Article pubs.acs.org/JAFC
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 C. Andersen,† Robert Burger,‡ Aaron S. Johnson,‡ and Mark R. Madson†,‡ †
Animal Drugs Research Center, U.S. Food and Drug Administration, Denver, Colorado 80225, United States Denver Laboratory, U.S. Food and Drug Administration, Denver, Colorado 80225, United States
‡
S Supporting Information *
ABSTRACT: A screening method for veterinary drug residues in fish, shrimp, and eel using LC with a high-resolution MS instrument has been developed and validated. The method was optimized for over 70 test compounds representing a variety of veterinary drug classes. Tissues were extracted by vortex mixing with acetonitrile acidified with 2% acetic acid and 0.2% ptoluenesulfonic acid. A centrifuged portion of the extract was passed through a novel solid phase extraction cartridge designed to remove interfering matrix components from tissue extracts. The eluent was then evaporated and reconstituted for analysis. Data were collected with a quadrupole-Orbitrap high-resolution mass spectrometer using both nontargeted and targeted acquisition methods. Residues were detected on the basis of the exact mass of the precursor and a product ion along with isotope pattern and retention time matching. Semiquantitative data analysis compared MS1 signal to a one-point extracted matrix standard at a target testing level. The test compounds were detected and identified in salmon, tilapia, catfish, shrimp, and eel extracts fortified at the target testing levels. Fish dosed with selected analytes and aquaculture samples previously found to contain residues were also analyzed. The screening method can be expanded to monitor for an additional >260 veterinary drugs on the basis of exact mass measurements and retention times. KEYWORDS: high-resolution mass spectrometry, aquaculture, veterinary drug residues, screening methods
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methods that utilize time-of-flight (ToF)8−11 or orbital ion trap (Orbitrap)12−14 HRMS. These methods have demonstrated the ability to detect, identify, and quantitate an increasing number of analytes. However, some of these early HRMS methods were sometimes limited by insufficient mass resolution to accurately measure exact mass in complex matrices and a lack of sensitivity to detect residue levels. The Q-Exactive is a hybrid quadrupoleOrbitrap HRMS instrument that has the advantage of operating at increased resolution and the ability to collect product ion spectra with or without initial precursor isolation using the quadrupole filter. Using a MS detector with this capability further enhances the need for the most universal extractant and cleanup method possible. Ideally, the method should also be simple and quick. Although a large number of veterinary drug residue LC-MS methods and cleanup strategies are available in the literature for fish and shrimp,5,15 specific lipid cleanup technologies have recently been introduced. Removal of lipids is especially important for high fat-containing matrices such as salmon and other fish. Fats (specifically phospholipids) can be significant HPLC column contaminants and can also contribute
INTRODUCTION Aquaculture is a growing industry anticipated to supply approximately 100 million tons, or >60%, of the fish destined for human consumption by 2030. Many types of veterinary drugs may be administered to fish in an aquaculture environment to treat disease or proactively prevent infection.1,2 Traditionally, analytical methods were developed to monitor for one residue, or for several analytes from a specific class of drugs, in a single species of fish or shellfish. More recently, multiclass methods have been developed using liquid chromatography (LC) with tandem mass spectrometry (MS),3−5 but these methods are still limited to targeted analytes. Using high-resolution mass spectrometry (HRMS) instruments, a virtually unlimited number of compounds can be