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Monitoring the trace amount of chemicals in various samples remains a challenge. This study was conducted to develop a new solid-phase microextraction (SPME) system (inside-tube SPME) for trace analysis of n-hexane in air and urine matrix. The inside-tube SPME system was prepared based on the phase separation technique. A mixture of carbon aerogel and polystyrene was loaded inside the needle using methanol as the anti-solvent. The air matrix of n-hexane was prepared in a Tedlar bag, and n-hexane vapor was sampled at a flow rate of 0.1 L/min. Urine samples spiked with n-hexane were used to simulate the sampling method. The limit of detection using the inside-tube SPME was 0.0003 μg/sample with 2.5 mg of adsorbent, whereas that using the packed needle was 0.004 μg/sample with 5 mg of carbon aerogel. For n-hexane analysis, the day-to-day and within-day coefficient variation were lower than 1.37%, with recoveries over 98.41% achieved. The inside-tube SPME is an inter-link device between two sample preparation methods, namely, a needle trap device and an SPME system. The result of this study suggested the use of the inside-tube SPME containing carbon aerogel (adsorbent) as a simple and fast method with low cost for n-hexane evaluation., (© 2020 John Wiley & Sons, Ltd.)

The aim of this study was to evaluate patients who had peripheral neuropathy and changes to their visual evoked responses resulting from exposure to n-hexane. Eighteen patients with acute or subacute neuropathy, who were working in a shoe factory, were investigated clinically and electrophysiologically. These evaluations were then repeated 9 months to 12 months after cessation of exposure to n-hexane. Results of the nerve conduction studies predominantly showed a decrease in motor and sensory conduction velocities. Between 9 and 12 months after cessation of exposure to n-hexane, 83.3% of patients had a complete clinical recovery. The electrophysiological studies also revealed improvement to the majority of motor and sensory nerve conduction velocities. The results of the visual evoked potential (VEP) studies were considered normal at admission, however, the P100 latencies at the 9-month to 12-month retest had improved (p < 0.05). As the abnormalities identified with clinical examination and nerve conduction studies, and the subclinical abnormalities revealed through VEP assessment, could be reversed after exposure to n-hexane had ceased, the clinical prognosis was usually good.

Although genomic screening studies have identified several genes associated with Parkinson's disease (PD), there is evidence that environmental factors are also involved in the pathogenesis of the disease and that hydrocarbon-solvents may be one of them. The genetic component is less evident in late-onset PD. To assess whether age and PD may affect the catabolism of the hydrocarbon n-hexane, a two-part study was performed. In the first part the urinary levels of its main metabolites, 2,5-hexanedione and 2,5-dimethylpyrroles, were measured in 108 patients and 108 healthy controls, matched by age and sex. Metabolite urinary excretion was significantly reduced in PD patients as compared with controls and was inversely related to age in both groups. In the second part the same comparison was made between 24 non-smoking and 10 smoking patients, matched to controls, after smoking of a hydrocarbon-rich cigarette. In these subjects also n-hexane and 2,5-hexanedione blood levels were measured. There was no appreciable difference in n-hexane blood levels between patients and controls in non-smokers, whereas there was a significant increase in patients over controls in smokers (p < 0.01). 2,5-hexanedione blood levels were significantly lower in patients than in healthy controls, both in non-smokers and in smokers, but the reduction was more pronounced in smokers (-46.3 % versus -10.7 %). The same was true for 2,5-hexanedione and 2,5-dimethylpyrrole urinary levels. This study suggests that aging and PD may be associated with a reduction in the capacity to eliminate the hydrocarbon n-hexane. This metabolic alteration may play a role in the pathogenesis of PD.

