Microcolumn high-performance liquid chromatographic assay for doxycycline in isolated alveolar macrophages

A procedure for the isolation of doxycycline from the alveolar macrophages is described. Due to the minimal amount of the sample, it was necessary to employ microcolumn HPLC. A great advantage of the methodology developed in the present paper consists in the direct injection of an aqueous supernatant over disintegrated macrophages without the use of liquid-liquid or solid-liquid extraction. Another advantage of the method is the fact that the injection of an aqueous solution on a microcolumn with a lipophilic stationary phase makes the analyzed substances concentrate, which increases the sensitivity of assay. Separation was performed on a Separon SGX C18 microcolumn, 150 x 1 mm, detection at 254 nm by UV, 1 microliter flow cell. Rabbits were treated intravenously with Vibramycin inj. sic. in a dose of 13 mg/kg body weight doxycycline. A procedure for washing macrophages from rabbit lungs was developed. Dry matter of macrophages ranged from 5.9 to 11.4 mg. In the samples of three rabbits the levels of doxycycline ranged from 110 to 270 ng per 1 mg of macrophages dry matter.