High Performance Liquid Chromatographic Determination of Clarithromycin in Lymphocytes Using a Post-column with Tris(2,2'-bipyridine) Ruthenium (III) Chemiluminescence Detection

Abstract

The concentration of antibiotics in cells must be determined for effective treatment of infectious diseases caused by obligate intracellular parasites, such as Chlamydia trachomatis and Legionella pneumophila. We confirmed the usefulness of high performance liquid chromatography (HPLC), which is already commonly used in medical facilities, for the measurement of antibiotics in cells. The measurement was carried out using a post-column with tris(2,2 -bipyridine) ruthenium (III) chemiluminescence detection. Clarithromycin (CAM) was used as the model antibiotic. The retention time of CAM on the column was 7.6 ± 0.4 min and the detection limit was 2.0 × 10 −3 ng on column. The linearity of the calibration curve was guaranteed until 2.0 ng on column. This level of performance is comparable to that of liquid chromatography-tandem mass spectrometry. The system was able to monitor changes in the concentration of CAM in lymphocytes. These findings suggest that HPLC, a general-purpose, already widely used detection system, could also contribute to the development of effective individual antibiotic treatment regimens at a wide variety of medical facilities.

Chemiluminescence and electrochemiluminescence detection of controlled drugs

“…Chiba et al developed a HPLC procedure with reverse‐phase separation and Ru(bipy) 3 2+ chemiluminescence detection for phencyclidine (PCP; Figure 21a), its hydroxylated metabolites (Figure 21b), and their glucuronide conjugates (after acid hydrolysis) 70. The chemiluminescence reagent was electrochemically oxidized to the ruthenium(III) state online prior to mixing with the column eluate 179. Limits of detection ranged from 3 × 10 −8 M to 6 × 10 −8 M. The procedure was successfully applied to urine from rats administered with PCP; sample preparation involved solid‐phase extraction 70…”

Determination and Stability Assessment of Clarithromycin in Human Plasma using RP-LC with Electrochemical Detection

“…In this context, various methods and techniques have been reported for determination of CLA in different matrices (7, 9 -11). Among those reported are liquid chromatography (LC) with ultraviolet (UV) detection (12 -14), LC-fluorometry (10,(15)(16)(17), capillary electrophoresis (18), LC-mass spectrometry (MS) and ultra-performance LC with tandem MS (ULC -MS-MS) and LC-nuclear magnetic resonance (NMR) (19)(20)(21)(22)(23)(24)(25). Other reported methods have been high-performance liquid chromatography (HPLC) with electrochemical detection (ECD) (26 -30).…”

“…Various methods in this context with different sample preparation, extraction procedures and chromatographic conditions along with ECD utilization have been reported for CLA alone or in combination with other analytes (26 -30). However, many of the published methods are lengthy (9,29), suffer from low recovery (28) or high detection limit (5,19,28) or apply a high column temperature (10,17,29,30). Additionally, some methods were assigned for analysis of the drug in rat plasma and tissue cells, where its concentration is much higher than in blood (5,18,26).…”