Objectives: Animal studies demonstrate that the formation of the neurotoxic metabolite, 2,5-hexanedione (HD) decreases during co-exposure to methyl ethyl ketone (MEK). The aim of the present study was to describe the influence of co-exposure to MEK on n-hexane toxicokinetics in humans., Methods: Four healthy male volunteers were exposed, on different occasions, to three different combinations of vapor of these solvents, namely: 50 ppm n-hexane alone, and in combination with 100 and 200 ppm MEK, for 2 h during light physical exercise (50 W). Arterialized capillary blood, venous blood, and urine were sampled at scheduled intervals before, during, and up to 24 h after the onset of the exposure. HD in venous blood and urine was analyzed by gas chromatography with electron capture detector after derivatization with O-(pentafluorobenzyl) hydroxylamine., Results: Serum HD decreased with increasing exposure to MEK at 2 h after the onset of the exposure, from an average concentration of 2.2 micromol/l in the n-hexane-alone condition to 1.2 and 0.44 micromol/l in the 100 and 200-ppm MEK conditions, respectively. The area under the concentration-time curve of HD in venous blood and the concentration of HD at 2 and 4 h after the end of exposure decreased with increasing MEK. These results suggest that combined exposure to MEK and n-hexane at occupationally realistic levels depresses the metabolism of n-hexane in a dose-dependent fashion., Conclusion: The internal exposure to the toxic metabolite of n-hexane decreased with co-exposure to MEK in a dose-dependent fashion. Estimation of external exposure by HD in serum or urine could be confounded by the co-exposure to MEK.

A sensitive method was developed for determination of n-hexane and cyclohexane in human body fluids by headspace capillary gas chromatography (GC) with cryogenic oven trapping. Whole blood and urine samples containing n-hexane and cyclohexane were heated in a 7.5 mL vial at 70 degrees C for 15 min, and 5 mL of the headspace vapor was drawn into a glass syringe. All vapor was introduced through an injection port of a GC instrument in the splitless mode into an Rtx-Volatiles middle-bore capillary column at an oven temperature of -40 degrees C for trapping volatile compounds. The oven temperature was programmed to 180 degrees C for GC with flame ionization detection. These conditions gave sharp peaks for both n-hexane and cyclohexane, a good separation of each peak, and low background impurities for whole blood and urine. The extraction efficiencies of n-hexane and cyclohexane were 13.2-30.3% for whole blood and 12.7-20.7% for urine. The coefficients of within-day variation in terms of extraction efficiency of both compounds were 5.0-9.5% for whole blood and 3.8-10.8% for urine; those of day-to-day variation for the compounds were not greater than 16.6%. The regression equations for n-hexane and cyclohexane showed good linearity in the range of 5-500 ng/0.5 mL for whole blood and urine. The detection limits (signal-to-noise ratio = 3) for both compounds were 1.2 and 0.5 ng/0.5 mL for whole blood and urine, respectively. The data on n-hexane or cyclohexane in rat blood after inhalation of each compound are also presented.

Since n-hexane metabolites are excreted as glucuronide conjugates, most conventional analytical procedures require preliminary hydrolysis, yielding to the 'total' 2,5-hexanedione (2,5-HD), but also giving rise to a number of artifacts. The whole pattern of n-hexane metabolites, both conjugated and unconjugated, as well as different methods of sample pretreatment have been evaluated by hyphenated techniques (liquid chromatography-mass spectrometry (LC-MS) and gas chromatography-mass spectrometry (GC-MS)). Aliquots of urine from rats exposed to n-hexane underwent enzymatic or acid hydrolysis or both; whereas one aliquot was applied to LC-MS, dichloromethane extracts were analyzed by GC-MS. In untreated urine, four glucuronides (-G) were identified and characterized by LC-MS: 2-hexanol-G, 5-hydroxy-2-hexanone-G, 4,5-dyhydroxy-2-hexanone-G, and 2,5-hexanediol-G. 'Free' 2,5-HD was detectable in non-hydrolyzed samples by both GC- and LC-MS. Whereas enzymatic hydrolysis did not increase the amount of 2,5-HD, acid hydrolysis led to increase 2,5-HD in variable amount and produced gamma-valerolactone as a result of a complete transformation of 4,5-dihydroxy-2-hexanone-G and the partial conversion from 5-hydroxy-2-hexanone-G. Further experiments showed that both 5-hydroxy-2-hexanone-G and 4,5-dihydroxy-2-hexanone-G, isolated by solid-phase extraction and hydrolyzed, yield comparable amount of 2,5-HD and gamma-valerolactone. In samples treated by acid hydrolysis, GC-MS only does not allow to understand the true source of 'total' 2,5-HD, which may be produced not only from 4,5-dihydroxy-2-hexanone-G but also from the more abundant 5-hydroxy-2-hexanone-G, which thus represents the main source of analytical artifacts. 'Free' 2,5-HD seems to be both suitable from an analytical point of view and meaningful for biological monitoring purposes, provided that conjugate metabolites are rapidly removed from the body leading to a negligible neurotoxic risk.

