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Article
Wide-scope screening method for multiclass veterinary drug residues in fish, shrimp and eel using liquid chromatography- quadrupole high resolution mass spectrometry Sherri B. Turnipseed, Joseph M. Storey, Jack J. Lohne, Wendy Cook Andersen, Robert Burger, Aaron S. Johnson, and Mark R. Madson J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b04717 • Publication Date (Web): 28 Dec 2016 Downloaded from http://pubs.acs.org on January 4, 2017
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Wide-scope screening method for multiclass veterinary drug
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residues in fish, shrimp and eel using liquid chromatography-
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quadrupole high resolution mass spectrometry
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Sherri B. Turnipseed1*, Joseph M. Storey1, Jack J. Lohne1, Wendy C. Andersen1, Robert Burger2,
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Aaron S. Johnson2, Mark R. Madson1, 2
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Animal Drugs Research Center, US Food and Drug Administration, Denver, CO 80225
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Denver Laboratory, US Food and Drug Administration, Denver, CO 80225
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* Corresponding author:
[email protected]; 303-236-3072
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ABSTRACT
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A screening method for veterinary drug residues in fish, shrimp and eel using LC with a high
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resolution MS instrument has been developed and validated. The method was optimized for over
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70 test compounds representing a variety of veterinary drug classes. Tissues were extracted by
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vortex mixing with acetonitrile acidified with 2% acetic acid and 0.2% p-toluene sulfonic acid.
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A centrifuged portion of the extract was passed through a novel solid phase extraction cartridge
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designed to remove interfering matrix components from tissue extracts. The eluent was then
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evaporated and reconstituted for analysis. Data were collected with a quadrupole- Orbitrap high
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resolution mass spectrometer using both nontargeted and targeted acquisition methods. Residues
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were detected based on the exact mass of the precursor and a product ion along with isotope
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pattern and retention time matching. Semi-quantitative data analysis compared MS1 signal to a
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one-point extracted matrix standard at a target testing level. The test compounds were detected
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and identified in salmon, tilapia, catfish, shrimp, and eel extracts fortified at the target testing
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levels. Fish dosed with selected analytes and aquaculture samples previously found to contain
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residues were also analyzed. The screening method can be expanded to monitor for an additional
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> 260 veterinary drugs based on exact mass measurements and retention times.
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KEYWORDS
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High resolution mass spectrometry, Aquaculture, Veterinary drug residues, Screening methods
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INTRODUCTION
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Aquaculture is a growing industry anticipated to supply approximately 100 million tons or over
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60% of the fish destined for human consumption by 2030. Many types of veterinary drugs may
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be administered to fish in an aquaculture environment to treat disease or proactively prevent
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infection.1, 2 Traditionally, analytical methods were developed to monitor for one residue, or for
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several analytes from a specific class of drugs, in a single species of fish or shellfish. More
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recently, multiclass methods have been developed using liquid chromatography (LC) with
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tandem mass spectrometry (MS),3-5 but these methods are still limited to targeted analytes.
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Using high resolution mass spectrometry (HRMS) instruments, a virtually unlimited
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number of compounds can be simultaneously analyzed because full-scan data are collected rather
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than preselected ion transitions corresponding to specific compounds. Selectivity is achieved by
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taking advantage of the instrument’s ability to provide very accurate mass measurements.
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Residue identification can be based on calculated exact masses of protonated molecules and
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fragment ions, relative isotopic abundances and retention times. This can lead to the
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development of methods that can monitor for a wide scope of residues and contaminants,
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allowing regulatory agencies to be more proactive in discovering possible adulteration of the
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food supply including aquacultured products.
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The use of HRMS technology for detecting drug residues in aquaculture and other foods has
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been reviewed6,
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and includes methods that utilize time-of-flight (ToF)8-11 or orbital ion trap
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(Orbitrap)12-14 HRMS. These methods have demonstrated the ability to detect, identify and
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quantitate an increasing number of analytes. However, some of these early HRMS methods were
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sometimes limited by insufficient mass resolution to accurately measure exact mass in complex
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matrices and a lack of sensitivity to detect residue levels.
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The Q-Exactive is a hybrid
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quadrupole-Orbitrap HRMS instrument that has the advantage of operating at increased
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resolution and the ability to collect product ion spectra with or without initial precursor isolation
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using the quadrupole filter.
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Using a MS detector with this capability further enhances the need for the most universal
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extractant and cleanup method possible. Ideally the method should also be simple and quick.
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Although a large number of veterinary drug residue LC-MS methods and cleanup strategies are
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available in the literature for fish and shrimp,5,
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recently been introduced. Removal of lipids is especially important for high fat-containing
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matrices such as salmon and other fish. Fats (specifically phospholipids) can be significant
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HPLC column contaminants and can also contribute to severe matrix suppression in signal
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response for MS detection. New commercially available clean-up techniques are designed to
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eliminate these interferences without severe loss of analyte recovery.16,
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clean-up products were evaluated along with the choice of initial extraction solution to optimize
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a procedure suitable for a wide variety of analytes at residue levels.
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specific lipid cleanup technologies have
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These new sample
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The aim of this research was to be able to screen and identify a wide scope of veterinary
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drug residues in several types of fish matrices. An optimized extraction method was used with an
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LC separation combined with a Q-Exactive (Orbitrap) HRMS.
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according to FDA guidelines18,
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addition to monitoring fish extracts for these test compounds, the full scan HRMS data could be
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compared to a compound database containing about 260 additional (> 330 total) veterinary drugs
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to significantly expand the number of residues that might be detected in any given sample.
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The method was validated
using the representative compounds listed in Table 1. In
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MATERIALS AND METHODS
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Standard Preparation
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Individual stock standards were made in methanol, except for β-lactams, which were dissolved in
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water or acetonitrile/water depending upon solubility. Oxolinic acid was prepared in acetonitrile.
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All stock standard solutions were made at a concentration of approximately 100 µg/mL as the
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free base or acid. Stock standards for the β-lactams were stored at - 25 °C; all others were stored
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at 4 °C. Two different spiking standard mixes (“stable” and “non-stable”) for positive ion
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compounds were made. A 1X target testing level (TTL) spike was made by adding portions (see
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below) of both mixtures to control tissue. The “unstable” analyte spiking mix contained the β-
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lactams, tetracyclines, cephapirin, and dye compounds as listed in Table 1. The unstable standard
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spiking mix was prepared by combining an amount (a volume equal to twenty times the TTL
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level for each compound, corrected for the exact concentration) of each individual stock standard
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and diluting to 20 mL with acetonitrile. To prepare a 1X TTL spike containing all of the unstable
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compounds, 20 µL of this standard mix was added to 2 g of control tissue. This “unstable”
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spiking mix standard is stable for six months at -25 °C. The “stable” standard spiking mix
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consists of all remaining positive ion compounds listed in Table 1. Similarly, the stable analyte
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mix was also prepared by combining an amount (a volume equivalent to 20X the TTL for each
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compound) of each individual stock standard and diluting to 40 mL with acetonitrile. To prepare
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a 1X TTL spike containing all of the stable compounds, 40 µL of this standard mix was added to
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2 g of control tissue. This “stable” spiking mix standard is stable for one year at -25 °C. A
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separate fortification mixture for the negative ion compounds was made in the same manner as
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the stable spiking mix. Samples were fortified with negative ion compounds by adding 40 µL of
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that standard mix to 2 g of control tissue.
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To prepare a solvent standard equivalent to 1X TTL in tissue, 100 µL of the stable
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standard mix and 50 µL of the unstable standard mix were combined and diluted to 5 mL with
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90:10 water: acetonitrile. (This assumes an extraction scheme of 2g→ 10 mL then 2 mL → 0.4
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mL). If an additional acetonitrile injection was needed for salmon, then an equivalent
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acetonitrile solvent standard was additionally prepared by diluting the previous solvent standard
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1:5 with acetonitrile. A negative ion 1X TTL equivalent solvent standard was prepared by
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diluting 100 µL of the negative mixed standard mix to 5 mL with 90:10 water:acetonitrile.
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Sample Extraction
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Homogenized tissue (2.0 ± 0.05 g) was weighed into a 50 mL polypropylene tube, and spiking
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standard mixes were added as appropriate and allowed to mix with the tissue for at least 5 min.
