Article pubs.acs.org/JAFC
Challenges in Implementing a Screening Method for Veterinary Drugs in Milk Using Liquid Chromatography Quadrupole Time-ofFlight Mass Spectrometry Sherri B. Turnipseed,*,† Jack J. Lohne,† Joseph M. Storey,† Wendy C. Andersen,† Susan L. Young,‡ Justin R. Carr,‡ and Mark R. Madson†,‡ †
Animal Drugs Research Center, U.S. Food and Drug Administration, , Denver Federal Center, Building 20, Denver, Colorado 80225, United States ‡ Denver Science Branch, U.S. Food and Drug Administration, Denver Federal Center, Building 20, Denver, Colorado 80225, United States ABSTRACT: High resolution mass spectrometry (HRMS) is a valuable tool for the analysis of chemical contaminants in food. Our laboratory has successfully developed methods to screen for veterinary drug residues using liquid chromatography quadrupole time-of-flight (Q-TOF). There have been, however, significant challenges as methods are transferred from the development stage to routine regulatory analysis. Having experimental retention time and product ion information for analytes greatly facilitates the ability to determine if residues found by the HRMS searching software are false detects. These data were collected for over 200 veterinary drug residues using LC Q-TOF MS. The screening levels of detection for over 150 veterinary drug residues in milk were determined, and over half of those tested can be detected at concentrations of 10 ng/mL or less; 72% can be found in milk when present at 100 ng/mL. Tentative identification of the product ions from these analytes is also presented. KEYWORDS: veterinary drug residues, quadrupole time-of-flight MS, screening, milk
■
INTRODUCTION Our laboratory has previously published a method1 for the screening and confirmation of a limited set of target compounds in milk using LC quadrupole time-of-flight (QTOF) MS to test the feasibility of this technique. Although the mass resolution for this instrument in the mass range of interest (R ∼ 10000 full width half-maximum at m/z 100−800) is not as high as can be obtained with other MS platforms such as the Orbitrap, it was sufficient to adequately measure the exact mass of these analytes in extracted milk samples. We found that LC Q-TOF MS could successfully detect all 25 target analytes tested in milk with precursor ion mass errors of 100 1000 >1000 >1000 NA >100 500 NA NA NA
C48H72O14 C50H74O14 C49H75NO13 C50H75NO14 C48H74O14 C37H53NO8 C43H63NO11
872.49221 898.50786 885.52384 913.51876 874.50786 639.37712 769.44011
895.4814e 921.4971e 886.5311 936.5080e 897.4970e 662.3663e 770.4474
17.8 18.2 14.4 17.5 18.9 18.0 18.7
>1000 >1000 1000 >1000 >1000 >1000 >1000
C12H15N3O2S C15H13N3O2S C12H15N3O3 C10H7N3S
265.08850 299.07285 249.11134 201.03607
266.0958 300.0801 250.1186 202.0433
10.6 12.6 8.7 5.6
10 100 5 5f
C12H18Cl2N2O C18H23NO3 C13H21NO3 C14H19N3O2
276.07962 301.16779 239.15214 261.14773
277.0869 302.1751 240.1594 262.1550
7.3 6.5 3.7 3.7
5 10 10g 10
C16H19N3O5S C16H19N3O4S C16H20N2O5S C19H18ClN3O5S C19H17Cl2N3O5S C19H19N3O5S C16H18N2O4S C16H18N2O4S C16H18N2O5S C15H20N2O3S C8H11NO5S C15H16N2O6S2