A liquid chromatography atmospheric pressure electrospray mass spectrometry (ESI-LC/MS) system was evaluated for the identification and characterization of n-hexane conjugated metabolites (glucuronides) in untreated urine samples. Chromatography of glucuronides was obtained under ion-suppressed reversed-phase conditions, by using high-speed (3 cm, 3 microns) columns and formic acid (2 mM) as modifier in the mobile phase. The mass spectrometer was operated in negative ion (NI) mode. For the first time, four glucuronides were identified by ESI-LC/MS in untreated urine samples of rats exposed to n-hexane: 2-hexanol-glucuronide, 5-hydroxy-2-hexanone-glucuronide, 2,5-hexanediol-glucuronide and 4,5-dihydroxy-2-hexanone-glucuronide. Confirmation of the conjugated metabolites was obtained by LC/MS/MS experiments. Gas chromatography/mass spectrometry (GC/MS) and atmospheric pressure chemical ionization (APCI) LC/MS analyses were performed on the same samples. An integrated approach GC/MS-LC/MS for the semi-quantitative analysis of n-hexane glucuronides, whose standards are not commercially available, is discussed and proposed here. In order to understand the fate of the metabolites during sample pre-treatment, a study about the effects of enzymatic and acid hydrolysis on urine samples was conducted on glucuronides isolated by solid-phase extraction. Combined analyses by GC/MS and LC/MS enabled us to distinguish 'true' n-hexane metabolites from compounds resulting from sample treatment and handling (i.e. enzymatic and acid hydrolysis, extraction and GC injection).

1. The described analytical procedure permits the simultaneous determination of the main n-hexane metabolites in urine. 2-Hexanone, 2-hexanol, 2, 5-hexanediol and 2, 5-hexanedione, were chosen to dose the rats used in this study. All urine samples were collected and analysed on a daily basis, before and after acidic hydrolysis (pH 0.1) by GC/MS. 2-Hexanone, 2, 5-dimethylfurane, gamma-valerolactone and 2, 5-hexanedione were determined before hydrolysis: 2-hexanol and 2, 5-hexanediol, after hydrolysis; and 5-hydroxy-2-hexanone and 4, 5-dihydroxy-2-hexanone were calculated by the difference between gamma-valerolactone and 2, 5-hexanedione with and without hydrolysis, respectively. 2. A metabolic scheme was proposed reflecting the biotransformations undergone by the four compounds assayed. We consider 2, 5-dimethylfurane as a "true metabolite' because the quantities detected were always greater before hydrolysis. 3. It has been reported that human and rat n-hexane metabolism follow a similar pattern. Therefore, as a practical application and without increasing either sample or time requirements, the simultaneous quantification of the different metabolites and their excretion profile could provide better information about the metabolic situation of exposed workers than the determination of 2, 5-hexanedione alone. According to our experimental results, 4, 5-dihydroxy-2-hexanone itself would be a good toxicity indicator.

1. To estimate the quantitative relation between exposure to airborne n-hexane and various markers of immune function, 35 male workers were examined and compared with unexposed controls. 2. Urinary 2,5-hexanedione concentrations were significantly higher in the exposed group than in the unexposed. 3. A significant suppression was observed in the serum immunoglobulin (IgG, IgM and IgA) levels between two populations. Also, a significant correlation was found between urinary 2,5-hexanedione concentrations and serum Ig level of the exposed group. 4. No significant difference between white blood cell counts was found in the two groups.

