TLC separation of hippuric, mandelic, and phenylglyoxylic acids from urine after mixed exposure to toluene and styrene.

A method using thin-layer chromatography is described to determine the concentration of hippuric acid, mandelic acid, and phenylglyoxylic acid present in the urine after occupational mixed exposure to toluene and styrene. These substances are known metabolites of toluene and styrene, and therefore the evaluation to mixed exposure to toluene and styrene may be carried out separating these metabolites beforehand. Procedures are proposed to separate the metabolites as follows: (1) separation of hippuric acid from mandelic acid, (2) separation of mandelic acid from phenylglyoxylic acid, and (3) separation of hippuric acid and mandelic acid from phenylglyoxylic acid. The developing reagent p-dimethylaminobenzaldehyde in acetic acid anhydride was used after separation on Kieselgel and Silicagel. The sensitivity of the method was 6 microgram of hippuric acid, 10 microgram of mandelic acid, and 7 microgram of phenylglyoxylic acid with an average recovery of 94%.     

Assessment of occupational exposure to organic solvents in paint industry

The paper brings the preliminary results of ambient and biological monitoring of exposure to xylene, toluene, and styrene in paint industry. Air samples were collected at seven stationary locations at work places of 31 male workers. Samples of venous blood were taken from all workers for styrene, toluene, and xylene analysis, and of urine for metabolic derivatives, mandelic, phenylglyoxylic, and o- and m-methylhippuric acids analysis. Blood was collected on Wednesday before and after work, and urine on Monday, Wednesday and Thursday before work and on Wednesday after work. Workers were exposed to moderate concentrations of xylene and low concentrations of toluene and styrene. During periodic medical surveillance Questionnaire 16 was applied as a screening test for early central nervous disturbances. Seven workers had positive findings and required further psychological tests as recommended by WHO. Risk assessment of combined long-term exposure to organic solvents in occupational environment requires a prospective epidemiological study with matched control group.     

Evaluation of hippuric,phenylglyoxylic and mandelic acids in urine as indices of styrene exposure

Summary In addition to those of phenylglyoxylic and mandelic acids, a distinct increase in hippuric acid level was observed in the urine of factory workers who were exposed to styrene at 50 to 200 ppm for 160 min. Comparison of the postexposure levels with respective non-exposure levels and supplements with rat exposure experiments revealed hippuric acid to be a poor indicator of styrene exposure at a moderate degree, while the other two acids are much more adequate indices. At a higher dose of styrene (e.g., 500 mg/kg i.p. or 100 ppm for 8 hrs for rats), however, the urinary levels of phenylglyoxylic and mandelic acids reach plateaus, while hippuric acid level remained proportional to the amount of styrene given.The biological half-life of styrene in human subjects is approximately 8 hrs as measured by the disappearance of phenylglyoxylic and mandelic acids from urine.This work was supported in part by a grant from the Fujiwara Memorial Foundation     

Comparative evaluation of urinalysis and blood analysis as means of detecting exposure to organic solvents at low concentrations

Summary One hundred and forty-three workers exposed to one or more of toluene, xylene, ethylbenzene, styrene, n-hexane, and methanol at sub-occupational exposure limits were examined for the time-weighted average intensity of exposure by diffusive sampling, and for biological exposure indicators by means of analysis of shift-end blood for the solvent and analysis of shift-end urine for the corresponding metabolite(s). Urinalysis was also performed in 20 nonexposed control men to establish the background level. Both solvent concentrations in blood and metabolite concentrations in urine correlated significantly with solvent concentrations in air. Comparison of blood analysis and urinalysis as regards sensitivity in identifying low solvent exposure showed that blood analysis is generally superior to urinalysis. It was also noted that estimation of exposure intensity on an individual basis is scarcely possible even with blood analysis. Solvent concentration in whole blood was the same as that in serum in the case of the aromatics, except for styrene. It was higher in blood than in serum in the case of n-hexane, and lower in the cases of styrene and methanol.     