simultaneously analyzed because full-scan data are collected rather than preselected ion transitions corresponding to specific compounds. Selectivity is achieved by taking advantage of the instrument’s ability to provide very accurate mass measurements. Residue identification can be based on calculated exact masses of protonated molecules and fragment ions, relative isotopic abundances, and retention times. This can lead to the development of methods that can monitor for a wide scope of residues and contaminants, allowing regulatory agencies to be more proactive in discovering possible adulteration of the food supply including aquacultured products. The use of HRMS technology for detecting drug residues in aquaculture and other foods has been reviewed6,7 and includes This article not subject to U.S. Copyright. Published 2016 by the American Chemical Society
Special Issue: 53rd North American Chemical Residue Workshop Received: Revised: Accepted: Published: 7252
October 21, 2016 December 22, 2016 December 28, 2016 December 28, 2016 DOI: 10.1021/acs.jafc.6b04717 J. Agric. Food Chem. 2017, 65, 7252−7267
Journal of Agricultural and Food Chemistry
Article
Table 1. Test Compounds analyte doramectin (DOR) emamectin B1a (EMA) ivermectin B1a (IVER) amoxicillin (AMOX) ampicillin (AMP) aspoxicillin (ASP) cloxacillin (CLOX) dicloxacillin (DICLOX) oxacillin (OXAC) penicillin G (PEN G) penillic acid albendazole (ALB) albendazole sulfoxide (ALB SULF) fenbendazole (FEN) fenbendazole sulfone (FEN SULF) cephapirin (CEPH) brilliant green (BG) crystal violet (CV) leucocrystal violet (LCV) leucomalachite green (LMG) malachite green (MG) ciprofloxacin (CIP) danofloxacin (DANO) difloxacin (DIFLOX) enrofloxacin (ENRO) norfloxacin (NOR) sarafloxacin (SAR) methyl testosterone (M TET) lincomycin (LIN) azithromycin (AZI) erythromycin A (ERY) erythromycin dehydrated spiramycin (SPIRO) tilmicosin (TIL) tylosin A (TYL) ketoconazole (KETO) metronidazole (MNZ) florfenicol amine (FFA) ormetoprim (ORM) trimethoprim (TRIMETH) ethoxyquin (ETHOX) flumequine (FLU) nalidixic acid (NAL) oxolinic acid (OXO) sulfacetamide (SAA) sulfachloropyridazine (SCP) sulfaclozine (SULC) sulfadiazine (SDZ) sulfadimethoxine (SDM) sulfadoxine (SDX) sulfaethoxypyridazine (SEP) sulfamerazine (SMR) sulfamethazine (SMZ) sulfamethoxazole (SMX) sulfamethoxypyridazine (SMP) sulfamonomethoxine (SULFMON) sulfapyridine (SPD) sulfaquinoxaline (SQX) sulfathiazole (STZ) chlortetracycline (CTC) doxycycline (DC)
class avermectin avermectin avermectin β-lactam β-lactam β-lactam β-lactam β-lactam β-lactam β-lactam β-lactam benzimidazole benzimidazole benzimidazole benzimidazole cephalosporin dye dye dye dye dye fluoroquinolone fluoroquinolone fluoroquinolone fluoroquinolone fluoroquinolone fluoroquinolone Hhormone lincomycin macrolide macrolide macrolide macrolide macrolide macrolide nitromidazole nitromidazole phenicol potentiator potentiator preservative quinolone quinolone quinolone sulfonamide sulfonamide sulfonamide sulfonamide sulfonamide sulfonamide sulfonamide sulfonamide sulfonamide Sulfonamide sulfonamide sulfonamide sulfonamide sulfonamide sulfonamide tetracycline tetracycline
TTL (μg/kg) a
200 200a 200a 100 25 25 25 25 25 25 NAc 50 50 50 50 25 1 1 1 1 1 5 5 5 5 5 5 0.8 50 50 50 NAc 50 50 50 10 10 50f 10 10 50 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 100g 100g
RT (min)
formula
MH+
11.3 9.6 12.0 1.9 4.2 2.6 9.0 9.6 8.5 7.5 4.9 7.0 5.0 8.4 7.3 3.4 9.5 9.0 5.6 8.2 8.1 4.6 5.0 5.4 5.1 4.7 5.4 9.4 3.8 5.4 6.6 7.2 5.5 6.0 7.0 7.2 2.0 1.33 4.6 4.3 7.7 7.82 7.6 6.5 2.2 5.8 6.9 2.9 7.0 6.1 6.2 4.1 4.8 6.1 5.1 5.6 4.0 7.1 3.9 5.6 5.9
