Five hundred µL

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of sample extracts was transferred into a 0.45 µm PVDF Syringeless Filter Device Mini-

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UniPrep vial (Whatman, GE Healthcare Life Science, UK) and pressed to filter. Sample

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extracts were ready to be injected into UHPLC/ESI Q-Orbitrap MS for analysis.

229 230

Method B: This is a procedure that combines salting-out acetonitrile/water

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extraction and SPE clean-up, which is known as salting-out supported liquid extraction

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(SOSLE).4 SOSLE performed in this study means a procedure that consists of two steps,

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i.e. Step 1 salting-out acetonitrile/water extraction, and Step 2 solid-phase extraction

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(SPE) clean-up throughout the text.

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Step 1 Extraction. The extraction step of this method was based on SOSLE4 with

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a slightly modification. Triplicate milk samples (5 g/sample) were weighed into 50 mL

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centrifuge tubes (VWR International, Canada). Two hundred fifty µL of 1 ppm working

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solution was spiked to each sample, equivalent to 50.0 µg/kg of veterinary drugs in

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sample. After 15 minutes, 5 mL of extraction buffer and 10 mL of acetonitrile were added

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to the sample. The extraction buffer contained 0.86 % oxalic acid and 0.74 % EDTA

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disodium salt, and its pH was adjusted to 3.0 using ammonium hydroxide. The sample

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mixture was capped and shaken for 30 seconds by hand and then centrifuged at 3000 rpm

243

(~2100 × g) for 5 minutes using a centrifuge. The supernatant was transferred into another

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50 mL centrifuge tube, to which 1 g of ammonium sulfate was added. The sample

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mixture was mixed for 2 min by hand, and then was left to stand for 2 min. A phase

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separation was observed from the mixture and two layers were obtained.

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Step 2 SPE Clean-up. Instead of diatomaceous earth cartridges as in the original

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SOSLE,4 polymeric reversed-phase sorbent such as OASIS HLB Plus cartridges were

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used for SPE clean-up. OASIS HLB Plus 225 mg cartridges, which were paired with 25

250

mL of syringe barrels, were set up for solid-phase extraction using a Visiprep 24-port

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SPE Vacuum Manifold (Sigma-Aldrich Corp). The cartridges were preconditioned

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sequentially with 10 mL of methanol, 10 mL of water and 2 mL of extraction buffer. The

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sample extracts from Step 1 were separated into three layers after centrifugation (3000

254

rpm or ~2100 × g, 3 min). The top acetonitrile layer (~ 10 mL) was transferred to a 16 ×

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125 mm disposable test tube and set aside for later to be loaded onto OASIS HLB

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cartridges. The lower aqueous layer was transferred onto the preconditioned Oasis HLB

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cartridges under vacuum at -2 to -3 inHg with a flow rate of ~1 mL/min. The very thin

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middle white layer (~1 or 2 mm), which was supposed to be fat from the sample, was

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discarded and should be avoided from being transferred onto the cartridges. Oasis HLB

260

cartridges were rinsed further with 2 mL of extraction buffer and allowed to run dry.

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Finally, the top acetonitrile layer, which served as eluting solvent as well, was dispensed

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onto the OASIS HLB cartridges. The flow (~1 mL/min) of the eluting solvent was

263

maintained under vacuum at -2 to -3 in Hg and the eluent was collected into a 16 × 125

264

mm disposable glass test tube. The OASIS HLB cartridges were run dry under vacuum.

265

The eluent was capped and inverted to mix a few times by hand. Two hundred fifty µL of

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sample extracts was transferred to a 0.45 µm PVDF Syringeless Filter Device Mini-

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UniPrep vial and then 250 µL of 0.1 M ammonium acetate was added. The Mini-UniPrep

268

unit was capped, mixed and pressed to filter. Sample extracts were ready to be injected to

269

UHPLC/ESI Q-Orbitrap MS for analysis.

270 271

Method C: This is a SOSLE method, which is modified from Method B. It

272

consisted of two steps, i.e. Step 1 salting-out acetonitrile/water extraction, and Step 2 SPE

273

clean-up.

274

Step 1 Extraction. This step is the same as that of Method B.

275

Step 2 SPE Clean-up. All procedures were the same as the Step 2 of Method B

276

until the last step. After the top acetonitrile layer was loaded onto the OASIS HLB

277

cartridges and the eluent was collected into a 16 × 125 mm disposable glass test tube, an

278

additional 5 mL of methanol was dispensed onto the cartridges to elute further. The

279

eluent was collected into the same test tube. The OASIS HLB cartridges were run dry

280

under vacuum. The eluent (~ 15 mL) was capped and inverted to mix a few times. Two

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hundred fifty µL of sample extracts was transferred to a 0.45 µm PVDF Syringeless Filter

282

Device Mini-UniPrep vial and then 250 µL of 0.1 M ammonium acetate was added. The

283

Mini-UniPrep unit was capped, mixed and pressed to filter. Sample extracts were ready to

284

be injected to UHPLC/ESI Q-Orbitrap MS for analysis.

285 286

Method D: This is a SOSLE method, which is modified from Method C. It

287

consisted of two steps, i.e. Step 1 salting-out acetonitrile/water extraction, and Step 2 SPE

288

clean-up.

289

Step 1 Extraction. This step is the same as that of Method B or C.

290

Step 2 SPE Clean-up. The procedure was the same as the Step 2 of Method C,

291

except the final extracts were concentrated three times. Right after the SPE step, 3 mL of

292

eluent was transferred into individual 5 mL PYREX brand centrifuge tubes, which was

293

pre-calibrated with 1 mL volume accuracy (VWR International, Canada). The sample

294

extracts was evaporated to 0.1-0.2 mL, which took approximately 20 min, using an N-

295

EVAP nitrogen evaporator (Organomation Associates Inc., USA) at 50 °C under a stream

296

of nitrogen. Then the extracts were reconstituted by making up to 0.5 mL with

297

acetonitrile and then to 1.0 mL with 0.1 M ammonium acetate. The extracts were

298

vortexed for 30 s. Five hundred µL of sample extracts was transferred into a 0.45 µm

299

PVDF Syringeless Filter Device Mini-UniPrep vial and pressed to filter. Sample extracts

300

were ready to be injected to UHPLC/ESI Q-Orbitrap MS for analysis.

301 302 303

To calculate an absolute recovery (%) or extraction efficiency of veterinary drugs from each individual method A to D, the UHPLC/ESI Q-Orbitrap MS response (peak

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area spiked before sample extraction) of a veterinary drug extracted from a sample was

305

compared to that (peak area spiked after sample extraction and clean-up) of a veterinary

306

drug prepared in a blank sample extract at 50 µg/kg equivalent in sample. That is:

307

Absolute Recovery (%) =

௉௘௔௞ ௔௥௘௔ ௦௣௜௞௘ௗ ௕௘௙௢௥௘ ௉௘௔௞ ௔௥௘௔ ௦௣௜௞௘ௗ ௔௙௧௘௥

× 100

308 309 310

Nested Experimental Design and Method Validation.

311

The Method D was adopted as the final method for validation in this study.

312

Fortification experiment. Milk samples (5.0 g/sample) were weighed into

313

individual 50 mL polypropylene centrifuge tubes (VWR International, Canada). Two

314

hundred fifty µL per four-level sample spike veterinary drug standard working solution(s)

315

was added into four centrifuge tubes to provide 10.0, 25.0, 50.0 and 80.0 µg/kg of

316

veterinary drugs equivalent in sample, followed by the addition of 25 µL of 5.0 µg/mL

317

internal calibration standard working solution (25.0 µg/kg equivalent in sample). Then

318

the samples were preceded for extraction and clean-up.

319 320

Preparation of Matrix-matched Calibration Standards and Calculation. Matrix-

321

matched calibration standards were prepared by adding standards and internal standards

322

to blank sample extracts that were processed through extraction and clean-up. A blank

323

milk sample (5.0 g) was weighed into a 50 mL centrifuge tube and the sample was

324

processed through the extraction and clean-up as described in Method D. Before

325

reconstitution, 100 µL of each six-level veterinary drug standard mix working solution

326

and 50 µL of 0.50 µg/mL internal calibration working solution were added the individual

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sample extracts to provide 1.0, 5.0, 20.0, 40.0, 60.0 and 100.0 µg/kg per standard and

328

25.0 µg/kg per internal standard equivalent in sample. The rest of the preparation was the

329

same as in Method D.