This paper reports the problem of interlaboratory quality control and the actual condition of biological monitoring of n-hexane among the seven major laboratories specializing in clinical chemistry tests, where more than 80% of the total urine specimens in Japan have been analyzed after the beginning of biological monitoring of n-hexane in 1989. First, transportation conditions of urine specimens and transportation effects on measurement results were studied. The seven major laboratories carried urine specimens at distances of more than 450 km from the University of Occupational and Environmental Health (UOEH) to the respective laboratories and had at least one transit terminal where specimens from various areas were collected and transferred to the analytical laboratories. All laboratories used cooler boxes with dry ice or ice bars and stored the specimens in freezers or refrigerators. Specimens arrived at the final destinations within 24 hours. The transportation from institutes for health examination to the laboratories did not affect the measurement results of 2,5-hexanedione (HD), a determinant of n-hexane biological monitoring. Interlaboratory cross-checks were performed among the seven major laboratories and four institutes for occupational health examination that have their own analytical laboratories. All of the laboratories analyzed HD by acid hydrolysis (pH < 1.0); interlaboratory differences were recognized at the first cross-check. After some laboratories intensified gas chromatography (GC) maintenance and changed to a new column, the interlaboratory variation lessened.(ABSTRACT TRUNCATED AT 250 WORDS)

Human polymorphonuclear leukocytes (PMN) were chosen to measure two cellular end points--chemotaxis and respiratory burst--and to verify whether they could function as biomarkers of early effect in detecting occupational exposure to n-hexane of apparently healthy shoe workers, without any electroneuromyographic (ENMG) abnormality. Chemotaxis, but not respiratory burst, was found to be impaired. A negative linear correlation between chemotaxis of PMN of those workers that had been exposed to n-hexane versus 2,5-hexanedione (2,5-HD) urinary concentrations were found. This negative trend is consistent with our previous in vitro experimental findings: it was observed that the progressive addition of 2,5-HD to PMN suspensions inhibited chemotaxis in a dose-dependent mode, while chemiluminescence was not modified. Now we have confirmed in vivo that chemotaxis is more sensitive than the respiratory burst response to 2,5-HD. Such results justify the interest in the behaviour of PMN harvested from workers exposed to n-hexane. Since significant inhibition of chemotactic activity was observed in some workers whose urinary 2,5-HD levels were lower than 5 mg l-1, which is the biological exposure index suggested by ACGIH, this study suggests that PMN chemotaxis may be proposed as a useful biomarker in detecting occupational exposure to low level of n-hexane.

The concentration of 9 environmental chemical pollutants in the general population was measured in blood and urine. For the 9 different pollutants, the blood samples tested varied from 88 for acetone to 431 for benzene. Urine samples varied from 48 for styrene to 213 for n-hexane. Six of these agents (benzene, toluene, styrene, n-hexane, acetone and carbon disulphide) were present in all or almost all (100-94%) blood samples. The three chlorides (chloroform, trichloroethylene and tetrachloroethylene) were present only in 60-85% of samples. After acetone, with blood concentrations in microgram/1 (mean 840 microgram/l), the highest mean blood levels were those of toluene (1097 ng/l), chloroform (955 ng/l) and n-hexane (642 ng/l). Trichloroethylene and free carbon disulphide showed similar values (458 and 438 ng/l, respectively). Finally, benzene, styrene and tetrachloroethylene showed the lowest values (262, 217 and 149 ng/l, respectively). There was generally a significant difference between rural and urban workers in terms of blood benzene (200 ng/l vs 264 ng/l), trichloroethylene (180 ng/l vs 763 ng/l) and tetrachloroethylene (62 ng/l vs 263 ng/l). In a group of subjects potentially exposed to industrial solvents, classed as chemical workers, blood benzene, toluene, chloroform and n-hexane were significantly higher than in rural and urban workers. Smokers showed a significantly higher blood concentration than non-smokers for benzene (381 ng/l vs 205 ng/1), toluene (1431 ng/l vs 977 ng/l), and n-hexane (838 ng/l vs 532 ng/l). All or almost all urine samples (100-92%) contained all the compounds except trichloroethylene and tetrachloroethylene, present in 79% and 76% of samples, respectively (table 2). Urinary concentrations of all compounds did not differ significantly between rural and urban workers. Benzene and toluene were significantly higher in in urine of smokers than of non-smokers. Chloroform and n-hexane showed significantly higher urinary than blood values. Excluding acetone, with urinary and blood concentrations in pg/l, chloroform, toluene and n-hexane showed the highest mean concentrations both in blood and in urine.