365.10454 349.10963 352.10929 435.06557 469.02660 401.10454 334.09873 334.09873 350.09364 308.11946 233.03579 384.04498
366.1118 350.1169 353.1166 436.0728 470.0339 402.1118 335.1060 335.1060 351.1009 309.1267 234.0431 385.0523
3.0 5.8 9.2 13.2 14.4 12.9 7.2 11.2 12.3 11 5.6 9.8
>100 200 100 1000 20f >1000 10 NA 10 100 >1000 1000 500 10 NA
3661
screening results at 10 ng/mL (X/ 20, %RSD)c
20 (43%) 20 (27%)
20 (14%) 20 (14%) NA 19 (28%)
15 (28%) 4 (21%)
20 (15%)
dx.doi.org/10.1021/jf405321w | J. Agric. Food Chem. 2014, 62, 3660−3674
Journal of Agricultural and Food Chemistry
Article
Table 1. continued compounds Cephalosporins desacetylcephapirin dehydrated Dyes brilliant green crystal violet leucocrystal violet leucomalachite green malachite green methylene blue Flukacides bithionol clorsulon closantel niclosamide nitroxinil oxyclozanide rafoxanide Ionophores lasalocid A monensin narasin nigericin salinomycin Lincosamides clindamycin lincomycin pirlimycin Macrolides clarithromycin desacetyl troleandomycin erythromycin A erythromycin anhydrate josamycin natamycin roxithromycin spiramycin tilmicosin tulathromycin tylosin A tylosin B (desmycosin) Miscellaneous acepromazine acetaminophen α-zearalenol amprolium caffeine clavulanate clopidol decoquinate detomidine dexamethasone diclazuril diminazene ethopabate fumagillin halofuginone imidocarb ketamine ketoconazole
formula
a
RT (min)
estimated screening LOD in milk (ng/mL)b
screening results at 10 ng/mL (X/ 20, %RSD)c
mass
precursor ion (m/z)
C15H13N3O4S2
363.03475
364.0420
1.6
NA
C27H32N2 C25H30N3 C25H31N3 C23H26N2 C23H25N2 C16H17N3S
384.25655 372.24397 373.25180 330.20960 329.20177 283.11432
385.2638 372.2434 374.2591 331.2169 329.2012 284.1216
14.9 13.6 7.8 11.8 12.2 8.6
NA NA NA NA NA NA
C12H6Cl4O2S C8H8Cl3N3O4S2 C22H14Cl2I2N2O2 C13H8Cl2N2O4 C7H3IN2O3 C13H6Cl5NO3 C19H11Cl2I2NO3
353.88426 378.90218 661.85217 325.98611 289.91883 398.87903 624.82053
352.8770i 377.8949i 660.8449i 324.9788i 288.9116i 397.8718i 623.8133i
16.9 10.8 17.8 16.5 13.1 16.2 18.2
500 10 >1000 >1000 NA 20 500
C34H54O8 C36H62O11 C43H72O11 C40H68O11 C42H70O11
590.38187 670.42921 764.50746 724.47616 750.49181
613.3711e 693.4184e 787.4967e 747.4654e 773.4810e
20.5 19.6 17.7 19.8 17.4
NA NA NA NA NA
C18H33ClN2O5S C18H34N2O6S C17H31ClN2O5S
424.17987 406.21376 410.16422
425.1871 407.2210 411.1715
8.4 5.1 8.1
5 5 5
20 (16%) 20 (15%) 20 (19%)
C38H69NO13 C39H65NO14 C37H67NO13 C37H65NO12 C42H69NO15 C33H47NO13 C41H76N2O15 C43H74N2O14 C46H80N2O13 C41H79N3O12 C46H77NO17 C39H65NO14
747.47689 771.44051 733.46124 715.45068 827.46672 665.30474 836.52457 842.51401 868.56604 805.56638 915.51915 771.44051
748.4842 772.4478 734.4685 716.4580 828.4740 666.3120 837.5318 843.5213 869.5733/435.2905j 806.5737 916.5264 772.4478
10.6 11.3 9.3 10.1 11.5 10.1 11.1 7.9 8.2 6.6 9.8 9.4
20g 100 10 5f 100 >100 100 100 10f 100 100 NA NA NA 5 >1000 5 NA NA 100 10 NA 5 5 5 10g
12 (57%)
3662
NA
20 (20%) 20 (12%)
20 (28%) 20 20 20 18
(17%) (44%) (20%) (27%)
dx.doi.org/10.1021/jf405321w | J. Agric. Food Chem. 2014, 62, 3660−3674