330 331

Matrix-matched standard calibration curves for each individual veterinary drug

332

were constructed using TraceFinder 3.2 (optimized for Environmental and Food Safety)

333

software. Concentration, µg/kg (ppb), versus the ratio (analyte area/IS area) of each

334

individual veterinary drug was plotted. Five commercially available deuterium labeled

335

standards, i.e. dimetridazole-d3, HMMNI-d3, ipronidazole-d3, ipronidazole-OH-d3, and

336

ronidazole-d3 were used as internal standards for quantifying their respective native

337

compounds, and dimetridazole-d3 was utilized for all other veterinary drugs that had no

338

isotopically labelled standards available. In general, a quadratic function was applied to

339

the calibration curves based on the line of best fit. Occasionally, linear regression may be

340

used for quantification. The 1/x weighting was used to improve the accuracy for the

341

quantification of veterinary drugs at low concentrations. Responses for the unknown

342

concentration or fortified samples were compared to the standard curves to calculate the

343

amount of veterinary drug residues, µg/kg (ppb), in samples. Matrix-matched calibration

344

standards were prepared fresh for each batch of samples.

345 346

The method was validated according to the nested experimental design, which

347

was described elsewhere.15 The main factors of variances associated with the method

348

performance or measurement uncertainty of an in-house validated method were

349

concentrations or spiked levels of analytes, matrix effects, day-to-day variation and

17



Page 18 of 50

350

within day variation of the method. The last two factors were designated as the

351

intermediate precision. In this study, there were a total of 5 batches of milk samples from

352

5 different local farms. For each matrix, samples were spiked at 10.0, 25.0, 50.0, and 80.0

353

µg/kg, in triplicate. Spike experiments were repeated on two different days or two

354

analysts. Overall recovery, intermediate precision and measurement uncertainty were

355

calculated using a combined computer program that consisted of SAS codes (SAS

356

Software Release 9.3, SAS Institute Inc., USA) along with a Microsoft Excel (Microsoft

357

Office 2010) workbook.

358

18


Page 19 of 50

359


RESULTS AND DISCUSSION

360

Ultra-high Performance Liquid Chromatography. For practical reasons, the

361

same UHPLC/ESI Q-Orbitrap instrument parameters used for the determination of 451

362

pesticides in fruits and vegetables12 were utilized for the analysis of veterinary drugs in

363

milk. This includes the same Q-Orbitrap parameters, LC column, LC mobile phases and

364

LC gradient profile. During the method development stage, 125 veterinary drugs and 5

365

isotopically labeled standards (Table 1, column 1) were injected individually onto the

366

UHPLC column (Hypersil Gold, 100 mm × 2.1 mm, 1.9 µm) and the Q-Orbitrap was

367

operated in Full MS/dd-MS2 positive mode. These data ensured that the correct retention

368

times were assigned to the respective compounds, and to determine the identities of

369

veterinary drugs in a mixture of standards. Full MS/dd-MS2 data are critical to

370

differentiate isobaric compounds. The mobile phases were methanol and water, each

371

containing 4 mM ammonium formate and 0.10% formic acid, and the gradient profile is

372

shown in Figure 1A. Veterinary drugs from a particular class or group demonstrated a

373

similar chemical property in terms of liquid chromatographic retention (Figure 1B). For

374

example, the very polar aminoglycosides were hardly retained on the column and all were

375

eluted as a group around 0.91 min. Endectocides and ionophores, which are relatively

376

non-polar, were well retained on the column and all were eluded between 9.31 and 10.68

377

min. The elution pattern of the 125 veterinary drugs showed up as clusters of individual

378

classes as depicted in Figure 1B. Aminoglycosides were excluded from the method due to

379

their poor chromatographic retention.

380

19



381

Page 20 of 50

The elution pattern on the reversed-phase column may possibly be perceived as an

382

indication of the extraction efficiency of the method for a class of veterinary drugs. In

383

other words, the retention time (RT) of a veterinary drug may be correlated to the method

384

extraction efficiency. For example when using Method D described in MATERIALS and

385

METHODS, the polar compounds with poor chromatographic retention (< 1.28 min),

386

remained as challenges to be extracted from milk samples. Amoxicillin, cefadroxil and

387

cephalosporin C (Table 1, Section D), which were eluded between 1.03 and 1.10 min,

388

were likely not recovered by the method. Desacetyl cephapirin, etanidazole, nimorazole

389

and sulfaguanidine (Table 1, Section A), which were eluded between 1.05 and 1.28 min,

390

had recoveries from 15.0 to 72.4%. Sulfanilamide (RT: 1.25 min and recovery: 97.1%)

391

was the only one exception from this observation. In general, the well-retained

392

compounds (RT > 1.5 min) showed good recoveries (Table 1, Section A and B). This is

393

to assume that the chromatographic retention of a veterinary drug as a result of its

394

polarity may serve as an indicator of its extraction efficiency of a method. However, this

395

assumption may not be necessary true vice versa since there are many other factors such

396

as matrix effects, solvent and solid phase extract sorbent used that contribute to the

397

extraction efficiency.

398 399

Under most circumstances, an extracted ion of a veterinary drug was presented as

400

a single LC peak. Most veterinary drugs had baseline separation from others, resulting

401

from the superior resolving power of both UHPLC and Q-Orbitrap MS. Some were co-

402

eluted because they were isobaric compounds such as ampicillin and cephradine (Table 1,

403

Section C). Veterinary drugs were eluted between 1.0 and 11.0 min and their peak shapes

20


Page 21 of 50


404

were of Gaussian distribution with a baseline peak-width of 5 to 10 seconds. The

405

retention times were reproducible under ± 0.2 min within- and between-batches for most

406

of the veterinary drugs. With the scan rate of 3 Hz for the resolution of 70,000 FWHM at

407

m/z 200, the Q-Orbitrap provided sufficient data points for quantification. For example, at

408

least 18 data points across the chromatographic peak were generated with a 6 seconds

409

(0.1 min) baseline peak-width.

410 411

Q-Orbitrap Mass Spectrometry. When operated in Full MS-SIM mode with a

412

range from m/z 80.0 to 1100.0 and 70,000 FWHM resolution at m/z 200, the Q-Orbitrap

413

acquired full MS scan data for the quantitation of veterinary drugs. The quantitative

414

results are provided in Table 1 (columns 10-12). When operated in Full MS/dd-MS2

415

mode, the Q-Orbitrap acquired product-ion spectra with accurate mass measurements for

416

identification according to a list of targeted exact masses (Table 1, columns 6-9). A

417

three-step normalized collision energy (NCE) of 40 ± 50% (i.e. the center energy 40

418

NCE; plus one above, 60 NCE; and one below, 20 NCE) was used for the fragmentation

419

of precursor ions in the high-energy collision dissociation (HCD) cell to produce product

420

ions for identification. The product ions created in the three-step were collected

421

sequentially in the HCD and transferred together to the Orbitrap mass analyzer for single

422

scan detection. Stepped NCE did not work for all of the veterinary drugs because not all

423

were optimally fragmented in the 40 ± 50% NCE range. As a routine practice from our

424

previous experience,12 the Full MS/dd-MS2 data of individual veterinary drugs were

425

acquired to determine their identities in a mixture. The dataset was also intentionally

426

collected to build a “Compound Database” or automated searchable exact mass spectral

21



Page 22 of 50

427

library for identification or screening purposes. However, in the current study, we

428

focused on the quantitative aspect of UHPLC/ESI Q-Orbitrap applications for veterinary

429

drugs.

430 431

Method Development on Sample Extraction. We began with a list of 125

432

veterinary drugs, which were included under the CFIA routine monitoring program for

433

determination of veterinary drug residues in milk. The plan was to develop a generic

434

extraction method to include 125 compounds in a single analysis, which previously was

435

achieved using multiple separate single-class methods, and to improve the monitoring

436

program efficiency. Method development was initiated with a conventional QuEChERS

437

but was gradually evolved into SOSLE, to include as many analytes as possible. Because

438

β-lactams, penicillins and tetracyclines are not stable molecules, their data for absolute

439

recovery comparisons during the method development are not included.

