Roland Burton

Metabolic profiling of valproic acid by cDNA-expressed human cytochrome P450 enzymes using negative-ion chemical ionization gas chromatography-mass spectrometry.

A sensitive negative ion chemical ionization (NCI) gas chromatographic-mass spectrometric (GC-MS) method was modified for the quantitation of valproic acid (VPA) metabolites generated from in vitro cDNA-expressed human microsomal cytochrome P450 incubations. The use of the inherent soft ionization nature of electron-capture NCI to achieve high sensitivity enabled us to conduct kinetic studies using small amounts of recombinant human P450 enzymes. The assay is based on the selective ion monitoring of the intense [M-181] fragments of pentafluorobenzyl (PFB) esters in the NCI mode, and has the following features: (1) a micro-extraction procedure to isolate VPA metabolites from small incubation volumes (100 microl); (2) a second step derivatization with tert.-butyldimethylsilylating reagents to enhance sensitivity for hydroxylated metabolites; (3) a short run-time (<30 min) while maintaining full separation of 15 VPA metabolites by using a narrow-bore non-polar DB-1 column plus a new temperature gradient; and (4) good reproducibility and accuracy (intra- and inter-assay RSDs <15%, bias <15%) by using seven deuterated derivatives of analytes as internal standards. The derivatives of mono-and diunsaturated metabolites, like the parent drug, produced abundant [M-181](-) ions while the hydroxylated metabolites gave an ion at m/z of 273, corresponding to the [M-181](-) ion of the tert.-butyldimethylsilyl ethers. In conclusion, the GC-NCI-MS analysis of valproate metabolites provided us with a high resolution and sensitivity necessary to conduct metabolic and kinetic studies of valproic acid in small volume samples typical of the in vitro cDNA-expressed micro-incubation enzymatic systems.

Capillary gas chromatography-mass spectrometry of valproic acid metabolites in serum and urine using tert.-butyldimethylsilyl derivatives

A quantitative method has been developed for valproic acid and twelve of its metabolites using capillary gas chromatography--mass spectrometry with selected-ion monitoring. The method is applicable to serum or urine and all metabolites are measured in a single run. Ions selected for quantitative purposes were the characteristic (M-57)+ ions of the tert.-butyldimethylsilyl (tBDMS) derivatives. The 4-hydroxyvalproic acid was measured as the gamma-lactone. Calibration curves were found to be linear and the sensitivities in the order of 0.1 microgram/ml. Patient data are presented. A comparison of tBDMS and trimethylsilyl (TMS) derivatives showed that tBDMS gave superior sensitivity for the unsaturated metabolites and a shorter analysis time. Mixed tBDMS-TMS derivatives were also investigated.

Valproic acid analysis in saliva and serum using selected ion monitoring (electron ionization) of the tert.-butyldimethylsilyl derivatives

A highly sensitive ion monitoring method for the determination of valproic acid in saliva and in serum has been developed based on the gas chromatographic--mass spectrometric analysis of the tert.-butyldimethylsilyl derivatives. Extraction methods are simple and the techniques for derivatization are rapid and convenient. Selected ion monitoring was carried out using electron ionization conditions and a common ion m/z 201 (M+--57) present in valproic acid and the internal standard octanoic acid. The lower limit of sensitivity that has acceptable precision for assay purposes is 0.1 mg/l based on a 200-microliter sample size. The ion monitoring method (derivatized) was compared to a gas chromatographic method (underivatized) for serum valproate assays and found to be essentially identical. The assay methodology was used in a kinetic study of valproic acid in two normal subjects. Saliva levels of drug were found to give reasonably good correlations with serum total and with serum free concentrations of drug in both individuals.U

A simple microcomputer interface for tail-flick determination

An inexpensive means of tail-flick latency data acquisition and analysis using an Apple IIe microcomputer and three simple interface circuits is described. Descriptions of the software and hardware are provided with schematics and program listings. The measurement, storage, and analysis of the data are integrated in one system.

Rapid assay for γ-aminobutyric acid in mouse brain synaptosomes using gas chromatography—mass spectrometry

A sensitive and efficient assay for γ-aminobutyric acid (GABA) was applied to fresh mouse whole brain synaptosomes where the extracted GABA was analyzed as its di(tert.-butyl(dimethylsilyl)) derivative by gas chromatography—mass spectrometry (GC—MS) using GABA-d6 as an internal standard. Endogenous levels of 20.01 ± 0.75 nmol GABA/mg protein were found. The method is characterized by a detection limit of about 10 fmol injected GABA derivative and coefficients of intra-day and inter-day variation of 0.95% and 7.7%, respectively. The rate of synaptosomal GABA synthesis was used to determine the activity of glutamate decarboxylase (GAD) as 314.9 ± 9.0 nmol GABA/mg protein/h. Both GABA levels and GAD activity were significantly elevated by therapeutic doses of the antiepileptic drug valproic acid.