High-performance liquid chromatographic assay for glucuronidation activity of 7-ethyl-10-hydroxycamptothecin (SN-38), the active metabolite of irinotecan (CPT-11), in human liver microsomes

A simple and sensitive assay for glucuronidation activity of 7-ethyl-10-hydroxycamptothecin (SN-38), the active metabolite of irinotecan (CPT-11), in human liver microsomes by high-performance liquid chromatography (HPLC) with fluorescence detection is reported. The method was validated for the determination of SN-38 glucuronide (SN-38G) with respect to specificity, linearity, recovery, stability, precision, accuracy, and limits of detection and quantitation. There was no interference from matrix and non-enzymatic reactions. The calibration curve for SN-38G was linear from 5 to 500 nM. Average recoveries ranged from 98 to 100% in spiked human liver microsome samples, and the SN-38G was stable at 4 degrees C for at least 72 h. The newly developed method was found to be more sensitive and selective than previous methods using thin layer chromatography and HPLC. The limit of quantitation for SN-38G was 5 nM (2.5 pmol/assay). The intra- and inter-day precision and accuracy were less than 7 and 4%, respectively. The intra- and inter-day precision of enzyme assay for UDP-glucuronosyltransferase (UGT) activity toward SN-38 in human liver microsomes was less than 4%. With this improved sensitivity, the kinetics of SN-38 glucuronidation in human liver microsomes could be determined more precisely. Therefore, this method is applicable to in vitro study on the side effects and drug interactions of CPT-11 using small amounts of biological sample.     

Mycosamine orientation of amphotericin B controlling interaction with ergosterol: sterol-dependent activity of conformation-restricted derivatives with an amino-carbonyl bridge

Amphotericin B (AmB 1) is known to assemble together and form an ion channel across biomembranes. The antibiotic consists of mycosamine and macrolactone moieties, whose relative geometry is speculated to be determinant for the drug's channel activity and sterol selectivity. To better understand the relationship between the amino-sugar orientation and drug's activity, we prepared conformation-restricted derivatives 2-4, in which the amino and carboxyl groups were bridged together with various lengths of alkyl chains. K+ influx assays across the lipid-bilayer membrane revealed that ergosterol selectivity was markedly different among derivatives; short-bridged derivative 2 almost lost the selectivity, while 3 showed higher ergosterol preference than AmB itself. Monte Carlo conformational analysis of 2-4 based on NOE-derived distances indicated that the amino-sugar moiety of 2 comes close to C41 because of the short bridge, whereas those of 3 and 4 are pointing outward. The mutual orientation of the amino-sugar moiety and macrolide ring is so rigid in derivatives 2 and 3 that these conformations should be unchanged upon complex formation in lipid membranes. These results strongly suggest that the large difference in sterol preference between derivatives 2 and 3 is ascribed to the different orientation of amino-sugar moieties. These findings allowed us to propose a simple model accounting for AmB-sterol interactions, in which hydrogen bonding between 2'-OH of AmB and 3beta-OH of ergosterol plays an important role.     

Ion-Pair Solvent Extraction of EDTA Anions with Tetraalkylammonium Ions in Various Organic Solvents

Summary.  The ion-pair solvent extraction behavior of ethylenediaminetetraacetate (EDTA) anion by various tetraalkylammonium ions was investigated at 25.0 ± 0.1°C. The extraction of EDTA exceeded 90% from the basic aqueous solution into the organic solvents such as n-hexane and benzene derivatives containing tri-n-octylmethylammonium chloride, but EDTA was hardly extracted from acidic solution. Among the chemical species of EDTA in aqueous solution, edta ⁴⁻ is the most extractable one. On the other hand, the extraction of EDTA was less than 1% into chloroform and 1,2-dichloroethane even from the basic aqueous solution. The effect of the structure of alkylammonium ion was also examined. Tetra-n-hexylammonium and tetra-n-octylammonium ions could not extract EDTA even from the basic aqueous solution, while the use of tri-n-octylmethylammonium and di-n-lauryldimethylammonium ions enhances the extraction of EDTA. These results suggest that the steric hindrance in the ion-pair of alkylammonium and EDTA anion in the organic phase affects the extractability of EDTA containing ion-pair. The solution structure of ion-pair in the organic phase was calculated by MMFF force field and the steric effect in the ion-pair was also suggested. From the extraction constants obtained, the possibility of the extraction separation of EDTA has been shown. Present address: Chemistry Department, Heilongjiang University, Harbin, China     

Isothermal differential scanning calorimetry on an exothermic phenomenon during thermal decomposition of hydromagnesite 4 MgCO3 · Mg(OH)2 · 4 H2O

An exothermic phenomenon and a simultaneous rapid evolution of a small amount of carbon dioxide at ?500°C during thermal decomposition of hydromagnesite 4 MgCO₃ · Mg(OH)₂ · 4 H₂O was studied by isothermal DSCTG in a carbon dioxide atmosphere. It was quantitatively confirmed that the exothermic phenomenon was due to crystallization of MgCO₃ from the amorphous phase and that the evolution of carbon dioxide was due to decomposition of the MgCO₃ by the heat of crystallization (?3.4 kcal mole^?1.     

Preparation and characterization of modified 3-aminopropylsilyl silica phases with 1,8-naphthalic anhydrides in HPLC

Modified 3-aminopropylsilyl silica stationary phases with 1,8-naphthalic anhydrides [3-(1,8-naphthalimido)propyl (NAIP), 3-(4-amino-1,8-naphthalimido)propyl (4A-NAIP), 3-(3-nitro-1,8-naphthalimido)propyl (3N-NAIP), and 3-(4-nitro-1,8-naphthalimido)propyl modified silyl silica gel (4N-NAIP)] are prepared in a single reaction. Chromatographic properties of these phases are characterized using various solutes including monosubstituted benzenes, alkyl benzenes, and polycyclic aromatic hydrocarbons (PAHs) in high-performance liquid chromatography. Even though the retention in these NAIPs is mainly based on the reversed-phase mode, 3N- and 4N-NAIP show a remarkable recognition toward aromatic hydrocarbons, which is attributed to their strong pi-pi interaction. Furthermore, the recognition ability is according to the molecular planarity and non-linearity of PAHs. Subsequently, 3N- and 4N-NAIP has a distinct selectivity from octadecyl silica and, therefore, will be a valuable alternative for a separation or a solid-phase extraction of aromatic compounds.     

Purification of enzymes by high-speed gel filtration on TSK-GEL SW columns

The purification of enzymes was investigated by high-speed gel filtration on TSK-GEL G3000SWG columns packed with porous silica gel deactivated by chemically bonded hydrophilic compounds. Crude β-galactosidase from bacterial cells and commercial urease were purified ca. 15-fold in a single gel filtration. These enzymes were eluted within an hour from the column and the recoveries of enzymatic activity were almost 100% although the operation was carried out at room temperature (22°). Samples up to 100 mg could be applied to the column without loss of separation efficiency.