440 441

Method A was based on the conventional QuEChERS, which was slightly

442

modified from a published method for the determination of 59 veterinary drug residues in

443

milk11 with references13,14 therein. Step 1 utilized the typical salting-out

444

acetonitrile/water extraction, while Step 2 invoked the use of dispersive solid-phase

445

extraction (d-SPE) for clean-up to remove non-polar compounds such as fat in milk. The

446

method was very simple and fast. It worked well for endectocides, ionophores,

447

macrolides, nitroimidazole, NSAIDS and sulfonamides with absolute recoveries over

448

70% (Figure 2, left), but the method showed poor repeatability for quantification with

449

relative standard deviations as high as 40-50% (Figure 2, right). Fluoroquinolones (Figure

22


Page 23 of 50


450

2-B1) had recoveries ranging from 0.5 to 43.5%, while β-lactams, penicillins and

451

tetracyclines were not recovered at all (data not shown), as similar observations have

452

been reported elsewhere.11

453 454


455 456

In order to recover β-lactams, penicillins and tetracyclines, and to improve the

457

recoveries of fluoroquinolones, we looked into other methodologies. Kaufmann et al

458

developed a procedure known as salting-out supported liquid extraction (SOSLE), which

459

combines the salting-out acetonitrile/water extraction (Step 1) and solid-phase extraction

460

(SPE) cleanup (Step 2) to extract the veterinary drugs in milk 4. In SOSLE, both

461

acetonitrile and water, from which a phase separation was induced afterwards by salting-

462

out using ammonium sulfate, were used to ensure the quantitative extraction of analytes

463

that were distributed in either aqueous or acetonitrile layer. The mixture of acetonitrile

464

and water was capable of extracting a wide range of analytes from the matrix because it

465

provided significantly higher extraction efficiency for both polar analytes as well as non-

466

polar compounds.4 The recoveries of tetracyclines and fluoroquinolones were

467

significantly increased as a result of the addition of complexing agents like EDTA and

468

the use of ammonium sulfate rather than magnesium sulfate to prevent chelation to

469

inorganic salts and to induce a phase separation of the acetonitrile and water mixture. The

470

resulting heavier aqueous phase was loaded onto SPE cartridges which contained coarse-

471

grained kieselguhr (also known as diatomaceous earth). The compounds retained on the

472

cartridges were eluted with the supernatant organic phase (acetonitrile). Final extraction

23



Page 24 of 50

473

and elution of the analytes was achieved through additional volumes of acetonitrile. The

474

combined eluents were evaporated to produce the injection-ready extracts.4

475 476

Due to the relatively large volume of the final extracts (25 mL) resulting from the

477

45 mL/8.3 g bed size of CHROMABOND XTR columns (diatomaceous earth cartridges,

478

MACHEREY-NAGEL GmbH & Co. KG, Düren,Germany) in the original method4 and

479

poor repeatability of recoveries from these cartridges in our experiments (data not

480

shown), we modified the method for a more practical application. By switching to

481

polymeric reversed-phase sorbent SPE cartridges (i.e. OASIS HLB Plus 225 mg

482

cartridges), the clean-up process required a significant smaller volume of organic solvent

483

to elute and the results showed improved repeatability. The polymeric reversed-phase

484

sorbent cartridges have been found to be efficient in extracting both polar and relatively

485

non-polar veterinary drugs in milk, eggs and honey.16,17 As described in MATERIALS

486

and METHODS, in Step 1, Method B used an extraction buffer (0.86 % oxalic acid and

487

0.74 % EDTA, pH 3) and acetonitrile to precipitate proteins in milk. After removal of

488

protein by centrifugation, ammonium sulfate was added to the supernatant to induce a

489

phase separation of the sample mixture. In Step 2, the resulting heavier aqueous phase

490

was loaded onto the SPE cartridge so to retain polar compounds (such as β-lactams,

491

penicillins, tetracyclines, etc), followed by the top acetonitrile layer, which contained

492

relatively non-polar compounds (such as endectocides, ionophores, etc), to serve as the

493

eluting solvent to elute veterinary drugs on the cartridges. Figure 2 (A2-H2) illustrates

494

that Method B significantly improve the repeatability of recoveries for all veterinary

495

drugs. Figure 2 (A1-H1) also demonstrates that Method B increases the recoveries of

24


Page 25 of 50


496

fluoroquinolones but a decrease in recoveries for the ionophores, macrolides, NSAIDS,

497

while the recoveries of endectocides, nitroimidozles, phenicols and sulfonamides

498

remained unchanged. It was observed that the recoveries of some macrolides such as

499

neospiramycin I and spiramycin I decreased significantly, which might be as a result of

500

their degradation under acid conditions (pH 3).18

501 502

To improve the recoveries, Method C was modified from Method B by increasing

503

the amount of the eluting solvent in Step 2. Besides the ~10 mL of acetonitrile (top layer

504

after the phase separation) as the eluting solvent, an additional 5 mL of methanol was

505

added to elute any strongly retained veterinary drug residues on the cartridges. Figure 2

506

(A1-H1) reveals that the recoveries of all veterinary drugs from Method C were increased

507

as compared to Method B. Most of the recoveries increased from 60 or 80% to ~100%,

508

and RSDs were less than 10%, with the exception of a few fluoroquinolones and

509

macrolides. The results indicated that the additional 5 mL of methanol was critical to

510

improve the recoveries in SOSLE when polymeric reversed-phase sorbent cartridges

511

were used for clean-up.

512 513

In both Methods B and C, the final extracts were diluted 1:1 rather than to be

514

concentrated. These methods may not be applicable to detect low concentration levels of

515

veterinary drugs in milk samples. To lower the detection limits into low parts-per-billion

516

levels (such as 1 ppb), in Step 2, additional concentration and reconstitution steps were

517

implemented in the development of Method D from Method C. As observed from Figure

518

2, the recoveries of some veterinary drugs from Method D were slightly lower and RSDs

25



Page 26 of 50

519

higher than the results provided by Method C. Method D proved to be well suited for

520

analyzing milk samples fortified at 1 ppb concentration level (Table 1, column 13). The

521

final concentrations of veterinary drugs in vials (prior to UHPLC/ESI Q-Orbitrap MS

522

injection) from Method A to D were 150, 12.5, 8.33 and 50 ppb (or ng/mL), respectively.

523

Method D resulted in 6 times concentrated sample extracts compared to Method C.

524

Apparently, the concentration and reconstitution steps caused losses of some compounds

525

and led to slightly increased variation. Overall, in terms of recoveries and RSDs, Method

526

C provided the best performance among four methods. However, in order to detect

527

veterinary drugs at low concentration levels, especially at 1 ppb level, Method D was

528

chosen to be validated for determination of veterinary drugs in milk. It is worth to note

529

that Method D is also able to recover β-lactams, penicillins and tetracyclines as shown in

530

Table 1.

531 532

In summary, as shown in Figure 2, Method A (except for β-lactams, penicillins

533

and tetracyclines), C or D can serve as independent methods to be validated for various

534

applications depending on testing scopes and instrument sensitivity. For example,

535

Method A (i.e. QuEChERS) can potentially be validated and used for analysis of

536

endectocides, ionophores, macroldies, nitromidazole, NSAIDS, phenicols, sulfonamides

537

in milk or other matrices because it is simple and fast. For highly sensitive LC-MS

538

systems, Method C (i.e. SOSLE) is the best choice for all veterinary drugs due to its

539

method performance in terms of recovery and repeatability. Method D (i.e. SOSLE)

540

proves to be the most sensitive among all, and it was found it fit for the needs of using

26


Page 27 of 50


541

UHPLC/ESI Q-Orbitrap MS to quantify veterinary drug residues in milk at low ppb

542

concentration levels.

