Authentic Reference Standards Research Articles

Uniformly labeled carbon-13 polychlorobiphenyl congeners as standards for isotope dilution analysis

A mixture of uniformly labeled /sup 13/C polychlorobiphenyl (PCB) congeners was prepared from benzene-U-/sup 13/C in three steps, iodination to form iodobenzene which was coupled using a copper catalyst producing biphenyl followed by ferric chloride catalyzed chlorination. The PCB-U-/sup 13/C mixture thus obtained contained at least 39 tetra-, penta-, hexa-, and heptachlorobiphenyls and was similar in congener composition to commercial PCB preparations. Semipreparative high-performance liquid chromatography was used to separate (on the milligram scale) ten fractions, eight of which contain essentially a single PCB congener. Relative retention time measurements on two capillary gas chromatographic columns using 18 authentic pentachlorobiphenyl reference standards permitted structures to be assigned to four pentachlorobiphenyl-U-/sup 13/C congeners. These isotopically enriched compounds 2,2',3,5,6-,2,2',3,4,5'-, 2,2',4,5,5'-, and 2,3',4,4',5-pentachlorobiphenyl-U-/sup 13/C congeners, are isotopic analogues of major constituents of widely used commercial PCB preparations and are useful standards for isotope dilution analysis of environmental samples.

Identification of phenolic acids in human urine by ion monitoring.

1. Two-different double derivatization techniques and two different g.l.c. systems were used to separate isomeric phenolic carboxylic acids in normal human urine. 2. Carboxylic acids were converted into n-butyl esters and phenolic functions into trifluoroacetic acid esters. 3. n-Butyl trifluoroacetoxybenzoates were separated by g.l.c. and detected by mass spectrometric single-ion monitoring. 4. Trifluoroacetates were hydrolysed under mild conditions, and liberated phenolic groups were subjected to flash methylation. 5. n-Butyl methoxybenzoates were separated by g.l.c. and detected by flame ionization. 6. All derivatives were identified by comparison of retention times and mass spectra with those of authentic reference standards. 7. The urine of a normal vegetarian volunteer was examined. 8. The presence of meta- and para-hydroxybenzoic acids, vanillic acid and isovanillic acid was confirmed by unambiguous techniques.

Separation of epimeric monohydroxylated metabolites of testosterone, androstenedione and thep-nitrobenzyl esters of hydroxylated 5β-cholanic acid using the same HPLC conditions

The separation of authentic reference standards of epimeric monohydroxylated derivatives of both testosterone and lithocholic acidp-nitrobenzyl esters on a 10 μm Partisil-10 column with a single mobile phase of hexane-tetrahydrofuran-isopropanol (80∶15∶5) is reported. Baseline separation of all epimeric derivatives of the bile acid PNB-esters was achieved, and the principal testosterone metabolites (6β-, 7α-, 16β-, and 15β- and 16α-hydroxy-testosterones) were separated from one another and the parent compound. This system was used to separate metabolites of both androstene-dione and [14C]-lithocholic acid.

Determination of 7-iodo-1,3-dihydro-1-methyl-5-(2′-fluorophenyl)-2H-1,4-benzodiazepin-2-one (ro 7-9957) and its major biotransformation products in blood and urine by electron capture-gas-liquid chromatography

A sensitive and specific electron capture-gas chromatographic assay was developed for the determination of 7-iodo-1,3-dihydro-1-methyl-5-(2′-fluorophenyl)-2H-1,4-benzodiazepin-2-one (I) and its major metabolites in blood and urine. The overall recovery of I and its N-desmethyl metabolite (II) from blood is apparently quantitative. The recovery of the major urinary metabolite, the N-desmethyl-3-hydroxy analog (IV), and the minor metabolites, the N-desmethyl analog (II) and the N-methyl-3-hydroxy analog (III) added to urine as authentic reference standards ranged from 80 to 85%. The sensitivity limits of detection are of the order of 2–3 ng of I and 4–5 ng of II per ml of blood or urine. The method was applied to the determination of blood levels and the urinary excretion pattern in a dog following oral and intravenous administration of a 1-mg/kg dose (total 13 mg), and in man following the intravenous administration of single 5- and 10-mg doses. The N-desmethyl metabolite II was more predominant in dog blood than was the orally or intravenously administered I, but II was barely measurable in human blood.

A method for the determination of urinary 15α-hydroxyestriol and estriol: Preliminary results of 15α-hydroxyestriol determinations in pregnancy urine

A quantitative gas Chromatographic method for the simultaneous determination of 15α-hydroxy-estriol (1,3,5(10)-estratriene-3,15α,16α,17β-tetrol = estetrol) and estriol in pregnancy urine is described. Its specificity was investigated by various Chromatographic methods and by comparing the mass-spectra of the steroids determined with those of authentic reference standards. The accuracy and precision of the method were studied. Preliminary results obtained with 85 normal urine samples at various stages of pregnancy are presented.

Isolation of estrone and 11β-hydroxy estrone from a feminizing adrenal carcinoma

This paper reports the isolation and characterization of estrone and 11β-hydroxy estrone from an adrenal carcinoma in a 25 year old male with gynecomastia. The tumor was homogenized and the extract divided into unconjugated neutral, phenolic, and conjugated fractions. The phenolic fraction was further divided into individual estrogens using paper chromatography. Characterization of estrone and 11β-hydroxy estrone was done by comparing the chromatographic behavior and functional groups before and after sodium borohydride reduction as well as acetylation with authentic reference standards. Final confirmation of the identity was obtained by infra-red analysis.