Blood and urine samples were collected from 57 male Japanese solvent workers [exposed to n-hexane (Hex-A), ethyl acetate, and toluene (Tol-A) at 1.5, 2.3, and 2.3 ppm as GM-TWA, respectively] and also from 20 male nonexposed workers at the end of a 8-h shift, and analyzed for n-hexane (Hex-B) and toluene (Tol-B) in blood, and n-hexane (Hex-U), toluene (Tol-U), 2,5-hexanedione [both with (HD-U/cHYD) and without hydrolysis (HD-U/sHYD)] and hippuric acid (HA-U) in urine. Regression analysis showed that both Hex-B and Tol-B correlated significantly with corresponding exposure to the solvents. Solvents in urine (Hex-U and Tol-U) also correlated with solvents in air but with smaller correlation coefficients than the solvents in blood. Both HD-U/cHYD and HD-U/sHYD showed significant correlation with Hex-A, but HA-U failed to do so with Tol-A. Based on the correlation among biological exposure indicators and solvent concentration in air, sensitivity as an exposure indicator was compared between the solvent in blood and the metabolite in urine in terms of the lowest solvent concentration at which the exposed can be separated (with statistical significance) from the nonexposed (the lowest separation concentration; LSC). The LSC was 3.9 ppm for Hex-B, 1 to 2 ppm for HD-U/sHYD and 10 to 30 ppm for HD-U/cHYD, suggesting that HD-U/sHYD is superior even to Hex-B in detecting low n-hexane exposure; this high sensitivity of HD-U/sHYD is due to the absence of HD-U/sHYD in the urine from the nonexposed.(ABSTRACT TRUNCATED AT 250 WORDS)

Blood and urine samples were collected at the end of an 8-h workshift from 30 male workers exposed to a mixture of n-hexane, ethyl acetate and toluene (each being about 2 ppm as geometric means) and also from 20 nonexposed male workers. Blood samples were analyzed for n-hexane and toluene, and urine samples were analyzed for n-hexane, toluene, 2,5-hexanedione (both with and without hydrolysis) and hippuric acid. Based on the correlation between biological exposure indicators and solvent concentrations in air, sensitivity as an exposure indicator was compared between solvents in blood and solvents or metabolites in urine in terms of the lowest solvent concentration at which the exposed subjects can be statistically separated from the nonexposed. Both n-hexane and toluene in blood were sensitive enough to detect the exposure at 6.1 ppm and 1.4 ppm, respectively. n-Hexane exposure below 2 ppm was detectable also by urinalysis for 2,5-hexadione without hydrolysis. Urinary hippuric acid, however, failed to detect low toluene exposure under the conditions studied. Of additional interest is the fact that toluene in urine correlated significantly with toluene in air, which apparently deserves further study for confirmation.

A sensitive and specific method for determination of 2,5-hexanedione in urine is described. Treatment of the urine specimen directly with n-butylamine yields n-butyl 2,5-dimethyl pyrrole. The latter is extracted into diisopropylether and analyzed by gas chromatography (GC) with flame ionization detection (FID), thermionic specific detection (TSD) (N mode), or mass spectrometric detection (MS). The minimum detectable quantities are 1 mg/L urine when employing FID with a coefficient of variation of less than 6%. Recovery of 2,5-hexanedione added to the urine at the level of 10 mg/L was 78.9%.

The apparent amount of 2,5-hexanedione, a biomarker of n-hexane expsoure in occupational health, in the urine of both exposed and non-exposed subjects varied not only as a function of the pH at which the urine sample was hydrolyzed but also depending on the capillary column used for gas chromatographic (GC) analysis of the urinary hydrolyzates after extraction with dichloromethane. The formation of a compound, identified by gas chromatography-mass spectrometry (GC-MS) as 2-acetylfuran, following acid hydrolysis was a major cause of confounding effects. This compound was hardly separated from 2.5-hexanedione on a capillary column such as DB-WAX, whereas separation could be achieved on a DB-1 capillary column. 2-Acetylfuran was formed when a urine sample was heated at a pH of less than 2 for hydrolysis, and the amount detected in urine did not differ between exposed and non-exposed subjects, indicating that the formation of 2-acetylfuran is independent of n-hexane exposure. When urinary hydrolysis is used, hydrolysis at a pH of less than 0.5, extraction with dichloromethane, and GC analysis on a non-polar capillary column are proposed to be the best analytical conditions for 2,5-hexanedione analysis in biological monitoring of exposure to n-hexane.

High-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 Ehrlich's 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.

