Toyohiro Taguchi

Quantitation of urinary metabolites of toluene, xylene, styrene, ethylbenzene, benzene and phenol by automated high performance liquid chromatography.

SummaryAn automated high performance liquid chromatographic method (HPLC) for the direct determination of urinary concentrations of hippuric acid (HA), and o-, m- and p-methyl hippuric acids (MHAs), metabolites of toluene and o-, m-, and p-xylenes, and of urinary phenyl glyoxylic acid (PGA) and mandelic acid (MA), metabolites of styrene or ethylbenzene, is described. Methanol was added to urine, the mixture was centrifuged and the supernatant was injected into HPLC. A stainless-steel column packed with octadecyl silanized silicate was used and the mobile phase was a mixed solution of 5 mM potassium phosphate monobasic/acetonitrile (90/10). The method is simple and specific. Urine can be analyzed without solvent extraction. Analysis can be performed satisfactorily within 45 min for samples containing HA, MHAs, PGA and MA, and within 15 min for those containing HA, PGA and MA. Another automated HPLC method for the determination of urinary concentrations of phenylsulfate (PhS) and phenylglucuronide (PhG), metabolites of benzene and phenol, is also described. Urine was centrifuged and the supernatant was injected into HPLC. A column packed with octadecyl silicate and a mobile phase of 50 mM of potassium phosphate monobasic/acetonitrile (85/15) were used. The whole analyses and quantitative determination can be performed within 15 min for samples containing PhS and PhG in the workers urine with a simple mobile phase. The accuracy and precision in the present methods by the use of automated HPLC were satisfactory.

Quantitative analysis of urinary glycine conjugates by high performance liquid chromatography: excretion of hippuric acid and methylhippuric acids in the urine of subjects exposed to vapours of toluene and xylenes

SummaryA new method for the direct determination of hippuric acid (HA) and o-, m- and p-methylhippuric acids (MHAs) in the urine, metabolites of toluene and o-, m- and p-xylenes by high performance liquid chromatography (HPLC) is described. A stainless-steel column packed with silica gel having dinitrophenyl residue and a mixed solution of methanol/water/acetic acid (80/20/0.2) containing tetra-n-butylammonium bromide (0.2% w/v) as mobile phase was used. Concentrations of HA and MHAs were estimated from their peak height at a wave length of 225 nm. Urine can be analyzed directly without solvent extraction or pretreatment to obtain complete separation of HA and o-, m- and p-MHAs. Urine samples from male workers exposed to toluene or xylenes were analyzed for HA or MHAs. The urinary levels of HA and MHAs increased by exposure to toluene and xylenes in proportion to the environmental concentrations of the solvents, although there is a considerable variation in metabolite concentrations. The slope of regression line between toluene and HA and that between m-xylene and m-MHA were similar. The urinary concentrations of HA and MHAs corresponding to 100 ppm (TLV) of toluene was 2.35 g/g creatinine and that of m-MHA corresponding to 100 ppm (TLV) of m-xylene was 2.05 g/g creatinine. The warning levels of the urinary metabolite concentrations of a group of workers and that of an individual worker corresponding to TLV of organic solvent concentration is discussed.

Quantitation of urinary chlorobenzene metabolites by HPLC: concentrations of 4-chlorocatechol and chlorophenols in urine and of chlorobenzene in biological specimens of subjects exposed to chlorobenzene

SummaryA simple method for the determination of 4-chlorocatechol (ClCh, 4-chloro-1,2-benzenediol) and chlorophenols (ClPh), metabolites of monochlorobenzene (ClBz), in urine by high performance liquid chromatography (HPLC) is described. Enzymatic hydrolysates of urine were applied to a stainless-steel column packed with octadecyl-silanized silica gel, and a mixed solution of 20 mM potassium phosphate monobasic : acetonitryl (75:25, v/v) was used as a mobile phase. The procedures for ether extraction and evaporation of extract could be omitted. The accuracy and precision of the present HPLC method were satisfactory. The excretion kinetics of ClCh and p-ClPh were investigated over 35 h after cessation of ClBz inhalation. Proportional relationships between concentrations of ClBz in air and of its metabolites in urine were observed. The slopes of regression lines predicting the levels of ClCh, p-ClPh and total ClPh in urine taken during the last 2 h of exposure to ClBz in air were 6.56, 1.13 and 2.83 mg/g creatinine for 1 ppm ClBz respectively. ClBz in the blood and the end exhaled air of subjects at the end of exposure were identified by gas chromatography (GC) and mass spectrometry. A proportional relationship was observed between the concentration of ClBz in air and that in blood. The validity of the threshold limit value (TLV) for ClBz as evaluated from the subjective and objective symptoms is discussed.

Urinary 2,5-hexanedione assay involving its conversion to 2,5-dimethylpyrrole.

SummaryHigh-performance liquid chromatography (HPLC), gas chromatography (GC) and spectrophotometry were used to examine 2,5-dimethylpyrrole, derived from 2,5-hexanedione in the acid-hydrolyzed urine of subjects exposed to n-hexane. The urine of a subject exposed to n-hexane was hydrolyzed with hydrochloric acid and then neutralized. Thereafter, 2,5-hexanedione in the hydrolysate was heated with ammonium carbonate to yield 2,5-dimethylpyrrole. A methanol extract of the 2,5-dimethylpyrrole formed was subjected to HPLC and an ethylacetate extract of it was subjected to GC, with a flame thermoionic detector (FTD). For spectrophotometry, the ethylacetate extract was allowed to react with Ehrlichs reagent and then the absorbance of the colored compound formed was read at 525 nm. Pyrrole condensation with ammonia is specific to 1,4-diketones and these three assay methods can be used for determination of 2,5-hexanedione in the urine of workers exposed to n-hexane. The hydrolytic conditions for urine of subjects exposed to n-hexane for analysis of 2,5-hexanedione were discussed.

High performance liquid chromatographic procedure for quantitative determination of urinary delta-aminolevulinic acid as indices of lead exposure

SummaryA high performance liquid chromatographic method for the determination of delta-aminolevulinic acids (ALA) in urine, is described. 2-Methyl-3 carbmethoxy-4-(3-propionic acid) pyrrole was produced by the condensation of ALA with methylacetoacetate (MAA) while heating. The methylacetoacetate could be replaced by ethylacetoacetate. The ALA pyrrole thus obtained was injected into high performance liquid chromatography (HPLC). A stainless-steel column packed with octadecyl silanized silica gel was used. The mixed solution of acetonitrile/50mM KH2PO4 (adjusted to pH 2.5 with 0.001 volumes of 85 g/dl H3PO4)(20/80) was a favorable mobile phase for the separation of ALA. Amino acetone pyrrole was produced by the condensation of amino acetone with methyl acetoacetate. The amino acetone pyrrole appeared later than ALA pyrrole on HPLC. The method is simple and specific. It has a lower detection limit of 0.2 ng on column. The analysis and quantitative determination of one specimen can be performed within 20 min. Mean ALA concentration of normal urine by HPLC was 1.18mg/g creatinine.