A rapid colorimetric method for the determination of phenylglyoxylic and mandelic acids: Its application to the urinalysis of workers exposed to styrene vapour

Ohtsuji, H., and Ikeda, M.(1970).Brit. J. industr. Med.,27, 150-154. A rapid colorimetric method for the determination of phenylglyoxylic and mandelic acids. Its application to the urinalysis of workers exposed to styrene vapour. A rapid colorimetric method has been developed for the determination of the styrene metabolites, phenylglyoxylic and mandelic acids, in urine. Ether extracts of acidified urine containing the two acids were evaporated to dryness in a test tube, and a mixture of sulphuric acid and formalin (100:1, v/v) was added for colour development. Factors necessary to convert optical extinction to gravimetric units were determined. When urinesamples from workers exposed to up to 30 ppm of styrene were analysed for phenylglyoxylic and mandelic acids together with hippuric acid, it was found that the phenylglyoxylic acid level provided the most sensitive index of styrene exposure and that the optical extinction at 350 nm was practically proportional to phenylglyoxylic acid concentration. No significant increase in hippuric acid levels was observed.     

The simultaneous determination of hippuric acid, o-, m-, p-methylhippuric acids, mandelic acid and phenylglyoxylic acid in urine by HPLC

A high-performance liquid chromatographic method is described for the simultaneous determination of six metabolites of aromatic hydrocarbons: hippuric acid (HA) from toluene; o-, m-, p-methylhippuric acids (o-, m-, p-MHA) from xylene; mandelic acid (MA) and phenylglyoxylic acid (PGA) from styrene and ethylbenzene. Metabolites were first extracted from urine by solid phase extraction with anion exchange resin, then isocratically separated on a C8 column with 3 microns particle size, 10 cm length and 3 mm internal diameter. Mobile phase was prepared diluting 16 mL of tetrahydrofuran, 14 mL of acetronitrile and 5 mL of methanol to 500 mL with phosphoric acid/potassium dihydrogen phosphate buffer 0.01 M (pH 2.7). The internal standard was 3-hydroxybenzoic acid. Chromatographic runs were completed in about 21 min. The accuracy and reproducibility obtained make this method useful for the biological monitoring of occupational exposure to toluene, xylene, styrene and ethylbenzene.     

Method for simultaneous determination of six metabolites of toluene, xylene and ethylbenzene, and its application to exposure monitoring of workers in a printing factory with gravure machines

For the biological monitoring of exposure to solvent composed of toluene, xylene, and ethylbenzene used in a printing factory with gravure machines, we developed a HPLC method for the simultaneous determination of urinary metabolites of this solvent, i.e. hippuric acid, o-, m-, and p-methylhippuric acid, mandelic acid and phenylglyoxylic acid. Except for phenylglyoxylic acid, urinary concentrations of the metabolites determined by the present method correlated well with the air concentrations of the respective solvent components. Hence the present method is useful in monitoring solvent exposure. In 91 workers of the printing factory and 53 control subjects, we also determined the concentrations of some phenolic metabolites and confirmed that o-cresol is a useful indicator for monitoring toluene exposure.     

Simultaneous determination by gas chromatography of the major metabolites in urine of toluene, xylenes and styrene

A gas chromatographic method has been developed to determine the following metabolites in urine simultaneously if necessary: hippuric acid from toluene or styrene; 3- and 4-methyl hippuric acids from xylenes; phenylglyoxylic acid and mandelic acid from styrene. Heptadecanoic acid is added to the urine as an internal standard and after ethyl acetate extraction from acidic solution, the trimethylsilyl (TMS) derivatives of the metabolites are formed and simultaneously analysed by gas chromatography on 3% OV-1 on 80/100 gas chrom Q (flame ionisation detector).     

Simultaneous determination by gas chromatography of the major metabolites in urine of toluene, xylenes and styrene.