C50H74O14 C49H75NO13 C48H74O14 C16H19N3O5S C16H19N3O4S C21H27N5O7S C19H18ClN3O5S C19H17Cl2N3O5S C19H19N3O5S C16H18N2O4S C16H18N2O4S C12H15N3O2S C12H15N3O3S C15H13N3O2S C15H13N3O4S C17H17N3O6S2 C27H33N2 C25H30N3 C25H31N3 C23H26N2 C23H25N2 C17H18FN3O3 C19H20FN3O3 C21H19F2N3O3 C19H22FN3O3 C16H18FN3O3 C20H17F2N3O3 C20H30O2 C18H34N2O6S C38H72N2O12 C37H67NO13 C37H65NO12 C43H74N2O14 C46H80N2O13 C46H77NO17 C26H28Cl2N4O4 C6H9N3O3 C10H14FNO3S C14H18N4O2 C14H18N4O3 C14H19NO C14H12FNO3 C12H12N2O3 C13H11NO5 C8H10N2O3S C10H9ClN4O2S C10H9ClN4O2S C10H10N4O2S C12H14N4O4S C12H14N4O4S C12H14N4O3S C11H12N4O2S C12H14N4O2S C10H11N3O3S C11H12N4O3S C11H12N4O3S C11H11N3O2S C14H12N4O2S C9H9N3O2S2 C22H23ClN2O8 C22H24N2O8
921.4971b 886.5311 897.4971b 366.1118 350.1169 494.1074 436.0729 470.0339 402.1118 335.1060 335.1060 266.0958 282.0907 300.0801 332.0700 424.0632 385.2638d 372.2434d 374.2591 331.2169 329.2012d 332.1405 358.1562 400.1467 360.1718 320.1405 386.1311 303.2319 407.2210 749.5158 734.4685 716.4580 843.5213 435.2903e 916.5264 531.1560 172.0717 248.0751 275.1503 291.1452 218.1539 262.0874 233.0921 262.0710 215.0485 285.0208 285.0208 251.0597 311.0809 311.0809 295.0859 265.0754 279.0910 254.0594 281.0703 281.0703 250.0645 301.0754 256.0209 479.1216 445.1605
7253
fragment ions 449.2298 82.0651 609.3398 114.0372 106.0651 160.0427 160.0427 160.0427 114.0372 114.0372 128.0528 159.0427 208.0175 159.0427 300.0437 152.0165 297.1386 251.1543 239.1543 194.0964 208.1121 245.1085 283.1241 299.0990 245.1085 276.1507 299.0990 97.0648 126.1277 158.1176 83.0491 158.1176 174.1125 174.1125 174.1125 82.0525 82.0525 104.0632 123.0665 123.0665 148.0757 202.0299 187.0502 216.0291 92.0495 92.0495 92.0495 92.0495 108.0444 92.0495 92.0495 92.0495 92.0495 92.0495 108.0444 92.0495 108.0444 92.0495 92.0495 444.0845 154.0499
777.4126 158.1176 753.4184 208.0427 114.0372 250.1186 277.0375 310.9985 160.0427 160.0427 160.0427 191.0148 240.0437 268.0539
302.1962 349.0853 160.0427 366.1118
243.0764 176.0706 289.0997 234.0696
292.0573 341.2012 356.2121 253.1699 239.1543 313.1699 288.1507 314.1663 356.1569 316.1820 302.1299 342.1413 109.1011 359.2214 591.4210 158.1176
576.3742
540.3136 522.3789
695.460
358.2278 315.1856
338.1499 342.1612
489.1455 128.0455 130.0651 259.1190 230.1162 176.1070 244.0768 215.0815 244.0604 108.0444 108.0444 108.0444 108.0444 156.0114 108.0444 108.0444 108.0444 108.0444 108.0444 126.0662 108.0444 156.0114 108.0444 108.0444
156.0114 156.0114 156.0114 156.0114 156.0768 156.0114 156.0114 156.0114 156.0114 156.0114 156.0114 156.0114 184.0869 156.0114 156.0114
410.1234
428.1340
230.0646
DOI: 10.1021/acs.jafc.6b04717 J. Agric. Food Chem. 2017, 65, 7252−7267
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Table 1. continued analyte oxytetracycline (OTC) tetracycline (TC) negative ion analyte chloramphenicol (CAP) florfenicol (FF) thiamphenicol (THIAM) toltrazuril (TOLT) toltrazuril sulfone (TOLT SULF) toltrazuril sulfoxide (TOLT SULFX) diflubenzuron (DIFLU) lufenuron (LUF) teflubenzuron (TEFLU)
class tetracycline tetracycline class phenicol phenicol phenicol toltrazuril toltrazuril toltrazuril benzylurea benzylurea benzylurea
TTL (μg/kg)
RT (min)
g
4.7 4.8
formula C22H24N2O9 C22H24N2O8
MH+ 461.1555 445.1605
100 100g TTL (μg/kg)
RT (min)
formula
MH−
0.3 5 5 50 50 50 50 50 50
6.5 6.0 4.6 10 9.9 9.2 10 10.4 10.3
C11H12Cl2N2O5 C12H14Cl2FNO4S C12H15Cl2NO5S C18H14F3N3O4S C18H14F3N3O6S C18H14F3N3O5S C14H9ClN2O2F2 C17H8Cl2F8N2O3 C14H6Cl2F4N2O2
321.00505 355.99319 353.99752 424.05843 456.04826 440.05335 309.02478 508.97115 378.96697
fragment ions 154.0499 154.0499
426.1183 410.1234 fragment ions
152.0353 185.0278 185.0278 316.98132 none 371.05781 242.98601 174.95972 195.95378
176.0353 335.9870 290.0259 404.97665
289.0184 325.9591 338.95451
257.0335
488.96492
Current FDA program recommends TTL of 10 μg/kg.20 bMNa+. cNA, not applicable (degradant). dM+. eMH22+. fFDA tolerance is 1 mg/kg for FFA as marker residue in aquaculture.20 gFDA tolerance is 2 mg/kg for sum of OTC, CTC, and TC in finfish and lobster.20
a
