672 Analyst, September, 1977, Vol. 102, pp. 672-67’7

Rapid Determination of Trichloroethanol and Tri chloroacetic Acid in Urine

Marianna Mantel and Raya Nothmann Israel Atomic Energy Commission, Soreq Nuclear Research Centre, Nuclear Chemistry Department, Yavne 70600, Israel

Trichloroethanol and trichloroacetic acid are determined in the same aliquot of urine by use of a simple spectrophotometric technique. The method is based on the Fujiwara reaction, i .e., the reaction between organic halides and pyri- dine in the presence of sodium hydroxide. Two spectrophotometric measure- ments are made, one at 530 nm for the determination of trichloroacetic acid, 16 min after colour development, and the other a t 367 nm for the determina- tion of total trichloro compounds and trichlorethanol, 3 h after colour development. The method is rapid and accurate and is therefore suitable for monitoring patients with trichloroethylene and chloral hydrate poisoning.

Keywords : Trichloroethanol determination ; trichloroacetic acid determination ; urine ; spectrophotornetry ; Fujiwara reaction

The determination of the presence of the two principal metabolites of trichloroethylene,l trichloroacetic acid and trichloroethanol, in urine has been used as an indication of potential poisoning by tri~hloroethylene.”~ Some workers based their evaluations on the determin- ation of trichloroacetic acid alone ,6--8 while others determined both metabolites individuallyg-11 or together as total trichloro compounds.lZ

Recently most investigators have concluded that the measurement of the two metabolites separately is necessary for the detection of potential poisoning. Therefore a sensitive method of analysis for trichloroacetic acid and trichloroethanol that can be carried out easily, rapidly and if possible on the spot is necessary in order to provide reliable and objective information. In this paper a simple, spectrophotometric method is described for the determination of trichloroacetic acid and trichloroethanol in the same aliquot of urine.

Most methods used for the determination of trichloroacetic acid are based on the Fujiwara reactionJ13 ie., on the measurement of the absorbance at 530 and 367 nm of the product of the reaction of trichloroacetic acid with pyridine in the presence of sodium hydroxide. Many methods have been p u b l i ~ h e d , ~ J ~ J * - ~ ~ which differ in respect of various parameters such as heating time, concentration of sodium hydroxide, water to pyridine ratio, time interval between colour development and measurement of absorbance.

The methods used for the determination of trichloroethanol are based on the measurement of the absorbance at 440 nm of the yellow colour produced by the compound in the Fujiwara reaction.16 However, most workersg~17 prefer to oxidise trichloroethanol with chromic acid or another strong oxidising agent, to trichloroacetic acid and then determine the latter compound. Recent publications indicate that the most used method is that of Tanaka and Ikeda,l7 which consists of oxidation of trichloroethanol to trichloroacetic acid by a strong oxidising agent (chromic acid and concentrated nitric acid) and spectrophotometric determinaton of the latter by the use of the Fujiwara reaction.

Recently, determination of trichloroacetic acid and trichloroethanol by gas chromatography was attempted.lsplg Some workers reported good results and high sensitivity, 0.1-1 pg ml-1 for trichloroethanol and 0.5-1 p g ml-l for trichloroacetic acid,18~20 while others claimed that the method is suitable only for trichloroethanol and does not give satisfactory results for trichloroacetic acid.3 This method requires more expensive equipment and specialised personnel.

In previous workz1 we studied the conditions under which the highest sensitivity can be obtained in the spectrophotometric determination of chloroform by the Fujiwara reaction. By applying these same conditions, Le., a water to pyridine ratio of 1 : 1 , 40% sodium hydr- oxide solution and measurement of the absorbance a t 530 and 367 nm (15 min and 3 h after colour development , respectively) a rapid, sensitive and simple method for the determin- ation of trichloroacetic acidJZ2 trichloroethanol and total trichloro compounds in urine was developed.

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MANTEL AND NOTHMANN 673 Experimental

Standard Solutions

analytical-reagent grade. dissolving these reagents in distilled water. necessary to add a few drops of 40% sodium hydroxide solution.