Journal of Agricultural and Food Chemistry
Article
Table 1. continued compounds Miscellaneous levamisole lidocaine maduramicin melamine nicarbazin (4,4′-dinitrocarbanilide) novobiocin ormetoprim progesterone pyrimethamine rifampin robenidine tiamulin toltrazuril trichlormethiazide trimethoprim tripelennamine virginiamycin M1 virginiamycin S1 xylazine Nitromidazoles dimetridazole metronidazole metronidazole hydroxide ornidazole ronidazole tindazole NSAIDs flunixin phenylbutazone flunixin hydroxide carprofen diclofenac ketoprofen mefenamic acid naproxen niflumic acid suxibuzone Peptides bacitracin A colistin B−polymix E2 colistin B−polymix E1 vancomycin Phenicols chloramphenicol chloramphenicol-d5 florfenicol thiamphenicol florfenicol amine Quinolones ciprofloxacin danofloxacin desethylene ciprofloxacin desethylene enrofloxacin difloxacin enofloxacin (enoxacin) enrofloxacin fleroxacin
formula
a
RT (min)
estimated screening LOD in milk (ng/mL)b
screening results at 10 ng/mL (X/ 20, %RSD)c
mass
precursor ion (m/z)
C11H12N2S C14H22N2O C47H80O17 C3H6N6 C13H10N4O5
204.07212 234.17321 916.53955 126.06539 302.06512
205.0794 235.1805 939.5288e 127.0727 301.0578i
5.4 6.3 21 1.1 15.5
20 5 1000 NA 100
C31H36N2O11 C14H18N4O2 C21H30O2 C12H13ClN4 C48H58N4O12 C15H13Cl2N5 C28H47NO4S C18H14F3N3O4S C8H8Cl3N3O4S2 C14H18N4O3 C16H21N3 C28H35N3O7 C43H49N7O10 C12H16N2S
612.23191 274.14298 314.22458 248.08287 882.40512 333.05480 493.32258 425.06571 378.90218 290.13789 255.17355 525.24750 823.35409 220.10342
613.2392 275.1503 315.2319 249.0902 883.4124 334.0621 494.3299 424.0584i 377.8949i 291.1452 256.1808 526.2547 824.3614 221.1107
16.3 6.8 16.5 8.6 ND 12 10.6 16.1 10.4 5.8 6.9 12.5 15.2 7.1
>1000 5 NA 5 NA >100 5 >100 10 5 100 5
C5H7N3O2 C6H9N3O3 C6H9N3O4 C7H10ClN3O3 C6H8N4O4 C8H13N3O4S
141.05383 171.06439 187.05931 219.04107 200.05455 247.06268
142.0611 172.0717 188.0666 220.0483 201.0618/223.0438e 248.0700
5.3 4.3 3.3 7.8 5.4 6.9
5 20 NA NA NA NA
20 (18%)
C14H11F3N2O2 C19H20N2O2 C14H11F3N2O3 C15H12ClNO2 C14H11Cl2NO2 C16H14O3 C15H15NO2 C14H14O3 C13H9F3N2O2 C24H26N2O6
296.07726 308.15248 312.07218 273.05566 295.01668 254.09429 241.11028 230.09429 282.06161 438.17909
297.0845 309.1598 313.0795 274.0629/272.0484i 296.0240 255.1016 242.1176/240.1030i 231.1016 283.0689/281.0543i 439.1864
13.8 15.9 12.9 15.3 15.7 13.6 16.2 13.7 15.6 15.4
5 200 200 >200 100 >200 200 10 200
20 (32%)
C66H103N17O16S C52H98N16O13 C53H100N16O13 C66H75Cl2N9O24
1421.74894 1154.74993 1168.76558 1447.43020
1422.7562/711.8817j 1177.7391e 1191.7548e 724.7224j
8.3 6.1 6.6 5.4
>500f NA NA >100
C11H12Cl2N2O5 C11H7D5Cl2N2O5 C12H14Cl2FNO4S C12H15Cl2NO5S C10H14FNO3S
322.01233 327.04371 357.00046 355.00480 247.06784
321.0051i 326.0364i 355.9932i 353.9975i 248.0751
9.8 9.8 9.2 7.2 1.5
5 NA 10 5 100
C17H18FN3O3 C19H20FN3O3 C15H16FN3O3 C17H20FN3O3 C21H19F2N3O3 C15H17FN4O3 C19H22FN3O3 C17H18F3N3O3
331.13322 357.14887 305.11757 333.14887 399.13945 320.12847 359.16452 369.13003
332.1405 358.1561 306.1248 334.1561 400.1467 321.1357 360.1718 370.1373
6.4 6.8 6.2 6.4 7.4 6.6 6.8 7.0
