Rapid, sensitive determination of unchanged promethazine in biological material using a nitrogen-selective flame ionization detector. Identification of metabolites by gas chromatography-mass spectrometry.

A rapid, sensitive method has been developed to study the kinetics of unchanged promethazine (PM) in biological material using a nitrogen-selective flame ionization detector (N-FID). Unchanged PM is distinguished from its desmethyl metabolite. Sample clean-up of several biological fluids (rat plasma, blood, urine, liver and kidney homogenates) was studied and gas chromatographic (GC) conditions optimized. Usually 50 microliters-1.0 ml samples are extracted into n-heptane by shaking with NaOH, re-extracted into H2SO4 and again extracted into n-heptane by addition of NaOH. Finally, the organic phase is separated, concentrated under N2 and PM determined by N-FID. However, a rapid, single-step method requiring only NaOH extraction into n-heptane may be used whenever GC background permits. Imipramine is used as an internal standard for calibration by peak height ratios in the overall range 5--1500 ng PM per sample. Recovery of both methods is high (97--99%) but precision of the single-step method is lower (relative S.D. 10% versus 3--4%). Use of sample volumes up to 1 ml allows accurate determination of concentrations as low as 10 ng/g. Examples of applications to commonly used animal models employing PM are given and simple adaptation for clinical samples suggested.