negative ion compounds was made in the same manner as the stable spiking mix. Samples were fortified with negative ion compounds by adding 40 μL of that standard mix to 2 g of control tissue. To prepare a solvent standard equivalent to 1X TTL in tissue, 100 μL of the stable standard mix and 50 μL of the nonstable standard mix were combined and diluted to 5 mL with 90:10 water/acetonitrile. (This assumes an extraction scheme of 2 g→ 10 mL then 2 mL → 0.4 mL). If an additional acetonitrile injection was needed for salmon, then an equivalent acetonitrile solvent standard was additionally prepared by diluting the previous solvent standard 1:5 with acetonitrile. A negative ion 1X TTL equivalent solvent standard was prepared by diluting 100 μL of the negative mixed standard mix to 5 mL with 90:10 water/acetonitrile. Sample Extraction. Homogenized tissue (2.0 ± 0.05 g) was weighed into a 50 mL polypropylene tube, and spiking standard mixes were added as appropriate and allowed to mix with the tissue for at least 5 min. The tissue was extracted with 8 mL of extraction solution consisting of 0.2% p-toluenesulfonic acid (p-TSA) monohydrate (w/v) and 2% glacial acetic acid (v/v) in acetonitrile. The samples were vortex mixed for 30 min using a multitube vortex mixer at a setting speed of 2500 rpm and then centrifuged for 7 min (4 °C) at minimum of 17,000 RCF (g). A portion (3 mL) of the extract was transferred to an Oasis PRiME HLB 6 cc (200 mg) extraction cartridge with a 15 mL polypropylene tube underneath. The samples were allowed to gravity drain (ca. 10 min) through the cartridges. The remaining few drops of extractant were gently pushed out through the SPE tube with a pipet bulb. (This should give just over 2 mL of liquid at this point.) If the tissue was salmon, 100 μL of the extract was transferred into an LC vial for an acetonitrile injection. The remaining portion of the extract was taken to near dryness under nitrogen stream at 55 °C (a drop of liquid remaining in the 15 mL tube was acceptable). The extract was then reconstituted with 400 μL of 10% acetonitrile in water (v/v), mixed, and centrifuged at a minimum of 28,900 RCF (g) for 7 min. Finally, an aliquot of 300 μL was carefully removed from the polypropylene tube, leaving particulates behind, and transferred into a LC vial. Overall there was no net dilution of concentration of the sample through the extraction procedure. (A sample fortified at 10 μg/ kg = 10 ng/mL in LC vial.) Incurred Samples. Incurred fish samples were provided by the Center for Veterinary Medicine Office of Research. Two tilapia were dosed with 1 mg/kg body weight of sulfadiazine with 1 day of depuration. Two other tilapia were dosed with 5 mg/kg body weight of sulfadiazine and 1 mg/kg body weight of trimethoprim and were sacrificed at 3 and 4 days. Two catfish were dosed with 5 mg/kg body weight of enrofloxacin with a 6 day withdrawal. Control tilapia and catfish were grown and harvested concurrently with these dosed animals. Catfish and salmon from an earlier (2014) dosing study with triphenylmethane dyes were also tested with this method. These fish were exposed to a water bath containing a mixture of 2 μg/L of
to severe matrix suppression in signal response for MS detection. New commercially available cleanup techniques are designed to eliminate these interferences without severe loss of analyte recovery.16,17 These new sample cleanup products were evaluated along with the choice of initial extraction solution to optimize a procedure suitable for a wide variety of analytes at residue levels. The aim of this research was to be able to screen and identify a wide scope of veterinary drug residues in several types of fish matrices. An optimized extraction method was used with an LC separation combined with a Q-Exactive (Orbitrap) HRMS. The method was validated according to U.S. Food and Drug Administration (FDA) guidelines18,19 using the representative compounds listed in Table 1. In addition to monitoring fish extracts for these test compounds, the full scan HRMS data could be compared to a compound database containing about 260 additional (>330 total) veterinary drugs to significantly expand the number of residues that might be detected in any given sample.
