Jean Nowlin

Analytical methods for the study of urinary thioether metabolites in the rat and guinea pig.

Methods are described for the isolation and identification of three classes of bivalent sulfur metabolites characterized as neutral methylthio ethers, ethyl acetate-soluble acidic thioethers and ethyl acetate-insoluble acidic thioethers from rat and guinea pig urine. After extraction of the metabolites by the ammonium carbonate-ethyl acetate procedure, the individual metabolites are separated by capillary gas chromatography and/or by high-performance liquid chromatography with both mu Bondapak C18 and Porasil columns. Identification of the metabolites is based on gas chromatography-mass spectrometry (electron impact) and on fast atom bombardment mass spectrometry. Interesting species differences in metabolism were observed. The major ethyl acetate-soluble acidic thioethers in rat urine are mercapturic acids. In contrast, in the guinea pig a new pathway involving mercaptopyruvic, mercaptolactic and mercaptoacetic acids is operative. The thioether metabolites of styrene oxide and phenanthrene are described, but the procedures have been applied in studies of several drugs and environmental chemicals in our laboratory.

The use of gas chromatographic-mass spectrometric-computer systems in pharmacokinetic studies

Pharmacokinetic studies involving plasma, urine, breast milk, saliva and liver homogenates have been carried out by selective ion detection with a gas chromatographic-mass spectrometric-computer system operated in the chemical ionization mode. Stable isotope labeled drugs were used as internal standards for quantification. The half-lives, the concentration at zero time, the slope (regression coefficient), the maximum velocity of the reaction and the apparent Michaelis constant of the reaction were determined by regression analysis, and also by graphic means.

Identification of metabolites of phenylacetic acid in rat brain by plasma desorption mass spectrometry

Abstract Mass spectra of acyl hydroxamates may be obtained directly, without prior derivatization or gas chromatography, by the new technique of plasma desorption mass spectrometry. Authentic alkyl and aryl hydroxamates showed the expected protonated molecular ions when isobutane was used as the carrier gas. Advantage was taken of this novel technique to identify phenylacetyl-CoA and phenylbutyric acid in brain extracts obtained from young rats after a loading dose of phenylacetic acid. The formation of these metabolic products lends strong support to our suggestion that brain dysfunction induced by phenylacetic acid in experimental phenylketonuria may be due to decreased availability of CoA and acetyl-CoA in the rapidly developing brain.

NEONATAL WITHDRAWAL FROM TRANSPLACENTALLY ACQUIRED MEPHOBARBITAL

Withdrawal symptoms consisting of severe tremors, irritability, opisthotonos and increased muscle tonus were observed in two siblings born to a mother requiring mephobarbital for control of seizures. Both infants manifested symptoms within the first minutes of life and continued to be symptomatic for 2-6 months after delivery. At birth the infants were small for gestation and had some of the physical findings (Hill, et al) observed in infants exposed to intrauterine anticonvulsant drugs. Somatic growth during the first 4 years and 18 months of life, respectively, was normal but mental development in the second infant at 18 months of age was slow (D.Q. 83).Placental transfer of mephobarbital was documented in both infants by selective ion detection using a gas chromatograph-mass spectrometer-computer system. Prolonged excretion of mephobarbital and phenobarbital was demonstrated in the second infant. At the time of delivery the maternal urine contained 17.22 μg/ml of phenobarbital and 1.15 μg/ml of mephobarbital. Urine collected from the infant between 10-27 hours of age contained 13.15 μg/ml of phenobarbital and .88 μg/ml of mephobarbital. Subsequent samples examined on 5, 8, 10, 15 and 22 days of age revealed continued excretion of phenobarbital (.13 μg/ml) through the last sampling period.

MATERNAL DRUG INGESTION AND LACTATION

Quantification and identification of drugs in human colostrum and breast milk have been accomplished by selective ion detection using a gas chromatograph-mass spectrometer-computer system. Drugs routinely administered to gravid females for acute as well as chronic indication in the pre and postpartum period were studied.Sixteen hours after the last maternal dose (30 mg q.i.d.) of phenobarbital the breast milk level was found to be 2.74 μg/ml. Oral ingestion of a short or intermediate acting barbiturate such as pentobarbital or butabarbital resulted in lower concentrations, i.e., .17 μg/ml 19 hours after ingestion and .37 μg/ml 1.5 hours after ingestion respectively. Intravenous administration of 15 mg of diazepam during labor resulted in a colostrum level of .10 μg/ml 25½ hours later. The level of diphenylhydantoin in breast milk 3 hours after a single 100 mg oral dose was 4.2 μg/ml with a decline to 1 μg/ml at 6 hours. Levels of .5 - 1.5 μg/ml were then obtained for the ensuing 18 hours. Additional drugs identified in breast milk but not quantified were secobarbital, ethosuximide, codeine, methadone, tolbutamide and caffeine.No obvious effects of the drugs were observed in these neonates, but breast milk may serve as a significant route for neonatal drug ingestion particularly when drugs are consumed daily or the drug consumed has prolonged activity.