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The tissue was extracted with 8 mL of extraction solution consisting of 0.2% p-toluene sulfonic
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acid (p-TSA) monohydrate (w/v) and 2% glacial acetic acid (v:v) in acetonitrile. The samples
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were vortex mixed for 30 min using a multi-tube vortex mixer at a setting speed of 2500 rpm
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then centrifuged for 7 min (4° C) at minimum of 17,000 RCF (x g). A portion (3 mL) of the
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extract was transferred to an Oasis PRiME HLB 6 cc (200 mg) extraction cartridge with a 15 mL
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polypropylene tube underneath. The samples were allowed to gravity drain (ca. 10 min) through
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the cartridges. The remaining few drops of extractant were gently pushed out through the SPE
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tube with a pipet bulb. (This should give just over 2 mL of liquid at this point). If the tissue was
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salmon, 100 µL of the extract was transferred into an LC vial for an acetonitrile injection. The
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remaining portion of the extract was taken to near dryness under nitrogen stream at 55° C (a drop
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of liquid remaining in the 15 mL tube was acceptable). The extract was then reconstituted with
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400 µL of 10% acetonitrile in water (v:v), mixed, and centrifuged at a minimum of 28,900 RCF
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(x g) for 7 min. Finally, an aliquot of 300 µL was carefully removed from the polypropylene
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tube, leaving particulates behind, and transferred into a LC vial. Overall there was no net
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dilution of concentration of the sample through the extraction procedure. (A sample fortified at
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10 µg/kg = 10 ng/mL in LC vial).
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Incurred Samples
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Incurred fish samples were provided by the Center for Veterinary Medicine Office of Research.
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Two tilapia were dosed with 1 mg/kg body weight of sulfadiazine with 1 day depuration. Two
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other tilapia were dosed with 5 mg/kg body weight of sulfadiazine and 1 mg/kg body weight of
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trimethoprim and were sacrificed at 3 and 4 days. Two catfish were dosed with 5 mg/kg body
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weight of enrofloxacin with a 6 day withdrawal. Control tilapia and catfish were grown and
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harvested concurrently with these dosed animals. Catfish and salmon from an earlier (2014)
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dosing study with triphenylmethane dyes were also tested with this method. These fish were
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exposed to a water bath containing a mixture of 2 µg/L of malachite green, crystal violet, and
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brilliant green for 1 hour followed by 1 hour depuration in clean water.
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Instrument Acquisition Methods
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Instrumentation
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The instrument used was a Thermo Q-Exactive Orbitrap high resolution mass spectrometer
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(HRMS) with a heated electrospray ionization source coupled to a Thermo Ultimate 3000 LC
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system. Thermo XCalibur software (V. 3.0.63) was used for data acquisition and preliminary
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data analysis; data analysis was also performed using TraceFinder software (V 3.2).
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MS Acquisition Programs
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The instrument was calibrated for mass accuracy according to the manufacturer’s
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recommendations at least once a week. The tuning method optimized signals for a majority of
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the test compounds with the LC conditions described below. General MS acquisition parameters
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were as follows: spray voltage, 4kV (positive), 2.5kV (negative); S-Lens RF level, 50; capillary
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temperature, 350 ˚C; auxiliary gas temperature, 325 ˚C; gas flow rate (N2, arbitrary units):
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sheath, 50; auxiliary, 10; sweep, 2. Other general MS parameters include: acquisition time, 0-
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12.5 min; Lock mass, OFF; Chrom peak, 15 s.
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Two different types of acquisition programs were used to analyze the fish extracts. All
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Ion Fragmentation (AIF) was used for initial data acquisition. AIF is a nontargeted method in
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which a full scan MS is followed by a MS2 scan where all precursors are allowed into the high
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collision dissociation (HCD) cell to form product ions simultaneously. The settings for AIF
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were MS1: 70K resolution, 3e6 automatic gain control target, maximum inject time 200 ms, m/z
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150-1000 scan range; MS2: 70K resolution, 3e6 automatic gain control target, maximum inject
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time 200 ms, m/z 80-1000 scan range, normalized collision energies of 10, 30, and 50.
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A second set of data (separate injection of fish extract) was obtained using Data
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Dependent MS2 (DDMS2) data acquisition. With this program, MS2 data were collected when a
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precursor ion from a predefined “inclusion list” was detected (from a full MS1 scan) above a set
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threshold. When that occurred, the quadrupole filtered the precursor ion into the HCD cell using
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a limited m/z window to produce fragment ions related to that compound. The inclusion list for
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positive ion analytes contains approximately 290 of the compounds from the larger database,
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including all test compounds and other analytes for which a retention time was known (1.5 min
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windows were used in the list). Some of the more obscure analytes in the larger compound
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database were not included so the instrument did not waste analysis time triggering spectra for
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those compounds. Analytes can be added to the inclusion list as needed. The DDMS2 inclusion
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list for negative ion compounds was much smaller containing only compounds included in the
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validation (Table 1), although this list can also be expanded as needed. The operating parameters
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for this data acquisition for MS1 are: 70K resolution, 1e5 automatic gain control target, maximum
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inject time 200 ms, m/z 150-1000 scan range. The operating parameters for DDMS2 are: 17.5K
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resolution, 1e5 automatic gain control target, maximum inject time 50 ms, loop count 3, isolation
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width 4 m/z, normalized collision energy 10, 30, 50. Other data dependent settings are: underfill
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ratio 0.5%, calculated intensity threshold 1e4, apex trigger 3-6 s, dynamic exclusion 6 s.
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Negative ion data were collected with separate injections using AIF and DDMS2 acquisition.
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Chromatography
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LC separation was performed using a Supelco Ascentis Express C18 (7.5 cm x 2.1 mm, 2.7 µm)
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fused-core reversed-phase column. The mobile phase consisted of 0.1 % formic acid in water
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(A) and acetonitrile (B) at a flow rate of 0.3 mL/min. The LC gradient program was initialized at
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5% B and held for 1.5 min then ramped to 50% B from 1.5 to 8.5 min, followed by a ramp to
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99% B from 8.5 to 9 min, and then was held at 99% B from 9 to 12 min. The mobile phase was
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returned to 5% B from 12 to 12.5 min and the column was re-equilibrated for an additional 2
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min. The total LC runtime was 14.5 min; MS data were collected for 12.5 min (no divert valve
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was used). The column temperature compartment was kept at 30 ˚C, and the autosampler tray
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temperature was maintained at 10 ˚C. The LC injection volumes were 10 µL or 20 µL for AIF or
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DDMS2 MS acquisition programs, respectively. Multiple injections of a high level dye standard
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may be needed to condition a new column in order to detect low levels of leuco crystal violet and
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leuco malachite green.
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Data Analysis
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Several stages of data analysis were performed. First the AIF data were analyzed to determine if
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the test compounds were identified and present at concentrations above the threshold cutoff level
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(>50% TTL). AIF data could also be compared to the larger veterinary drug compound database
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to determine if additional analytes (beyond the test compounds) were in a sample. The product
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ion spectra from DDMS2 data could also be evaluated. Negative ion data were evaluated using
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the same processes.
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Limit testing and confirmation of identity for test compounds using AIF data
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AIF data from full MS1 scans were used for initial screening of test compounds. A Thermo
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TraceFinder “Quantitative Method” was established to provide data for the test compounds listed
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in Table 1, including a few degradants (e.g., penillic acid and dehydrated erythromycin). The
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test compounds in the “Quantitative Method” were a subset of analytes imported from the larger
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compound database (N > 330) which contains information for retention time and exact masses of
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fragment ions.
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In order to be qualitatively identified, the precursor ions must be present (signal-to-noise
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>3) and match theoretical exact mass within a 5 ppm mass tolerance. The data analysis program
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searched for residues within a time window of 60 s (30 s on each side of specified retention
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time), but a more narrow retention time match (± 0.1 min) to a standard injected the same day
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was typically observed. Fragment detection was also required with at least one fragment ion
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with 500 count minimum intensity threshold within a 10 ppm maximum mass deviation window.
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These criteria are consistent with the FDA guidance.19 The isotope match feature was also
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enabled with a 70% fit threshold, 5 ppm mass deviation, and 10% intensity deviation allowance.
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A sample would be considered presumptive positive for a test compound if the qualitative
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criteria were met and the signal was ≥ 50% as compared to the matrix-extracted standard
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fortified at the TTL.
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Expanded screening for additional veterinary drug residues
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A Thermo TraceFinder “Screening Method” was used to search for additional residues beyond
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the test compounds.
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containing > 330 potential veterinary drug residues, including metabolites and minor
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components. New compounds are continuously being added to the database, and experimental
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data for retention time and fragment ions are included for a majority of these compounds.
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Criteria used in the “Screening Method” were 3 ppm mass tolerance for the precursor ion, greater
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than 100 signal-to-noise ratio, and a signal of greater than 5000 counts for initial detection. In
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order to identify a residue by the screening method, a retention time window match within 60 s
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and a minimum of one fragment ion with intensity threshold of > 500 counts and mass tolerance
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within 10 ppm were required. The retention time and fragment ion criteria could be ignored if
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not defined in the database. The isotope pattern match option was used to filter out false detects.
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Additional qualitative data analysis from DDMS2
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The extracts were analyzed in a separate LC-MS injection using DDMS2 data acquisition. After a
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full MS1 scan, product ion spectra were collected after precursor isolation for analytes in an
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inclusion list if the signal from the MS1 ion met data dependent triggering requirements. Residue
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findings were evaluated for identification criteria using the same data analysis methods described
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above for AIF data. Product ion spectra produced by DDMS2 data acquisition could also be
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manually evaluated using XCalibur QualBrowser software for consistency with solvent and/or
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matrix-extracted standards. Alternatively, spectra could be compared to commercially available
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libraries.
