Documentation

Method Abstract 205

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


CONDITIONS FOR SEPARATION OF APRAMYCIN GENTAMICIN AND NEOMYCIN

TIME (Min)

1700-
1101 %

1700-
1102 %

1700-
1103 %

0

60

40 

0

15

13.2

8.8

78

15.1

12

10

78

30

12

10

78

30.1

0

22

78

31 0 22 78
31.1 60 40 0
37 60 40 0

FLOW RATE: 0.8 mL/min, COLUMN TEMP.: 40 ºC, INJECTION VOLUME: 10 mL



 
 

MA 205


APRAMYCIN ANALYSIS

TIME (Min)

1700-
1101 %

1700-
1102 %

1700-
1103 %

0

67

33

0

5

67

33

0

15

14.7

7.3

78

20

14.7

7.3

78

20.1

0

22

78

21 0 22 78
21.1 67 33 0

SAMPLE: 11.1 nmole Apramycin on column




 

MA 205


GENTAMICIN ANALYSIS

TIME (Min)

1700-
1101 %

1700-
1102 %

1700-
1103 %

0

43

31

26

20

9

13

78

30

9

13

78

30.1

0

22

78

31

0

22

78

31.1 43 31 26

SAMPLE: 5 x 10-6 g Gentamicin complex on column



 

MA 205
 


NEOMYCIN ANALYSIS

TIME (Min)

1700-
1101 %

1700-
1102 %

1700-
1103 %

0

60

40 

0

15

13.2

8.8

78

25

11

11

78

25.1

0

22

78

26

0

22

78

26.1 60 40 0

SAMPLE: 2.75 nmole Neomycin


MA 205/


    

 

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