A gas chromatographic method has been developed to determine the following metabolites in urine simultaneously if necessary: hippuric acid from toluene or styrene; 3- and 4-methyl hippuric acids from xylenes; phenylglyoxylic acid and mandelic acid from styrene. Heptadecanoic acid is added to the urine as an internal standard and after ethyl acetate extraction from acidic solution, the trimethylsilyl (TMS) derivatives of the metabolites are formed and simultaneously analysed by gas chromatography on 3% OV-1 on 80/100 gas chrom Q (flame ionisation detector).     

Cytogenetic markers, DNA single-strand breaks, urinary metabolites, and DNA repair rates in styrene-exposed lamination workers

The effect of occupational exposure to styrene on frequencies of chromosomal aberrations and binucleated cells with micronuclei and on single-strand break levels in peripheral blood lymphocytes was studied in 86 reinforced plastic workers and 42 control individuals (including 16 maintenance workers with intermittent, low-dose exposure). In these individuals, the irradiation-specific DNA repair rates and the repair rates of 8-oxoguanines were investigated. We assessed the exposure by measuring the concentrations of styrene in air and in blood and of mandelic acid, phenylglyoxylic acid, 4-vinyl phenol conjugates and regioisomeric phenyl hydroxyethyl mercapturic acids in urine. All these parameters correlated with one another. No clear relationship was found between the styrene exposure and the frequencies of chromosomal aberrations. Binucleated cells with micronuclei were moderately related to the parameters of styrene exposure. We found a negative correlation between all exposure parameters and single-strand breaks. The positive correlation between exposure parameters and DNA repair rates suggests that particular DNA repair pathways may be induced by styrene exposure.     

Mutual metabolic suppression between benzene and toluene in man

Summary The exposure intensity during a shift and the metabolite levels in the shift-end urine were examined in male workers exposed to either benzene (65 subjects; the benzene group), toluene (35 subjects; the toluene group), or a mixture of both (55 subjects; the mixture group). In addition, 35 non-exposed male workers (the control group) were similarly examined for urinary metabolites to define background levels. A linear relationship was established between the intensity of solvent exposure and the corresponding urinary metabolite levels (i.e. phenol, catechol and quinol from benzene, and hippuric acid and o-cresol from toluene) in each case when one of the three exposed groups was combined with the control group for calculation. Comparison of regression lines in combination with regression analysis disclosed that urinary levels of phenol and quinol (but not catechol) were lower in the mixture group than in the benzene group when the intensities of exposure to benzene were comparable, indicating that the biotransformation of benzene to phenolic compounds (excluding catechol) in man is suppressed by co-exposure to toluene. Conversely, metabolism of toluene to hippuric acid was suppressed by benzene co-exposure. Conversion of toluene to o-cresol was also reduced by benzene, but to a lesser extent. The significance of the present findings on the mutual suppression of metabolism between benzene and toluene is discussed in relation to solvent toxicology and biological monitoring of exposure to the solvents.     

Toluene vapor exposure and urinary excretion of hippuric acid among workers in China.

A factory survey was conducted in three provinces in China from 1985 to 1989. The time-weighted average toluene concentrations in breathing zone air were monitored by diffusive sampling, whereas hippuric acid (HA) concentrations in shift-end urine samples were measured by high performance liquid chromatography (HPLC). Exposed workers (456 men and women) were those for whom toluene (up to 548 ppm toluene) accounted for greater than or equal to 90% of total exposure (by vapor concentration in ppm), whereas 517 nonexposed controls were recruited from the same factories or from factories of the same region. There was a linear correlation between the intensity of toluene exposure and HA concentration in the shift-end urine. Comparison of the results with findings in the literature shows that the toluene-induced increase in urinary HA concentration among workers in China is significantly smaller than the published values, whereas HA concentrations in urine samples from nonexposed controls are comparable to the levels previously reported.     

