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ANTONIO L. DOADRIO Y COLS. AN. R. ACAD. NAC. FARM.

2.3. Apparatus and instrument

Kontron high-pressure liquid chromatograph equipped with a
Kontron 420 pump. An automatic injector with 6 valves Kontron
Auto Sampler 460. A variable-wavelength UV detector of Kontron
Uvikon 735 LC. Phenomenex C18 column (25 x 0.46 cm), fulled with
particles of 5 mm, besides this column of separation, a precolumn
and a protective column fulled with the same material that the first
column were used. A Kontron Station Data with D450 software was
used to control the detector and injector, on the other hand it allowed
the integration of the chromatogram peaks and it cuantificated the
results.

Paper Whatman discs, discs AA of 7 mm diameter were used to
absorb the antibiotic solutions in the agar-plate experiments.

2.4. Analytical procedure

The extraction of cefotaxime in blood was carried out by means
of protein precipitants in 10% trichloroacetic acid (8) 1.5 mL of this
reagent was added to 0.5 mL of blood. This mixture was centrifuged
at 3000 rpm for 5 min. The supernatant was collected through a
Pasteur pipet and filtered through MFS disks of nylon of 0.4 mm of
diameter. The clean supernatant was assayed by HPLC.

The extraction of cefotaxime in organs such as liver, spleen,
kidney, lung and heart, was carried out by means of trichloroacetic
acid 20%. 2 mL of this reagent were added to 0.5 g of dry weight of
each organ. Each mixture was done homogeneous and was
centrifuged at 3000 rpm for 10 min. The supernatant was collected
through a Pasteur pipet and filtered through MFS disks of nylon of
0.4 mm of diameter. It was later assayed by HPLC.

Isocratic HPLC conditions used along this wak were: the mobile
phase consisted of a 20:80% solution (v/v) of methanol in 0.01 M
PO4H2K. The pH of the final solution was adjusted to 3.2 with
phosphoric acid. The flow rate was 1 mL/min. Chromatograph was
loaded with 40 µL of each sample. Chromatograms were run at room
temperature. Samples were detected at 254 nm.

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