Development of Two Innovative 96-Microwell-Based Spectrophotometric Assays with High Throughput for Determination of Fluoroquinolone Antibiotics in their Pharmaceutical Formulations

Abstract

. Fluoroquinolone antibiotics (FQAs) have broad-spectrum antibacterial activity, high potency,
variable indications, and excellent pharmacokinetic profiles. Many spectrophotometric assays have been described for the determination of FQAs in their pharmaceutical formulations; however, most of these assays
have limited throughput for application in quality control laboratories. This study describes the development and validation of two innovative 96-microwell-based spectrophotometric assays, with high throughput
for the quality control of seven FQAs. These FQAs were levofloxacin (LEV), norfloxacin (NOR), ciprofloxacin (CIP), gemifloxacin (GEM), danofloxacin (DAN), enrofloxacin (ENR), and marbofloxacin (MAR). The
first assay (assay I) was developed for LEV, NOR, CIP, and GEM via the formation of red metal complexes
(MCs) with FeCl3
. The second assay (assay II) was developed for LEV, DAN, ENR, and MAR via the formation of red charge transfer complexes (CTCs) with 2,3-dich1oro-5,6-dicyano-1,4-benzoquinone (DDQ). The
reactions of FQAs with both FeCl3 and DDQ reagents were performed in transparent 96-microwell plates,
and the absorbance of the colored complexes was measured at 460 nm using an absorbance microplate
reader. The best reaction conditions were established for both assays; under which, Beer’s law was obeyed
in the range of 10-100 µg/well with good correlation coefficients (0.9943-0.9982). The limits of detection were
in the range of 4.5-7.5 µg/well, and the limits of quantification were in the range of 14.9–25.0 µg/well. Both
assays showed high precision, as the values of the relative standard deviations (RSD) did not exceed 3.4%.
The accuracy of both assays was proven by recovery studies, as the recovery values were in the range of 98.1-
102.6% (± 0.9-2.7%). The assays were applied for the determination of all seven FQAs in their tablets with
good accuracy and precision. The assays are simple, economical, and have the capacity for high-throughput
testing; therefore, they are convenient and beneficial for the regular determination of FQAs in pharmaceutical formulations. An additional advantage of these assays is that all FQAs can be determined on a single
system without modifications in the detection wavelength.