Simultaneous determination of 11 antibiotics and their main metabolites from four different groups by reversed-phase high-performance liquid chromatography-diode array-fluorescence (HPLC-DAD-FLD) in human urine samples

A new, accurate and sensitive reversed-phase high-performance liquid chromatography (RP-HPLC) as analytical method for the quantitative determination of 11 antibiotics (drugs) and the main metabolites of five of them present in human urine has been worked out, optimized and validated. The analytes belong to four different groups of antibiotics (sulfonamides, tetracyclines, penicillins and anphenicols). The analyzed compounds were sulfadiazine (SDI) and its N⁴-acetylsulfadiazine (NDI) metabolite, sulfamethazine (SMZ) and its N⁴-acetylsulfamethazine (NMZ), sulfamerazine (SMR) and its N⁴-acetylsulfamerazine (NMR), sulfamethoxazole (SMX), trimetroprim (TMP), amoxicillin (AMX) and its main metabolite amoxicilloic acid (AMA), ampicillin (AMP) and its main metabolite ampicilloic acid (APA), chloramphenicol (CLF), thiamphenicol (TIF), oxytetracycline (OXT) and chlortetracycline (CLT). For HPLC analysis, diode array (DAD) and fluorescence (FLD) detectors were used. The separation of the analyzed compounds was conducted by means of a Phenomenex^® Gemini C₁₈ (150 mm × 4.6 mm I.D., particle size 5 μm) analytical column with LiChroCART^® LiChrospher^® C₁₈ (4 mm × 4 mm, particle size 5 μm) guard column. Analyzed drugs were determined within 34 min using formic acid 0.1% in water and acetonitrile in gradient elution mode as mobile phase. A linear response was observed for all compounds in the range of concentration studied. Two procedures were optimized for sample preparation: a direct treatment with methanol and acetonitrile and a solid phase extraction procedure using Bond Elut^® Plexa™ columns. The method was applied to the determination of the analytes in human urine from volunteers under treatment with different pharmaceutical formulations. This method can be successfully applied to routine determination of all these drugs in human urine samples.