Simultaneous determination of aconitum alkaloids in rat body fluids by high-performance liquid chromatography

A sensitive, reliable and accurate high-performance liquid chromatography (HPLC) method coupled with photodiode array detector (DAD) were developed for the simultaneous quantitative determination of aconitine, mesaconitine and hypaconitine in rat plasma and urine by optimizing the extraction, separation and analytical conditions. The analyses were chromatographed on a ZORBAX Eclipse XDB-C18 column (150 mm ~ 4.6 mm i.d.; 5 ƒÊm particle size) with gradient elution using solvents ofacetonitrile and ammonium acetate buffer (pH 10.0). The detection wavelength was 240 nm. Intra-assay and inter-assay precision of the analyses were less than 10% and the average recovery rates obtainedwere in the range of 85.63 - 90.94% for all analysis of the three aconitum alkaloids with relative standard deviations (RSD) below 14%. Positive linear relationships were observed in correlation coefficients that exceeded 0.95. The limits of detection (signal-to-noise ratio of 3) were 2.64, 1.58 and 2.75 ng for aconitine, mesaconitine and hypaconitine, respectively. The method can provide a scientific and technical platform to determine the concentration of aconitum alkaloids in plasma during a pilot pharmacokinetic study in rats

Carbapenem and cefoxitin resistance of Klebsiella pneumoniae strains associated with porin OmpK36 loss and DHA-1 β-lactamase production

Clinical isolates of carbapenem-resistant Klebsiella pneumoniae (K. pneumoniae) strains are being increased worldwide. Five pan-resistant K. pneumoniae strains have been isolated from respiratory and ICU wards in a Chinese hospital, and reveal strong resistance to all β-lactams, fluoroquinolones and aminoglycosides. Totally 27 β-lactamase genes and 2 membrane pore protein (porin) genes in 5 K. pneumoniae strains were screened by polymerase chain reaction (PCR). The results indicated that all of 5 K. pneumoniae strains carried blaTEM-1 and blaDHA-1 genes, as well as base deletion and mutation of OmpK35 or OmpK36 genes. Compared with carbapenem-sensitive isolates by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), the resistant isolates markedly lacked the protein band of 34-40 kDa, which might be the outer membrane proteins of OmpK36 according to the electrophoresis mobility. In addition, the conjugation test was confirmed that blaDHA-1 mediated by plasmids could be transferred between resistant and sensitive strains. When reserpine (30 µg/mL) and carbonyl cyanide m-chlorophenylhydrazone (CCCP) (50 µg/mL) were added in imipenem and meropenem, the MICs had no change against K. pneumoniae strains. These results suggest that both DHA-1 β-lactamase and loss or deficiency of porin OmpK36 may be the main reason for the cefoxitin and carbapenem resistance in K. pneumoniae strains in our hospital