Simultaneous determination of ceftazidime and pyridine in human plasma by LC-UV.

A sensitive, accurate and precise liquid chromatography (LC) method for the simultaneous determination of ceftazidime and pyridine in human plasma has been developed and validated. Acetonitrile (ACN) was employed to precipitate the proteins in the plasma samples. Chromatographic separation was performed with a Kinetex® C18 (150 mm × 3 mm, 2.6 µm) column with gradient elution. Ammonium formate 20 mM and ACN were mixed in a ratio of 98:2 (v/v) for mobile phase A and 85:15 (v/v) for mobile phase B. Both were adjusted to pH 4.5 with formic acid. The flow rate was 0.4 mL/min. UV detection was performed at 254 nm. Calibration curves were linear in the range from 0.3 to 225 µg/mL for ceftazidime and from 0.2 to 10 µg/mL for pyridine with correlation coefficients ≥ 0.999. Within- and between-run precision and accuracy were satisfactory with coefficients of variation (CV) ≤ 8.0% and deviations ≤ 7.0%, respectively. The method fulfilled all validation criteria prescribed by the European Medicines Agency guidelines. Next, it has been used successfully to analyze plasma samples of patients who received ceftazidime under intermittent and continuous administration. With intermittent administration, the concentration of the antibiotics reached a peak and then dropped quickly, which may be below the minimal inhibitory concentration (MIC). With continuous administration, the concentration of the antibiotics remained stable over 24 h, certainly above the MIC. Although the same tendency in ceftazidime concentration changes over time was observed, a difference in concentration amongst the patients was noticeable. The concentration of pyridine in plasma was negligible.

Kinetic study of CYP3A4 activity on verapamil by capillary electrophoresis.

The use of capillary electrophoresis (CE) for the determination of CYP3A4 activity with verapamil as a substrate was investigated. CYP3A4 activity was determined by the quantitation of the product, norverapamil, based on separation by CE. The separation conditions were as follows: capillary, 80.5 cm (75 microm i.d., 72 cm effective length); 50 mM sodium phosphate buffer (pH 8.8); 20 kV (100 microA) applied voltage; UV detection at 200 nm; capillary temperature, 25 degrees C. With the developed CYP3A4 activity assay and the Lineweaver-Burk equation, the Michaelis-Menten parameters Km and Vmax for formation of norverapamil from verapamil in the presence of CYP3A4 were determined and were 22.8+/-2.5 microM and 7.67+/-0.26 pmol/min/pmol (or 983 pmol/min/mg) CYP3A4, respectively.