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Method Abstract
for Post-column Liquid Chromatography 205
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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.
Take one part feed:10 parts
Extraction solution (w/v) Catalog No. 1700-1118
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 (0.45um Nylon) and placed in an
autosampler vial.
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COLUMN: |
ALKION™ cation-exchange, K+ form, 4
x 150 mm,
Catalog No. 9410917
ALKION™ Guard column, 3 x 20 mm, Catalog No.
9493020 |
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TEMPERATURE: |
40 °C |
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FLOW RATE: |
0.8 mL/min |
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MOBILE PHASE:
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K01, Potassium buffer
K02, Potassium titrant
K03, Potassium ionic strength adjuster |
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POST-COLUMN SYSTEM: |
Pinnacle PCX |
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REACTOR VOLUME: |
0.15 mL |
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TEMPERATURE: |
45 °C |
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REAGENT: |
o-Phthalaldehyde/Thiofluor
+ Brij 35® |
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FLOW RATE: |
0.3 mL/min |
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DETECTION: |
Fluorometer
 ex:
330 nm
em:
465 nm |
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Sample: 11.1 nmole Apramycin on column
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0 |
67
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33
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0
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5 |
67
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33
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0
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15 |
14.7
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7.3
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78
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20 |
0
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7.3
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78
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20.1 |
0
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22
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78
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21 |
0
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22
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78
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21.1 |
67
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33
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0
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Sample: 5 x 10-6 g Gentamicin complex on
column
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0 |
43
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31
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26
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20 |
9
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13
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78
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30 |
9
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13
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78
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30.1 |
0
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22
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78
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31 |
0
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22
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78
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31.1 |
43
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31
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26
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Sample: 2.75 nmole Neomycin on column
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0 |
60
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40
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0
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15 |
13.2
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8.8
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78
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25 |
11
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11
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78
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25.1 |
0
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22
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78
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26 |
0
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22
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78
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26.1 |
60
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40
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0
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“ALKION” and Thiofluor are trademarks of Pickering
Laboratories, Inc.
“Brij 35” is a registered trademark of ICI
Americas, Inc.
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