The trichloroethanol (Cl,CCH,OH) and trichloroacetic acid (Cl,CCO,H) used were of Stock solutions of concentration 10 pg ml-l were prepared by

For the dissolution of trichloroethanol it is

Apparatus

of 10-mm light path. Absorbance readings were taken with a Beckmann DB spectrophotometer with quartz cells

Procedure A 5-ml sample of urine (or a smaller volume made up to 5 ml with distilled water) was intro-

duced into a 25-ml flask having a glass stopper and 5 ml of pyridine and 10 ml of 40% m/m sodium hydroxide solution were added. After shaking the flask and contents well and heating on a water-bath a t 70 "C for 15 min, the solution was cooled to room temperature, transferred to a 40-ml separating funnel and shaken well. The two layers were allowed to separate and the aqueous layer discarded. The red-coloured pyridine layer was transferred to a 10-ml calibrated flask and diluted to volume with distilled water. The absorbance of this solution was read after 15 min at 530 nm and after 3 h at 367 nm in cells of 10-mm light path.

Calibration Graphs Volumes of 0 . 5 4 ml of the stock solutions of trichloroacetic acid and trichloroethanol were

diluted to 5 ml with urine from a non-exposed person and treated according to the above procedure. The absorbance of the trichloroacetic acid solutions was read at 530 and 367 nm and of the trichloroethanol solutions a t 367 nm only. Thus, two calibration graphs (at 530 and 367 nm) were obtained for trichloroacetic acid and one (at 367 nm) for trichloroethanol.

Results and Discussion Calculation of Results

The amount of trichloroacetic acid in the sample was calculated from the absorbance of the red pyridine layer a t 530 nm. The value for total trichloro compounds corresponded to the absorbance at 367 nm. The amount of trichloroethanol in the sample was calculated from the absorbance a t 367 nm. The absorbance due to the calculated amount of trichloroacetic acid was read from its standard calibration graph a t this wavelength and substracted from the total absorbance. The remainder was the absorbance due to trichloroethanol.

Optimum Conditions The Fujiwara reaction is not specific and is extremely sensitive to many factors,23 e.g., the

presence of organic solvents other than pyridine, the volume and concentration of the sodium hydroxide solution, heating time, temperature during the reaction and time elapsed between mixing and reading the absorbance. Variations of these factors may lead to different results for the same organic halide. Therefore, in order to obtain reliable results it is necessary to adopt a standard procedure and rigorously to maintain all of these parameters constant. The following parameters were studied.

Concentration of sodi%m hydroxide Ten-millilitre volumes of sodium hydroxide solutions of concentrations ranging from 2 to

40% m/vn were added to solutions containing 60 pg of trichloroacetic acid and treated as described under Procedure. The results obtained are shown in Fig. 1. At con- centrations below 6% m/m no phase separation was observed and no red colour developed. Nevertheless the solutions absorbed at 367 nm. With concentrations higher than 6% m/m two phases were formed, a colourless aqueous layer and a red pyridine layer. The absorbance of both layers was measured at 367 nm. The absorbance of the pyridine layer increased with

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674 MANTEL AND NOTHMANN: RAPID DETERMINATION OF TRICHLOROETHANOL Analyst, vd. 102

1.4

1.2

Q) 1.0

f 0.8 e - 0 -

51 0.6 f2 < 0.4

- - -

L -

Concentration of sodium hydroxide, % m/m

Fig. 1. Effect of the concentration of sodium hydroxide on the absorbance of a 6 pg ml-1 solution of trichloroacetic acid : A, single phase; B, pyridine layer; C, aqueous layer.

increasing sodium hydroxide concentration, while that of the aqueous layer decreased. The highest absorbance of the pyridine layer was obtained with 40% mlm sodium hydroxide solu- tion, with almost no absorbance by the aqueous layer.

