ABSTRACT
Modern isolation techniques by direct extraction with organic solvents or after protein precipitation by various sedimenting or salting-out agents are characterized by low efficiency and do not permit to liberate derivatives of barbituric acid from their complexes with blood proteins. The use of enzymatic hydrolysis makes it possible to break bonds between barbiturates and protein and thereby improve the efficiency of isolation. We performed enzymatic hydrolysis of the model phenobarbital-blood and barbamyl-blood complexes with the use of trypsin, pepsin, chymotrypsin, and papain. The degree of phenobarbital extraction with trypsin and barbamyl was estimated at 62.1 +/- 1.2% and 75.1 +/- 1.6% respectively; in other words, it was 32.7 +/- 1.0% and 51.1 +/- 1.0% higher than that achieved by traditional methods. Certain validation characteristics of the new method are presented.
Subject(s)
Amobarbital/blood , Barbiturates/blood , Blood Proteins/chemistry , Endopeptidases/chemistry , Forensic Medicine/methods , Hypnotics and Sedatives/blood , Phenobarbital/blood , Amobarbital/chemistry , Amobarbital/isolation & purification , Barbiturates/chemistry , Barbiturates/isolation & purification , Chymotrypsin/chemistry , Humans , Hydrolysis , Hypnotics and Sedatives/chemistry , Hypnotics and Sedatives/isolation & purification , Papain/chemistry , Pepsin A/chemistry , Phenobarbital/chemistry , Phenobarbital/isolation & purification , Trypsin/chemistryABSTRACT
A rapid and accurate method for quantification of amobarbital and phenobarbital was developed using gas chromatography-mass spectrometry (GC-MS) without derivatization. Though the compounds measured without derivatization showed low sensitivity because of adsorption, addition of 3% formic acid to the solvent improved the sensitivity for the analytes. Taking account of matrix effect, solid-phase and liquid-liquid extraction from serum were examined. The correlation coefficients of the calibration curves were 0.9995 or better, and the accuracy and precision of intraday and interday assays were in line with Food and Drug Administration (FDA) criteria.
Subject(s)
Amobarbital/blood , Formates/chemistry , Gas Chromatography-Mass Spectrometry/methods , Hypnotics and Sedatives/blood , Phenobarbital/blood , Solvents/chemistry , Amobarbital/isolation & purification , Calibration , Carboxylic Acids/chemistry , Gas Chromatography-Mass Spectrometry/standards , Hypnotics and Sedatives/isolation & purification , Phenobarbital/isolation & purification , Reproducibility of Results , TemperatureABSTRACT
Amobarbital [5-ethyl-5-(3-methylbutyl)barbituric acid], USP, was found to contain an impurity that was not associated with hydrolysis and decomposition of the barbiturate ring. The impurity was isolated by semipreparative HPLC and was identified as 5-ethyl-5-(2-methylbutyl)barbituric acid (1) by MS (electron impact and chemical ionization) and 1H NMR. The substitution pattern on the alkyl side chain was verified by using the achiral NMR shift reagent tris(6,6,7,7,8,8,8-heptafluoro-2,2- dimethyl-3,5-octanedionato)europium(III). Older samples of amobarbital, USP, contained greater than 6% of 1, whereas recent samples of amobarbital, USP, contained less than 1% of 1. Because the pharmacological profiles of 1 and amobarbital in rodents are comparable, the impurity probably does not constitute a clinically significant problem for humans.