Simultaneous determination of p-aminobenzoic acid, acetyl-p-aminobenzoic acid and p-aminohippuric acid in serum and urine by capillary gas chromatography with use of a nitrogen-phosphorus detector.

In various studies during recent years, the use of p-aminobenzoic acid has been described in screening tests for exocrine pancreatic function. A synthetic three-unit compound N-benzoyl-L-tyrosyl-p-aminobenzoic acid has been administered orally and hydrolysed in the small intestine in the presence of chymotrypsin to N-benzoyl-L-tyrosine and p-aminobenzoic acid. This study describes a convenient procedure in which, after a selective extraction and derivatization with diazomethane, capillary gas chromatography is used combined with nitrogen-sensitive detection. With the proposed procedure, p-aminobenzoic acid and its major metabolites, acetyl-p-aminobenzoic acid and p-aminohippuric acid, can be monitored in serum and in urine samples.

Urinary excretion of purine and pyrimidine metabolites in the neonate.

Random urine samples from 614 neonates were screened for metabolites of purine and pyrimidine metabolism using an adapted column chromatographic method. A restricted number of metabolites and a number of unidentified peaks appeared on the chromatograms. No inborn errors of this metabolism were found. The chromatograms were identical in term and in premature or dysmature neonates, except for the presence of more unidentified peaks in the latter group. The pattern was not influenced by the type of feeding or i.v. nutrition. Metabolites of different medications were identified. One female neonate with an increased excretion of uracil was shown to be heterozygous for ornithine carbamyl transferase deficiency.

Improved micromethod for assay of serum angiotensin converting enzyme.

We describe conditions for determining angiotensin converting enzyme (EC 3.4.15.1) in serum, by liquid chromatography. Serum (10 microL) is assayed with the artificial substrate hippuryl-glycyl-glycine (30 mmol/L) in a 50 mmol/L "HEPES" buffer solution with a high salt content (300 mmol of NaCl and 400 mmol of Na2SO4 per liter), at pH 8.0. The resulting enzymic activity is ninefold that of the currently popular spectrophotometric assay of Cushman and Cheung as modified by Lieberman (Am. J. Med. 59: 365-372, 1975). The hippuric acid end product is separated from the substrate by reversed-phase liquid chromatography and measured spectrophotometrically at 228 nm. o-Methyl hippuric acid is used as internal standard. The mean value for 100 normal control subjects was 317 (SD 96) nmol of hippuric acid released per milliliter of serum per minute. The enzyme activity is greater in newborns (p less than 0.05) and has a tendency to decrease with age. This partly automated method, which is optimized with regard to activity and detection, can be used in clinical routine.

Simultaneous high performance liquid-chromatographic determination of carbamazepine, carbamazepine-10,11-epoxide, ethosuximide, phenobarbital, phenytoin, primidone and phenylethylmalonamide in plasma.

A common methodology is reported for the determination of five major anticonvulsants (carbamazepine, ethosuximide, phenobarbital, phenytoin, primidone) and their active metabolites (carbamazepine-10,11-epoxide, phenylethylmalonamide) in 30 microliters of plasma. After a single step of deproteinisation and extraction with acetonitrile, leading to an almost complete recovery of all the analytes, 5 microliters is injected on a reversed-phase column (Lichrosorb RP-18, 5 microns). The anticonvulsants are eluted isocratically at a column temperature of 50 degrees C with a mobile phase consisting of acetonitrile/phosphate buffer p' 6.9 (40/60 by vol), and monitored at 208 nm. Quantitation, using peak height or peak area, is based on the ratio of analyte to internal standard (allylisobutylbarbital) referenced to a serum-based multiple drug standard. The composition and pH of the mobile phase, temperature of the column, choice of wavelength of detection and size of the column material are crucial for the optimal separation of these five drugs and their two active metabolites in a chromatographic time of only 12 min, without sacrificing high sensitivity and column life.