Wafer to pyridine ratio Solutions containing 60 pg of trichloroacetic acid were prepared in volumes of water varying

from 1 to 9 rnI. Pyridine was added to give a total volume of 10 ml and these solutions were treated as described under Procedure. Maximum absorbance at 367 nm was obtained with a 1 : I water t o pyridine ratio. Tem@&we during the reaction aazd heating time

ance values. ation less sensitive.

AbsorPfion spectm and the efect of tiwe The absorption spectra of the Fujiwara reaction products obtained with trichloroacetic acid

and trichloroethanol were measured in the range 330-630 nm within 15 min after separation of the pyridine layer and up to 5 h later. It can

Heating the reaction mixture for 15 min at 70 "C in a water-bath gave the maximum absorb- Prolonging the heating time or increasing the temperature made the determin-

The spectra obtained are shown in Fig. 2.

c A 6

530 430 530 630 Wavelengthhm

Fig. 2. Absorption spectra of the pyridine layer (time after development of colour in parentheses) : A, a 3.8 p g ml-1 solution of trichloroacetic acid (15 min) ; B, a 3.8 p g ml-l solution of trichloroacetic acid (3 h) ; and C, a 4.5 pg rnl-l solution of trichloro- ethanol (3 h).

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September, 1977 AND TRICHLOROACETIC ACID I N URINE 675

be seen that the products obtained with trichloroacetic acid have two absorbance maxima, at 530 and at 367 nm. The intensity of the latter peak increases with time whereas that of the peak a t 530 nrn decreases and almost disappears after 3 4 h. The product obtained with trichloroethanol shows only one absorbance maximum, at 367 nm, which remains unchanged with time. Under the conditions in which the reaction was carried out in the work reported here only a small maximum was obtained a t this wavelength. Fig. 3 shows the variation with time of the absorbance of a 3.8 p g ml-1 solution of trichloroacetic acid and an 8.0 pg ml-1 solution of trichloroethanol.

A second maximum at 410 nm has been reported.l*

0.6

al c

0.3 z n a

0

Ti rn e/m i n

Fig. 3. Effect of time on absorbance for: A, a 3.8 p g m1-I solution of trichloroacetic acid at 367 nm; B, a 3.8 pg ml-' solution of trichloro- acetic acid at 530 nm; and C, an 8.0 p g ml-l solution of trichloroethanol at 367 nm.

The molar absorptivities of the Fujiwara reaction products obtained with trichloroacetic acid and trichloroethanol were calculated from the respective calibration graphs at 530 and 367 nm, at times of 15 min, 1 and 3 h after the development of the red colour. Table I shows the results obtained.

Influence of Urhe The analysis of urine samples to which known amounts of trichloroacetic acid and trichloro-

ethanol had been added showed low results, especially for trichloroethanol. In order to investigate this effect calibration graphs were prepared by diluting the stock solutions of trichloroacetic acid and trichloroethanol with urine instead of distilled water. The samples were treated as described under Procedure and the moIar absorptivities calculated. The results are given in Table I. I t can be seen that the presence of urine lowers the molar absorptivity, i.e., the sensitivity, by about 25% for trichloroethanol and to a lesser extent for trichloroacetic acid.

TABLE I VARIATKON WITH TIME AFTER COLOUR DEVELOPMENT OF THE MOLAR ABSORPTIVITIES OF TEE FUJIWARA REACTION PRODUCTS OF TRICHLOROACETIC ACID AND TRICHLOROETHANOL, USING

DISTILLED WATER OR URINE FOR DILUTION OF THE SAMPLES

Molar absorptivities f

L I At 367 nrn

At 530 nm using f A 'I distilled water Using distilled water Using urine

h 1 f

A > Compound '15 min l h 3 h 15 min I h 3 h

Trichloroacetic acid . . . .10000 0 0 17300 21300 25600 15500 18600 21 500

Trichloroethanol . . 0 0 0 7300 7 300 7 300 5 600 5 600 5 600 Total trichloro

compounds .. NM* NM NM NM NM 32700 NM NM 27000

* NM = not measured.