543 544

Matrix Effects. Sample matrix can either enhance or suppress the ionization of

545

veterinary drugs and its effects can vary from sample-to-sample, which ultimately affects

546

the UHPLC/ESI Q-Orbitrap MS quantitative results. To evaluate matrix effects, the

547

responses (peak areas) of veterinary drugs in sample extracts were compared to those

548

veterinary drug standards prepared in solvent buffer at 50 µg/kg equivalent in sample. In

549

general, about 74.0-76.0% veterinary drugs had ion suppression < 30% or ion

550

enhancement ≤ 20%, 2.1-4.2% were subjected to ion suppression ≥ 30%, and 20.8-24.0%

551

were subjected to ion enhancement > 20% in milk (Figure 3). Veterinary drugs that were

552

suppressed ≥ 30% included flunixin, roxithromycin and thiamphenicol. Veterinary drugs

553

that were enhanced > 20% included doramectin, eprinomectin B1a, ivermectin, HMMNI,

554

metronidazole, metronidazole-OH, nimorazole, ternidazole, sulfacetamide, sulfadiazine,

555

sulfaguanidine, sulfamerazine, sulfamethoxypyridazine, sulfathiazole, sulfisomidine,

556

sulfanilamide, 4-epitetracycline, chlortetracycline, oxytetracycline, cefapirin and

557

desacetyl cephapirin. Therefore, matrix-matched standard calibration curves along with

558

isotopically labeled standards (Figure 4) were used to minimize or compensate for matrix

559

effects to improve the accuracy of the UPLC/ESI Q-Orbitrap MS quantification. Five

560

commercially available deuterium labeled standards, i.e. dimetridazole-d3, HMMNI-d3,

561

ipronidazole-d3, ipronidazole-OH-d3, and ronidazole-d3 were used as internal standards

562

for quantifying their respective native compounds, and dimetridazole-d3 was utilized for

563

all other veterinary drugs. The calibration curves were observed to be linear or quadratic

27



Page 28 of 50

564

with coefficient of determinations (R2) ≥ 0.97. Because of matrix effects, ion source

565

contamination or other unidentified factors, the responses of some veterinary drugs either

566

decreased or increased slightly over time. To average out the response changes during the

567

course, matrix-matched standard calibration curves were constructed based on the two

568

injections, i.e. before and after spike samples, so as to improve the method performance

569

(Figure 4).

570 571

Insert Figures 3 and 4 here

572 573

Quantification and Method Performance. The UHPLC/ESI Q-Orbitrap MS

574

method was validated according to a nested design, reported elsewhere. To evaluate the

575

method performance characteristics including accuracy expressed as overall recovery,

576

intermediate precision and measurement uncertainty (MU),15 four factors such as

577

concentrations or spiked levels of veterinary drugs, matrix effects, day-to-day variation

578

and within-day variation (described in MATERIALS and METHODS), were considered.

579

For the 89 veterinary drugs that were chemically stable and were evaluated through the

580

fully nested experimental design, their method performance results are summarized in

581

Tables 1 Section A, and are illustrated in Figure 5. About 70.8% of the veterinary drugs

582

in milk had recoveries between 71% and 120%; 88.8% had intermediate precision ≤ 20%;

583

and 93.3% showed measurement uncertainty ≤ 50%. The Codex CAC/GL 71-200919

584

recommended that method performance criteria such as recovery of 70-120% and

585

repeatability ≤ 20% for quantitative analytical methods to support maximum residue limit

28


Page 29 of 50


586

for veterinary drug residues in foods. Therefore, Method D demonstrated the optimized

587

performance to quantify a majority of veterinary drugs listed in Table 1 Section A.

588


589 590 591

For the 13 veterinary drugs, primarily penicillins and β-lactams that were not

592

chemically stable and not able to go through the fully nested experimental design but

593

were still quantifiable, their method performance was summarized according to one-day

594

experiment and the results are listed in Table 1 Section B. In general, the method for

595

those veterinary drugs demonstrated a good repeatability with the exception of cefapirin

596

and some additional compounds that had a recovery 120% (Table 1, Section

597

B).

598 599

Table 1 Section C lists three veterinary drugs, ampicillin, cephradine and tylosin

600

B that can be screened but not quantified. This is becasue ampicillin and cephradine are

601

isobaric compounds and cannot be chromatographically separated, and tylosin B has no

602

standard commercially available to prepare calibration curves for quantification.

603 604

Aminoglycosides (a total of 15, Table 1 Section D) were poorly retained due to

605

their polarity on the reversed-phase C18 column with retention times ~ 0.91 min.

606

Amoxicillin, cefadroxil, cefalonium, cephalexin and cephalosporin C (a total of 5, Table

607

1 Section D) showed low sensitivity or were not recovered during the extraction

29



Page 30 of 50

608

procedure. Therefore, those veterinary drugs (a total of 20) were not included in the final

609

method.

610 611

Overall, the UHPLC/ESI Q-Orbitrap MS along with SOSLE can serve as a

612

reliable and practical method for quantifying or screening a total of 105 veterinary drugs

613

in a single extraction process and within a 14-min analytical runtime.

614 615

Veterinary Drug Identification. According to Codex CAC/GL 71-200919 for

616

veterinary drug analysis and identification, when high resolution mass spectrometers are

617

used in a confirmatory method, the high resolution provides more reliable identification

618

of the mass and may be used to predict the elemental composition of each fragment. For a

619

single stage high resolution mass spectrometer, each structurally significant fragment

620

detected is assigned a value of two identification points, while each product ion generated

621

in a tandem high resolution mass spectrometer is assigned an identification point value of

622

2.5. In addition, at least one ion ratio must also be measured to eliminate the potential for

623

product ions of the same mass arising from isobaric compounds of similar structure.

624

SANCO/12495/201320 required ≥2 diagnostic ions (preferably the precursor ion and its

625

product ion) with mass accuracy 100 veterinary

725

drug residues in bovine muscle by ultrahigh performance liquid chromatography-tandem

726

mass spectrometry. J. Chromatogr. A 2012, 1258, 43-54.

Stubbings, G.; Bigwood, T. The development and validation of a multiclass liquid

Martinez Vidal, J. L.; Frenich, A. G.; Aguilera-Luiz, M. M.; Romero-Gonzaez, R.

Wang, J.; Leung, D. The challenges of developing a generic extraction procedure

Wang, J.; Chow, W.; Chang, J.; Wong, J. W. Ultra-high performance liquid

Geis-Asteggiante, L.; Lehotay, S. J.; Lightfield, A. R.; Dutko, T.; Ng, C.; Bluhm,

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727

14.

Lehotay, S. J.; Lightfield, A. R.; Geis-Asteggiante, L.; Schneider, M. J.; Dutko,

728

T.; Ng, C.; Bluhm, L.; Mastovska, K. Development and validation of a streamlined

729

method designed to detect residues of 62 veterinary drugs in bovine kidney using ultra-

730

high performance liquid chromatography--tandem mass spectrometry. Drug Test Anal.

731

2012, 4 Suppl 1, 75-90.

732

15.

733

chromatography with electrospray ionization tandem mass spectrometry and estimation

734

of measurement uncertainty. J. AOAC Int. 2007, 90, 550-567.

735

16.

736

and honey using both ultra-performance liquid chromatography/quadrupole time-of-flight

737

mass spectrometry and high-performance liquid chromatography/tandem mass

738

spectrometry. Rapid Commun. Mass Spectrom. 2007, 21, 3213-3222.

739

17.

740

chromatography/electrospray ionization-tandem mass spectrometry. J. AOAC Int. 2004,

741

87, 45-55.

742

18.

743

spectrometry, in food, biological and environmental matrices. Mass Spectrom. Rev. 2009,

744

28, 50-92.

745

19.

746

regulatory food safety assurance programme associated with the use of veterinary drugs

747

in food producing animals. Adopted 2009. Revision 2012, 2014. FAO/WHO Codex

748

Alimentarius International Food Standards.

Wang, J.; Wotherspoon, D. Determination of pesticides in apples by liquid

Wang, J.; Leung, D. Analyses of macrolide antibiotic residues in eggs, raw milk,

Wang, J. Confirmatory determination of six penicillins in honey by liquid

Wang, J. Analysis of macrolide antibiotics, using liquid chromatography-mass

CAC/GL 71-2009. Guidelines for the design and implementation of national

37



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749

http://www.codexalimentarius.org/standards/list-of-standards/. Accessed on July 30,

750

2015

751

20.

752

in food and feed. Document No. SANCO/12571/2013 Supersedes Document No.

753

SANCO/12495/2011 Implemented by 01/01/2014

754

http://www.eurl-pesticides.eu/library/docs/allcrl/AqcGuidance_Sanco_2013_12571.pdf .

Method validation and quality control procedures for pesticide residues analysis

Accessed on July 30, 2015.

755 756

21.

757

Thevis, M. Sensitive determination of prohibited drugs in dried blood spots (DBS) for

758

doping controls by means of a benchtop quadrupole/Orbitrap mass spectrometer. Anal.