Biological exposure index (BEI) of n-hexane was studied for accuracy using a physiologically based pharmacokinetic (PB-PK) model. The kinetics of n-hexane in alveolar air, blood, urine, and other tissues were simulated for different values of alveolar ventilations and also for constant and variable exposures. The kinetics of 2,5-hexanedione, the toxic n-hexane metabolite, were also simulated. The ranges of n-hexane concentrations in biological media and the urinary concentrations of 2,5-hexanedione are discussed in connection with a mean n-hexane exposure of 180 mg/m3 (50 ppm) (threshold limit value [TLV] suggested by American Conference of Governmental Industrial Hygienists [ACGIH] for 1988-89). The experimental and field data as well as those predicted by simulation with the PB-PK model were comparable. The physiological-pharmacokinetic simulations are used to propose the "dynamic" BEIs of n-hexane and 2,5-hexanedione. The use of simulation with PB-PK models enables a better understanding of the limits, advantages, and issues associated with biological monitoring of exposures to industrial solvents.

To make clear how the n-hexane metabolism is modified by co-exposure with MEK, rats were exposed to various concentrations of MEK mixed with a fixed concentration of n-hexane. Twenty-four male Wistar rats were divided into four equal groups. Each group was exposed for 8 h to 2000 ppm n-hexane, 2000 ppm n-hexane plus 200 ppm MEK, 2000 ppm n-hexane plus 630 ppm MEK and 2000 ppm n-hexane plus 2000 ppm MEK, respectively. Free metabolites and the sum of free and conjugated metabolites of n-hexane were analyzed by gas chromatography. The main metabolite was 2-hexanol during the exposure and 2,5-hexanedione (2,5-HD) after the exposure in any group. The main metabolites, 2-hexanol and 2,5 HD, decreased in inverse proportion to the co-exposed MEK concentrations. The results suggest that augmentation of n-hexane neurotoxicity by MEK co-exposure could not be explained only by 2,5-HD. In addition, 2,5-HD is recommended as an index for biological monitoring of n-hexane exposure. However, one should be careful to evaluate the exposed n-hexane concentration by urinary 2,5-HD, because n-hexane metabolism could be largely modified by co-exposure with MEK.

A gas chromatographic method for analyzing the urinary metabolites of n-hexane (2-hexanol, 2,5-hexanedione, 2,5-dimethylfuran and gamma-valerolactone), of 2-methylpentane (2-methyl-2-pentanol), of 3-methylpentane (3-methyl-2-pentanol), and of cyclohexane (cyclohexanol) was developed. Processing of urine and the gas chromatographic conditions are described. The recovery rate of all hexane metabolites, except 2,5-dimethylfuran, ranged between 92 and 100%. The variation coefficient of metabolites determination was between 1.5 and 5%, apart from 2.5-dimethylfuran determination for which the variation coefficient was 15%. The detection limits ranged between 0.2 and 0.7 mg/l and between 0.05 and 0.1 mg/l when a packed or capillary column was used. Results obtained from a packed and capillary column are discussed.

Male Wistar rats were exposed to 2000 ppm n-hexane or were treated with a single dose of 2,5-hexanedione (200 mg kg-1). Analysis of the urine collected after both treatments revealed the formation of 4,5-dihydroxy-2-hexanone via 5-hydroxy-2-hexanone or 2,5-hexanedione, respectively. Identification of this new n-hexane metabolite included enzymatic hydrolysis of the excreted glucuronide, derivatization of the keto group with O-methylhydroxylamine, and subsequent GC-MS analysis. The chemical structure derived from the mass spectra obtained was confirmed by further analysis of the methoxime-TMS derivatives and of deuterated 4,5-dihydroxy-2-hexanone excreted after treating rats with 2,5-[2H10]-hexanedione. 4,5-Dihydroxy-2-hexanone is proposed to be identical with a urinary constituent that is excreted by rats after n-hexane exposure and is converted to 2,5-dimethylfuran or 2,5-hexanedione, respectively, depending on the conditions of urine treatment prior to GC-MS analysis.