Exposure-excretion relationship of styrene and acetone in factory workers: A comparison of a lipophilic solvent and a hydrophilic solvent

A factory survey was conducted in the second half of a working week on 41 exposed male workers, who were engaged in fiber-reinforced plastics work and exposed to the mixed vapors of styrene and acetone. Nonexposed workers, 20 men, were recruited from the same factory. Styrene and acetone in respiratory zone air were monitored for a 8-h shift with carbon cloth- and water-equipped personal diffusive samplers, respectively. Blood and urine samples were collected at the shift-end. Acetone and styrene concentrations in whole blood, serum and urine were measured by head-space gas chromatography, and phenylglyoxylic acid in urine by high-performance liquid chromatography. All biological exposure indicators analyzed correlated significantly with the intensity of exposure to the corresponding solvent during the shift. The slopes of the regression lines indicate that a very small fraction of styrene absorbed will be excreted into urine as styrene per se, and that styrene is quite effectively excreted into urine after metabolic conversion. In contrast, the slopes of regression lines for acetone suggest that acetone distributes both in the blood and urine quite evenly. When the distribution of the solvent in serum was compared with that in the whole blood, it was found that almost all of styrene in blood is present in the serum, whereas acetone distributed very evenly in the cellular and noncellular fractions of the blood.     

Biological monitoring of styrene: a review

Recent literature about the biological monitoring of styrene-exposed workers is reviewed. Styrene primarily exhibits its toxicity on the central and peripheral nervous systems, although its mutagenicity and chromosome damaging ability also may be relevant. Uptake, transformation and excretion of styrene show that beside the usual biological indicators, such as urinary mandelic and phenylglyoxylic acids (main metabolites), other indicators also may be of interest. These include styrene in expired air, in blood or in urine. Moreover, intermediate or final metabolites such as styrene glycol or mandelic acid in blood also have been proven to be useful in the interpretation of individual values. The most widely used analytical methods for these indicators are gas or high performance liquid chromatography. Correlations between exposure and the different biological indicators mentioned above show that the most reliable indicators are mandelic acid (MA) in urine sampled at the end of the work shift (but not the first day of the week) and the sum of mandelic and phenylglyoxylic acids (MA + PGA) in urine sampled 16 hr after exposure (before the next shift). The biological exposure limit values corresponding to the threshold limit value-time-weighted average (TLV-TWA) of 50 ppm of styrene are 850 mg MA/g creatinine in the end-of-shift sample and 330 mg MA + PGA/g creatinine in the next-morning sample. Other biological indexes, such as styrene glycol (phenyl ethylene glycol) in blood or styrene in urine, look promising but require further research in field situations.     

Toluene in blood as a marker of choice for low-level exposure to toluene

The validity of two new biological exposure markers of toluene in blood (TOL-B) and toluene in urine (TOL-U) was examined in comparison with that of the traditional marker of hippuric acid in urine (HA-U) in 294 male workers exposed to toluene in workroom air (TOL-A), mostly at low levels. The exposure was such that the geometric mean for toluene was 2.3 ppm with a maximum of 132 ppm; the workers were also exposed to other solvents such as hexane, ethyl acetate, styrene, and methanol, but at lower levels. The chance of cutaneous absorption was remote. Higher correlation with TOL-A and better sensitivity in separating the exposed workers from the nonexposed subjects were taken as selection criteria. When workers exposed to TOL-A at lower concentrations (< 50 ppm, < 10 ppm, < 2 ppm, etc.) were selected and correlation with TOL-A was examined, TOL-B showed the largest correlation coefficient which was significant even at TOL-A of < 1 ppm, whereas correlation of HA-U was no longer significant when TOL-A was < 10 ppm. TOL-U was between the two extremes. The sensitivities of TOL-B and TOL-U were comparable; HA-U showed the lowest sensitivity. Thus, it was concluded that TOL-B is the indicator of choice for detecting toluene exposure at low levels.     