759

Bioanal. Chem. 2012, 403, 1279-1289.

760

22.

761

screening method for the detection of antibiotic residues in muscle tissues using liquid

762

chromatography and high resolution mass spectrometry with a LC-LTQ-Orbitrap

763

instrument. Food Addit. Contam. A 2011, 28, 1340-1351.

764

23.

765

http://www.hc-sc.gc.ca/dhp-mps/vet/mrl-lmr/mrl-lmr_versus_new-nouveau-eng.php.

766

Thomas, A.; Geyer, H.; Schänzer, W.; Crone, C.; Kellmann, M.; Moehring, T.;

Hurtaud-Pessel, D.; Jagadeshwar-Reddy, T.; Verdon, E. Development of a new

Canadian Maximum Residue Limits (MRLs) for Veterinary Drugs in Foods.

Accessed on July 30, 2015.

38


Page 39 of 50


Table 1. Antibiotics, Exact Mass and UHPLC Retention Time for Data Processing and Quantification, and UHPLC/ESI Q-Orbitrap MS Method Performance Results a

Antibiotics g

1

Total Molecular formula number

Class

2

3

Section A: Compounds with Full validation

89

Abamectin B1a Doramectin Emamectin B1a Eprinomectin B1a Ivermectin Moxidectin Selamectin Cinoxacin Ciprofloxacin Danofloxacin Difloxacin Enoxacin Enrofloxacin Flumequine Lomefloxacin Marbofloxacin Nalidixic Acid Norfloxacin Ofloxacin Orbifloxacin Oxolinic Acid Pipemidic Acid Sarafloxacin Sparfloxacin Lasalocid Monensin Narasin Nigericin Salinomycin Erythromycin Neospiramycin I Oleandomycin Roxithromycin Spiramycin I Tilmicosin Tylosin A

7

Endectocides Endectocides Endectocides Endectocides Endectocides Endectocides Endectocides Fluoroquinolones Fluoroquinolones Fluoroquinolones Fluoroquinolones Fluoroquinolones Fluoroquinolones Fluoroquinolones Fluoroquinolones Fluoroquinolones Fluoroquinolones Fluoroquinolones Fluoroquinolones Fluoroquinolones Fluoroquinolones Fluoroquinolones Fluoroquinolones Fluoroquinolones Ionophores Ionophores Ionophores Ionophores Ionophores Macrolides Macrolides Macrolides Macrolides Macrolides Macrolides Macrolides

4

C48H72O14 C50H74O14 C49H75NO13 C50H75NO14 C48H74O14

17

C37H53NO8 C43H63NO11 C12H10N2O5 C17H18FN3O3 C19H20FN3O3 C21H19F2N3O3 C15H17FN4O3

C19H22FN3O3 C14H12FNO3 C17H19F2N3O3 C17H19FN4O4 C12H12N2O3 C16H18FN3O3 C18H20FN3O4 C19H20F3N3O3 C13H11NO5 C14H17N5O3 C20H17F2N3O3 C19H22F2N4O3

5

C34H54O8 C36H62O11 C43H72O11 C40H68O11

7

C42H70O11 C37H67NO13 C36H62N2O11 C35H61NO12 C41H76N2O15 C43H74N2O14

C46H80N2O13 C46H77NO17

Exact mass

Retention Time, min

[M + H]

5

9.99 10.26 9.31 9.86 10.57 10.41 10.63 4.90 3.74 3.78 4.12 3.49 3.83 6.35 3.93 3.23 6.12 3.62 3.49 3.94 5.18 3.02 4.26 4.55 10.06 10.09 10.68 10.61 10.46 6.86 4.92 6.27 7.56 5.24 6.06 6.75

+

+

+

++

Overall b,e recovery (%)

Intermediate c,e precision (%)

Measurement d,e uncertainty (%)

Peak height Canadian MRLsk f at LCL (µg/kg) (µg/kg)

[M + NH4]

[M + Na]

6

7

8

9

10

11

12

13

873.49948 899.51513

895.48143 921.49708

770.44739 263.06625 332.14050 358.15615 400.14672 321.13575 360.17180 262.08740 352.14672 363.14631 233.09207 320.14050 362.15106 396.15295 262.07100 304.14042 386.13107 393.17327

890.52603 916.54168 903.55767 931.55258 892.54168 657.41094 787.47394 280.09280 349.16705 375.18270 417.17327 338.16229 377.19835 279.11395 369.17327 380.17286 250.11862 337.16705 379.17761 413.17950 279.09755 321.16697 403.15762 410.19982

591.38915 671.43649 765.51474 725.48344 751.49909

608.41569 688.46304 782.54129 742.50999 768.52564

734.46852

751.49507 716.46919 705.45320 854.55840 860.54783 886.59987 933.55298

437.25338 450.26121 443.76920 457.76665 438.26121 320.69584 385.72733 132.03676 166.57389 179.58171 200.57700 161.07151 180.58954 131.54734 176.57700 182.07679 117.04967 160.57389 181.57917 198.58011 131.53914 152.57385 193.56918 197.09028 296.19821 336.22188 383.26101 363.24536 376.25318 367.73790

120.1 118.5 117.1 120.8 117.3 112.7 110.5 115.9 38.9 48.0 82.5 30.8 62.5 119.4 53.1 44.7 120.9 34.0 47.4 74.0 118.6 24.6 73.5 80.4 102.1 111.4 104.3 100.8 107.5 55.0 12.4 97.6 115.9 29.7 68.8 104.9

8.9 9.6 7.5 5.9 12.0 21.8 22.2 3.9 7.9 12.6 10.5 7.5 9.8 4.4 6.7 8.3 4.7 8.5 8.5 5.9 4.2 10.4 6.2 5.7 12.8 7.7 18.6 30.6 7.6 26.7 14.5 5.4 13.5 11.5 9.2 5.4

18.7 19.2 15.4 13.5 24.8 43.8 48.1 8.6 16.1 25.2 22.1 15.8 20.4 11.8 14.1 18.3 12.8 17.0 17.7 12.8 9.1 21.4 13.3 12.1 25.7 18.0 37.4 61.7 15.2 66.3 32.7 11.1 27.1 26.8 19.5 11.8

438000 (5) 78600 (1) 381000 (1) 195000 (1) 524000 (5) 494000 (1) 74700 (5) 475000 (1) 283000 (1) 448000 (1) 228000 (1) 329000 (1) 427000 (1) 425000 (1) 371000 (1) 147000 (1) 422000 (1) 318000 (1) 438000 (1) 777000 (1) 419000 (1) 122000 (1) 172000 (1) 740000 (1) 710000 (1) 409000 (1) 2120000 (5) 1280000 (1) 426000 (1) 249000 (1) 379000 (1) 546000 (1) 781000 (1) 228000 (1) 1080000 (1) 96700 (1)

886.53112

914.52603 875.51513 640.38439

699.44264 688.42665 837.53185

843.52128 869.57332 916.52643

908.51306 936.50798 897.49708 662.36634

792.42933 285.04819 354.12244 380.13809 422.12867 343.11769 382.15374 284.06934 374.12867 385.12825 255.07401 342.12244 384.13301 418.13490 284.05294 326.12236 408.11302 415.15522 613.37109 693.41843 787.49668 747.46538 773.48103 756.45046 721.42458 710.40860 859.51379 865.50323 891.55526 938.50837

[M + 2H]

350.22496

344.71696 419.26956 422.26428 435.29030

458.76685


14

20 40

10

50


2‐methyl‐4(5)‐nitroimidazole Nitroimidazoles Dimetridazole Nitroimidazoles Dimetridazole‐d3i Nitroimidazoles Etanidazole Nitroimidazoles HMMNI Nitroimidazoles HMMNI‐d3 Nitroimidazoles Ipronidazole Nitroimidazoles Ipronidazole‐d3 Nitroimidazoles Ipronidazole‐OH Nitroimidazoles Ipronidazole‐OH‐d3 Nitroimidazoles Metronidazole Nitroimidazoles Metronidazole‐OH Nitroimidazoles Nimorazole Nitroimidazoles Ornidazole Nitroimidazoles Ronidazole Nitroimidazoles Ronidazole‐d3 Nitroimidazoles Ternidazole Nitroimidazoles Tinidazole Nitroimidazoles 5‐hydroxyflunixin NSAIDS Flunixin NSAIDS Phenylbutazone NSAIDS Penicillin G Penicillins Penicillin V Penicillins Florfenicol Phenicols Thiamphenicol Phenicols