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MATERIALS AND METHODS
Standard Preparation. Individual stock standards were made in methanol, except for β-lactams, which were dissolved in water or acetonitrile/water depending upon solubility. Oxolinic acid was prepared in acetonitrile. All stock standard solutions were made at a concentration of approximately 100 μg/mL as the free base or acid. Stock standards for the β-lactams were stored at −25 °C; all others were stored at 4 °C. Two different spiking standard mixes (“stable” and “nonstable”) for positive ion compounds were made. A 1X target testing level (TTL) spike was made by adding portions (see below) of both mixtures to control tissue. The “nonstable” analyte spiking mix contained the β-lactams, tetracyclines, cephapirin, and dye compounds as listed in Table 1. The nonstable standard spiking mix was prepared by combining an amount (a volume equal to 20 times the TTL level for each compound, corrected for the exact concentration) of each individual stock standard and diluting to 20 mL with acetonitrile. To prepare a 1X TTL spike containing all of the nonstable compounds, 20 μL of this standard mix was added to 2 g of control tissue. This “nonstable” spiking mix standard is stable for 6 months at −25 °C. The “stable” standard spiking mix consists of all remaining positive ion compounds listed in Table 1. Similarly, the stable analyte mix was also prepared by combining an amount (a volume equivalent to 20X the TTL for each compound) of each individual stock standard and diluting to 40 mL with acetonitrile. To prepare a 1X TTL spike containing all of the stable compounds, 40 μL of this standard mix was added to 2 g of control tissue. This “stable” spiking mix standard is stable for 1 year at −25 °C. A separate fortification mixture for the 7254
DOI: 10.1021/acs.jafc.6b04717 J. Agric. Food Chem. 2017, 65, 7252−7267
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dye standard may be needed to condition a new column to detect low levels of leucocrystal violet and leucomalachite green. Data Analysis. Several stages of data analysis were performed. First, the AIF data were analyzed to determine if the test compounds were identified and present at concentrations above the threshold cutoff level (>50% TTL). AIF data could also be compared to the larger veterinary drug compound database to determine if additional analytes (beyond the test compounds) were in a sample. The product ion spectra from DDMS2 data could also be evaluated. Negative ion data were evaluated using the same processes. Limit Testing and Confirmation of Identity for Test Compounds Using AIF Data. AIF data from full MS1 scans were used for initial screening of test compounds. A Thermo TraceFinder “Quantitative Method” was established to provide data for the test compounds listed in Table 1, including a few degradants (e.g., penillic acid and dehydrated erythromycin). The test compounds in the “Quantitative Method” were a subset of analytes imported from the larger compound database (N > 330), which contains information for retention time and exact masses of fragment ions. To be qualitatively identified, the precursor ions must be present (signal-to-noise > 3) and match theoretical exact mass within a 5 ppm mass tolerance. The data analysis program searched for residues within a time window of 60 s (30 s on each side of specified retention time), but a narrower retention time match (±0.1 min) to a standard injected the same day was typically observed. Fragment detection was also required with at least one fragment ion with 500 count minimum intensity threshold within a 10 ppm maximum mass deviation window. These criteria are consistent with FDA guidance.19 The isotope match feature was also enabled with a 70% fit threshold, 5 ppm mass deviation, and 10% intensity deviation allowance. A sample would be considered presumptive positive for a test compound if the qualitative criteria were met and the signal was ≥50% as compared to the matrixextracted standard fortified at the TTL. Expanded Screening for Additional Veterinary Drug Residues. A Thermo TraceFinder “Screening Method” was used to search for additional residues beyond the test compounds. Data collected using AIF were compared to a compound database containing >330 potential veterinary drug residues, including metabolites and minor components. New compounds are continuously being added to the database, and experimental data for retention time and fragment ions are included for a majority of these compounds. Criteria used in the “Screening Method” were 3 ppm mass tolerance for the precursor ion, >100 signal-to-noise ratio, and a signal of >5000 counts for initial detection. To identify a residue by the screening method, a retention time window match within 60 s and a minimum of one fragment ion with an intensity threshold of >500 counts and a mass tolerance within 10 ppm were required. The retention time and fragment ion criteria could be ignored if not defined in the database. The isotope pattern match option was used to filter out false detects. Additional Qualitative Data Analysis from DDMS2. The extracts were analyzed in a separate LC-MS injection using DDMS2 data acquisition. After a full MS1 scan, product ion spectra were collected after precursor isolation for analytes in an inclusion list if the signal from the MS1 ion met data-dependent triggering requirements. Residue findings were evaluated for identification criteria using the same data analysis methods described above for AIF data. Product ion spectra produced by DDMS2 data acquisition could also be manually evaluated using XCalibur QualBrowser software for consistency with solvent- and/or matrix-extracted standards. Alternatively, spectra could be compared to commercially available libraries. Validation. The method was validated according to the FDA Office of Food and Veterinary Medicine Guidelines for Chemical Method Validations v. 2 for “limit testing”18 with veterinary drugs from a variety of chemical classes in representative matrices. A summary of the fortification samples generated for the validation is included in the Supporting Information. Salmon and tilapia were used for the initial validation. The majority of the replicates were analyzed at the TTL (1X) to generate variance data at that level to set an appropriate threshold cutoff to determine if samples should be considered presumptive positive. Fortification samples at 2X, 0.5X, and 0.1X of
