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.

LC_FS0506

Peaks

	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

Conditions

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
Standard/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. Want even better performance when analyzing metal-sensitive compounds? Check out Inert LC columns at www.restek.com/inert.

Download PDF

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

LC_FS0502

Peaks

	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

	Peaks	tR (min)	Conc. (ng/mL)	Precursor Ion	Product Ion	Product Ion
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

Conditions

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
Standard/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
Notes	Want even better performance when analyzing metal-sensitive compounds? Check out Inert LC columns at www.restek.com/inert.

Download PDF

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

LC_FS0504

Peaks

	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.

Conditions

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
Standard/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. Want even better performance when analyzing metal-sensitive compounds? Check out Inert LC columns at www.restek.com/inert.

Download PDF

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

LC_FS0505

Peaks

	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

	Peaks	tR (min)	Conc. (ng/mL)	Precursor Ion	Product Ion	Product Ion
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

Conditions

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
Standard/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
Notes	Want even better performance when analyzing metal-sensitive compounds? Check out Inert LC columns at www.restek.com/inert.

Download PDF

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

LC_FS0500

Peaks

	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

	Peaks	tR (min)	Conc. (ng/mL)	Precursor Ion	Product Ion	Product Ion
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

Conditions

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
Standard/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
Notes	Want even better performance when analyzing metal-sensitive compounds? Check out Inert LC columns at www.restek.com/inert.

Download PDF

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

LC_FS0501

Peaks

	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

	Peaks	tR (min)	Conc. (ng/mL)	Precursor Ion	Product Ion	Product Ion
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

Conditions

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
Standard/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

Download PDF

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

LC_FS0503

Peaks

	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

Conditions

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
Standard/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
Notes	Want even better performance when analyzing metal-sensitive compounds? Check out Inert LC columns at www.restek.com/inert.

Download PDF

Want even better performance when analyzing mycotoxins and other metal-sensitive compounds?

Learn more at www.restek.com/inert

Author

• 
  Restek Corporation

  Restek is a leading provider of chromatography columns, accessories, and certified reference materials. Trust Restek for reliable, high-quality analytical solutions.

  View all posts

FSSS2276

Version: B