Electrokinetic chromatographic estimation of the enantioselective binding of nomifensine to human serum albumin and total plasma proteins.

This report is the first evidence of enantioselective binding of nomifensine to human serum albumin (HSA) and plasma proteins. The overall process with HSA included: (i) consistent experimental design along two independent sessions; (ii) incubation of nomifensine-HSA designed mixtures; (iii) ultrafiltration for separating the unbound enantiomers fraction; (iv) electrokinetic chromatography (EKC) using heptakis-2,3,6-tri-O-methyl-ß-cyclodextrin as chiral selector to provide experimental data for enantiomers (first, E1, and second, E2, eluted ones); and (v) a recent direct equation allowing univariate tests and robust statistics to provide consistent parameters and uncertainty. A significant enantioselectivity to HSA (2.7 ± 0.1) was encountered, related to a 1:1 stoichiometry and log affinity constants of 3.24 ± 0.10 and 3.67 ± 0.08 for E1 and E2, respectively. The protein binding (PB) estimated at physiological concentration levels was 40 ± 5 and 63 ± 4% for E1 and E2, respectively. The use of synthetic human sera allowed in vitro estimation of the total plasma PB for the racemate (61 ± 5%; coincident with in vivo values), and its enantiomers (58 ± 7 and 64 ± 4% for E1 and E2, respectively). Comparison allowed the relative importance of HSA respect to other plasma proteins for binding nomifensine to be established.

Application of protein-liposome conjugate as a pseudo-stationary phase in capillary electrophoresis.

Liposomes have very similar structure to cell plasma membranes. Using liposomes as stationary phase in liquid chromatography (LC) or micellar electrokinetic chromatography (MEKC) has been demonstrated to be a good, dynamic method for the study of the interaction between cell membranes and important biomolecules. There has been no report on integrating plasma membrane proteins with phospholipids as pseudo-stationary phase in MEKC. In this paper, a novel mode of capillary electrophoresis (CE) is developed, that is, protein-liposome conjugate. This protein-liposome biomimetic membrane is demonstrated for the first time to be applicable as pseudo-stationary phase in MEKC. The protein is able to significantly improve chromatographic performance and stability. The experimental phenomena are further confirmed in terms of specific capacity factors and free binding energy. This new CE mode is used to investigate the interaction between dopamine transporter and dopamine-nomifensine.

An integrated approach for profiling oxidative metabolites and glutathione adducts using liquid chromatography coupled with ultraviolet detection and triple quadrupole-linear ion trap mass spectrometry.

The use of liquid chromatography (LC) coupled with triple quadrupole linear ion trap (Qtrap) mass spectrometry (MS) for both quantitative and qualitative analysis in drug metabolism and pharmacokinetic studies is of great interest. Here, a new Qtrap-based analytical methodology for simultaneous detection, structural characterization and semi-quantitation of in vitro oxidative metabolites and glutathione trapped reactive metabolites was reported. In the current study, combined multiple ion monitoring and multiple reaction monitoring were served as surveying scans to trigger product ion spectral acquisition of oxidative metabolites and glutathione adduct, respectively. Then, detection of metabolites and recovery of their MS/MS spectra were accomplished using multiple data mining approaches. Additionally, on-line ultraviolet (UV) detection was employed to determine relative concentrations of major metabolites. Analyses of metabolites of clozapine and nomifensine in rat liver microsomes not only revealed multiple oxidative metabolites and glutathione adducts, but also identified their major oxidative metabolism and bioactivation pathways. The results demonstrated that the LC/UV/MS method enabled Qtrap to perform the comprehensive profiling of oxidative metabolites and glutathione adducts in vitro.

Colorimetric methods for the assay of nomifensine maleate.

Two colorimetric methods are reported for the assay of nomifensine maleate. The methods are based on coupling between the diazotised form of nomifensine maleate and (i) N-(1-naphthyl)-ethylene diamine dihydrochloride (Bratton-Marshall reagent) and (ii) p-aminosalicylic acid (PAS). The optimum conditions for the reactions were investigated. The coupled products exhibit maximum absorbance at 470 and 435 nm for the Bratton-Marshall and PAS reagents, respectively. With PAS, a linear relationship has been established between absorbance (Amax) and concentration of nomefensine maleate over the range 2-12 micrograms ml-1. Similarly, with the Bratton-Marshall reagent, a linear relationship exists in the concentration range 2-16 micrograms ml-1. The calculated mean percent recoveries for nomifensine maleate in the commercial capsules (Merital 25 mg) respectively. Similarly, for the added recoveries, the percentage obtained were 99.01 +/- 0.46 and 100.03 +/- 1.03, respectively.