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Validation
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The method was validated according to the FDA Office of Food and Veterinary Medicine
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Guidelines for Chemical Method Validations v 2. for “limit testing”18 with veterinary drugs from
Data collected using AIF were compared to a compound database
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a variety of chemical classes in representative matrices. A summary of the fortification samples
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generated for the validation is included in the supplemental material. Salmon and tilapia were
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used for the initial validation. The majority of the replicates were analyzed at the TTL (1X) to
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generate variance data at that level to set an appropriate threshold cutoff to determine if samples
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should be considered presumptive positive. Fortification samples at 2X, 0.5X, and 0.1X of the
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TTL were also analyzed along with blank matrix samples to determine the lowest minimum
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detectable and confirmation levels, as well as the rates of false positive and false negative results.
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The method was also applied, with fewer overall replicates, to catfish, shrimp and eel in order to
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demonstrate matrix extension to these species. Incurred samples of tilapia (SDZ and SDZ +
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TMP), catfish (ENRO, dyes), and salmon (dyes), as well as regulatory samples that had been
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found to contain residues, were also tested. A second set of validation samples was generated for
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negative ion compounds. Shrimp and salmon were the primary matrices validated for negative
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ion analytes, as these are the species in which phenicol or benzyl urea residues are expected to be
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found, respectively. RESULTS AND DISCUSSION
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Compounds and Testing Levels
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A list of veterinary drug residues of importance in aquacultured species (along with their target
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testing levels) is given in Table 1. The target testing level (TTL) or “1X” is not necessarily
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considered an official tolerance or action level. The majority of these compounds are not
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approved for use in fish in the U.S., so technically any amount found and identified would be
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violative. These values are meant to provide a reasonable concentration at which a residue may
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be expected to be consistently detected and identified using this screening method. For ease of
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sample preparation and data reporting, it is helpful if TTLs for the test compounds are all within
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a practical range (5-50 µg/kg). Some analytes will have TTLs higher (tetracyclines) or lower
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(methyl testosterone and dyes) than that range due to previously established levels of interest. 20
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In addition, for some compounds, such as amoxicillin and the avermectins, the TTLs may be
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>100 µg/kg due to higher detection limits for these analytes in this method. The minimum
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detectable limits for all compounds extracted from fish was determined, and for most compounds
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was found to be considerably lower (0.1X) than the TTL.
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Optimization of Extraction Procedure
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Extraction Solvent
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The list of analytes shown in Table 1 includes triphenylmethane dyes and avermectins. These
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two classes of compounds greatly increase the difficulty of choosing an acceptable sample
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extractant and clean-up procedure. Previous work4 summarizes the difficulty of including dyes in
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a multi-residue method. In addition, it is desirable that any chosen extractant be able to extract
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very polar analytes such as florfenicol amine and metronidazole as well as several non-polar
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compounds. The goal was to develop a simple cleanup procedure to apply to as many of the
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compounds in this list as possible.
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Methanol or acetonitrile as the primary organic component in a meat or fish extractant is
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most widely reported in the literature. Water (with or without buffer salts) is not effective for
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extracting non-polar analytes.
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precipitate proteins in tissue. The use of acetonitrile only (with no other purification steps) as a
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veterinary drug extractant for tissue has been published.21 Acetonitrile has also been used (in
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combination with secondary hexane partitioning) to extract residues from catfish, salmon and
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trout.22 However, when 100% acetonitrile (without acid modifiers) was tried for our list of
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analytes, recoveries for crystal violet and fluoroquinolones were very low (< 10%). Current
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veterinary drug residue methods used by USDA23, 24 for bovine muscle use 4:1 acetonitrile:water
Acetonitrile is usually preferred because of its ability to
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for extraction.
The method has some advantages, including extracting a wide range of
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compounds and allowing very rapid sample throughput. However, although this extractant
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appeared to extract avermectins successfully in bovine muscle, the 80% acetonitrile solution was
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too polar to successfully extract ivermectin from high-fat salmon. In addition, the final extract
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composition described in the USDA method (70% acetonitrile) gave poor peak shape for polar,
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early eluting analytes using the rapid reversed phase chromatographic gradient developed for this
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screening method.
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chromatography, but would sacrifice sensitivity.
Further dilution with water of the final extract would improve
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Our initial investigations focused on using acetonitrile containing acid modifiers as an
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extractant. Acetonitrile with 0.1% acetic acid has been used25 to extract residues from salmon
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and was successful in extracting emamectin and malachite green. Acetonitrile with 1% acetic
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acid has also been used26 for shrimp with similar results. In initial experiments with salmon,
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degradation of several β-lactams was observed when 0.2% or 1% formic acid in acetonitrile was
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used as an extractant. Formic acid in water is known27 to cause rapid degradation of monobasic
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penicillins. Further, formic acid in acetonitrile did not extract avermectins well from salmon and
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had an adverse effect on macrolides. For these reasons, acetic acid was chosen to acidify the
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acetonitrile extractant. By increasing acetic acid from 0.1 to 2% in the acetonitrile extractant, the
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recoveries for fluoroquinolones, avermectins and penicillins doubled. Previous work in our
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laboratory4 showed that the addition of p-TSA improved the recovery of dyes and
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fluoroquinolone compounds so this acid was added to the 2% acetic acid in acetonitrile
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extractant. Provided that acetic acid was also present in the extractant, the p-TSA did not seem
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to appreciably degrade penicillins. The final extraction solvent chosen was acetonitrile with 2%
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acetic acid with 0.2% p-TSA. A comparison of recoveries for representative analytes using
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variable amounts of acid modifiers in tilapia (using Oasis PRiME HLB) is shown in Figure 1.
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Sample Clean-up
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Two new types of cleanup techniques designed specifically for high-fat samples were
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evaluated. Agilent’s EMR® Lipid system is a dispersive SPE technique involving two primary
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sorbents: a water-activated sorbent designed to specifically trap fats containing >5-carbon
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aliphatic chains and a second sorbent containing magnesium sulfate and sodium chloride. A
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published report16 using this technique for the analysis of veterinary drug residues in bovine liver
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was initially evaluated for salmon. Although this procedure worked well for many analytes in
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Table 1, some were not recovered. The 5% formic acid solution used as the extractant in the
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application note did not work well for some penicillins or for the avermectins in salmon. The
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magnesium sulfate sorbent also greatly lowered the recovery of some tetracyclines.
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A Waters Oasis ®PRiME HLB SPE cartridge was also evaluated as a clean-up tool. As
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described in a published method17 for the analysis of veterinary residues in pork tissue, a portion
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of tissue extract is introduced into the SPE cartridge (no conditioning is required) and collected
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by gravity drain. For this method, two changes were made to accommodate the analyte list in
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Table 1. First the extractant of formic acid, acetonitrile and water was changed to the final
327
optimized extractant described above. Second, to improve sensitivity, a portion of the extract
328
was evaporated (instead of diluted per original method) and reconstituted with 9:1
329
water/acetonitrile to allow for successful reversed-phase chromatography. For the newest triple
330
quadrupole mass spectrometers, dilution may become less of an issue, but for full scan data
331
acquisition obtained using the Orbitrap, a 10 minute evaporation step was included to
332
concentrate the extract. The larger 200 mg SPE sorbent size was used to collect a larger volume
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of extractant for evaporation and concentration. Various HPLC filters were evaluated for use in
334
filtering the final extract before injection. Although polyvinylidene fluoride filters worked the
335
best (compared to nylon or PTFE), we observed inconsistent or no recovery after filtration in
336
solvent spikes for some analytes. For this reason the final extract was not filtered but was
337
instead centrifuged to help remove any remaining particulates after the evaporation and
338
reconstitution step.
339
For salmon, some compounds (avermectins, brilliant green and crystal violet) were lost
340
when extracts were evaporated and reconstituted most likely because they partitioned into
341
insoluble lipid material.
342
compounds in salmon as described earlier in the experimental section. Because the relevant
343
compounds for the acetonitrile injection elute late in the reversed phase chromatographic system,
344
good peak shapes and consistent retention times are achieved despite the high organic content.
345
This extra injection was also required for nonpolar negative ion compounds (lufenuron,
346
teflubenzuron, toltrazuril) fortified in salmon. A portion of the acetonitrile extract (before
347
drying) was also tested with the other fish matrices. In general, this step was not necessary with
348
the less fatty fish as residues were adequately recovered in the evaporated and reconstituted
349
extract.
