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.


Restek Raptor LC Columns

PeakstR (min)Conc.
(ng/mL)
Precursor IonProduct IonProduct Ion
1.Desacetyl cephapirin0.70150382.03111.92124.21
2.Sulfanilamide0.85200172.9893.0775.23
3.Amoxicillin0.92100366.24349.10208.07
4.Cephapirin1.3650424.17292.08124.14
5.Tildipirosin1.38200734.59561.45204.15
6.Desfuroyl ceftiofur cysteine disulfide1.40300549.16183.02126.00
7.Lincomycin1.5050407.32359.23389.28
8.Sulfadiazine1.5720251.18156.0492.08
9.Cefquinome1.73200529.19134.10125.12
10.Ampicillin1.7850350.19106.07160.06
11.Sulfathiazole1.7910256.16156.0392.08
12.Marbofloxacin1.8110363.2072.11320.10
13.Cefalexin1.82100348.10158.05174.05
14.Sulfapyridine1.8610250.13156.1092.08
15.Norfloxacin1.9620320.23276.20233.13
16.Ofloxacin1.9810362.21318.20261.15
17.Sulfamerazine2.0020265.08156.0392.08
18.Cefalonium2.01100459.16337.03123.10
19.Oxytetracycline2.0225461.27426.15443.32
20.Ciprofloxacin2.0420332.18288.22245.15
21.Cefacetrile2.09300362.07258.08178.01
22.Tulathromycin A2.11100806.65577.42420.31
23.Tetracycline2.2125445.28154.07427.32
24.Danofloxacin2.2320358.22340.16314.21
25.Enrofloxacin2.3210360.29316.22245.13
26.Orbifloxacin2.3510396.22352.17226.12
27.Thiamphenicol*2.38200354.16290.04184.98
28.Sulfamethazine2.3910279.23186.08124.08
29.Sulfamethizole2.5210271.17156.02108.02
30.Sulfamethoxypyridazine2.5610281.14156.03126.07
31.Sarafloxacin2.5910386.20342.20368.15
32.Difloxacin2.6510400.23356.17299.13
33.Cefazolin2.75100455.10323.06295.09
34.Spiramycin2.96200843.64540.36699.48
35.Pirlimycin3.0520411.32363.18327.21
36.Chlortetracycline3.0825479.27154.07371.06
37.Sulfachlorpyridazine3.1220285.05156.03108.09
38.Gamithromycin3.28100777.63619.52601.45
39.Sulfadoxine3.3910311.17156.03108.09
40.Sulfamethoxazole3.4620254.18155.98147.06
41.Cefoperazone3.52100646.26143.07148.02
42.Florfenicol*3.55200356.10336.02184.98
43.Sulfaethoxypyridazine3.5620295.17267.07156.03
44.Tilmicosin3.64100869.72696.50522.42
45.Sulfisoxazole3.7620268.14156.03113.10
46.Oxolinic acid3.945262.10244.06215.96
47.Chloramphenicol*4.00200321.16151.99257.04
48.Ceftiofur4.1150524.14241.08125.24
49.Erythromycin4.3125734.64576.40558.38
50.Sulfadimethoxine4.3610311.17156.09108.09
51.Sulfaquinoxaline4.4220301.18156.04108.02
52.Tylosin**4.67100916.62772.49598.36
53.Penicillin G5.07100335.18176.07160.07
54.Flumequine5.345262.15244.11202.03
55.Penicillin V5.56100351.10160.06114.07
56.Oxacillin5.99100402.15160.05114.06
57.Virginiamycin M16.1350526.43508.31355.10
58.Tylvalosin6.19501042.71814.46640.39
59.Cloxacillin6.39100436.15277.06160.05
60.Nafcillin6.6025415.19199.09171.06
61.Dicloxacillin6.96100470.11160.05311.02
*Acquired in negative ion mode
Multiclass Veterinary Antibiotics on Raptor™ C18 by LC-MS/MS
LC_FS0506
ColumnRaptor™ 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
0.000.49010
4.500.46535
7.000.44555
7.010.49010
9.000.49010
DetectorMS/MS
Ion Mode:ESI+/ESI-
Mode:Scheduled MRM
InstrumentUHPLC
Notes1. 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

PeakstR (min)Conc.
(ng/mL)
Precursor IonProduct IonProduct Ion
1.Tildipirosin1.01200734.59561.45204.15
2.Lincomycin1.1150407.32359.23389.28
3.Tulathromycin A1.19100806.65577.42420.31
4.Spiramycin1.41200843.64540.36699.48
5.Pirlymycin1.4920411.32363.18327.21
6.Gamithromycin1.50100777.63619.52601.45
7.Tilmicosin1.58100869.72696.50522.42
8.Erythromycin1.8025734.64576.40558.38
9.Tylosin1.87100916.62772.49598.36
10.Tylvalosin2.30501042.71814.46640.39
11.Virginiamycin M12.4550526.43508.31355.10
Macrolide, Lincosamide, and Streptogramin Antibiotics on Raptor™ C18 by LC-MS/MS
LC_FS0502
ColumnRaptor™ 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
0.000.49010
3.000.42080
3.010.49010
5.000.49010
DetectorMS/MS
Ion Mode:ESI+
Mode:MRM
InstrumentUHPLC

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

PeakstR (min)Conc.
(ng/mL)
Precursor IonProduct IonProduct Ion
1.Oxytetracycline1.2825461.27426.15443.32
2.Tetracycline1.3425445.28154.07427.32
3.Thiamphenicol*1.48200354.16290.04184.98
4.Chlortetracycline1.5625479.27154.07371.06
5.Florfenicol*1.86200356.10336.02184.98
6.Chloramphenicol*1.95200321.16151.99257.04
*Acquired in negative ion mode.
Amphenicol and Tetracycline Antibiotics on Raptor™ C18 by LC-MS/MS
LC_FS0504
ColumnRaptor™ 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
0.000.49010
3.000.42080
3.010.49010
5.000.49010
DetectorMS/MS
Ion Mode:ESI+/ESI-
Mode:MRM
InstrumentUHPLC
NotesTetracyclines and amphenicols were analyzed with ESI+ and ESI- mode, respectively.