13

C4H5N3O2 C5H7N3O2 C5H4N3O2D3 C7H10N4O4 C5H7N3O3 C5H4N3O3D3 C7H11N3O2 C7H8N3O2D3 C7H11N3O3 C7H8N3O3D3 C6H9N3O3 C6H9N3O4 C9H14N4O3 C7H10N3O3Cl C6H8N4O4 C6H5N4O4D3 C7H11N3O3 C8H13N3O4S

3

2 2

C14H11F3N2O3 C14H11F3N2O2 C19H20N2O2 C16H18N2O4S C16H18N2O5S C12H14Cl2FNO4S C12H15Cl2NO5S

1.68 2.72 2.72 1.11 1.99 2.01 4.64 4.62 3.91 3.88 2.24 1.65 1.28 3.77 2.19 2.19 2.94 2.83 7.57 7.88 7.97 6.13 6.50 3.92 3.13

128.04545 142.06110 145.07963 215.07748 158.05602 161.07454 170.09240 173.11093 186.08732 189.10584 172.07167 188.06658 227.11387 220.04835 201.06183 204.08036 186.08732 248.06995

313.07945 297.08454 309.15975 335.10600 351.10092

145.07200 159.08765 162.10618 232.10403 175.08257 178.10109 187.11895 190.13748 203.11387 206.13239 189.09822 205.09313 244.14042 237.07489

218.08838 221.10691 203.11387 265.09650 330.106 314.11109 326.18630 352.13255 368.12747

358.00774

375.03429

356.01208

373.03862

150.02740 164.04305 167.06157 237.05943 180.03796 183.05649 192.07435 195.09287 208.06926 211.08779 194.05361 210.04853 249.09581 242.03029 223.04378 226.06230 208.06926 270.05190 335.0614 319.06648 331.14170 357.08795 373.08286 379.98968 377.99402

64.52636 71.53419 73.04345 108.04238 79.53165 81.04091 85.54984 87.05910 93.54730 95.05656 86.53947 94.53693 114.06057 110.52781 101.03455 102.54382 93.5473 124.53861 157.04337 149.04591 155.08352 168.05664 176.05410 179.50751 178.50968


Page 40 of 50

95.7 100.8

4.3 3.1

9.1 6.5

252000 (1) 134000 (1)

66.4 101.1

12.1 3.4

26.5 7.0

217000 (1) 278000 (5)

100.6

2.3

5.4

536000 (1)

101.4

2.8

6.4

283000 (1)

102.5 76.9 54.3 114.9 102.0

6.7 24.5 9.7 3.7 3.0

13.6 49.1 25.2 7.9 5.9

213000 (1) 131000 (1) 146000 (1) 166000 (1) 82400 (1)

103.9 114.1 67.3 119.8 57.8 74.5 96.0 119.7 111.1

5.6 4.2 35.3 16.0 34.7 20.6 13.9 3.8 9.7

12.2 9.1 74.0 34.0 71.0 73.9 31.3 9.0 19.9

267000 (1) 359000 (1) 71500 (1) 6 1570000 (1) 280000 (1) 304000 (20) 6 (0.01 IU/mL)l 117000 (20) 100000 (1) 55000 (1)

Page 41 of 50

Dapsone Sulfabenzamide Sulfacetamide Sulfachloropyridazine Sulfadiazine Sulfadimethoxine Sulfadoxine Sulfaethoxypyridazine Sulfaguanidine Sulfamerazine Sulfameter Sulfamethazine Sulfamethizole Sulfamethoxazole Sulfamethoxypyridazine Sulfamonomethoxine Sulfamoxole Sulfanilamide Sulfanitran Sulfaphenazole Sulfapyridine Sulfaquinoxaline Sulfathiazole Sulfisomidine Sulfisoxazole Trimethoprim 4‐epitetracycline Chlortetracycline Doxycycline Oxytetracycline Tetracycline Cefoxitin Desacetyl cephapirin


Sulfonamides Sulfonamides Sulfonamides Sulfonamides Sulfonamides Sulfonamides Sulfonamides Sulfonamides Sulfonamides Sulfonamides Sulfonamides Sulfonamides Sulfonamides Sulfonamides Sulfonamides Sulfonamides Sulfonamides Sulfonamides Sulfonamides Sulfonamides Sulfonamides Sulfonamides Sulfonamides Sulfonamides Sulfonamides Sulfonamides Tetracyclines Tetracyclines Tetracyclines Tetracyclines Tetracyclines β‐Lactams β‐Lactams

26

C12H12N2O2S C13H12N2O3S C8H10N2O3S C10H9ClN4O2S C10H10N4O2S

C12H14N4O4S C12H14N4O4S C12H14N4O3S C7H10N4O2S C11H12N4O2S C11H12N4O3S C12H14N4O2S C9H10N4O2S2 C10H11N3O3S C11H12N4O3S C11H12N4O3S C11H13N3O3S C6H8N2O2S

C14H13N3O5S C15H14N4O2S C11H11N3O2S C14H12N4O2S C9H9N3O2S2 C12H14N4O2S C11H13N3O3S C14H18N4O3

5

C22H24N2O8 C22H23ClN2O8 C22H24N2O8 C22H24N2O9 C22H24N2O8

2

C16H17N3O7S2 C15H15N3O5S2

3.21 4.33 1.77 3.71 1.98 4.90 4.09 4.52 1.05 2.91 3.22 3.46 3.30 3.82 3.53 3.89 3.28 1.25 6.06 4.61 2.71 5.11 2.50 2.33 4.06 3.33 3.09 4.86 5.58 3.80 3.74 3.84 1.10

249.06922 277.06414 215.04849 285.02075 251.05972 311.08085 311.08085 295.08594 215.05972 265.07537 281.07029 279.09102 271.03179 254.05939 281.07029 281.07029 268.07504 173.03792

336.06487 315.09102 250.06447 301.07537 256.02089 279.09102 268.07504 291.14517 445.16054 479.12157 445.16054 461.15546 445.16054

428.05807 382.05259

266.09577 294.09069 232.07504 302.04730 268.08627 328.10740 328.10740 312.11249 232.08627 282.10192 298.09684 296.11757 288.05834 271.08594 298.09684 298.09684 285.10159 190.06447 353.09142 332.11757 267.09102 318.10192 273.04744 296.11757 285.10159 308.17172 462.18709 496.14812 462.18709 478.18201 462.18709

271.05117 299.04608 237.03043 307.00270 273.04167 333.06280 333.06280 317.06788 237.04167 287.05732 303.05223 301.07297 293.01374 276.04133 303.05223 303.05223 290.05698 195.01987 358.04681

337.07297 272.04642 323.05732 278.00284 301.07297 290.05698 313.12711 467.14249 501.10351 467.14249 483.13740 467.14249 445.08462 450.04001 399.07914 404.03453

125.03825 139.03571 108.02788 143.01401 126.03350 156.04406 156.04406 148.04661 108.03350 133.04132 141.03878 140.04915 136.01954 127.53333 141.03878 141.03878 134.54116 87.02260 168.53607 158.04915 125.53588 151.04132 128.51409 140.04915 134.54116 146.07622 223.08391 240.06442 223.08391 231.08137 223.08391 214.53267 191.52993


107.8 113.3 104.3 105.9 101.8 114.3 110.7 105.7 72.4 102.0 108.6 104.4 107.2 112.3 104.6 106.9 89.8 97.1 117.8 113.4 98.1 109.5 98.1 83.5 109.7 47.6 96.8 53.7 59.8 30.7 43.6 100.5 15.0

4.2 4.9 10.4 3.7 6.2 4.0 3.9 3.6 14.3 9.3 4.0 5.2 4.5 3.8 4.5 3.8 6.7 16.9 4.9 3.9 4.6 4.2 5.6 3.3 3.7 6.7 10.5 13.2 12.2 20.6 9.6 7.5 54.8

9.1 10.6 21.2 8.7 12.6 8.5 8.6 8.5 28.8 20.1 8.8 10.9 9.2 8.3 9.9 8.5 13.5 36.4 11.1 8.6 9.3 9.3 11.7 7.6 8.5 13.4 29.0 31.2 30.1 41.5 22.7 16.5 110.3