350
Method Evaluation
This necessitated a separate acetonitrile injection to detect those
351
Although this method is meant to be used as a screen, some evaluation was made of its
352
quantitative performance (analyte recoveries and method precision). The majority of the test
353
compounds fortified in tilapia had recoveries (as compared to a single solvent standard at the
354
target testing level) of greater than 70%. Recovery values for selected analytes in tilapia are
355
shown in Figure 2. Some compounds such as avermectins gave high recoveries (150-220%)
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compared to a solvent standard, indicating matrix signal enhancement might occur especially
357
when monitoring sodiated molecules. A few compounds (such as penillic acid and erythromycin
358
dehydrate) were formed by decomposition during the acidic extraction process, so their apparent
359
recoveries were quite high as compared to a solvent standard. The compounds with lower
360
recoveries tended to be β-lactams, benzimidazoles, and macrolides. No changes were made to
361
the extraction method to accommodate the negative ion compounds.
362
Strategy for screening using HRMS
363
The strategy for this method development was to optimize and validate sample preparation and
364
MS acquisition methods for representative compounds that are most likely to be used in
365
aquaculture, and then use the full scan HRMS capability along with a compound database of
366
veterinary drugs to screen samples for >260 additional compounds.
367
compound databases with exact mass data for thousands of analytes are commercially available,
368
this method is focused on more limited set of analytes that are likely to be used as veterinary
369
drugs. In addition, a majority of the compounds in this database have been analyzed with this
370
LC-MS method so that relevant retention time data have been obtained. The chromatographic
371
parameters were also designed to accommodate a large number of compounds with very
372
different polarities in a relatively short analysis time. The retention times of the test compounds,
373
along with exact masses of precursor and product ions, are listed in Table 1. Exact mass data for
374
product ions which have been verified as the correct theoretical value are also included.11,
375
Searching against larger commercial databases containing additional compounds such as
376
pesticides, forensic chemicals, and other contaminants can always be performed, but this can
377
lead to a higher percentage of false detects.11
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Although very large
28
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378
Two types of MS acquisition methods were evaluated. Initially, nontargeted AIF was
379
used to analyze the extracts. With AIF data acquisition, all precursor ions (MS1) eluting into the
380
MS at a given time are allowed into the HCD cell to form product ions. This results in a
381
“product ion” spectrum that is a mixture of ions from different analytes and matrix components.
382
However, using the HRMS instrument’s ability to extract data within a narrow mass range, a
383
specific product ion in that spectrum can be detected within the retention time window of the
384
precursor ion to identify the compound.
385
generating screening data as shown below. A more targeted approach, DDMS2, was also used to
386
generate product ions specific to selected precursor ion from compounds on an inclusion list.
387
Data Independent Analysis (which allows subsets of precursors into the HCD cell) was also
388
investigated, but was not found to have any clear advantages over AIF at this time.
389
Results for Validation Samples
390
AIF data
391
With this screening method, data from fortified samples were analyzed to determine if the test
392
compounds could be detected, identified, and measured at a concentration near the TTL. Area
393
counts from extracted ion chromatograms of the precursor ions were compared to those from a
394
matrix-extracted standard fortified at the TTL (1X). Figure 3 shows examples of extracted ion
395
chromatograms of selected positive ion residues in tilapia fortified at the TTL.
AIF proved to be a simple, reliable method of
396
Semi-quantitative limit testing determines if a residue is present at or above the
397
concentration of interest, so it is important to measure the variance of the signals generated from
398
residues present in samples at this concentration. A threshold cutoff value can then be set to
399
determine when to call a sample “presumptive positive”.4, 18 For all matrices tested, 10 replicate
400
samples fortified at the TTL were extracted and analyzed on the same day. One extracted sample
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401
was assigned as the standard (100% relative recovery) and the relative recoveries of the other
402
replicates were determined by comparison to that single standard (data included as supplemental
403
Table 2). The precision was also determined. For most residues the average recoveries for these
404
data were greater than 90% and standard deviations were less than 15%.
405
cloxacillin (CLOX) in salmon samples fortified at the TTL had an average recovery of 91% with
406
a standard deviation of 6%. The limit threshold cutoff value for that compound in salmon
407
calculates to 80%. This means any salmon sample with a signal for CLOX greater than 80% of
408
the signal in a matrix extracted TTL standard could (with 95% confidence) contain CLOX at a
409
concentration at or above the TTL. For some residues, however, the standard deviations were
410
higher and the threshold cutoff values were therefore lower. In order to avoid false negatives
411
and simplify data analysis by treating all test compounds the same, a threshold cutoff value of
412
≥50% TTL seemed reasonable for all analytes to be considered presumptive positive.
For example,
413
In addition to a residue having the correct exact mass precursor ion (5 ppm) and a signal
414
of ≥50% compared to that analyte in a matrix-extracted standard of the same matrix fortified at
415
the TTL, other criteria for a residue to be presumptive positive for the test compounds include
416
retention time matching and the presence of at least one fragment ion with the correct exact mass
417
(within 10 ppm).19 Isotope matching can also be used for comparison, but is not required in the
418
identification criteria. The allowed retention time window was set fairly wide (30 s each side of
419
specified time) to accommodate drift in the method due to column age or slight changes in
420
mobile phase composition. The measured retention times were generally much tighter than the
421
allowed 30 s (usually within 0.1 min). The data were examined closely to determine if a peak
422
was within the established experimental time when compared to matrix-extracted standards
423
analyzed on the same day. Figure 4 shows selected product ions and the precursor isotope
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424
pattern for ciprofloxacin in catfish (5 µg/kg). The ciprofloxacin data show that the software is
425
able to isolate and evaluate product ions generated by AIF to determine if the criteria for
426
identification are met.
427
standard), the number of false negatives for samples fortified at the TTL and number of false
428
positives (for the test compounds) in matrix blanks could be determined and are shown in Table
429
2. In general, the performance of these test compounds in this screen was very good using the
430
AIF acquisition program with false positives detected for only one compound and less than 2%
431
false negatives for a majority of the compounds in all matrices. A few compounds (amoxicillin,
432
florfenicol amine, avermectins, and lufenuron) had higher rates (5-20%) of false negatives at the
433
TTL level. Sulfacetamide performed poorly in the screening method with low variable recovery
434
and high background and was removed from the list of validated test compounds.
With these criteria (including signal ≥50% compared to the TTL
435
Since the TTLs are somewhat arbitrary (any amount of a non-approved drug identified
436
could be considered violative), the minimum detectable limits using AIF data were also
437
determined by fortifying samples with the test compounds at lower levels. The same criteria,
438
with the exception of the ≥ 50% of signal compared to TTL, were applied to determine if the
439
residues could be detected and identified. In most cases, analytes were still identified in extracts
440
from fish and shellfish that had been fortified at 0.1X the TTL.
441
Because this method is designed to analyze a wide variety of compounds with very
442
different polarities, the performance for some drugs may not compare well to methods that were
443
developed specifically for those analytes. For example, some of the avermectins do not meet all
444
the criteria for presumptive positive with this screening method below a fortification level of 100
445
µg/kg, even though the current testing level for these compounds at FDA is 10 µg/kg.20 There
446
are several reasons for this including the fact that these compounds are much more nonpolar than
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most veterinary drugs, so extraction efficiency and low solubility may reduce recoveries.
448
Penicillins, on the other hand, are most soluble in water; to extract these compounds
449
simultaneously with avermectins proves difficult. The extraction presented here was the best
450
compromise. In addition, although using the generic acetonitrile/0.1% formic acid LC gradient
451
works well for a majority of the veterinary drugs in the >330 compound database, a buffer
452
containing ammonium formate would have facilitated the formation of ammonium adducts for
453
the avermectins (rather than the sodiated ions which are more difficult to dissociate into product
454
ions). The sodiated precursor ions of ivermectin and doramectin were observed in extracts at
455
levels below 200 µg/kg (0.1 X or 20 µg/kg), meaning the method could potentially be used to
456
detect these residues at lower levels. The incidence of false positives that can occur when only
457
using the measured exact mass of the precursor ion would be less for these compounds because
458
their m/z values (m/z 897, 921) are above those of most background contaminants. However, the
459
absence of product ions prevented confirmation as presumptive positive under the described
460
criteria.
461
detected for this compound at the 0.1X level.
Emamectin, in contrast, forms a protonated molecule, and product ions could be
462
This HRMS screening method was also validated for several negative ion compounds. It
463
was possible to detect chloramphenicol in shrimp and salmon using negative ion AIF data
464
acquisition below the established TTL (0.3 µg/kg); florfenicol and thiamphenicol were detected
465
at 0.1X of the 5 µg/kg TTL. Although the precursor ion of toltrazuril sulfone was detected at
466
low levels, it did not form consistent product ions so could not be considered presumptive
467
positive even at the TTL. The more nonpolar negative ion compounds were detected and
468
identified by AIF at low levels from fortified salmon and catfish in the acetonitrile portion
469
(lufenuron, teflubenzuron), or in the final 10% acetonitrile extract (toltrazuril metabolites) or
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both (diflubenzuron, toltrazuril ). All negative ion compounds could be detected using the
471
extraction procedure in fortified shrimp, tilapia, and eel although the recoveries for lufenuron
472
were variable.
