MA 205 / AMINOGLYCOSIDE ANTIBIOTICS IN FEED
APRAMYCIN, GENTAMICIN , NEOMYCIN
Aminoglycoside antibiotics are among those commonly used in animal feeds to manage intestinal microorganisms. The beneficial effects include improved growth and generally healthier animal populations. Use of antibiotics creates a demand for analytical procedures to verify concentrations in pre-mixes and feeds and in some instances for residue analysis in animal products.
This note describes a simple, robust analytical method for the family of Aminoglycoside antibiotics in feeds and animal products. The sample is homogenized with a generic extraction solution and the crude soluble portion is directly injected into an HPLC ion-exchange column. The column effluent is then mixed with an OPA/Thiofluor™ reagent which forms highly fluorescent derivatives with the primary amine moieties of the antibiotics.
Call if you need to analyze other Aminoglycosides.
Extraction Procedure:
Add one part feed to 10 parts Extraction solution Catalog No. 1700-1118 (w/v) and homogenize for five minutes. Centrifuge for 10 minutes. Three layers will form: the pellet, a supernatant emulsion and a soft layer of floating fat. Carefully lift the floating fatty layer with a spatula and discard. Transfer the emulsion to a sealable vial. Coagulate the emulsion by placing the vial in a boiling water bath for 15 minutes. Centrifuge for 10 minutes. The clear supernate is filtered (0.45 um Nylon) and placed in an autosampler vial.
METHOD
Analytical Conditions
Column: ALKION™ cation-exchange, K+ form, 4 x 150 mm, Catalog No. 9410917; ALKION™ Guard column, 3 x 20 mm, Catalog No. 9493020
Temperature: 40 °C
Flow Rate: 0.8 mL/min
Mobile Phase: 1700-1101, Potassium buffer, K01
1700-1102, Potassium titrant, K02
1700-1103, Potassium ionic strength adjuster, K03
Post-column Conditions
Post-column System: Pinnacle PCX
Reactor Volume: 0.15 mL
Temperature: 45 ˚C
Reagent: o-Phthalaldehyde/Thiofluor + Brij 35®
Flow Rate: 0.3 mL/min
Detection: Fluorometer λex: 330 nm, λem: 465 nm
TIME (Min)
1700-
1700-
1700-
0
60
40
0
15
13.2
78
15.1
12
78
30
12
78
30.1
0
78
FLOW RATE: 0.8 mL/min, COLUMN TEMP.: 40 ºC, INJECTION VOLUME: 10 mL
APRAMYCIN ANALYSIS
TIME (Min)
1700-
1700-
1700-
0
67
33
0
5
67
0
15
14.7
78
20
14.7
78
20.1
0
78
SAMPLE: 11.1 nmole Apramycin on column
GENTAMICIN ANALYSIS
TIME (Min)
1700-
1700-
1700-
0
43
31
26
20
9
78
30
9
78
30.1
0
78
31
0
78
SAMPLE: 5 x 10-6 g Gentamicin complex on column
NEOMYCIN ANALYSIS
TIME (Min)
1700-
1700-
1700-
0
60
40
0
15
13.2
78
25
11
78
25.1
0
78
26
0
78
SAMPLE: 2.75 nmole Neomycin
CONDITIONS FOR SEPARATION OF APRAMYCIN GENTAMICIN AND NEOMYCIN
1101 %
1102 %
1103 %
8.8
10
10
22
31
0
22
78
31.1
60
40
0
37
60
40
0
1101 %
1102 %
1103 %
33
7.3
7.3
22
21
0
22
78
21.1
67
33
0
1101 %
1102 %
1103 %
13
13
22
22
31.1
43
31
26
1101 %
1102 %
1103 %
8.8
11
22
22
26.1
60
40
0
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ALKION” and Thiofluor are trademarks of Pickering Laboratories, Inc.; “Brij 35” is a registered trademark of ICI Americas, Inc.
REFERENCES:
Driver, J. L., Thex, N., Raynie, D., Ofitserova, M., Pickering, M. Single-Laboratory Validation for the Quantification of Neomycin B and Neomycin C in Animal Feeds by Liquid Chromatography Fluorescence Detection with Post-column Derivatization. (2009) J.AOAC Int, 92, 34-41