Featured Application: Multiclass Veterinary Antibiotics on Raptor C18 by LC-MS/MS

One Analysis, One Column, Less than 9 Minutes for Over 60 Multiclass Antibiotics

• Highly efficient peak separation and fast analysis times.
• Versatility and global applicability for antibiotic residue analysis—capable of individual class panel optimization for quantitation:

- Macrolide, Lincosamide, and Streptogramin (Figure 1)

- Amphenicol and Tetracycline (Figure 2)

- Quinolone (Figure 3)

- Penicillin, Cephalosporin, and Tetracycline (Figure 4)

- Sulfonamide (Figure 5)
  (For Ionophore, use on Raptor Biphenyl. [Figure 6])

The use of antibiotics on food-producing animals is a public health and safety concern due to the potential of generating drug-resistant bacteria. Many countries in the European Union and Canada have banned the use of antibiotics for nontherapeutic purposes, and the United States is implementing a policy to reduce the use of medically important antibiotics for growth promotion. To regulate the proper use of veterinary antibiotics, the U.S. FDA has set maximum residue limits (MRL) for a variety of animal tissue and food products (21 CFR Part 556). A sensitive, efficient, and reliable analytical method for different classes of antibiotics is necessary to meet this regulation, and the Raptor C18 LC column is the ideal choice.

Peaks tR (min) Conc. (ng/mL) Precursor Ion Product Ion Product Ion 1. Desacetyl cephapirin 0.70 150 382.03 111.92 124.21 2. Sulfanilamide 0.85 200 172.98 93.07 75.23 3. Amoxicillin 0.92 100 366.24 349.10 208.07 4. Cephapirin 1.36 50 424.17 292.08 124.14 5. Tildipirosin 1.38 200 734.59 561.45 204.15 6. Desfuroyl ceftiofur cysteine disulfide 1.40 300 549.16 183.02 126.00 7. Lincomycin 1.50 50 407.32 359.23 389.28 8. Sulfadiazine 1.57 20 251.18 156.04 92.08 9. Cefquinome 1.73 200 529.19 134.10 125.12 10. Ampicillin 1.78 50 350.19 106.07 160.06 11. Sulfathiazole 1.79 10 256.16 156.03 92.08 12. Marbofloxacin 1.81 10 363.20 72.11 320.10 13. Cefalexin 1.82 100 348.10 158.05 174.05 14. Sulfapyridine 1.86 10 250.13 156.10 92.08 15. Norfloxacin 1.96 20 320.23 276.20 233.13 16. Ofloxacin 1.98 10 362.21 318.20 261.15 17. Sulfamerazine 2.00 20 265.08 156.03 92.08 18. Cefalonium 2.01 100 459.16 337.03 123.10 19. Oxytetracycline 2.02 25 461.27 426.15 443.32 20. Ciprofloxacin 2.04 20 332.18 288.22 245.15 21. Cefacetrile 2.09 300 362.07 258.08 178.01 22. Tulathromycin A 2.11 100 806.65 577.42 420.31 23. Tetracycline 2.21 25 445.28 154.07 427.32 24. Danofloxacin 2.23 20 358.22 340.16 314.21 25. Enrofloxacin 2.32 10 360.29 316.22 245.13 26. Orbifloxacin 2.35 10 396.22 352.17 226.12 27. Thiamphenicol* 2.38 200 354.16 290.04 184.98 28. Sulfamethazine 2.39 10 279.23 186.08 124.08 29. Sulfamethizole 2.52 10 271.17 156.02 108.02 30. Sulfamethoxypyridazine 2.56 10 281.14 156.03 126.07 31. Sarafloxacin 2.59 10 386.20 342.20 368.15 32. Difloxacin 2.65 10 400.23 356.17 299.13 33. Cefazolin 2.75 100 455.10 323.06 295.09 34. Spiramycin 2.96 200 843.64 540.36 699.48 35. Pirlimycin 3.05 20 411.32 363.18 327.21 36. Chlortetracycline 3.08 25 479.27 154.07 371.06 37. Sulfachlorpyridazine 3.12 20 285.05 156.03 108.09 38. Gamithromycin 3.28 100 777.63 619.52 601.45 39. Sulfadoxine 3.39 10 311.17 156.03 108.09 40. Sulfamethoxazole 3.46 20 254.18 155.98 147.06 41. Cefoperazone 3.52 100 646.26 143.07 148.02 42. Florfenicol* 3.55 200 356.10 336.02 184.98 43. Sulfaethoxypyridazine 3.56 20 295.17 267.07 156.03 44. Tilmicosin 3.64 100 869.72 696.50 522.42 45. Sulfisoxazole 3.76 20 268.14 156.03 113.10 46. Oxolinic acid 3.94 5 262.10 244.06 215.96 47. Chloramphenicol* 4.00 200 321.16 151.99 257.04 48. Ceftiofur 4.11 50 524.14 241.08 125.24 49. Erythromycin 4.31 25 734.64 576.40 558.38 50. Sulfadimethoxine 4.36 10 311.17 156.09 108.09 51. Sulfaquinoxaline 4.42 20 301.18 156.04 108.02 52. Tylosin** 4.67 100 916.62 772.49 598.36 53. Penicillin G 5.07 100 335.18 176.07 160.07 54. Flumequine 5.34 5 262.15 244.11 202.03 55. Penicillin V 5.56 100 351.10 160.06 114.07 56. Oxacillin 5.99 100 402.15 160.05 114.06 57. Virginiamycin M1 6.13 50 526.43 508.31 355.10 58. Tylvalosin 6.19 50 1042.71 814.46 640.39 59. Cloxacillin 6.39 100 436.15 277.06 160.05 60. Nafcillin 6.60 25 415.19 199.09 171.06 61. Dicloxacillin 6.96 100 470.11 160.05 311.02 *Acquired in negative ion mode LC_FS0506 Column Raptor C18 (cat.# 9304A12) Dimensions: 100 mm x 2.1 mm ID Particle Size: 2.7 µm Pore Size: 90 Å Guard Column: Raptor C18 EXP guard column cartridge 5 mm, 2.1 mm ID, 2.7 µm (cat.# 9304A0252) Temp.: 35 °C Sample Diluent: Water Conc.: 5–300 ng/mL Inj. Vol.: 2 µL Mobile Phase A: 0.1% Formic acid in water B: 0.1% Formic acid in acetonitrile Time (min) Flow (mL/min) %A %B 0.00 0.4 90 10 4.50 0.4 65 35 7.00 0.4 45 55 7.01 0.4 90 10 9.00 0.4 90 10 Detector MS/MS Ion Mode: ESI+/ESI- Mode: Scheduled MRM Instrument UHPLC Notes 1. Positive and negative polarity data were collected simultaneously from a single injection. 2. Amphenicol compounds (chloramphenicol, thiamphenicol, and florfenicol) were detected with negative polarity. 3. The MRM was scheduled at +/- 20 to 30 seconds for each analyte. 4. Multiclass antibiotics include penicillin, cephalosporin, tetracycline, sulfonamide, macrolide, lincosamide, streptogramin, amphenicol, and quinolone. **The retention time for Tylosin is noted in the peak list; however, it was not included in the chromatogram.

