RESUMO
OBJECTIVE: To evaluate the effect of different types of microplate and loading volumes on the detection results of multi-function microplate reader, and to optimize the analysis method. METHODS: A multi-function microplate reader was used to perform spectrum scanning on each of 5 detection holes of common and ultraviolet(UV) microplates, and the applicable detection wavelength range was those with light transmittance greater than 80.00%. The optical density measurement was carried out on each 12 detection holes of common and UV microplates at different wavelengths, then the matching of the detection holes was compared. Potassium permanganate was quantitatively analyzed by common microplate and UV microplate, while acetone was analyzed by UV microplate, and then detection limit, lower limit of quantitation(LLQ), accuracy and precision at different loading volumes and concentrations were calculated and compared. RESULTS: The shortest applicable analyzing wavelengths for common and UV microplates were(362±2) and(230±3) nm respectively, while the longest applicable analyzing wavelengths were both 1 000 nm. The light transmittance of UV microplate was higher than that of common microplate when the analyzing wavelengths were lower than 400 nm(P<0.01). The deviation and range of light transmittance of detection holes analyzed by UV microplates were smaller than that of common microplates when the analyzing wavelengths were 350-1 000 nm(P<0.05). The detection limit and LLQ of potassium permanganate by multi-function microplate reader was not associated with the types of microplate. The adding standard recoveries of potassium permanganate by UV microplate was higher than that by common microplate(P<0.05). The adding standard recoveries of potassium permanganate by loading volumes of 200 and 250 μL was lower than that by loading volumes of 150 μL(P<0.01), while adding standard recovery of acetone by loading volumes of 200 μL was lower than that by loading volumes of 150 μL(P<0.05). CONCLUSION: When using a multi-function microplate reader to detect chemicals, it is recommended to use UV microplate with wavelengths at the range of 230-1 000 nm, and loading volumes of 200-250 μL.
RESUMO
Ciprofloxacin HCl (CIP) and Metronidazole (MET) are antibacterial drugs used in combination for treatment of mixed aerobic/anaerobic infections. An UPLC-MS/MS method was developed for the simultaneous estimation of CIP and MET in spiked human plasma using sildenafil citrate as an internal standard (IS). Protein precipitation was used for analyte extraction. The chromatographic separation was completed within 6 min using a mobile phase of 0.1% formic acid in water and acetonitrile (70: 30, v/v), Zorbax C18, 100 x 4.6 mm, 3.5 μm analytical column, at a flow rate of 0.5 mL min-1. Multiple reaction monitoring (MRM) transitions were measured in the positive ion mode. Validation of the method showed standard curves to be linear in the range of 10-4000 ng mL-1 for CIP and 30-12000 ng mL-1 for MET with mean correlation coefficient exceeding 0.999. In human plasma, CIP and MET were stable for at least 36 days at –70 ± 5 °C, 6 hours at ambient temperature and after three freeze thaw cycles. After extraction from plasma, the samples were stable in auto sampler at 22 °C for 6 hours. The method was simple, specific, sensitive, precise, accurate and suitable for bioequivalence and pharmacokinetic studies.