Unexpected adduct ion formation under chemical ionization conditions.

As part of our efforts to characterize the in vivo metabolic fate of the antihypertensive agent alpha-methyldopa, we have examined the urine of alpha-methyldopa-treated rats with the aid of a direct insertion probe chemical ionization mass spectral assay. The mass spectrum of the sample obtained by chromatographic purification followed by treatment with ethanolic hydrochloric acid and pentafluoropropionic anhydride displayed an intense ion at m/z 812, consistent with the beta-ethoxy-N,O,O,O-tetrakispentafluoropropionyl derivative of 6-hydroxy-alpha-methyl-norepinephrine, a potential aromatic hydroxylation product of the known alpha-methyldopa metabolite alpha-methyl-norepinephrine. Comparison of this spectrum with the spectrum obtained with the corresponding synthetic 6-hydroxy-alpha-methylnorepinephrine, however, ruled out this possibility. A more thorough examination of the mass spectral data established that the ion at m/z 812 observed with the metabolic species was due to the formation of an unexpected adduct ion between a known metabolite of alpha-methyldopa and an impurity ion formed from a common constituent of urine. This paper summarizes the characterization of this adduct ion.

Synthetic and preliminary hemodynamic and whole animal toxicity studies on (R,S)-, (R)-, and (S)-2-methyl-3-(2,4,5-trihydroxyphenyl)alanine.

The synthesis, resolution, and absolute configuration assignment of 2-methyl-3-(2,4,5-trihydroxphenyl)alanine (6-OH-alpha-Me-Dopa) are reported. Hemodynamic studies in the rat have shown that this structural analogue and potential metabolite of the clinically useful drug (S)-alpha-Me-Dopa possesses weak hypotensive activity which resides in the R enantiomer. LD50 studies in mice have established that 6-OH-alpha-Me-Dopa is over four times more toxic than alpha-Me-Dopa. Chronic exposure to 6-OH-alpha-Me-Dopa leads to renal and hepatic lesions. The case of oxidation of this hydroquinone to the electrophilic quinone species may contribute to its enhanced toxicity compared to alpha-Me-Dopa.

Oxidative and cardiovascular studies on natural and synthetic catecholamines.

The cyclic voltammometric behavior of epinephrine, norepinephrine, dopamine, epinine, alpha-methyldopamine, beta-methyldopamine, beta-methylepinine, and beta-methoxyepinine has been examined in order to evaluate substituent effects on cyclization rates of the electrochemically generated quinones. We observed that alpha and beta substituents caused a modest enhancement of cyclization rates while an N-methyl group dramatically increased cyclization rates. No correlation was observed between calculated amine pKa values, suggesting that differences in cyclization rates between the primary and secondary amine series were due to inherent nucleophilicity, a measure of which would be gas-phase proton affinities. The acute pressor effects of the newly synthesized catecholamines were compared with the native amines.

The in vitro metabolism of halothane (2-bromo-2-chloro-1,1,1-trifluoroethane) by hepatic microsomal cytochrome P-450.

Incubation of halothane with rabbit liver microsomes in the presence of NADPH and oxygen resulted in the formation of trifluoroacetic acid (TFAA). No TFAA was detected in the absence of either NADPH or oxygen. TFAA also was found when a 9000 x g supernatant fraction (with NADPH and oxygen) was used instead of the microsomal fraction. The 105,000 x g supernatant fraction (with NADPH and oxygen), however, did not produce detectable levels of TFAA. Phenobarbital pretreatment of the animals resulted ina significant increase in both the formation of TFAA and the cytochrome P-450 content of microsomes. A good correlation was found between formation of TFAA and microsomal cytochrome P-450 levels. The amount of TFAA formed in hepatic microsomes was significantly inhibited by carbon monoxide (CO), SKF 525-A (2-diethylaminoethyl-2,2,-diphenylvalerate HCl) and p-chloromercuribenzoic acid. On the other hand, TFAA was formed when NADH was used as an electron donor instead of NADPH. In conclusion, these results clearly demonstrate the metabolism of halothane by the hepatic microsomal mixed-function oxidase system.

Formaldehyde formation as a metabolite of methoxyflurane.

The production of formaldehyde as a metabolite of enzymatic biotransformation of methoxyflurane was observed in vitro in hepatic microsomal preparations derived from Japanese monkeys, rabbits and Wistar strain rats. Production rates of formaldehyde were 0.80-2.50 n moles/mg protein/min in the induced group which was pretreated with phenobarbitone, and 0.15-0.68 n moles/mg protein/min in the non-induced group. This reaction needed the presence of NADPH and oxygen. Formaldehyde production was almost completely inhibited by addition of 25% CO. Further degradation of formaldehyde did not occur in microsomal suspension. No detectable species difference was observed among the animals used in this study. These results indicate that the biotransformation of methoxyflurane is preceded by interaction with cytochrome p-450 in hepatic microsomes in the presence of NADPH and oxygen.

Esterification of trifluoroacetic acid with phenyldiazomethane for quantitative gas chromatographic analysis. Methods involving separation from biological materials.

Trifluoroacetic acid in biological materials has been quantitatively determined by gas chromatography. Benzyl trifluoroacetate has been prepared by the reaction of the acid with phenyldiazomethane, and has been successfully analyzed by gas chromatography without any interference from other peaks. The procedure has been used to determine trifluoroacetic acid in microsomal suspension incubated with halothane, a gaseous anaesthetic.

The gas chromatographic mass spectrometric determination of trifluoroacetic acid in biological fluid. Application to halothane metabolism.

The methyl ester of trifluoroacetic acid was prepared by reaction with N,N'-dimethylformamide dimethylacetal and was successfully passed through a gas chromatograph. Trifluoroacetic acid was detected by the use of gas chromatography and low and high resolution gas chromatography mass spectrometry in an acidic extract of an incubation medium containing microsomes, reduced nicotinamide adenine dinucleotide phosphate, oxygen and halothane. However, trifluoroacetic acid could not be detected when nicotinamide adenine dinucleotide or oxygen was omitted from the incubation system. From these results, it was proved that halothane is oxidatively metabolized to trifluoroacetic acid by hepatic microsomes.