malachite green, crystal violet, and brilliant green for 1 h followed by 1 h of depuration in clean water. Instrument Acquisition Methods. Instrumentation. The instrument used was a Thermo Q-Exactive Orbitrap high-resolution mass spectrometer (HRMS) with a heated electrospray ionization source coupled to a Thermo Ultimate 3000 LC system. Thermo XCalibur software (v. 3.0.63) was used for data acquisition and preliminary data analysis; data analysis was also performed using TraceFinder software (v. 3.2). MS Acquisition Programs. The instrument was calibrated for mass accuracy according to the manufacturer’s recommendations at least once a week. The tuning method optimized signals for a majority of the test compounds with the LC conditions described below. General MS acquisition parameters were as follows: spray voltage, 4 kV (positive), 2.5 kV (negative); S-Lens RF level, 50; capillary temperature, 350 °C; auxiliary gas temperature, 325 °C; gas flow rates (N2, arbitrary units), sheath, 50; auxiliary, 10; sweep, 2. Other general MS parameters included acquisition time, 0−12.5 min; lock mass, OFF; and chrom peak, 15 s. Two different types of acquisition programs were used to analyze the fish extracts. All ion fragmentation (AIF) was used for initial data acquisition. AIF is a nontargeted method in which a full scan MS is followed by a MS2 scan where all precursors are allowed into the high collision dissociation (HCD) cell to form product ions simultaneously. The settings for AIF were as follows: MS1, 70K resolution; 3e6 automatic gain control target; maximum inject time, 200 ms; m/z 150−1000 scan range. MS2, 70K resolution; 3e6 automatic gain control target; maximum inject time, 200 ms; m/z 80−1000 scan range; normalized collision energies of 10, 30, and 50. A second set of data (separate injection of fish extract) was obtained using Data Dependent MS2 (DDMS2) data acquisition. With this program, MS2 data were collected when a precursor ion from a predefined “inclusion list” was detected (from a full MS1 scan) above a set threshold. When that occurred, the quadrupole filtered the precursor ion into the HCD cell using a limited m/z window to produce fragment ions related to that compound. The inclusion list for positive ion analytes contains approximately 290 of the compounds from the larger database, including all test compounds and other analytes for which a retention time was known (1.5 min windows were used in the list). Some of the more obscure analytes in the larger compound database were not included so that the instrument did not waste analysis time triggering spectra for those compounds. Analytes can be added to the inclusion list as needed. The DDMS2 inclusion list for negative ion compounds was much smaller, containing only compounds included in the validation (Table 1), although this list can also be expanded as needed. The operating parameters for this data acquisition for MS1 are 70K resolution, 1e5 automatic gain control target, maximum inject time of 200 ms, and m/z 150−1000 scan range. The operating parameters for DDMS2 are 17.5K resolution, 1e5 automatic gain control target, maximum inject time of 50 ms, loop count of 3, isolation width of 4 m/z, and normalized collision energies of 10, 30, and 50. Other data-dependent settings are as follows: underfill ratio, 0.5%; calculated intensity threshold, 1e4; apex trigger, 3−6 s; dynamic exclusion, 6 s. Negative ion data were collected with separate injections using AIF and DDMS2 acquisition. Chromatography. LC separation was performed using a Supelco Ascentis Express C18 (7.5 cm × 2.1 mm, 2.7 μm) fused-core reversedphase column. The mobile phase consisted of 0.1% formic acid in water (A) and acetonitrile (B) at a flow rate of 0.3 mL/min. The LC gradient program was initialized at 5% B and held for 1.5 min and then ramped to 50% B from 1.5 to 8.5 min, followed by a ramp to 99% B from 8.5 to 9 min, and then was held at 99% B from 9 to 12 min. The mobile phase was returned to 5% B from 12 to 12.5 min, and the column was re-equilibrated for an additional 2 min. The total LC run time was 14.5 min; MS data were collected for 12.5 min (no divert valve was used). The column temperature compartment was kept at 30 °C, and the autosampler tray temperature was maintained at 10 °C. The LC injection volume was 10 or 20 μL for the AIF or DDMS2 MS acquisition program, respectively. Multiple injections of a high-level 7255
DOI: 10.1021/acs.jafc.6b04717 J. Agric. Food Chem. 2017, 65, 7252−7267
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Figure 1. Comparison of selected analyte recoveries in tilapia at 1X TTL with different levels of acid modifiers (normalized to 0.2% p-TSA and 2% acetic acid (AA) recoveries).
Figure 2. Recoveries for selected analytes in tilapia fortified at the target testing level (1X) as compared to a 1X solvent standard. the TTL were also analyzed along with blank matrix samples to determine the lowest minimum detectable and confirmation levels, as well as the rates of false-positive and false-negative results. The method was also applied, with fewer overall replicates, to catfish, shrimp, and eel to demonstrate matrix extension to these species. Incurred samples of tilapia (SDZ and SDZ + TMP), catfish (ENRO, dyes), and salmon (dyes), as well as regulatory samples that had been found to contain residues, were also tested. A second set of validation samples was generated for negative ion compounds. Shrimp and salmon were the primary matrices validated for negative ion analytes, as these are the species in which phenicol or benzyl urea residues are expected to be found, respectively.
