A time-resolved fluoroimmunoassay for alpha-fetoprotein in serum and amniotic fluid

Abstract

Methicillin-resistant Staphylococcus aureus infection is a serious clinical problem worldwide. Ceftaroline, daptomycin, linezolid in combination with rifampicin are particularly used in this indication. To allow monitoring of these antibiotics, an on-line solid phase extraction coupled to high-performance liquid chromatography–tandem mass spectrometry assay requiring a 100 μL aliquot of human plasma has been developed. Besides, significance of 25-O-desacetylrifampicin concentrations was evaluated.Sample pre-treatment is limited to protein precipitation with methanol. After centrifugation 10 μL of supernatant are injected into the chromatographic system, which consists of an on-line solid phase extraction followed by a separation on a phenyl-hexyl column and detected by a tandem mass spectrometer. Plasma drug concentrations were determined by multiple reaction monitoring in positive ion mode, and assay performance was evaluated. 25-O-Desacetylrifampicin activity, was compared to rifampicin using a microbiological method.Sample preparation using methanol precipitation followed by solid-phase extraction yielded good recovery and ionization efficiency, with chromatographic separation achieved within 3 min per sample. Within-run and between-run precisions ranged respectively from 1.22% to 9.35% and from 1.61% to 9.36%. Lower limits of quantification were 0.04 mg/L for linezolid, 0.1 mg/L for rifampicin, 0.2 mg/L for ceftaroline and 0.5 mg/L for daptomycin. It appears that 25-O-desacetylrifampicin displays a substantial intrinsic bactericidal activity against S. aureus.This assay provides simple, rapid, sensitive and accurate quantification of the four antibiotic drugs and one metabolite and can be routinely used to monitor drug concentration in methicillin-resistant S. aureus infected patients.