Int Arch Occup Environ Hlth 36,153-160 (1976) © by Springer-Verlag 1976

Measurement of Toluene and Xylene Metabolites by Gas Chromatography K ENGSTROM, K HUSMAN, and J RANTANEN Department of Toxicology and Biochemistry, Institute of Occupational Health (Acting Head: J

Rantanen, M D ), Haartmaninkatu 1,

SF-00290 Helsinki 29, Finland

Summary

A new method for analyzing urinary hippuric and methyl hip-

puric acids by gas chromatography was developed

As well the direct analysis of hippuric acids as the determination of their alkaline hydrolytic

products, benzoic and toluic acids, are described different methods are compared and discussed

Results of the two

The method was applied to

workers occupationally exposed to paints containing mainly toluene and xylene The urinary hippuric acid concentrations were related to the level of toluene and xylene in blood. Key words: Toluene-hippuric acid work

Gas chromatography

Xylene-methylhippuric acid

Urinalysis of metabolites

Painting

Blood analysis of

solvents.

INTRODUCTION Most of the urinary benzoic components, whereas

acid is derived from the dietary

a smaller part is excreted as

intermediate amino acid metabolism (Quick,

1931)

a result of The benzoic

acid excreted in urine is mainly conjugated to glycine (Quick, 1931) In human absorbed toluene and m and p-xylene are also converted

to benzoic

and respectively

acids and excreted mainly as hippuric methylhippuric acids

in urine

to m

and p-toluic

acid or respectively

(Teisinger and Sbr 6va,

as

1955 ;

Fabre et

al , 1960) Numerous reports have demonstrated that excretion of hippuric acids is correlated to environmental toluene and p and m-xylene exposure (Ohtsuji and Ikeda, 1969 ; the

Pagnotto

and Liberman,

and Sbr 8 va,

1967 ;

Ogata et al ,

1970 ;

Teisinger

1955 ;

Mikulski et al , 1972). Several spectrophotometric procedures for analyzing hip-

puric acids (Pagnotto and Liberman, 1967 ; Ogata et al , 1970) and a fluorometric one (Ellman et al , 1961) have been described

These methods

are simple and sensitive but do not

allow differential analysis of hippuric

and methylhippuric

153

acid without a preceding paper or thin-layerchromatographic step, which is laborious and time-consuming Separate determination is, however, possible with gas chromatography (Buchet and Lauwerys, 1973). Our aim was to find a gas chromatographic procedure for analyzing urinary hippuric acids, specially for monitoring workers using paints containing toluene and/or xylene as solvent GLC-analysis was performed both by direct silylation of hippuric acids and by splitting off the glycine moiety before silylation of benzoic and toluic acids In order to evaluate the methods, urinalysis of occupationally exposed painters was correlated with blood concentrations of toluene and xylene.

MATERIAL AND METHODS Reagents

The chemicals used were as follows: diethylether p a ,

ethylacetate p a , hippuric acid p a , sodium-hydroxide p a , sodiumchloride p a , sulphuric acid p a and m and p-xylene (Merck, Darmstadt, West Germany), pyridine (Baker, Devenp.a ter, Holland) and silyl-8 (Pierce Chemical Company, Rockford, Illinois, USA) m and p-methylhippuric acids 1. Treatment of Urine Samples 2 ml of urine was pipetted into a test

tube, acidified with 40 V1 of 6 N HCl and saturated with Na Cl. 10 ml of ethylacetate was added and shaken for 10 min in a mechanical shaker 2 ml of the ethylacetate phase was evap100 pl of pyridine-silyl-8 (1:1) was added orated to dryness and kept for a few minutes at room temperature The sample containing 1-2 pl of the reaction mixture was injected into the gas chromatograph 250-2000 mg/l of hippuric acid in water and 100-1000 mg/l of methylhippuric acid in normal urine were used as standards The urinary results were corrected for specific gravity and creatinine (Jaffe reaction). Hydrolysis of Hippuric Acids

Hydrolysis of the urinary hippuric

acids was performed in alkaline solution according to van der Heiden et al (1971) 2 ml of urine and 2 ml 11 N NaOH were pipetted into a test tube and kept in electrical bath at 1100 C for 1 h The hydrolysate was neutralized with 5 ml 6 N H2 50 4 . Thereafter the hydrolysis product was treated as described above, except that diethylether instead of ethylacetate was used for the extraction. Gas Chromatography

The determinations were carried out with a

Hewlett Packard type 5700 A gas chromatograph (HP, Route 41, Avendale, Penna 19311) Glass column (length 130 cm, internal 1 Kindly supplied by Mr Rainer Sj 6holm (M Sc ), Dept Abo Akademi, Turku, Finland.

