Green Automated Solid Phase Extraction to Measure Levofloxacin in Human Serum via Liquid Chromatography with Fluorescence Detection for Pharmacokinetic Study

Abstract

A simple, selective, rapid, sensitive and less costly green automated solid phase extraction bio-analytical high-performance liquid chromatographic-based technique with fluorescence detection (Aut-SPE-BA-HPLC-FL) for the quantification of levofloxacin in human serum samples has been developed and validated. The serum samples were loaded into the chromatographic system without prior treatment and then injected into short (20 mm × 4.6 mm, 20 µm) protein-coated (PC) µBondapak CN (µBCN) silica pre-column (PC-µBCN-pre-column). Levofloxacin was retained and pre-concentrated on the head of the PC-µBCN-pre-column, while proteins and other polar components were eliminated using phosphate buffer saline (PBS), pH 7.4, as the first mobile phase in the extraction step. Levofloxacin is then transferred to the analytical column; ZORBAX Eclipse XDB-C18 (150 mm × 46 mm, 5 µm), through the aid of a column-switching valve technique, on-throughs the elution mode using the second mobile phase containing a methanol and phosphate buffer (0.05 M, pH 5) in a ratio of 70:30 (v/v). Levofloxacin signals were detected using a fluorescence detector operated at excitation/emission wavelengths of 295/500 nm. The proposed Aut-SPE-BA-HPLC-FL methodology showed linearity over a levofloxacin concentration range of 10–10,000 ng/mL (r2 = 0.9992), with good recoveries ranging from 87.12 to 97.55%. Because of the validation qualities in terms of linearity, recovery, precision, accuracy, selectivity and robustness, the Aut-SPE-BA-HPLC-FL method has been used in some clinical trials for therapeutic drug monitoring and the pharmacokinetic study of levofloxacin in human serum.