473
DDMS2 data
474
Data Dependent MS2 acquisition, where a narrow range of precursor ions are allowed through
475
the quadrupole into the HCD cell and fragmented, generated product ion spectra more specific to
476
the precursor. These spectra can be used to generate product ion ratios and/or search against
477
library spectra. With this method, DDMS2 worked well for a majority of compounds tested.
478
Figure 5 illustrates how isobaric compounds flumequine and oxolinic acid (FLU and OXO) can
479
be identified on the basis of their difference in precursor ion exact mass, retention times, and
480
unique product ion spectra obtained by DDMS2. Some analytes, however, were missed by
481
DDMS2 data acquisition because the threshold signal level needed to trigger the product ion
482
spectra was not reached.
483
recoveries. The number of false negatives based on DDMS2 acquisition is shown in Table 2.
484
Approximately 20 of the positive ion test compounds had a false negative rate of >10% at the
485
TTL. Many of these analytes did produce product ion spectra using DDMS2 in extracts from fish
486
that had been fortified at a slightly higher level (2X TTL).
487
compounds did not generate DDMS2 product ion spectra at the TTL, even though product ions
488
were detected using AIF. Because AIF data gave more reliable results for the presence of
489
confirmatory product ions, DDMS2 spectra were collected to obtain additional qualitative
490
information but were not required for initial screening and identification.
491
Results for Incurred and Regulatory Fish Samples
492
Test Compounds
Generally this occurred for residues with low TTL and/or poor
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Similarly, some negative ion
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493
In addition to fortified samples, incurred and violative regulatory samples were also tested with
494
this screening method. Initially data from these samples were evaluated to look for the test
495
compounds that had been validated. The results for residues found in incurred and violative
496
samples are shown in Table 3 and 4, respectively. As expected, high levels of sulfadiazine
497
(SDZ) were found in tilapia dosed with that compound. An average of 230 µg/kg SDZ was
498
measured in fish dosed at 1 mg/kg body weight (1 day withdrawal), and up to 650 µg/kg SDZ in
499
fish dosed with 5 mg/kg body weight (3 day withdrawal). However, only low levels (< 1 µg/kg)
500
of trimethoprim were detected in tilapia dosed with a combination of trimethoprim and SDZ, and
501
DDMS2 product ion spectra were only triggered for trimethoprim in one of the two dosed fish.
502
Sulfadiazine was also identified in five regulatory tilapia samples that had previously been found
503
to be violative for that residue. The concentrations of SDZ in these fish estimated using a one-
504
point matrix-extracted TTL standard for comparison were similar to the amounts reported
505
previously using LC-MS/MS methodology.4 Sulfamethoxazole and trimethoprim were identified
506
in another sample of tilapia. Sulfamethazine (SMZ) was found in regulatory eel samples; one
507
sample had levels of SMZ > 100 µg/kg, and the other was below the threshold testing level (
260 additional compounds contains information to
545
match precursor ion and, for many analytes, retention time and fragment ions. When data from
546
the incurred and violative regulatory samples were compared to compounds in the larger
547
database, several other analytes were detected and these results are also shown in Tables 3 and 4.
548
In fish extracts containing high levels of sulfonamides, the N4 acetyl metabolites were also
549
detected. These metabolite products have been described8, and were included in the DDMS2
550
inclusion list so that unique product ion spectra could be obtained.
551
Another additional compound that was found in many of these samples was ethoxyquin
552
dimer, a known by-product of ethoxyquin in fish.32 When these samples were first analyzed, a
553
reference standard for ethoxyquin dimer was not available for comparison, and the compound
554
was detected by matching the exact m/z of the precursor.
555
purchased and it was determined that the retention time and product ions also matched that
556
compound. The metabolite desethylene-enrofloxacin (des-ENRO) that had been found earlier in
557
milk samples from cows dosed with ENRO33 was also found in the catfish dosed with this drug.
558
Thiabendazole was also identified in one regulatory eel sample when the data were compared to
559
the larger veterinary drug database, although the estimated levels were quite low (< 1 µg/kg
560
when compared to a solvent standard). One regulatory eel sample was also analyzed by AIF
561
using negative ion data acquisition, but no compounds were detected.
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26
562
It is worth noting that the rate of false detections increased when the data were compared
563
to more potential analytes in a larger compound database. Searching by exact mass of precursor
564
ion only can lead to a significant number of false hits (40-50) even with the narrower mass
565
window of 3 ppm. Adding criteria for retention time and product ion match greatly reduced the
566
number of potential positives to a reasonable number (< 5). Some compounds were often
567
detected in many samples (including matrix controls) at low abundances (~104 counts) with the
568
correct retention time and fragment ions using the screening data analysis program with AIF
569
data, but DDMS2 spectra could not be obtained. These include small mass compounds with
570
common fragment ions (e.g., lidocaine, benzocaine) and hormones (hydrocortisone,
571
prednisolone, etc.).
572
These results from the analysis of incurred and violative regulatory aquaculture samples
573
demonstrate the ability of the HRMS screening method to confirm the findings of more
574
traditional LC-MS/MS methods for a wide range of veterinary drug residues. In addition, the
575
capability to detect metabolites such as N4 acetyl-sulfonamides, des-ENRO, and ethoxyquin
576
dimer provides verification that the veterinary drugs were administered to the fish and are not an
577
artifact of post-production processing or analysis. The identification of other unexpected drug
578
residues in these samples, even at low levels, also demonstrates the potential for expanding the
579
scope of monitoring for chemical contaminants in aquacultured fish and shellfish using HRMS
580
screening methods.
581
ACKNOWLEDGEMENTS
582
The authors would like to thank Charles Gieseker of the FDA Center for Veterinary Medicine
583
Office of Research for providing the incurred fish tissues.
584
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SUPPORTING INFORMATION
585 586
The supporting information includes two additional tables of information. The first supporting
587
information table describes the number and type of samples analyzed for method validation. The
588
second supporting table consists of the limit test threshold cutoff values for each residue in the
589
different seafood matrices using the average relative recovery and standard deviation data from
590
replicate extracts fortified at the target testing level. REFERENCES
591 592 593
1. Boison, J. O.; Turnipseed, S. B., A review of aquaculture practices and their impacts on chemical food safety from a regulatory perspective. J. AOAC Int. 2015, 98, 541-549.
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2.
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3. Li, H.; Kijak, P. J.; Turnipseed, S. B.; Cui, W., Analysis of veterinary drug residues in shrimp: a multi-class method by liquid chromatography-quadrupole ion trap mass spectrometry. J. Chromatogr. B 2006, 836, 22-38.
598 599 600 601
4. Storey, J. M.; Clark, S. B.; Johnson, A. S.; Andersen, W. C.; Turnipseed, S. B.; Lohne, J. J.; Burger, R. J.; Ayres, P. R.; Carr, J.; Madson, M. R., Analysis of sulfonamides, trimethoprim, fluoroquinolones, quinolones, triphenylmethane dyes and methyltestosterone in fish and shrimp using liquid chromatography mass spectrometry. J. Chromatogr. B 2014, 972, 38-47.
602 603 604
5. Dasenaki, M. E.; Thomaidis, N. S., Multi-residue determination of 115 veterinary drugs and pharmaceutical residues in milk powder, butter, fish tissue and eggs using liquid chromatography–tandem mass spectrometry. Anal. Chim. Acta 2015, 880, 103-121.
605 606 607
6. Turnipseed, S. B.; Lohne, J. J.; Boison, J. O., Review: Application of high resolution mass spectrometry to monitor veterinary drug residues in aquacultured products. J. AOAC Int. 2015, 98, 550-558.
608 609 610 611 612
7. Verdon, E.; Hurtaud-Pessel, D.; Thota, J. R., A role for high-resolution mass spectrometry in the high-throughput analysis and identification of veterinary medicinal product residues and of their metabolites in foods of animal origin. In High-Throughput Analysis for Food Safety, Wang, P. G.; Vitha, M. F.; Kay, J. F., Eds. John Wiley & Sons, Inc: Hoboken, NJ, 2014.
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8. Turnipseed, S. B.; Clark, S. B.; Storey, J. M.; Carr, J. R., Analysis of veterinary drug residues in frog legs and other aquacultured species using liquid chromatography quadrupole time-of-flight mass spectrometry. J. Agric. Food Chem. 2012, 60, 4430-4439.
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9. Dasenaki, M. E.; Bletsoue, A. A.; Koulis, G. A.; Thomaidis, N. S., Qualitative multiresidue screening method for 143 veterinary drugs and pharmaceuticals in milk and fish
World Bank Report, Fish to 2030: Prospects for Fisheries and Aquaculture. 2013.
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618 619
tissue using liquid chromatography quadrupole-time-of-flight mass spectrometry. J. Agric. Food Chem. 2015, 63, 4493-4508.
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10. Boix, C.; Ibanez, M.; Sancho, J. V.; Leon, N.; Yusa, V.; Hernandez, F., Qualitative screening of 116 veterinary drugs in feed by liquid chromatography–high resolution mass spectrometry: Potential application to quantitative analysis. Food Chem. 2014, 160, 313-320.