1220000 (1) 329000 (1) 149000 (1) 215000 (1) 253000 (1) 814000 (1) 1120000 (1) 747000 (1) 976000 (1) 657000 (1) 556000 (1) 822000 (1) 174000 (1) 612000 (1) 865000 (1) 303000 (1) 494000 (1) 52900 (5) 179000 (5) 652000 (1) 436000 (1) 311000 (1) 186000 (1) 345000 (1) 460000 (1) 1360000 (1) 288000 (40) 173000 (40) 198000 (40) 158000 (20) 151000 (5) 437000 (40) 2600000 (20)

10 10

10 10 10 10

10

10

10 10 10

100 100 100


Section B: Compounds of Unstable but quantifiable

h

13 C19H18ClN3O5S

6.77

436.07285

453.09939 458.05479

218.54006

Penicillins

C19H17Cl2N3O5S

7.00

470.03387

487.06042 492.01582

235.52057

Oxacillin

Penicillins

C19H19N3O5S

6.56

402.11182

419.13837 424.09376

201.55955

Cefamandole Cefapirin

β‐Lactams β‐Lactams

C18H18N6O5S2

4.27 2.42

463.08529 424.06315

480.11183 485.06723 441.08970 446.04510

232.04628 212.53521

Cefazolin Cefoperazone


C14H14N8O4S3

C25H27N9O8S2

3.30 3.76

455.03729 646.14968

472.06384 477.01923 663.17623 668.13162

228.02228 323.57848

Cefotaxime

β‐Lactams

C16H17N5O7S2

3.49

456.06422

473.09077 478.04616

228.53575

Cefquinome Ceftiofur


C23H24N6O5S2 C19H17N5O7S3

2.78 5.10

529.13224 524.03629

546.15879 551.11418 541.06284 546.01823

265.06976 262.52178

Cefuroxime

β‐Lactams

C16H16N4O8S

3.36

425.07616

442.10271

447.05811

213.04172

Cephacetrile j Cephalothin


C13H13N3O6S C16H16N2O6S2

2.46 5.00

340.05978 397.05225

357.08633

362.04173

414.07880

419.03420

170.53353 199.02977

C39H65NO14 C16H19N3O4S C16H19N3O4S

6.42 3.92 3.92

772.44778 350.11690 350.11690

789.47433 794.42973 367.14345 372.09885 367.14345 372.09885

Cloxacillin

Penicillins

Dicloxacillin

3

10

C17H17N3O6S2

Section C: Compounds for Screening

3

Tylosin B Ampicillin Cephradine

1 1 1

Macrolides Penicillins β‐Lactams

Concentration Recoveryb (%) (µg/kg) 112.2 10.0 122.2 25.0 101.9 50.0 102.0 80.0 146.2 50.0 79.3 80.0 119.7 10.0 115.7 25.0 91.0 50.0 101.2 80.0 136.6 80.0 56.7 25.0 50.0 50.0 55.1 80.0 134.3 80.0 134.3 50.0 122.9 80.0 94.6 25.0 88.2 50.0 92.0 80.0 99.8 80.0 121.5 10.0 130.6 25.0 121.1 50.0 124.6 80.0 116.9 25.0 94.7 50.0 100.7 80.0 106.5 80.0 10.0 109.8 122.2 25.0 113.5 50.0 113.0 80.0

Page 42 of 50

c

RSD (%) 7.1 2.3 5.5 6.7 0.9 11.0 7.0 4.8 7.9 2.3 3.0 23.7 9.5 8.4 2.3 5.6 2.2 2.9 6.7 2.0 0.5 2.1 6.5 4.1 4.8 4.4 5.7 4.3 3.4 5.4 4.7 5.3 1.4

82400 (5)

271000 (40) 69600 (5)

41900 (60) 113000 (20)

48800 (60) 166000 (40) 166000 (20)

206000 (60) 118000 (5)

100

38200 (20)

101000 (60) 42600 (5)

386.72753 no standard but screening 175.56209 co‐elute with cephradine but screening at 80 µg/kg. 175.56209 co‐elute with ampicillin but screening at 80 µg/kg.


20

10

Page 43 of 50

Section D: Compounds Excluded from the Method Amikacin Aminoglycosides Aminoglycosides Apramycin Aminoglycosides Dihydrostreptomycin Aminoglycosides G418 Gentamycin C1 Aminoglycosides Aminoglycosides Gentamycin C1A Gentamycin C2 Aminoglycosides Aminoglycosides Hygromycin B Aminoglycosides Kanamycin A Aminoglycosides Kanamycin B Neomycin Aminoglycosides Aminoglycosides Paromomycin Aminoglycosides Spectinomycin Aminoglycosides Streptomycin Aminoglycosides Tobramycin Amoxicillin Penicillins Cefadroxil β‐Lactams Cefalonium β‐Lactams Cephalexin β‐Lactams Cephalosporin C β‐Lactams a


20 15

1 4

C22H43N5O13 C21H41N5O11 C21H41N7O12 C20H40N4O10 C21H43N5O7 C19H39N5O7 C20H41N5O7 C20H37N3O13 C18H36N4O11 C18H37N5O10 C23H46N6O13 C23H45N5O14 C14H24N2O7 C21H39N7O12 C18H37N5O9 C16H19N3O5S C16H17N3O5S C20H18N4O5S2 C16H17N3O4S C16H21N3O8S

0.92 0.91 0.92 0.91 0.91 0.90 0.91 0.91 0.92 0.91 0.92 0.92 0.97 0.91 0.92 1.03 1.05 2.86 3.94 1.10

586.29301 540.28753 584.28860 497.28172 478.32353 450.29223 464.30788 528.23991 485.24533 484.26132 615.31956 616.30358 333.16563 582.27295 468.26640 366.11182 364.09617 459.07914 348.10125 416.11221

603.31956 557.31408 601.31515 514.30827 495.35007 467.31877 481.33442 545.26646 502.27188 501.28787 632.34611 633.33013 350.19218 599.2995 485.29295 383.13837 381.12272 476.10569 365.12780 433.13876

608.27496 562.26948 606.27054 519.26366 500.30547 472.27417 486.28982 550.22186 507.22728 506.24326 637.30151 638.28552 355.14757 604.25489 490.24835 388.09376 386.07811

481.06108 370.08320 438.09416

293.65014 270.64741 292.64794 249.1445 239.6654 225.64975 232.65758 264.6236 243.12631 242.6343 308.16342 308.65543 167.08645 291.64011 234.63684 183.55955 182.55172 230.04321 174.55426 208.55974

not chromatographically retained not chromatographically retained not chromatographically retained not chromatographically retained not chromatographically retained not chromatographically retained not chromatographically retained not chromatographically retained not chromatographically retained not chromatographically retained not chromatographically retained not chromatographically retained not chromatographically retained not chromatographically retained not chromatographically retained low sensitivity or not recovered low sensitivity or not recovered low sensitivity or not recovered low sensitivity or not recovered low sensitivity or not recovered

Number or text in bold font style and underlined indicates ionization form or charge state for data processing or quantification.

b

Bold and underlined are antibiotics with recoveries not in the range of 71 to 120 %.

c

Bold and underlined are antibiotics with intermediate precision > 20 %.

d

Bold and underlined are antibiotics with MU > 50 %.

e

For data in red color font, the method performance was based on three spike levels, i.e. 25.0, 50.0 and 80.0 µg/kg due to its poor sensitivity.

For data in sky blue color font, the method performance was based on two spike levels, i.e. 50.0 and 80.0 µg/kg due to its poor sensitivity. f

LCL: lowest concentration level of the matrix‐matched calibration curve.

g

Column number.

h

The method performance of unstable antibiotics was estimated from one experiment when working solution was prepared fresh.

i

Dimetridazole-d3 was used as an internal standard for compounds that have no isotopically labelled standards for quantification.

j

Data was processed according to the mass of a fragment m/z 337.03170.

k

Canadian Maximum Residue Limits (MRLs) for Veterinary Drugs in Foods (http://www.hc‐sc.gc.ca/dhp‐mps/vet/mrl‐lmr/mrl‐lmr_versus_new‐nouveau‐eng.php)

l

IU=international unit. 0.006 μg of penicillin G is equivalent to 0.01 IU.