154

of Org

Chemistry,

Table 1

Reproducibility and recovery of the different methods

Sample

Amount added to

Amount

Recovery

SD

found

Coeff

of

variation

urine (n= 10) mg/l

mg/l

mg/

%

Hippuric acid

2000

1825

Methylhippuric acid

2000

1960

91

32

2

98

66

3

Benzoic acid

2000

Toluic acid

2000

1885

94

39

2

1930

97

40

2

Without hydrolysis

After hydrolysis

diameter 1/8 inch) was used and the column was packed with Varaport 30 (80/100 mesh), coated with 10 % OV-17 (Varian aerograph, Walnut Greek, California, USA) The temperatures for hippuric acids were as follows: oven 2300C, injector 250 0 C, detector 250 0 C, and for benzoic and toluic acids, respectNitroively: oven 135 0 C, injector 200 0 C, and detector 250 0 C gen (30 ml/min) was used as carrier gas and the flame was produced with an air-hydrogen mixture (flow values: 300 and 60 ml/min, respectively). Blood Analysis Blood toluene, m and p-xylene were analyzed 1 ml of sample was kept at 900 C with a head space technique in a head space bottle for 30 min The sample was transfered to the gas chromatograph according to G 6 ke (1972) The gas chromatographic conditions were as follows: stainless steel tubing (length 50 cm, internal diameter 1/8 inch) filled with Uc-W98 (10% on Chromosorb W AW-DMCS, 80/100 mesh) The temperature of the oven was 75 0 C, of the injector and detector 1500°C and 200 0 C respectively The coefficient of variation for an amount of 1 ppm (vol/vol) was 5% (n=10) with a detection limit of 0 005 ppm (vol/vol).

RESULTS Methodical Results A linear relationship between the peak height and concentration of each different acid was obtained The identification was performed by comparing retention times with those of known standards Table 1 shows the data for recovery and reproducibility experiments obtained by the two different methods. 155

0 8

a

o

a

0 0

C

C o C

x

Methyl hippuric acid concentration lmg/gof creatinine in urinel Fig 1

Urinary methylhippuric acid excretion correlated with toluene concentration in blood

To study the possible differences between the two methods an analysis was performed directly as hippuric acids and after alkaline hydrolysis as benzoic and toluic acids from urines of 16 workers occupationally exposed to toluene and xylene. No significant difference was found to prevail between the total amounts of the two acids in the concentration ranges 252-2043 mg of hippuric and 10-2180 mg of methylhippuric acid/l urine (t-test for differences of paired observations gave for hippuric acid t= 0 19, P > 0 5, n-1=15, and for methylhippuric acid t=O 008, P > 0 5, n-1 =15). Urinalysis

The methods were applied to urines of painters oc-

cupationally exposed to different concentrations of toluene and m and p-xylene The urinary proofs were collected at the end of an 8-h working day, and simultaneous blood samples were taken, for analysis of xylene and toluene A positive linear relationship

(r=O 91),

P < O 001, n= 20) between xylene

in blood and urinary methylhippuric acid was obtained (Fig 1). For toluene and hippuric acid the correlation was not good (r=0 64 , P > O 01, n= 20) (Fig 2) For unexposed controls (39 trainmen) we found a mean hippuric acid concentration of 1037 mg/l (range 126-4844 mg/l) (corrected for specific gravity of 1.018) or 739 mg/g creatinine (range 86-2340 mg/g).

156

S o

1O 0

aa_ 0

. 05

CT = 0 000 x CH O14

O

*

t

U C o 0

R= 0 64 (P< O01)

O *

U

0

00 %

0

Os O l I An 10 20 x 10+2 Hippuric acid concentration l mg/g ofcreatinine inurinel

Fig 2

Urinary hippuric acid excretion correlated with toluene concentration in blood

DISCUSSION Detoxication of toluene is started by oxidation to benzoic acid, which then is eliminated mainly as hippuric acid Bray et al (1951) investigated the conjugation of benzoic acid in rabbits and found 19% to be excreted as glucuronide conjugate and the rest as hippuric acid On the contrary toluene is entirely eliminated as hippuric acid (Smith et al , 1954) Quick (1931) demonstrated that when a sufficiently large dose of benzoic acid was administered, part of the benzoic acid (about 10-12%) appeared in human urine as glucuronide conjugate. 10-20% of the benzoic acid was eliminated in human urine as glucuronide conjugate after a 5-h exposure to toluene (concentration 271-1177 mg/m3) (Teisinger and Sbrova, 1955). Bray et al (1949) found the main metabolites of m and p-xylene in rabbits to consist of m and p-toluic acids and excreted almost entirely as glycine conjugates Practically no ester glucuronide formation was observed The o-toluic acid was excreted mainly in unconjugated form and in form of ester glucuronide, while only a small amount is conjugated to glycine Fabre et al (1960) isolated and identificated urinary xylene metabolites in guinea pig, rabbit and rat and

157

'c c~

1

.