623 624 625 626
11. Turnipseed, S. B.; Lohne, J. J.; Storey, J. M.; Andersen, W. C.; Young, S. L.; Carr, J.; Madson, M. R., Challenges in implementing a screening method for veterinary drugs in milk using liquid chromatography quadrupole time-of-flight mass spectrometery. J. Agric. Food Chem. 2014, 62, 3660-3674.
627 628 629 630
12. Gomez Perez, M. L.; Romero-Gonzalez, R.; Plaza-Bolanos, P.; Genin, E.; Vidal, J. L.; Frenich, A. G., Wide-scope analysis of pesticide and veterinary drug residues in meat matrices by high resolution MS:detection and identification using Exactive-Orbitrap. J. Mass Spectrom. 2014, 49, 27-36.
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13. Wang, X.; Liu, Y.; Su, Y.; Yang, J.; Wang, Z.; He, L., High-throughput screening and confirmation of 22 banned veterinary drugs in feedstuffs using LC-MS/MS and high-resolution orbitrap mass spectrometry. J. Agric. Food Chem. 2014, 62, 516-527.
634 635 636 637
14. Wang, J.; Leung, D.; Chow, W.; Chang, J.; Wong, J. W., Development and validation of a multiclass method for analysis of veterinary drug residues in milk using ultrahigh performance liquid chromatography electrospray ionization quadrupole orbitrap mass spectrometry. J. Agric. Food Chem. 2015, 63, 9175-9187.
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15. Samanidou, V.; Bitas, D.; Charitonos, S.; Papadoyannis, I., On the extraction of antibiotics from shrimps prior to chromatographic analysis. Chromatogr. 2016, 3, 8.
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16. Zhao, L.; Lucas, D., Multi-residue analysis of veterinary drugs in bovine liver by LCMS/MS. Agilent Application Note 2015.
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17. Young, M.; Tran, K., Oasis PRiME HLB cartridge for effective cleanup of meat extracts prior to multi-residue veterinary drug UPLC-MS analysis. Waters Application Note 2015.
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18. FDA, Guidelines for the Validation of Chemical Methods for the FDA Foods and Veterinary Medicine Program, 2nd Edition. 2015.
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19. FDA, Acceptance criteria for confirmation of identity of chemical residues using exact mass data for the FDA Foods and Veterinary Medicine Program. 2015.
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20.
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21. Robert, C.; Gillard, N.; Brasseur, P.-Y.; Pierret, G.; Ralet, N.; Dubois, M.; Delahaut, P., Rapid multi-residue and multi-class qualitative screening for veterinary drugs in foods of animal origin by UHPLC-MS/MS. Food Addit. Contam. A 2013, 30, 443-457.
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22. Smith, S.; Gieseker, C.; Reimschuessel, R.; Decker, C.-S.; Carson, M. C., Simultaneous screening and confirmation of multiple classes of drug residues in fish by liquid chromatography-ion trap mass spectrometry. J. Chromatogr. A 2009, 1216, 8224-8232.
FDA, Chemotherapeutics in Seafood Compliance Program 7304.018. 2016.
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23. Geis-Asteggiante, L.; Lehotay, S. J.; Lightfield, A. R.; Dutko, T.; Ng, C.; Bluhm, L., Ruggedness testing and validation of a practical analytical method for >100 veterinary drug residues in bovine muscle by ultrahigh performance liquid chromatography–tandem mass spectrometry. J. Chromatogr. A 2012, 1258, 43-54.
659 660 661
24. Schneider, M. J.; Lehotay, S. J.; Lightfield, A. R., Validation of a streamlined multiclass, multiresidue method for determination of veterinary drug residues in bovine muscle by liquid chromatography–tandem mass spectrometry. Anal. Bioanal. Chem. 2015, 407, 4423-4435.
662 663 664
25. Hernando, M. D.; Mezcua, M.; Suárez-Barcena, J. M.; Fernández-Alba, A. R., Liquid chromatography with time-of-flight mass spectrometry for simultaneous determination of chemotherapeutant residues in salmon. Anal. Chim. Acta 2006, 562, 176-184.
665 666 667
26. Villar-Pulido, M.; Gilbert-López, B.; García-Reyes, J. F.; Martos, N. R.; Molina-Díaz, A., Multiclass detection and quantitation of antibiotics and veterinary drugs in shrimps by fast liquid chromatography time-of-flight mass spectrometry. Talanta 2011, 85, 1419-1427.
668 669 670
27. Mastovska, K.; Lightfield, A. R., Streamlining methodology for the multiresidue analysis of beta-lactam antibiotics in bovine kidney using liquid chromatography-tandem mass spectrometry. J. Chromatogr. A 2008, 1202, 118-123.
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28. Geis-Asteggiante, L.; Nuñez, A.; Lehotay, S. J.; Lightfield, A. R., Structural characterization of product ions by electrospray ionization and quadrupole time-of-flight mass spectrometry to support regulatory analysis of veterinary drug residues in foods. Rapid Commun. Mass Spectrom. 2014, 28, 1061-1081.
675 676 677 678
29. Roybal, J. E.; Walker, C. C.; Pfenning, A. P.; Turnipseed, S. B.; Storey, J. M.; Gonzales, S. A.; Hurlbut, J. A., Concurrent determination of four flouroquinolones in catfish, shrimp, and salmon by liquid chromatography with fluorescence detection. J. AOAC Int. 2002, 85, 12931301.
679 680 681 682
30. Andersen, W. C.; Casey, C. R.; Schneider, M. J.; Turnipseed, S. B., Expansion of scope of AOAC First Action Method 2012.25; Single laboratory validation of triphenylmethane dye and leuco metabolite analysis in shrimp, tilapia, catfish and salmon by LC-MS/MS. J. AOAC Int. 2015, 98, 636-648.
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32. He, P.; Ackman, R. G., Residues of ethoxyquin and ethoxyquin dimer in ocean-farmed salmonids determined by high-pressure liquid chromatography. J. Food Sci. 2000, 65, 13121314.
687 688 689
33. Turnipseed, S. B.; Storey, J. M.; Clark, S. B.; Miller, K., Analysis of veterinary drugs and metabolites in milk using quadrupole- time of flight liquid chromatography mass spectrometry. J. Agric. Food Chem. 2011, 59, 7569-7581.
FDA, Code of Federal Regulations. Title 21 2015, 573.380 Ethoxyquin in animal feeds.
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692
FIGURE CAPTIONS
693
Figure 1. Comparison of selected analyte recoveries in tilapia at 1X TTL with different levels of
694
acid modifiers (normalized to 0.2% p-TSA and 2% acetic acid (AA) recoveries).
695
Figure 2. Recoveries for selected analytes in tilapia fortified at the target testing level (1X) as
696
compared to a 1X solvent standard.
697
Figure 3. Extracted ion chromatograms (5 ppm window) from MS1 AIF data for representative
698
test compounds in tilapia fortified at target testing level
699
Figure 4. AIF product ions (A) and precursor isotope pattern (B) for CIP (5 µg/kg) in catfish
700
extract compared to theoretical values.
701
Figure 5. DDMS2 data for OXO and FLU in shrimp at target testing level (10 µg/kg). (A)
702
Extracted ion chromatograms for MH+ (B) Chromatograms for MS2 of m/z 262 (C) Product ion
703
spectra
704
Figure 6. AIF data for regulatory eel sample #1. (A) Test compounds that were presumptive
705
positive (signal ≥50% target testing level). (B) Test compounds identified, but at concentrations
706
< 50% target testing level. (C) Other compounds identified when compared to larger veterinary
707
drug database.
708
Figure 7. DDMS2 product ion spectra for residues in regulatory eel sample #2. (A) lincomycin
709
(B) ethoxyquin dimer.