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which a residue may be expected to be consistently detected and identified using this screening method. For ease of sample preparation and data reporting, it is helpful if TTLs for the test compounds are all within a practical range (5−50 μg/kg). Some analytes will have TTLs higher (tetracyclines) or lower (methyl testosterone and dyes) than that range due to previously established levels of interest.20 In addition, for some compounds, such as amoxicillin and the avermectins, the TTLs may be >100 μg/kg due to higher detection limits for these analytes in this method. The minimum detectable limits for all compounds extracted from fish was determined, and for most compounds were found to be considerably lower (0.1X) than the TTL. Optimization of Extraction Procedure. Extraction Solvent. The list of analytes shown in Table 1 includes triphenylmethane dyes and avermectins. These two classes of compounds greatly increase the difficulty of choosing an acceptable sample extractant and cleanup procedure. Previous work4 summarizes the difficulty of including dyes in a multiresidue method. In addition, it is desirable that any chosen extractant be able to extract very polar analytes such as
RESULTS AND DISCUSSION
Compounds and Testing Levels. A list of veterinary drug residues of importance in aquacultured species (along with their target testing levels) is given in Table 1. The target testing level (TTL) or “1X” is not necessarily considered an official tolerance or action level. The majority of these compounds are not approved for use in fish in the United States, so technically any amount found and identified would be violative. These values are meant to provide a reasonable concentration at 7256
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Figure 3. Extracted ion chromatograms (5 ppm window) from MS1 AIF data for representative test compounds in tilapia fortified at target testing level.
Figure 4. AIF product ions (A) and precursor isotope pattern (B) for CIP (5 μg/kg) in catfish extract compared to theoretical values.
florfenicol amine and metronidazole as well as several nonpolar compounds. The goal was to develop a simple cleanup procedure to apply to as many of the compounds in this list as possible. Methanol or acetonitrile as the primary organic component in a meat or fish extractant is most widely reported in the literature. Water (with or without buffer salts) is not effective for extracting nonpolar analytes. Acetonitrile is usually preferred because of its ability to precipitate proteins in tissue.
The use of acetonitrile only (with no other purification steps) as a veterinary drug extractant for tissue has been published.21 Acetonitrile has also been used (in combination with secondary hexane partitioning) to extract residues from catfish, salmon, and trout.22 However, when 100% acetonitrile (without acid modifiers) was tried for our list of analytes, recoveries for crystal violet and fluoroquinolones were very low (2e 0.5
5 >100 25
0f 100f 0f
77f 100f 8f
0f 0f 0f
diflubenzuron lufenuron teflubenzuron
benzylurea
50 50 50
0.5 0.5 0.5
10 10 10
0f 8f 0f
69f 34f 11f
0f 0f 0f
0.15 0.5 0.5
Current FDA program recommends TTL of 10 μg/kg.20 bMarker compounds were degradants; amounts were compared to matrix-extracted standard fortified with parent compound at TTL. cFDA tolerance is 1 mg/kg for florfenicol amine as marker residue in aquaculture.20 dFDA tolerance is 2 mg/kg for sum of oxytetracycline, chlortetracycline, and tetracycline in finfish and lobster.20 eNo fragments were observed for toltrazuril sulfone. fFor negative ion analytes: N = 35 fortified at 1X and N = 15 blanks.
a
4:1 acetonitrile/water for extraction. The method has some advantages, including extracting a wide range of compounds and allowing very rapid sample throughput. However, although this extractant appeared to extract avermectins successfully in bovine muscle, the 80% acetonitrile solution was too polar to successfully extract ivermectin from high-fat salmon. In addition, the final extract composition described in the USDA method (70% acetonitrile) gave poor peak shape for polar, early-eluting analytes using the rapid reversed-phase chromatographic gradient developed for this screening method. Further dilution with water of the final extract would improve chromatography, but would sacrifice sensitivity. Our initial investigations focused on using acetonitrile containing acid modifiers as an extractant. Acetonitrile with 0.1% acetic acid has been used25 to extract residues from salmon and was successful in extracting emamectin and malachite green. Acetonitrile with 1% acetic acid has also been used26 for shrimp with similar results. In initial experiments with salmon, degradation of several β-lactams was observed when 0.2 or 1% formic acid in acetonitrile was used as an extractant. Formic acid in water is known27 to cause rapid degradation of monobasic penicillins. Furthermore, formic acid in acetonitrile did not extract avermectins well from salmon and had an adverse effect on macrolides. For these reasons, acetic acid was chosen to acidify the acetonitrile
