Purification and characterization of [3H]mepyramine (histamine H1 antagonist)-binding protein from rat liver: a highly homologous protein with cytochrome P450 2D.

A protein having a high-affinity binding site for [3H]mepyramine (MBP) was purified to homogeneity from rat liver membranes. The purified MBP has a single type of binding site for [3H]mepyramine with Kd value of 18.5 nM, and its molecular weight was determined to be 56,000 by SDS polyacrylamide gel electrophoresis. Amino acid sequences of twelve tryptic peptides derived from MBP are highly homologous with those of rat debrisoquine 4-hydroxylase (cytochrome P450 2D1) and other rat P450 2D subfamily members. In immunoblotting analysis, an antibody against rat P450 2D1 stained a band corresponding to MBP with Mr of 56,000; its migration position was clearly different from that of rat P450 2D1. Substrates and inhibitors of debrisoquine 4-hydroxylase potently displace [3H]-mepyramine binding to MBP. Quinine and quinidine showed 400 and 80 times, respectively, higher affinity for MBP than for debrisoquine 4-hydroxylase. These results suggest that MBP is a novel P450 2D family member.

Liquid chromatography of antihistamines using post-column tris(2,2'-bipyridine) ruthenium(III) chemiluminescence detection.

The separation and detection of five antihistamine drugs commonly found within over-the-counter allergy and cold pharmaceutical products was performed by HPLC with chemiluminescence (CL) detection. Comparable detection limits at 5-10 pmol were found for the antihistamines by both UV at 214 nm and tris(2,2'-bipyridine) ruthenium(III) CL. However, urine samples were found not to generate as large an unretained peak by CL detection as compared to those peaks by UV detection at 214 and 254 nm. For example, the pheniramine peak representing 0.15 microgram/ml was almost totally obscured at 214 nm. Quantitative results received for three antihistamine commercial samples ranged from 4 to 8% error in accuracy when an internal standard was used to compensate for short term detector drift.

Application of Empore C-8 extraction disks for screening urine in systematic toxicological analysis.

Solid-phase extraction (SPE) by means of disposable columns has become a widely accepted technique for sample pretreatment in toxicology, both for directed analyses and for screening analyses. However, the sample capacity in SPE is usually limited to a few millilitres. Therefore, we have investigated to what extent these problems can be overcome by using Empore extraction disks, consisting of chemically modified C-8 reversed-phase silica, embedded in an inert polytetrafluoroethylene (PTFE) matrix. Human urine was selected as the matrix and dexetimide and mepyramine were initially used as test drugs because these drugs were available in tritiated form. Additional drugs investigated included codeine, hexobarbital, imipramine, methamphetamine, and nitrazepam. In these investigations, the sample capacity for untreated urine was at least 25 mL, and analyte quantities up to 250 micrograms could be retained by these filters. Washing with water/methanol mixtures was successful in removing substantial amounts of endogenous interferences, and methanol proved to be an acceptable eluent. Thus, these disks seem to have interesting potential for toxicological analysis in that sample concentration and cleanup can be achieved at the same time.

Ascorbate increases apparent "specific" binding of [3H]pyrilamine to glass fiber filters.

Although filtration through glass fiber filters provides a convenient method for separating bound from unbound radioligand, binding of the ligand to the filters is often a problem. Ascorbate (0.1%) has been shown to decrease binding of serotonergic acids to these filters. In the present study, relatively high binding of [3H]pyrilamine, an H1-antagonist, to glass fiber filters was observed. The addition of ascorbate increased, rather than decreased, binding of [3H]pyrilamine to the filters. Thus, the effect of ascorbate on binding of radioligands to glass fiber filters appears to be dependent upon the particular ligand involved.