Figure 1: Macrolide, Lincosamide, and Streptogramin Antibiotics on Raptor C18 by LC-MS/MS

Peaks tR (min) Conc. (ng/mL) Precursor Ion Product Ion Product Ion 1. Tildipirosin 1.01 200 734.59 561.45 204.15 2. Lincomycin 1.11 50 407.32 359.23 389.28 3. Tulathromycin A 1.19 100 806.65 577.42 420.31 4. Spiramycin 1.41 200 843.64 540.36 699.48 5. Pirlymycin 1.49 20 411.32 363.18 327.21 6. Gamithromycin 1.50 100 777.63 619.52 601.45 7. Tilmicosin 1.58 100 869.72 696.50 522.42 8. Erythromycin 1.80 25 734.64 576.40 558.38 9. Tylosin 1.87 100 916.62 772.49 598.36 10. Tylvalosin 2.30 50 1042.71 814.46 640.39 11. Virginiamycin M1 2.45 50 526.43 508.31 355.10 LC_FS0502 Column Raptor C18 (cat.# 9304A12) Dimensions: 100 mm x 2.1 mm ID Particle Size: 2.7 µm Pore Size: 90 Å Guard Column: Raptor C18 EXP guard column cartridge 5 mm, 2.1 mm ID, 2.7 µm (cat.# 9304A0252) Temp.: 40 °C Sample Diluent: Water Conc.: 20–200 ng/mL Inj. Vol.: 2 µL Mobile Phase A: 0.1% Formic acid in water B: 0.1% Formic acid in acetonitrile Time (min) Flow (mL/min) %A %B 0.00 0.4 90 10 3.00 0.4 20 80 3.01 0.4 90 10 5.00 0.4 90 10 Detector MS/MS Ion Mode: ESI+ Mode: MRM Instrument UHPLC