extractant. By increasing acetic acid from 0.1 to 2% in the acetonitrile extractant, the recoveries for fluoroquinolones, avermectins, and penicillins doubled. Previous work in our laboratory4 showed that the addition of p-TSA improved the recovery of dyes and fluoroquinolone compounds, so this acid was added to the 2% acetic acid in acetonitrile extractant. Provided that acetic acid was also present in the extractant, the p-TSA did not seem to appreciably degrade penicillins. The final extraction solvent chosen was acetonitrile with 2% acetic acid with 0.2% p-TSA. A comparison of recoveries for representative analytes using variable amounts of acid modifiers in tilapia (using Oasis PRiME HLB) is shown in Figure 1. Sample Cleanup. Two new types of cleanup techniques designed specifically for high-fat samples were evaluated. Agilent’s EMR Lipid system is a dispersive SPE technique involving two primary sorbents: a water-activated sorbent designed to specifically trap fats containing >5-carbon aliphatic chains and a second sorbent containing magnesium sulfate and sodium chloride. A published study16 using this technique for the analysis of veterinary drug residues in bovine liver was initially evaluated for salmon. Although this procedure worked well for many analytes in Table 1, some were not recovered. The 5% formic acid solution used as the extractant in the application note did not work well for some penicillins or for 7259
DOI: 10.1021/acs.jafc.6b04717 J. Agric. Food Chem. 2017, 65, 7252−7267
Journal of Agricultural and Food Chemistry
Article
Figure 5. DDMS2 data for OXO and FLU in shrimp at target testing level (10 μg/kg): (A) extracted ion chromatograms for MH+; (B) chromatograms for MS2 of m/z 262; (C) product ion spectra.
the avermectins in salmon. The magnesium sulfate sorbent also greatly lowered the recovery of some tetracyclines. A Waters Oasis PRiME HLB SPE cartridge was also evaluated as a cleanup tool. As described in a published method17 for the analysis of veterinary residues in pork tissue, a portion of tissue extract is introduced into the SPE cartridge (no conditioning is required) and collected by gravity drain. For this method, two changes were made to accommodate the analyte list in Table 1. First, the extractant of formic acid, acetonitrile, and water was changed to the final optimized extractant described above. Second, to improve sensitivity, a portion of the extract was evaporated (instead of diluted per original method) and reconstituted with 9:1 water/acetonitrile to allow for successful reversed-phase chromatography. For the newest triple-quadrupole mass spectrometers, dilution may become less of an issue, but for full scan data acquisition obtained using the Orbitrap, a 10 min evaporation step was included to concentrate the extract. The larger 200 mg SPE
sorbent size was used to collect a larger volume of extractant for evaporation and concentration. Various HPLC filters were evaluated for use in filtering the final extract before injection. Although polyvinylidene fluoride filters worked the best (compared to nylon or PTFE), we observed inconsistent or no recovery after filtration in solvent spikes for some analytes. For this reason the final extract was not filtered but was instead centrifuged to help remove any remaining particulates after the evaporation and reconstitution step. For salmon, some compounds (avermectins, brilliant green, and crystal violet) were lost when extracts were evaporated and reconstituted most likely because they partitioned into insoluble lipid material. This necessitated a separate acetonitrile injection to detect those compounds in salmon as described earlier in the experimental section. Because the relevant compounds for the acetonitrile injection elute late in the reversed phase chromatographic system, good peak shapes and consistent retention times are achieved despite the high organic 7260
DOI: 10.1021/acs.jafc.6b04717 J. Agric. Food Chem. 2017, 65, 7252−7267
Journal of Agricultural and Food Chemistry
Article
Table 3. Screening Results for Incurred Fish Samples incurred sample
dosing drug (X), mg/kg body weight
depuration time
N
found by QqQ30 (μg/kg)
test compounds presumptive positive AIFa (μg/kg)
additional compounds found by AIF N4 acetyl-SDZ ETHOX dimerb N4 acetyl-SDZ ETHOX dimerb N4 acetyl-SDZ ETHOX dimerb N4 acetyl-SDZ
tilapia inc 1
SDZ (1)
1 day
2
not analyzed
SDZ (220)
tilapia inc 2
SDZ (1)
1 day
2
not analyzed
SDZ (240)
tilapia inc 3
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
des-ENRO
catfish inc 2
ENRO (5)
6 days
4
not analyzed
catfish inc 3
MG (2)c CV (2) BG (2)
1h
1
salmon inc 4
MG (2)c CV (2) BG (2)
1h
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 (620) CIP (30) ENRO (601) CIP (41) LCV (2.7) LMG (0.8)
none
ETHOX dimerb
tilapia inc 4
des-ENRO
spectra obtained by DDMS2 SDZ N4 acetyl-SDZ SDZ N4 acetyl-SDZ SDZ N4 acetyl-SDZ SDZ TRIMETH N4 acetyl-SDZ ENRO, CIP ETHOX ENRO, CIP ETHOX LCV ETHOX
none
comments ETHOX found by AIF at