N-hexane and toluene in the urine of occupationally exposed subjects. Measurement and significance of their presence. The determination of n-hexane and toluene in urine was performed in 23 subjects who were occupationally exposed to n-hexane and in 8 subjects exposed to toluene, by means of the head space technique. A Hewlett-Packard 5880 A gas chromatograph supplied with Hewlett-Packard 5970 A Mass Selective Detector was employed. The Authors found significant correlations between urine concentration of the substance and environmental concentration (for n-hexane r = 0,866; for toluene r = 0,770.

Exposure to n-hexane, a component of many industrial solvent mixtures, is known to cause polyneuropathy in man. The concentration of metabolites in urine following exposure may be useful in biological monitoring. In a comparative study experimental animals (rat, rabbit and monkey) were subjected to single inhalatory treatments of 6, 12 and 24 h with 5,000 ppm of pure n-hexane. At the end of the treatments and at intervals thereafter, urine, and in rats also blood, were collected and analyzed for n-hexane and its metabolites. While the urine of rats contained 2-hexanol, 3-hexanol, methyl n-butyl ketone, 2,5-dimethylfuran, y-valerolactone and 2,5-hexanedione, rabbit and monkey urine were found to contain only 2-hexanedione, rabbit and monkey urine were to contain only 2-hexanol, 3-hexanol, methyl n-butyl ketone and 2,5-hexanedione. Within 72 h of the end of exposure, the principal metabolite was 2,5-dimethylfuran in rats and 2-hexanol in rabbits and monkeys. In all three species the excretion rates of methyl n-butyl ketone, 3-hexanol and 2-hexanol peaked several hours earlier than 2,5-hexanedione (and gamma-valerolactone and 2,5-dimethylfuran in rats). In all species 2,5-hexanedione was still detectable in urine 60 h following exposure. n-Hexane metabolites in rat blood were 2-hexanol, methyl-n-butyl ketone, 2,5-dimethylfuran and 2,4-hexanedione. The first two, as well as n-hexane itself, were found in maximum concentration immediately after termination of exposure, while 2,5-dimethylfuran and 2,5-hexanedione, with the longer exposure times, peaked some hours later. The data from urine collected at the end of exposure were compared with those obtained in a parallel study in humans occupationally exposed to a mixture of hexane isomers. Humans chronically exposed to 10-140 ppm n-hexane had 2,5-hexanedione concentrations in urine ranging from 0.4 to 21.7 mg/l, i.e., in the same proportion as rats exposed once for 6 or 12 h to 5,000 ppm.

Environmental exposure to commercial hexane (n-hexane, 2-methylpentane, and 3-methylpentane) was tested in several work places in five shoe factories by taking three grap-air samples during the afternoon shift. Individual exposure ranges were 32-500 mg/m3 for n-hexane, 11-250 mg/m3 for 2-methylpentane, and 10-204 mg/m3 for 3-methylpentane. The metabolites of commercial hexane in the urine of 41 workers were measured at the end of the work shift. 2-Hexanol, 2,5-hexanedione, 2,5-dimethylfuran, and gamma-valerolactone were found as n-hexane metabolites and 2-methyl-2-pentanol and 3-methyl-2-pentanol as 2-methylpentane and 3-methylpentane metabolites. The presence of metabolites in the urine was correlated with occupational exposure to solvents. n-Hexane exposure was correlated more positively with 2-hexanol and 2,5-hexanedione than with 2,5-dimethylfuran and gamma-valerolactone. A good correlation was also found between total n-hexane metabolites and n-hexane exposure. 2-Methyl-2-pentanol and 3-methyl-2-pentanol were highly correlated with 2-methylpentane and 3-methylpentane exposure. The results suggest that the urinary excretion of hexane metabolites may be used for monitoring occupational exposure to n-hexane and its isomers.

Lung uptake and excretion of n-hexane were studied in ten workers in a shoe factory. Simultaneous samples of inhaled and alveolar air were collected with the aid of a Rhan-Otis valve, personal samplers, and charcoal tubes. Alveolar excretion was monitored during a six hour postexposure period. Uptake was calculated from lung ventilation, the retention coefficient, and environmental concentrations. The amount of exhaled n-hexane was calculated from the decay curve. According to the experimental data, alveolar retention was about 25% of the inhaled n-hexane, corresponding to a lung uptake of about 17%. The postexposure alveolar excretion was about 10% of the total uptake. The main metabolites of n-hexane were identified and measured by capillary GC/MS in spot urine samples collected before, at the end, and 15 hours after the same working shift. Urinary concentrations were low, though related to n-hexane in the air. 2,5-Hexanedione in the end of shift samples gave the best estimate of overall exposure. About 3 mg/g creatinine of 2,5-hexanedione would correspond to about 50 ppm of n-hexane in the air (mean daily exposure).