Figure 2: Amphenicol and Tetracycline Antibiotics on Raptor C18 by LC-MS/MS

Peaks tR (min) Conc. (ng/mL) Precursor Ion Product Ion Product Ion 1. Oxytetracycline 1.28 25 461.27 426.15 443.32 2. Tetracycline 1.34 25 445.28 154.07 427.32 3. Thiamphenicol* 1.48 200 354.16 290.04 184.98 4. Chlortetracycline 1.56 25 479.27 154.07 371.06 5. Florfenicol* 1.86 200 356.10 336.02 184.98 6. Chloramphenicol* 1.95 200 321.16 151.99 257.04 *Acquired in negative ion mode. LC_FS0504 Column Raptor C18 (cat.# 9304A12) Dimensions: 100 mm x 2.1 mm ID Particle Size: 2.7 µm Pore Size: 90 Å Guard Column: Raptor C18 EXP guard column cartridge 5 mm, 2.1 mm ID, 2.7 µm (cat.# 9304A0252) Temp.: 40 °C Sample Diluent: Water Conc.: 25–200 ng/mL Inj. Vol.: 2 µL Mobile Phase A: 0.1% Formic acid in water B: 0.1% Formic acid in acetonitrile Time (min) Flow (mL/min) %A %B 0.00 0.4 90 10 3.00 0.4 20 80 3.01 0.4 90 10 5.00 0.4 90 10 Detector MS/MS Ion Mode: ESI+/ESI- Mode: MRM Instrument UHPLC Notes Tetracyclines and amphenicols were analyzed with ESI+ and ESI- mode, respectively.

Figure 3: Quinolone Antibiotics on Raptor C18 by LC-MS/MS

Peaks tR (min) Conc. (ng/mL) Precursor Ion Product Ion Product Ion 1. Marbofloxacin 1.28 10 363.20 72.11 320.10 2. Norfloxacin 1.32 20 320.23 276.20 233.13 3. Ofloxacin 1.32 10 362.21 318.20 261.15 4. Ciprofloxacin 1.35 20 332.18 288.22 245.15 5. Danofloxacin 1.40 20 358.22 340.16 314.21 6. Enrofloxacin 1.44 10 360.29 316.22 245.13 7. Orbifloxacin 1.47 10 396.22 352.17 226.12 8. Sarafloxacin 1.55 10 386.20 342.20 368.15 9. Difloxacin 1.57 10 400.23 356.17 299.13 10. Oxolinic acid 2.23 5 262.10 244.06 215.96 11. Flumequine 2.78 5 262.15 244.11 202.03 LC_FS0505 Column Raptor C18 (cat.# 9304A12) Dimensions: 100 mm x 2.1 mm ID Particle Size: 2.7 µm Pore Size: 90 Å Guard Column: Raptor C18 EXP guard column cartridge 5 mm, 2.1 mm ID, 2.7 µm (cat.# 9304A0252) Temp.: 40 °C Sample Diluent: Water Conc.: 5–20 ng/mL Inj. Vol.: 2 µL Mobile Phase A: 0.1% Formic acid in water B: 0.1% Formic acid in acetonitrile Time (min) Flow (mL/min) %A %B 0.00 0.4 90 10 3.00 0.4 40 60 3.01 0.4 90 10 5.00 0.4 90 10 Detector MS/MS Ion Mode: ESI+ Mode: MRM Instrument UHPLC

Figure 4: Penicillin, Cephalosporin, and Tetracycline Antibiotics on Raptor C18 by LC-MS/MS

Peaks tR (min) Conc. (ng/mL) Precursor Ion Product Ion Product Ion 1. Desacetyl cephapirin 0.68 150 382.03 111.92 124.21 2. Amoxicillin 0.89 100 366.24 349.10 208.07 3. Cephapirin 1.05 50 424.17 292.08 124.14 4. Desfuroyl ceftiofur cysteine disulfide 1.06 300 549.16 183.02 126.00 5. Cefquinome 1.16 200 529.19 134.10 125.12 6. Ampicillin 1.18 50 350.19 106.07 160.06 7. Cefalexin 1.19 100 348.10 158.05 174.05 8. Oxytetracycline 1.26 50 461.27 426.15 443.32 9. Cefalonium 1.29 100 459.16 337.03 123.10 10. Tetracycline 1.32 50 445.28 154.07 427.32 11. Cefacetrile 1.37 300 362.07 258.08 178.01 12. Cefazolin 1.50 100 455.10 323.06 295.09 13. Chlortetracycline 1.54 50 479.27 154.07 371.06 14. Cefoperazone 1.69 100 646.26 143.07 148.02 15. Ceftiofur 1.85 50 524.14 241.08 125.24 16. Penicillin G 2.18 100 335.18 176.07 160.07 17. Penicillin V 2.33 100 351.10 160.06 114.07 18. Oxacillin 2.44 100 402.15 160.05 114.06 19. Cloxacillin 2.56 100 436.15 277.06 160.05 20. Nafcillin 2.63 25 415.19 199.09 171.06 21. Dicloxacillin 2.76 100 470.11 160.05 311.02 LC_FS0500 Column Raptor C18 (cat.# 9304A12) Dimensions: 100 mm x 2.1 mm ID Particle Size: 2.7 µm Pore Size: 90 Å Guard Column: Raptor C18 EXP guard column cartridge 5 mm, 2.1 mm ID, 2.7 µm (cat.# 9304A0252) Temp.: 40 °C Sample Diluent: Water Conc.: 25–300 ng/mL Inj. Vol.: 2 µL Mobile Phase A: 0.1% Formic acid in water B: 0.1% Formic acid in acetonitrile Time (min) Flow (mL/min) %A %B 0.00 0.4 90 10 3.00 0.4 20 80 3.01 0.4 90 10 5.00 0.4 90 10 Detector MS/MS Ion Mode: ESI+ Mode: MRM Instrument UHPLC