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

PeakstR (min)Conc.
(ng/mL)
Precursor IonProduct IonProduct Ion
1.Marbofloxacin1.2810363.2072.11320.10
2.Norfloxacin1.3220320.23276.20233.13
3.Ofloxacin1.3210362.21318.20261.15
4.Ciprofloxacin1.3520332.18288.22245.15
5.Danofloxacin1.4020358.22340.16314.21
6.Enrofloxacin1.4410360.29316.22245.13
7.Orbifloxacin1.4710396.22352.17226.12
8.Sarafloxacin1.5510386.20342.20368.15
9.Difloxacin1.5710400.23356.17299.13
10.Oxolinic acid2.235262.10244.06215.96
11.Flumequine2.785262.15244.11202.03
Quinolone Antibiotics on Raptor™ C18 by LC-MS/MS
LC_FS0505
ColumnRaptor™ 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
0.000.49010
3.000.44060
3.010.49010
5.000.49010
DetectorMS/MS
Ion Mode:ESI+
Mode:MRM
InstrumentUHPLC

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

PeakstR (min)Conc.
(ng/mL)
Precursor IonProduct IonProduct Ion
1.Desacetyl cephapirin0.68150382.03111.92124.21
2.Amoxicillin0.89100366.24349.10208.07
3.Cephapirin1.0550424.17292.08124.14
4.Desfuroyl ceftiofur cysteine disulfide1.06300549.16183.02126.00
5.Cefquinome1.16200529.19134.10125.12
6.Ampicillin1.1850350.19106.07160.06
7.Cefalexin1.19100348.10158.05174.05
8.Oxytetracycline1.2650461.27426.15443.32
9.Cefalonium1.29100459.16337.03123.10
10.Tetracycline1.3250445.28154.07427.32
11.Cefacetrile1.37300362.07258.08178.01
12.Cefazolin1.50100455.10323.06295.09
13.Chlortetracycline1.5450479.27154.07371.06
14.Cefoperazone1.69100646.26143.07148.02
15.Ceftiofur1.8550524.14241.08125.24
16.Penicillin G2.18100335.18176.07160.07
17.Penicillin V2.33100351.10160.06114.07
18.Oxacillin2.44100402.15160.05114.06
19.Cloxacillin2.56100436.15277.06160.05
20.Nafcillin2.6325415.19199.09171.06
21.Dicloxacillin2.76100470.11160.05311.02
Penicillin, Cephalosporin, and Tetracycline Antibiotics on Raptor ™ C18 by LC-MS/MS
LC_FS0500
ColumnRaptor™ 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
0.000.49010
3.000.42080
3.010.49010
5.000.49010
DetectorMS/MS
Ion Mode:ESI+
Mode:MRM
InstrumentUHPLC

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

PeakstR (min)Conc.
(ng/mL)
Precursor IonProduct IonProduct Ion
1.Sulfanilamide0.83200172.9893.0775.23
2.Sulfadiazine1.4520251.18156.0492.08
3.Sulfathiazole1.6010256.16156.0392.08
4.Sulfapyridine1.6710250.13156.1092.08
5.Sulfamerazine1.7920265.08156.0392.08
6.Sulfamethazine2.0710279.23186.08124.08
7.Sulfamethizole2.1110271.17156.02108.02
8.Sulfamethoxypyridazine2.1610281.14156.03126.07
9.Sulfachlorpyridazine2.5520285.05156.03108.09
10.Sulfadoxine2.7510311.17156.03108.09
11.Sulfamethoxazole2.7820254.18155.98147.06
12.Sulfaethoxypyridazine2.8420295.17267.07156.03
13.Sulfisoxazole2.9820268.14156.03113.10
14.Sulfadimethoxine3.3710311.17156.09108.09
15.Sulfaquinoxaline3.4020301.18156.04108.02
Sulfonamide Antibiotics on Raptor™ C18 by LC-MS/MS
LC_FS0501
ColumnRaptor™ 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
0.000.49010
3.500.46040
3.510.49010
5.000.49010
DetectorMS/MS
Ion Mode:ESI+
Mode:MRM
InstrumentUHPLC

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

PeakstR (min)Conc.
(ng/mL)
Precursor IonProduct IonProduct Ion
1.Lasalocid A1.92100613.42377.28595.40
2.Monensin2.12100693.50675.44461.30
3.Salinomycin2.19100773.57431.24531.39
4.Maduramicin2.30100939.65877.58473.31
5.Narasin2.58100787.59431.27531.35
Ionophore Antibiotics on Raptor™ Biphenyl by LC-MS/MS
LC_FS0503
ColumnRaptor™ 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
0.000.53565
3.000.52575
3.010.53565
5.000.53565
DetectorMS/MS
Ion Mode:ESI+
Mode:MRM
InstrumentUHPLC