Simultaneous determination of pancuronium, vecuronium and their related compounds using LC-ESI-MS.

A simultaneous determination method of quaternary amino steroidal muscle relaxants, pancuronium (PAN), vecuronium (VEC), and 17-monodesacetyl pancuronium (17-OH-PAN), 3,17-bisdesacetyl pancuronium (3,17-OH-PAN), 3-monodesacetyl vecuronium (3-OH-VEC), 3,17-bisdesacetyl vecuronium (3,17-OH-VEC) in human serum was developed using liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS). The weak cation exchange cartridge was useful for the extraction of these compounds. Under optimized LC-ESI-MS conditions, these compounds were almost fully separated within 6.5 min. Linear responses over the concentration range 0.25-50.0 ng/mL were demonstrated for each compound. The developed method successfully detected VEC, 3-OH-VEC and 3,17-OH-VEC in serum intravenously administered with VEC. The level of 3-OH-VEC was higher than other compounds. This suggested that 3-OH-VEC was useful as a forensic probe in VEC administration.

Determination of chlorhexidine (CHD) and nonylphenolethoxylates (NPEOn) using LC-ESI-MS method and application to hemolyzed blood.

Rapid and reliable methods for identification of chlorhexidine (CHD) and nonylphenolethoxylates (NPEOn) in antiseptic and hemolyzed blood using electrospray ionization mass spectrometry (ESI-MS) were developed. Fragmental analysis provides accurate evidence for the presence of CHD in the samples. For the determination of CHD in hemolyzed blood, the method was also developed using LC-ESI-MS. Linearity of calibration curve was obtained over the concentration range of 0.1-11 microg/mL with residuals from -4.3 to 6.7%. We applied the methods to the case of suicidal injection of antiseptic and successfully detected CHD and NPEOn from hemolyzed blood. The CHD concentration was 352 microg/mL.

Effects of axial methionine coordination on the in-plane asymmetry of the heme electronic structure of cytochrome c.

The paramagnetic susceptibility ( chi) tensors of the oxidized forms of thermophile Hydrogenobacter thermophilus cytochrome c(552) (Ht cyt c(552)) and a quintuple mutant (F7A/V13 M/F34Y/E43Y/V78I; qm) of mesophile Pseudomonas aeruginosa cytochrome c(551) (Pa cyt c(551)) have been determined on the basis of the redox-dependent (1)H NMR shift changes of the main-chain NH and C(alpha)H proton resonances of non-coordinated amino acid residues and the NMR structures of the reduced forms of the corresponding proteins (J. Hasegawa, T. Yoshida, T. Yamazaki, Y. Sambongi, Y. Yu, Y. Igarashi, T. Kodama, K. Yamazaki, Y. Kyogoku, Y. Kobayashi (1998) Biochemistry 37:9641-9649; J. Hasegawa, S. Uchiyama, Y. Tanimoto, M. Mizutani, Y. Kobayashi, Y. Sambongi,Y. Igarashi (2000) J Biol Chem 275:37824-37828). From the chi tensors determined, we obtained the contact shifts for heme methyl proton resonances, which provided the heme electronic structures of the oxidized forms of Ht cyt c(552) and qm. We also characterized the heme electronic structure of the cyanide adducts of the proteins, where the axial Met was replaced by an exogenous cyanide ion, through the analysis of (1)H NMR spectra. The results indicated that the heme electronic structures of both the proteins in their oxidized forms with axial His and Met coordination are largely different to each other, while those in their cyanide adducts are similar to each other. These results demonstrated that the orientation of the axial Met sulfur lone pair, with respect to heme, predominantly contributes to the spin delocalization into the porphyrin-pi system of heme in the oxidized proteins with axial His and Met coordination.

Relationship between redox function and protein stability of cytochromes c.

Electrochemical, 1H NMR, and optical studies on mesophile Pseudomonas aeruginosa cytochrome c551, its single (F34Y) and quintuple (F7A/V13M/F34Y/E43Y/V78I) mutants, and thermophile Hydrogenobacter thermophilus cytochrome c552 at wide temperature range demonstrated that the stable protein exhibits the low redox potential predominantly due to the enthalpic contribution to the redox reaction. The overall stability of the oxidized form was shown to determine the stability of the Fe-methionine coordination bond, which then directly regulates the redox function.

Influence of amino acid side chain packing on Fe-methionine coordination in thermostable cytochrome C.

Paramagnetic NMR and optical studies of the oxidized forms of mesophile Pseudomonas aeruginosa cytochrome c(551) and its quintuple mutant (F7A/V13M/F34Y/E43Y/V78I), and thermophile Hydrogenobacter thermophilus cytochrome c(552) demonstrated that the amino acid side chain packings in the protein interior influence the coordination bond between the heme iron and the axial methionine in the proteins. The strength of heme axial coordinations was found to correlate with the overall protein thermostability.