Assay of midazolam and brotizolam in plasma by a gas chromatographic and a radioreceptor technique
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A gas Chromatographic method (GLC) using a capillary column, electron capture (EC) detection and a solid injection system for the determination of midazolam and brotizolam in plasma is described. Furthermore, the application of a radioreceptor technique (RRA), using a dry and stable benzodiazepine receptor preparation and3H-flunitrazepam as a labelled ligand for the assay of both drugs is presented.
A comparison was made between the RRA and the GLC method. For the gas Chromatographic method, linear calibration graphs (r>0.999) were obtained in the concentration range of 2.0–100 ng/ml. Extraction yield was 81±6% in the same concentration range. For the RRA technique good linearity (r>0.995) was obtained in the range of 0.1–0.5 ng of brotizolam, and 1.0–5.0 ng of midazolam. Differences between duplicates were less than 8%. The relative affinity of the 1-hydroxymethyl and 4-hydroxy metabolites of brotizolam and midazolam were 0.33 and 0.14 respectively for brotizolam and 0.64 and 0.23 for midazolam. Extraction yields of parent drugs and metabolites were similar.
Application of the methods to pharmacokinetic studies of the drugs indicated that they were sufficiently sensitive to measure plasma concentrations for at least three times the elimination half-lives. There was a good correlation between results obtained with the gas Chromatographic method and those obtained with the receptor technique. Correlation coefficients were 0.94 (based on 37 samples) for brotizolam and 0.99 (based on 33 samples) for midazolam, and there were no significant differences between pharmacokinetic parameters obtained with the two methods. It was concluded that after a single dose of brotizolam only low concentrations of 1-methylbrotizolam were present in plasma of young healthy subjects, whereas after midazolam administration 1-methylmidazolam was present in measurable amounts during the first few hours.
KeywordsMidazolam Extraction Yield Calibration Graph Linear Calibration Relative Affinity
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