125

1500

125


UHPLC Gradient Profile

A

0 1 2 3 4 5 B

Time 0.00 8.00 9.00 11.00 11.10 14.00

A% 88.0 5.0 0.0 0.0 88.0 88.0

B% 12.0 95.0 100.0 100.0 12.0 12.0

Retention Time Distribution

12

Aminoglycosides (15) Endectocides (7)

10 Retention Time (min)

µL/min 300 300 300 300 300 300

Fluoroquinolones (17) Ionophores (5)

8

Macrolides (8)

Nitroimidazoles (13)

6

NSAIDs (3) Penicillins (7)

4

Phenicols (2) Sulfonamides (26)

2

Tetracyclines (5) β-Lactams (17)

0

0

50 Antibiotic IDs

100

Figure 1


Page 44 of 50

Page 45 of 50


A1

Endectocides

A2

120.0

Abamectin B1a

80.0

Doramectin

60.0

Emamectin B1a

40.0

Eprinomectin B1a

20.0

Doramectin Emamectin B1a

20.0

Eprinomectin B1a

Ivermectin

0.0 M-A

M-B

M-C

M-A

Selamectin

Fluoroquinolones

B1

Ivermectin

0.0

Moxidectin

M-D

Method

M-B

M-C

Fluoroquinolones

B2

60.0

40.0

20.0

0.0 M-A

M-B

M-C

M-D

Cinoxacin Ciprofloxacin Danofloxacin Difloxacin Enoxacin Enrofloxacin Flumequine Lomefloxacin Marbofloxacin Nalidixic Acid Norfloxacin Ofloxacin Orbifloxacin Oxolinic Acid Pipemidic Acid Sarafloxacin Sparfloxacin

80.0 70.0 60.0 50.0

RSD (%)

80.0

40.0 30.0 20.0 10.0 0.0 M-A

M-B

Ionophores

C1

M-C

M-D

Cinoxacin Ciprofloxacin Danofloxacin Difloxacin Enoxacin Enrofloxacin Flumequine Lomefloxacin Marbofloxacin Nalidixic Acid Norfloxacin Ofloxacin Orbifloxacin Oxolinic Acid Pipemidic Acid Sarafloxacin Sparfloxacin

Method

Method

Ionophores

C2

120.0

50.0

100.0

40.0

80.0

Lasalocid

60.0

Monensin

40.0

Narasin

RSD (%)

Recovery (%)

Selamectin

90.0

100.0

Nigericin

20.0 M-A

M-B

M-C

Lasalocid

30.0

Monensin 20.0

Narasin Nigericin

10.0

Salinomycin

0.0

Salinomycin

0.0

M-D

M-A

M-B

Method

D1

D2

Macrolides

100.0

Neospiramycin I

60.0

Oleandomycin

40.0

Roxithromycin Spiramycin I

20.0

Tilmicosin 0.0 M-B

M-C Method

M-D

Tylosin A

RSD (%)

Erythromycin

80.0

M-A

M-C

M-D

Method

120.0

Recovery (%)

Moxidectin

M-D

Method

120.0

Recovery (%)

Abamectin B1a

40.0

RSD (%)

Recovery (%)

100.0

Endectocides

60.0

Macrolides

45.0 40.0 35.0 30.0 25.0 20.0 15.0 10.0 5.0 0.0

Erythromycin Neospiramycin I Oleandomycin Roxithromycin Spiramycin I Tilmicosin M-A

M-B

M-C Method


M-D

Tylosin A


120.0 Recovery (%)

E2

Nitroimidazoles 140.0

100.0 80.0 60.0 40.0 20.0 0.0 M-A

M-B

M-C

M-D

Method

2-MN Dimetridazole Etanidazole HMMNI Ipronidazole Ipronidazole-OH Metronidazole Metronidazole-OH Nimorazole Ornidazole Ronidazole Ternidazole Tinidazole

NSAIDS

F1

30.0 25.0 20.0 15.0 10.0 5.0 0.0 M-A

M-B

M-D

80.0

60.0

Flunixin

40.0

RSD (%)

60.0

80.0

Phenylbutazone

40.0

Flunixin Phenylbutazone

20.0

20.0 0.0

0.0 M-A

M-B

M-C

M-D

M-A

M-B

Method

M-C

M-D

Method

Phenicols

G1

Phenicols

G2

140.0

30.0

120.0

25.0

80.0 60.0

Florfenicol

40.0

Thiamphenicol

RSD (%)

100.0

20.0 15.0

Florfenicol

10.0

Thiamphenicol

5.0

20.0 0.0

0.0 M-A

M-B

M-C

M-D

M-A

M-B

Method

H1

M-C

M-D

Method

H2

Sulfonamides

140.0

Sulfonamides

70.0

Dapsone

Dapsone

Sulfabenzamide

Sulfabenzamide

Sulfacetamide

Sulfacetamide

Sulfachloropyridazine

120.0

Sulfachloropyridazine

60.0

Sulfadiazine

Sulfadiazine Sulfadimethoxine

Sulfadimethoxine

Sulfadoxine

Sulfadoxine 100.0

50.0

Sulfaethoxypyridazine

Sulfaethoxypyridazine Sulfaguanidine

Sulfaguanidine

Sulfamerazine

Sulfamerazine Sulfameter

80.0

Sulfamethazine Sulfamethizole Sulfamethoxazole 60.0

Sulfamethoxypyridazine

Sulfameter

40.0

Sulfamethazine

RSD (%)

Recovery (%)

2-MN Dimetridazole Etanidazole HMMNI Ipronidazole Ipronidazole-OH Metronidazole Metronidazole-OH Nimorazole Ornidazole Ronidazole Ternidazole Tinidazole

NSAIDS

F2

100.0

Recovery (%)

M-C

Method

120.0 Recovery (%)

Nitroimidazoles 35.0

RSD (%)

E1

Page 46 of 50

Sulfamethizole Sulfamethoxazole 30.0

Sulfamethoxypyridazine Sulfamonomethoxine

Sulfamonomethoxine

Sulfamoxole

Sulfamoxole Sulfanilamide

40.0

Sulfanitran

Sulfanitran

Sulfaphenazole

Sulfaphenazole

Sulfapyridine 20.0

Sulfanilamide

20.0

Sulfaquinoxaline

Sulfapyridine 10.0

Sulfaquinoxaline Sulfathiazole

Sulfathiazole

Sulfisomidine

Sulfisomidine Sulfisoxazole

0.0 M-A

M-B

M-C

Method

M-D

Trimethoprim

Sulfisoxazole

0.0 M-A

M-B

M-C

Method

Figure 2


M-D

Trimethoprim

Page 47 of 50


Matrix Effects

Frequency (%)

80.0

60.0

40.0

20.0

0.0 Ion suppression ≥ 30%

Ion suppression < 30% or enhancement ≤ 20%

Ion enhancement > 20%

Milk A

2.1

76.0

21.9

Milk B

4.2

74.0

21.9

Milk C

2.1

74.0

24.0

Milk D

4.2

75.0

20.8

Milk E

3.1

76.0

20.8

Figure 3



Figure 4


Page 48 of 50

Page 49 of 50


A

Overall Recovery (%) 80.0

70.8

Frequency (%)

70.0 60.0 50.0 40.0 30.0

25.8

20.0 10.0

3.4

0.0 ≤70%

B

80.0 70.0

68.5

71-120%

>120%

Intermediate Precison (%)

Frequency (%)

60.0 50.0 40.0 30.0 16.9

20.0

11.2

10.0

3.4

0.0 ≤10%

C

11-15%

16-20%

>20%

Measurement Uncertainty (%) 70.0

64.0

Frequency (%)

60.0 50.0 40.0 30.0 16.9

20.0

7.9

10.0

11.2

0.0

≤ 20 %

21-30%

31-40%

>40%

Figure 5



Page 50 of 50

TOC Graphic

Retention Time Distribution 12

Aminoglycosides (15) Endectocides (7)

Retention Time (min)

10

Fluoroquinolones (17) Ionophores (5)

8

Macrolides (8) Nitroimidazoles (13)

6

NSAIDs (3) Penicillins (7)

4

Phenicols (2) Sulfonamides (26)

2

Tetracyclines (5) β-Lactams (17)

0 0

20

40

60

80

100

120

Antibiotic IDs


Development and Validation of a Multiclass Method for Analysis of

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