Figure 5: Sulfonamide Antibiotics on Raptor C18 by LC-MS/MS

Peaks tR (min) Conc. (ng/mL) Precursor Ion Product Ion Product Ion 1. Sulfanilamide 0.83 200 172.98 93.07 75.23 2. Sulfadiazine 1.45 20 251.18 156.04 92.08 3. Sulfathiazole 1.60 10 256.16 156.03 92.08 4. Sulfapyridine 1.67 10 250.13 156.10 92.08 5. Sulfamerazine 1.79 20 265.08 156.03 92.08 6. Sulfamethazine 2.07 10 279.23 186.08 124.08 7. Sulfamethizole 2.11 10 271.17 156.02 108.02 8. Sulfamethoxypyridazine 2.16 10 281.14 156.03 126.07 9. Sulfachlorpyridazine 2.55 20 285.05 156.03 108.09 10. Sulfadoxine 2.75 10 311.17 156.03 108.09 11. Sulfamethoxazole 2.78 20 254.18 155.98 147.06 12. Sulfaethoxypyridazine 2.84 20 295.17 267.07 156.03 13. Sulfisoxazole 2.98 20 268.14 156.03 113.10 14. Sulfadimethoxine 3.37 10 311.17 156.09 108.09 15. Sulfaquinoxaline 3.40 20 301.18 156.04 108.02 LC_FS0501 Column Raptor C18 (cat.# 9304A12) Dimensions: 100 mm x 2.1 mm ID Particle Size: 2.7 µm Pore Size: 90 Å Guard Column: Raptor C18 EXP guard column cartridge 5 mm, 2.1 mm ID, 2.7 µm (cat.# 9304A0252) Temp.: 40 °C Sample Diluent: Water Conc.: 10–200 ng/mL Inj. Vol.: 2 µL Mobile Phase A: 0.1% Formic acid in water B: 0.1% Formic acid in acetonitrile Time (min) Flow (mL/min) %A %B 0.00 0.4 90 10 3.50 0.4 60 40 3.51 0.4 90 10 5.00 0.4 90 10 Detector MS/MS Ion Mode: ESI+ Mode: MRM Instrument UHPLC

Figure 6: Ionophore Antibiotics on Raptor Biphenyl by LC-MS/MS

Peaks tR (min) Conc. (ng/mL) Precursor Ion Product Ion Product Ion 1. Lasalocid A 1.92 100 613.42 377.28 595.40 2. Monensin 2.12 100 693.50 675.44 461.30 3. Salinomycin 2.19 100 773.57 431.24 531.39 4. Maduramicin 2.30 100 939.65 877.58 473.31 5. Narasin 2.58 100 787.59 431.27 531.35 LC_FS0503 Column Raptor Biphenyl (cat.# 9309A12) Dimensions: 100 mm x 2.1 mm ID Particle Size: 2.7 µm Pore Size: 90 Å Guard Column: Raptor Biphenyl EXP guard column cartridge 5 mm, 2.1 mm ID, 2.7 µm (cat.# 9309A0252) Temp.: 40 °C Sample Diluent: Water:methanol (10:90) Conc.: 100 ng/mL Inj. Vol.: 2 µL Mobile Phase A: 0.5% Formic acid in water B: 0.5% Formic acid in acetonitrile Time (min) Flow (mL/min) %A %B 0.00 0.5 35 65 3.00 0.5 25 75 3.01 0.5 35 65 5.00 0.5 35 65 Detector MS/MS Ion Mode: ESI+ Mode: MRM Instrument UHPLC