I

a

15

I

I

I

1

I

1

10

& l

|

·

Time(min)

a

|

|{

5

I ||i

i EI

I ,,al L

I

A

l

l

10

O b

m

l

l

i

I

10

5

5 Time(min)

I

I

I

I

I

O

O

Fig 3a und b Gas chromatograms of a urine extract containing hippuric, m and p-meth(a) With hydrolysis; (b) without hydrolysis ylhippuric acid

found the toluic acids to be combined to glycine and glucuronic acid. By alkaline hydrolysis it is possible to liberate the free acid from conjugates of glycine and glucuronic acid, thus allowing the determination of the total excretion of benzoic In the present work no marked difference and toluic acids was found to prevail between the urinary concentrations of hippuric acids (t for hippuric acid = O 19 and for methylhippuric acid = O 008, n-1= 15) as measured with the two differbefore and after hydrolysis) Determination ent methods (i e after hydrolysis seems, however, more recommendable because the difference may be more distinct at higher doses, exceeding the conjugation capacity of liver.

158

As compared to direct analysis the hydrolytic method is not more laborious, because the time needed for hydrolysis is gained in the evaporation of samples (Bp ethylether = 340 C, Bp ethylacetate = 770 C) Another advantage of the recommended method is the better chromatographic resolution of benzoicand toluic acids (Fig 3a) as compared to that of hippuric and In storage experiments for one methylhippuric acids (Fig 3b) month at +40 C, the loss of the urinary hippuric acids at posthydrolytic analysis was found to be less than 20 %, while the loss was much higher when determined unhydrolyzed. To test the method, the relationship was studied between the toluene concentration in blood and the excretion concentration of hippuric acid in urine and the xylene concentration in blood and the urinary excretion of methylhippuric acid respectively Poor correlation was obtained between the blood toluene and urinary hippuric acid (r=O 64), because of the great variations in hippuric acid content in urine of unexposed persons This may be due to the low level of exposure (air contents < 50 ppm) in the present study and correlation may be better at higher exposures Parallel observations were (1973) who found no dependence of made by Szadkowski et al excretion of hippuric acid upon the concentration of toluene (1970) and in air or in blood On the other hand Ogata et al Pagnotto and Liberman (1967) have got good correlations between urinary hippuric acid and concentration of toluene in air. The correlation (r=O 91) between xylene concentration in blood and urinary methylhippuric acid content was found to be much greater, thus suggesting that the determination of methylhippuric acid might be a more valuable indicator in the hygienic control of workers exposed to xylene On the basis of the present results urinary hippuric acid is not a reliable indicator for exposure to toluene at least at low level. Acknowledgements assistance.

Thanks are due to Mrs

Hilkka Jrventaus for technical

REFERENCES Bray, H G , Humphris, B G , Thorpe, W V : Metabolism of derivatives of toluene, o-, m and p-xylenes Biochem J 45, 241-244 (1949) Bray, H G , Thorpe, W V , White, K : The formation of benzoic acid from benzamide, toluene, benzylalcohol and benzaldehyd and its conjugation with glycine and glucuronic acid in the rabbit Biochem J 48, 88-96 (1951) Buchet, J P , Lauwerys, R R : Measurement of urinary hippuric and m-methylhippuric acids by gas chromatography Brit J industr Med 30, 125-128 (1973)

159

Ellman, G L , Burkhalter, A , Ladou, J : A fluorometric method for the (1961) 57, 813-81 determination of hippuric acid J Lab clin Med Fabre, R , Truhaut, R , Laham, S : Recherches sur le mtabolisme compare des xyldnes ou dimethylbenzenes Arch Mal prof 21, 314-328 (1960) G6ke, G : A device for headspace analysis for use with dual column gas Chromatographia 5, 622-623 (1972)

chromatographs

van der Heiden, C , Wadman, S K , Ketting, D , de Bree, P K : Urinary and feacal excretion of metabolites of tyrosine and phenylalanine in a patient with cystic fibrosis and severely impaired amino acid absorption. Clin chim Acta 31, 133-141

(1971)

van der Heiden, C , Wauters, E A K , Ketting, D , Duran, M , Wadman, S K : Gas chromatographic analysis of urinary tyrosine and phenylalanine metabolites in patients with gastrointestinal disorders 34, 269-289 (1971)

Clin chim Acta

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26,

Mikulski, P J , Wiglusz, R , Bublewska, A , Uselis, J : Investigation of Brit J industr Med. exposure of ships' painters to organic solvents 29, 450-453 (1972) Ogata, M , Tomokuni, K , Takatsuka, Y : Urinary excretion of hippuric acid and m or p-methylhippuric acid in the urine of persons exposed to vapours of toluene and m

or p-xylene as a test of exposure

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industr Med

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(1931)

Smith, J N , Smithies, R H , Williams, R T : The metabolism of alkyla) Glucoronic acid excretion following the administration of benzenes alkylbenzenes Biochem J

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Szadkowski, D , Pett, R , Angerer, J , Manz, A , Lehnert, Losungsmittelbelastung am Arbeitsplatz

II

G : Chronische

Schadstoffspiegel im Blut

und Metabolitenelimination im Harn in ihrer Bedeutung als berwachungs31, kriterien bei toluolexponierten Tiefdruckern Int Arch Arbeitsmed 265-276 (1973) Teisinger, J , Sbrova, J : L'elimination de l'acide benzoique dans l'urine et son rapport avec la concentration maximum tolerable de toluene dans l'air

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16, 216-220 (1955)

Received August 7, 1975 / Accepted September 15,

160

1975