Application of high-pressure liquid chromatography in the analysis of urinary metabolites of aromatic solvents

The introduction of HPLC methods in industrial toxicology represents a valuable tool for the routine monitoring of workers occupationally exposed to aromatic solvents. The HPLC method described here permits the simultaneous determination of metabolites of ethylbenzene, styrene, toluene, xylene isomers, benzene, phenol and cresol isomers in diluted urine samples. Pretreatment of urine samples with steam distillation is necessary only for determination of phenol and cresols because of their low concentrations. A comparison between a GLC and the HPLC procedure for mandelic and phenylglyoxylic acids confirmed the satisfactory performance of the HPLC method.     

Capillary gas chromatographic method for mandelic and phenylglyoxylic acids in urine

Summary In support of an occupational investigation of styrene exposure, a capillary gas chromatographic method was developed for the quantitation of the styrene metabolites mandelic and phenylglyoxylic acids. The method was based on that of Guillemin and Bauer [7], in which phenylglyoxylic acid was converted to mandelic acid by reduction before instrumental analysis. The earlier method had to be modified for use with capillary columns; the resulting method was sensitive, selective and reproducible. The detection limit was approximately 0.001 mg/ml urine. Approximately <5% relative precision was achieved in the range of 0.05–2 mg/ml urine. Mandelic acid was resolved from other components of urine and from by-products of derivatization.     

Kinetic interpretation of the exposure test for styrene

Summary The excretion kinetics of mandelic and phenylglyoxylic acids investigated over three subsequent days after cessation of styrene inhalation could be expressed by biphasic functions, similar for both metabolities; the half-times for the first and the second phases were 2.5 and 30 h, respectively. The possibility of styrene accumulation in exposure repeated daily was assessed by kinetic modelling; it appears negligible if measurements are based on urine samples collected at the end of the working shift. The above contention has been examined in workers exposed to styrene in the polyester industry: concentrations of styrene in air monitored continuously varied from 26 to 130 mg/m3. The relationship between styrene concentration and rate of urinary excretion of the total amount of mandelic and phenylglyoxylic acids was rectilinear and demonstrated a reasonable agreement between experimental and industrial data. The trends of concentrations within the day and week gave no indication of substantial styrene cumulation under repeated industrial exposure.     

Evaluation of organic solvent ototoxicity by the upper limit of hearing.

To clarify the effects of organic solvents on hearing, we measured the upper limit of hearing in 93 male workers exposed to organic solvents in 7 factories that produced plastic buttons or baths. Medical examinations, environmental monitoring (i.e., concentration in breathing-zone air), and biological monitoring (i.e., concentration in urine) of the organic solvents were also done. Although the organic solvent concentrations in the environmental monitoring were lower than the occupational exposure limit, the upper limit of hearing was reduced in workers who were exposed for 5 y or more. This reduction was dose-dependent and was related to styrene concentrations in breathing-zone air and mandelic acid concentrations in urine. Even individuals who had normal medical examinations showed a reduced upper limit of hearing. The upper limit of hearing may serve as an early detection indicator of health effects in workers constantly exposed to styrene.     

A new derivatization procedure for the analysis of hippuric acid and m-methyl-hippuric acid by gas chromatography

Summary The industrial solvents, toluene and xylene, have physicochemical properties that can be hazardous to the workers exposed. Since hippuric acid and m-methyl-hippuric acid represent the products of toluene and xylene biotransformation in urine, they are used as biological markers in studies on occupational exposure to these solvents. Several methods have been used to determine hippuric acid and m-methyl-hippuric acid —either based on gas chromatography or on high-performance liquid chromatography. In this study we propose the derivatization of hippuric acid and methyl-hippuric acid using methanol in acid medium (HCl), a low-cost reagent with a low level of toxicity. The method has been routinely used in our laboratory for 1 year and has proven to be a reliable procedure for the biological control of occupational exposure to toluene and/or xylene.