710
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Table 1. Test Compounds Analyte Doramectin (DOR) Emamectin B1a (EMA)
Class Avermectin Avermectin
TTL (µg/kg)
RT (min)
Formula
MH+
Fragment Ions
200
a
11.3
C50H74O14
921.4971
200
a
9.6
C49H75NO13
886.5311
a
Ivermectin B1a (IVER)
Avermectin
200
Amoxicillin (AMOX)
β-lactam
Ampicillin (AMP)
b
b
449.2298
777.4126
82.0651
158.1176
609.3398
753.4184
302.1962
12.0
C48H74O14
897.4971
100
1.9
C16H19N3O5S
366.1118
114.0372
208.0427
349.0853
β-lactam
25
4.2
C16H19N3O4S
350.1169
106.0651
114.0372
160.0427
Aspoxicillin (ASP)
β-lactam
25
2.6
C21H27N5O7S
494.1074
160.0427
250.1186
366.1118
Cloxacillin (CLOX)
β-lactam
25
9.0
C19H18ClN3O5S
436.0729
160.0427
277.0375
Dicloxacillin (DICLOX)
β-lactam
25
9.6
C19H17Cl2N3O5S
470.0339
160.0427
310.9985
Oxacillin (OXAC)
β-lactam
25
8.5
C19H19N3O5S
402.1118
114.0372
160.0427
243.0764
Penicillin G (PEN G)
β-lactam
25
7.5
C16H18N2O4S
335.1060
114.0372
160.0427
176.0706
NA
4.9
C16H18N2O4S
335.1060
128.0528
160.0427
289.0997 234.0696
c
Penillic acid
β-lactam
Albendazole (ALB)
Benzimidazole
50
7.0
C12H15N3O2S
266.0958
159.0427
191.0148
Albendazole sulfoxide (ALB SULF)
Benzimidazole
50
5.0
C12H15N3O3S
282.0907
208.0175
240.0437
Fenbendazole (FEN)
Benzimidazole
50
8.4
C15H13N3O2S
300.0801
159.0427
268.0539
Fenbendazole sulfone (FEN SULF)
Benzimidazole
50
7.3
C15H13N3O4S
332.0700
300.0437
Cephapirin (CEPH)
Cephalosporin
25
3.4
C17H17N3O6S2
424.0632
152.0165
292.0573
Brilliant Green (BG)
Dye
1
9.5
C27H33N2
385.2638 d
297.1386
341.2012
d
251.1543
356.2121
Crystal violet (CV)
Dye
1
9.0
C25H30N3
372.2434
Leucocrystal violet (LCV)
Dye
1
5.6
C25H31N3
374.2591
239.1543
253.1699
358.2278
Leucomalachite green (LMG)
Dye
1
8.2
C23H26N2
331.2169
194.0964
239.1543
315.1856
208.1121
313.1699
d
Malachite green (MG)
Dye
1
8.1
C23H25N2
329.2012
Ciprofloxacin (CIP)
Fluoroquinolone
5
4.6
C17H18FN3O3
332.1405
245.1085
288.1507
Danofloxacin (DANO)
Fluoroquinolone
5
5.0
C19H20FN3O3
358.1562
283.1241
314.1663
Difloxacin (DIFLOX)
Fluoroquinolone
5
5.4
C21H19F2N3O3
400.1467
299.0990
356.1569
Enrofloxacin (ENRO)
Fluoroquinolone
5
5.1
C19H22FN3O3
360.1718
245.1085
316.1820
338.1499 342.1612
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Analyte
Class
Norfloxacin (NOR)
Fluoroquinolone
TTL (µg/kg)
RT (min)
5
4.7
C16H18FN3O3
320.1405
276.1507
302.1299
Formula
MH+
Fragment Ions
Sarafloxacin (SAR)
Fluoroquinolone
5
5.4
C20H17F2N3O3
386.1311
299.0990
342.1413
Methyl Testosterone (M TET)
Hormone
0.8
9.4
C20H30O2
303.2319
97.0648
109.1011
Lincomycin (LIN)
Lincomycin
50
3.8
C18H34N2O6S
407.2210
126.1277
359.2214
Azithromycin (AZI)
Macrolide
50
5.4
C38H72N2O12
749.5158
158.1176
591.4210
Erythromycin A (ERY)
Macrolide
50
6.6
C37H67NO13
734.4685
83.0491
158.1176
c
Erythromycin dehydrated
Macrolide
NA
7.2
C37H65NO12
716.4580
158.1176
Spiramycin (SPIRO)
Macrolide
50
5.5
C43H74N2O14
843.5213
174.1125
540.3136
174.1125
522.3789
e
Tilmicosin (TIL)
Macrolide
50
6.0
C46H80N2O13
435.2903
Tylosin A (TYL)
Macrolide
50
7.0
C46H77NO17
916.5264
174.1125
Ketoconazole (KETO)
Nitromidazole
10
7.2
C26H28Cl2N4O4
531.1560
82.0525
489.1455
Metronidazole (MNZ)
Nitromidazole
10
2.0
C6H9N3O3
172.0717
82.0525
128.0455
1.33
C10H14FNO3S
248.0751
104.0632
130.0651
4.6
C14H18N4O2
275.1503
123.0665
259.1190
f
Florfenicol Amine (FFA)
Phenicol
50
Ormetoprim (ORM)
Potentiator
10
576.3742
695.460
230.0646
Trimethoprim (TRIMETH)
Potentiator
10
4.3
C14H18N4O3
291.1452
123.0665
230.1162
Ethoxyquin (ETHOX)
Preservative
50
7.7
C14H19NO
218.1539
148.0757
176.1070
Flumequine (FLU)
Quinolone
10
7.82
C14H12FNO3
262.0874
202.0299
244.0768
Nalidixic Acid (NAL)
Quinolone
10
7.6
C12H12N2O3
233.0921
187.0502
215.0815
Oxolinic Acid (OXO)
Quinolone
10
6.5
C13H11NO5
262.0710
216.0291
244.0604
Sulfacetamide (SAA)
Sulfonamide
10
2.2
C8H10N2O3S
215.0485
92.0495
108.0444
156.0114
Sulfachloropyridazine (SCP)
Sulfonamide
10
5.8
C10H9ClN4O2S
285.0208
92.0495
108.0444
156.0114
Sulfaclozine (SULC)
Sulfonamide
10
6.9
C10H9ClN4O2S
285.0208
92.0495
108.0444
156.0114
Sulfadiazine (SDZ)
Sulfonamide
10
2.9
C10H10N4O2S
251.0597
92.0495
108.0444
156.0114
Sulfadimethoxine (SDM)
Sulfonamide
10
7.0
C12H14N4O4S
311.0809
108.0444
156.0114
156.0768
Sulfadoxine (SDX)
Sulfonamide
10
6.1
C12H14N4O4S
311.0809
92.0495
108.0444
156.0114
Sulfaethoxypyridazine (SEP)
Sulfonamide
10
6.2
C12H14N4O3S
295.0859
92.0495
108.0444
156.0114
Sulfamerazine (SMR)
Sulfonamide
10
4.1
C11H12N4O2S
265.0754
92.0495
108.0444
156.0114
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TTL (µg/kg)
RT (min)
Sulfonamide
10
4.8
C12H14N4O2S
279.0910
Sulfamethoxazole (SMX)
Sulfonamide
10
6.1
C10H11N3O3S
254.0594
92.0495
108.0444
156.0114
Sulfamethoxypyridazine (SMP)
Sulfonamide
10
5.1
C11H12N4O3S
281.0703
108.0444
126.0662
156.0114
Sulfamonomethoxine (SULFMON)
Sulfonamide
10
5.6
C11H12N4O3S
281.0703
92.0495
108.0444
156.0114
Sulfapyridine (SPD)
Sulfonamide
10
4.0
C11H11N3O2S
250.0645
108.0444
156.0114
184.0869
Sulfaquinoxaline (SQX)
Sulfonamide
10
7.1
C14H12N4O2S
301.0754
92.0495
108.0444
156.0114
Sulfathiazole (STZ)
Sulfonamide
10
108.0444
156.0114 428.1340
Analyte
Class
Sulfamethazine (SMZ)
Chlortetracycline (CTC) Doxycycline (DC) Oxytetracycline (OTC)
Tetracycline Tetracycline Tetracycline
Tetracycline (TC)
Tetracycline
Negative Ion Analytes
Class
Chloramphenicol(CAP)
Formula
MH+
Fragment Ions 92.0495
108.0444
3.9
C9H9N3O2S2
256.0209
92.0495
100
g
5.6
C22H23ClN2O8
479.1216
444.0845
100
g
5.9
C22H24N2O8
445.1605
154.0499
410.1234
100
g
4.7
C22H24N2O9
461.1555
154.0499
426.1183
g
4.8
C22H24N2O8
445.1605
154.0499
410.1234
100 TTL (µg/kg)
RT (min)
Phenicol
0.3
Florfenicol (FF)
Phenicol
Thiamphenicol (THIAM)
MH-
6.5
Formula C11H12Cl2N2O5
321.00505
152.0353
176.0353
5
6.0
C12H14Cl2FNO4S
355.99319
185.0278
335.9870
Phenicol
5
4.6
C12H15Cl2NO5S
353.99752
185.0278
290.0259
Toltrazuril (TOLT)
Toltrazuril
50
10
C18H14F3N3O4S
424.05843
316.98132
404.97665
Toltrazuril Sulfone (TOLT SULF)
Toltrazuril
50
9.9
C18H14F3N3O6S
456.04826
Toltrazuril Sulfoxide (TOLT SULFX)
Toltrazuril
50
9.2
C18H14F3N3O5S
440.05335
371.05781
Diflubenzuron (DIFLU)
Benzylurea
50
10
C14H9ClN2O2F2
309.02478
242.98601
289.0184
Lufenuron (LUF)
Benzylurea
50
10.4
C17H8Cl2F8N2O3
508.97115
174.95972
325.9591
Teflubenzuron (TEFLU)
Benzylurea
50
10.3
C14H6Cl2F4N2O2
378.96697
195.95378
338.95451
a