The urinary concentration of some solvents (acetone, cyclohexane, 1,2 dichloropropane, n-hexane, methyl ethyl ketone, perchloroethylene, styrene, toluene, 1,1,1, trichloroethane) was measured by means of a gas chromatography Hewlett-Packard 5890 supplied with a flame ionization detector (GC-FID, DANI HS 3950). The coefficient of variation of the method was lower than 5%. The sensitivity of the GC-FID was very similar to what of mass spectrometer detector (GC-MSD, HP 5970 A).

Peripheral neuropathies caused by exposures to the industrial solvents n-hexane and MBK exhibit strinkingly similar characteristics. In in vivo studies, the metabolites of MBK and n-hexane identified in blood and urine of guinea pigs were 2-hexanol (partly as glucuronide in urine); and 2,5-hexanedione which was detected only in MBK treated groups. Phenobarbital pretreatment increased 2-hexanol urinary excretion in both solvent treatment groups. In in vitro studies, hepatic reduction of MBK required the cytosol fraction to form 2-hexanol; whereas the oxidation of MBK and n-hexane required the microsomal fraction to form 2,5-hexanedione and 2-hexanol, respectively. The in vivo and in vitro biotransformation of MBK and n-hexane to a common metabolite (2-hexanol) suggests that the neurotoxic action of these solvents may be metabolite related.

Urinary metabolites were tested in 41 shoe-factory workers exposed to a mixture of 10 solvents among which "commercial hexane" was the prevailing component. Cyclohexanol, 2-methyl-2-pentanol, 3-methyl-2-pentanol, and trichloroethanol were determined in connection with exposure to cyclohexane, 2-methylpentane, 3-methylpentane, and trichloroethylene, respectively. 2-Hexanol, 2,5-hexanedione, 2,5-dimethylfuran, and gamma-valerolactone were all determined in connection with n-hexane exposure only. 2,5-Hexanedione was the principal n-hexane metabolite found in the workers' urine. This finding of the experimentally proven neurotoxin 2,5-hexanedione in the urine of shoe-factory workers exposed to "commercial hexane" is consistent with the idea that this compound is responsible for the development of neuropathy in this group of individuals.

The concentration of n-hexane in urine was determined in 30 subjects occupationally exposed to n-hexane (median value 59.6 mg/m3) in a shoe factory. The measurement of the substance was performed by means of a Hewlett-Packard 5880 gas chromatograph supplied with a Hewlett-Packard 5970 Mass Selective Detector. The analyses were performed by the head space method (constant volume method, after determination of the urine partition coefficient by the multiple phase equilibration method). The authors found a significant correlation between the n-hexane urine concentrations (microgram/l, Cu) and the n-hexane environmental concentrations (mg/m3, Ci) (r = 0.84; Cu = 0.0669 X Ci + 0.8396).

Male Wistar rats were exposed to 1000 ppm n-hexane, and the excreted urinary metabolites were analyzed by capillary gas chromatography-mass spectrometry (GC-MS). 1-Hexanol, 2-hexanol, 3-hexanol, 2-hexanone, 2,5-hexanedione, 2,5-dimethyltetrahydrofuran, 2,5-dimethyl-2,3-dihydrofuran and gamma-valerolactone were identified by their retention times and their mass spectra. Quantitative gas chromatographic analyses were performed using an FID. Experiments on the hydrolysis of conjugated n-hexane metabolites revealed that enzymatic hydrolysis (in addition to acid hydrolysis) was not required, as treatment with HCl hydrolyzed conjugates sensitive to acid as well as conjugates sensitive to beta-glucuronidase. By incorporating acid hydrolysis only and by using C18-cartridges for sample extraction, a method was developed that allowed the determination of n-hexane metabolites with a sample preparation time of only 45 min. Assay precision was assessed by repeated analyses of the same urine sample. Coefficients of variation for the individual metabolites ranged from between 1.8 and 3.3.