DETERMINATION OF SEVERAL N-METHYL-D-ASPARTATE RECEPTOR BLOCKERS IN PLASMA AND BRAIN BY A SELECTIVE HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHIC METHOD WITH COLUMN-SWITCHING

A general procedure is presented for the determination of several N-me thyl-D-aspartate (NMDA) receptor open-channel and subtype-selective bl ockers, which have been evaluated and developed as neuroprotective dru gs for the treatment of brain stroke and trauma. The method involves d eproteination of plasma with ethanol, or homogenization of brain sampl es in ethanol, dilution of the supernatant with ammonium acetate and d irect injection into an HPLC column-switching system. Although the inv estigated NMDA receptor blockers are all tertiary amines, they have qu ite different structures. However, they are all concentrated on the fi rst column (Purospher RP-18, 125x4 mm), whereas polar interfering comp ounds are washed out with 1% ammonium acetate-acetic acid-acetonitrile (100:1:5, v/v/v). Due to the special selectivity of the Purospher RP- 18 material, the analytes and the internal standard are then selective ly eluted with 25% acetonitrile (without any buffer in the mobile phas e) and transferred to the analytical column (Superspher 60 RP-select B , 250x4 mm), where they are separated by gradient elution and detected by UV or fluorescence detection. The low degree of interference allow ed the development of sensitive methods with quantification limits of 5 ng/ml for animal plasma (0.4 mi used), 0.5 ng/ml for human plasma (1 mi used) and 50 ng/g for brain tissue (200 mg used). (C) 1998 Elsevie r Science B.V.