Current FDA program recommends TTL of 10 µg/kg20 b MNa+ c NA= Not applicable (degradant) d M+ e MH22+ f FDA tolerance is 1 mg/kg for FFA as marker residue in aquaculture20 g FDA tolerance is 2 mg/kg for sum of OTC, CTC, and TC in finfish and lobster 2020
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156.0114
Fragment Ions 257.0335
None
488.96492
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34
Table 2. Screening validation results
Analyte Doramectin Emamectin B1a Ivermectin B1a Amoxicillin Ampicillin Aspoxicillin Cloxacillin Dicloxacillin Oxacillin Penillic acid Albendazole Albendazole sulfoxide Fenbendazole Fenbendazole sulfone Cephapirin Brilliant Green Crystal violet Leucocrystal violet Leucomalachite green Malachite green Ciprofloxacin Danofloxacin
Class Avermectin
β-lactam
Benzimidazole
Cephalosporin Dye
Fluoroquinolone
TTL (µg/kg) 200 a 200 a 200 a 100 25 25 25 25 25 25 b 50 50 50 50 25 1 1 1 1 1 5 5
Lowest level confirmed by AIF (X) 0.5 0.1 0.5 1 0.1 1 0.1 0.5 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.5 0.5 0.5 0.1 0.5 0.5 0.5
Minimum Detectable Concentration (µg/kg) 100 20 100 100 2.5 25 2.5 12.5 2.5 2.5 5 5 5 5 2.5 0.5 0.5 0.5 0.1 0.5 2.5 2.5
ACS Paragon Plus Environment
% False Neg. at 1X (AIF) N= 77 13 1 5 6 1 0 0 0 0 0 0 0 0 0 0 1 0 3 0 0 0 0
% False Neg. at 1X (DDMS2) N= 77 19 0 57 97 13 32 2 5 31 99 0 0 0 0 0 84 14 73 42 2 10 90
% False Pos. in blanks (AIF) N= 47 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
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Analyte Difloxacin Enrofloxacin Norfloxacin Sarafloxacin Methyl testosterone Lincomycin Azithromycin Erythromycin dehyr Spiramycin Tilmicosin Tylosin A Ketoconazole Metronidazole Florfenicol Amine Ormetoprim Trimethoprim Ethoxyquin Flumequine Nalidixic Acid Oxolinic Acid Sulfacetamide Sulfachloropyridazine Sulfaclozine Sulfadiazine Sulfadimethoxine Sulfadoxine
Class
Hormone Lincomycin Macrolide
Nitromidazole Phenicol Potentiator Preservative Quinolone
Sulfonamide
TTL (µg/kg) 5 5 5 5 0.8 50 50 b 50 50 50 50 10 10 50c 10 10 50 10 10 10 10 10 10 10 10 10
Lowest level confirmed by AIF (X) 0.1 0.1 0.5 0.1 0.5 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 1 0.1 0.1 0.1 0.1 0.1
Minimum Detectable Concentration (µg/kg) 0.5 0.5 2.5 0.5 0.4 1 5 5 2.5 5 5 1 1 5 1 1 5 1 1 1 10 1 1 1 1 1
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% False Neg. at 1X (AIF) N= 77 0 0 1 0 0 0 1 0 0 0 0 0 0 9 0 0 10 0 0 0 21 0 0 1 0 0
% False Neg. at 1X (DDMS2) N= 77 0 0 0 0 2 0 12 0 1 0 0 0 13 51 0 0 10 0 0 0 100 8 0 31 0 0
% False Pos. in blanks (AIF) N=47 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 9 0 0 0 0 0
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36
Lowest level confirmed by AIF (X) 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.5 0.1 0.1 0.1 0.1 0.5 0.1 0.1 0.1 > 2e 0.5 0.5 0.5 0.5
Minimum Detectable Concentration (µg/kg) 1 1 1 1 1 1 1 1 5 10 10 10 10 0.15 0.5 0.5 0.5 > 100 25 10 10 10
% False Neg. at 1X (AIF) N= 77 0 1 0 0 0 0 0 0 6 0 0 0 0 0f 0f 0f 0f 100 f 0f 0f 8f 0f
% False Neg. at 1X (DDMS2) N= 77 0 17 14 0 8 0 4 1 16 0 0 0 2 94 f 0f 34 f 77 f 100 f 8f 69 f 34 f 11 f
% False Pos. in blanks (AIF) N=47 0 0 0 0 0 0 0 0 0 0 0 0 0 0f 0f 0f 0f 0f 0f 0f 0f 0f
TTL Analyte Class (µg/kg) Sulfaethoxypyridazine 10 Sulfamerazine 10 Sulfamethazine 10 Sulfamethoxazole 10 Sulfamethoxypyridazine 10 Sulfamonomethoxine 10 Sulfapyridine 10 Sulfaquinoxaline 10 Sulfathiazole 10 Tetracycline Chlortetracycline 100 d Doxycycline 100 d Oxytetracycline 100 d Tetracycline 100 d Chloramphenicol Phenicol 0.3 Florfenicol 5 Thiamphenicol 5 Toltrazuril Toltrazuril 50 Toltrazuril Sulfone 50 Toltrazuril Sulfoxide 50 Benzylurea Diflubenzuron 50 Lufenuron 50 Teflubenzuron 50 a Current FDA program recommends TTL of 10 µg/kg20 b Marker compounds were degradants; amounts were compared to matrix-extracted standard fortified with parent compound at TTL. c FDA tolerance is 1 mg/kg for florfenicol amine as marker residue in aquaculture20 d FDA tolerance is 2 mg/kg for sum of oxytetracycline, chlortetracycline, and tetracycline in finfish and lobster 20 e f No fragments were observed for toltrazuril sulfone For negative ion analytes: N= 35 fortified at 1X and N = 15 blanks
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Table 3. Screening results for incurred fish samples Incurred sample
Dosing Drug (X) mg/kg body weight SDZ (1)
Depuration Time
N=
Found by QqQ (µg/kg)30
1 day
2
Not analyzed
Test Compounds Presumptive Positive AIF (µg/kg)a SDZ (220)
SDZ (1)
1 day
2
Not analyzed
SDZ (240)
SDZ (5) TRIMETH (1) SDZ (5) TRIMETH (1)
3 days
2
Not analyzed
SDZ (650 )
4 days
2
Not analyzed
SDZ (280)
Catfish Inc#1
ENRO (5 )
6 days
4
Not analyzed
ENRO (620) CIP (30)
Des-ENRO
Catfish Inc#2 CatfishInc#3
ENRO (5 )
6 days
4
Not analyzed
MG (2) c CV (2) BG (2)
1 hr
1
MG (2) c CV (2) BG (2)
1 hr
1
LCV (4.3) MG (3.1) LMG (2.7) BG (1.2) BG (1.8) MG (1.8) LMG (0.8) LCV (0.4)
ENRO (601) CIP (41) LCV (2.7) LMG (0.8)
None
Tilapia Inc#1 Tilapia Inc#2 Tilapia Inc#3 Tilapia Inc#4
SalmonInc#4
Additional compounds found by AIF
Spectra obtained by DDMS2
Comments
N4 acetyl- SDZ ETHOX dimer b N4 acetyl- SDZ ETHOX dimer b N4 acetyl- SDZ ETHOX dimer b N4 acetyl- SDZ
SDZ N4 acetyl- SDZ SDZ N4 acetyl- SDZ SDZ N4 acetyl- SDZ SDZ TRIMETH N4 acetyl- SDZ ENRO, CIP ETHOX
ETHOX found by AIF at < 50% TTL
Des-ENRO
ENRO, CIP ETHOX LCV ETHOX
ETHOX found by AIF at < 50% TTL BG, CV, MG , and ETHOX found by AIF at < 50% TTL
ETHOX dimer c
none
BG and MGs found by AIF at < 50% TTL
TRIMETH found by AIF at < 50% TTL TRIMETH found by AIF at < 50% TTL ETHOX found by AIF at < 50% TTL
a
Average amount calculated as compared to one-point matrix-extracted standard with same matrix at 1X target testing level
b
Ethoxyquin dimer standard was obtained and compound was added to DDMS2 inclusion list after these data were collected
c
Fish were exposed to bath containing 2 µg/L MG, CV, and BG for 1 hr followed by 1 hr in clean water tank
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Table 4. Screening results for violative regulatory fish samples Regulatory Sample
N=
Found by QqQ (µg/kg) 4
Tilapia Reg#1 Tilapia Reg#2 Tilapia Reg#3 Tilapia Reg#4 Tilapia Reg#5 Tilapia Reg#6
2
SDZ (76)
2
SDZ (77)
Test compounds Presumptive Positive AIF (µg/kg) a ETHOX (105) SDZ (69) SDZ (52)
2
SDZ (9)
SDZ (6)
2
SDZ (4)
SDZ (3)
2
SDZ (5)
SDZ (3)
2
SMX (20) TRIMETH (6)
SMX (15) TRIMETH (6)
N4 acetyl-SMX ETHOX dimer
Catfish Reg#1 Catfish Reg#2 Eel Reg#1
2
LCV (1.1) ENRO (