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676 MANTEL AND NOTHMANN : RAPID DETERMINATION OF TRICHLOROETHANOL AEaZyst, I/‘d. 102 The analysis of spiked urine samples was repeated using calibration graphs prepared with

solutions obtained by the addition of each reagent to urine. Under these conditions the results obtained showed satisfactory recoveries (Table 11). An average accuracy of & 10% was obtained. From repeated analyses of three of the spiked samples the precision of the method was calculated to be &5.5%.

TABLE I1 ACCURACY OF T H E METHOD2*

Amount Amount added/ found/ CLg 1-1 tLg 1-1 w.0 rrichloro- Tnchloro-

Urine sample acetic Trichloro- acetic Trichloro- number acid ethanol acid ethanol

1 100 100 110 89 2 50 120 45 108 3 130 80 112 71 4 50 70 53 62

Recovery / CLg 1-1 - Trichloro-

acetic Trichloro- acid ethanol

-11 y - 12 - 18 -9 $ 3 -8

Accuracy, %

T------ Trichloro- acetic Trichloro- acid ethanol

10.0 11.0 10.0 10.0 13.8 11.2 6.0 11.4

Average : f 10.0 f 10.9

Applications As mentioned before the measurement of trichloroacetic acid and trichloroethanol con-

centrations in the urine of persons exposed to trichloroethylene can be used as a measure of trichloroethylene poisoning. This solvent is widely used in industry as a de-greaser as it is not explosive and it is non-flammable. Inhaled in large amounts or for extended periods of time it has been found to be toxic to humans, resulting in shortness of breath and in heart, neuro- logical and digestive disorders. Upon inhalation, trichloroethylene is rapidly absorbed by the lungs and converted in the body by way of chloral hydrate into trichloroacetic acid and tri- chloroethanol, which are both excreted in urine.l The concentrations of trichloroacetic acid and trichloroethanol in urine that would correspond to the threshold of toxic symptoms in humans have not been definitely determined. The difficulty may be due to inexact reporting of symptoms by exposed persons in answer to the questions of medical investigators but to no less extent to the lack of simple and accura.te methods for the determination of the two metabolites in urine. The proposed method fulfils the requirements as it permits the determin- ation of the individual metabolites.

It is recommended that spot urine samples be collected rather than 24-h specimens with which the kinetics of metabolite excretion may be obscured.2 The necessary corrections for differences in urine concentrations are made by expressing the data as functions of the creatinine excretion rate in urine,25 which is practically constant.

Owing to its simplicity the method can be carried out in sitzt and factory or workshop workers, who are constantly exposed to trichloroethylene, can be routinely checked. If speed rather than high sensitivity is required the spectrophotometric readings can be taken after 1 h (see Table I). On the other hand, owing to the fact that after development of the maximum colour (after about 3 h) the absorbance remains constant for at least 24 h, a large number of samples can be prepared and measured at a later time.

Because of its high accuracy the method can be used not only as a toxicological diagnostic test but also for trichloroethylene and chloral hydrate metabolism studies in animals after injection of different doses. The fluctuations in the results can be attributed with certainty to variations in concentration, rather than to errors in measurement. Thus, the proposed method may help to establish a more exact relationship between metabolite concentrations in urine and toxic symptoms.

References 1. 2. 3.

Browning, E., “Toxicity and Metabolism of Industrial Solvents,” Elsevier, Amsterdam, 1965, p. 189. Lowry, L., Vandervort, R., and Polakoff, P., J . Occup. Med., 1974, 16, 98. Ikeda, M., Ohtsuji, H., Imamura, T., and Komoike, J., BY. J . Ind. Med., 1972, 29, 328.

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September, 1977 AND TRICHLOROACETIC ACID I N URINE

4. 5. 6. 7. 8. 9.

10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22.

23. 24. 25.

677 Kimmerle, G., and Eben, A., Arch. Tox., 1973, 30, 127. Ogata, M., Takatsuka, Y., and Tomokuni, K., BY. J . Ind. Med., 1971, 28, 386. Parker, D., Report No. Sc-M-67-431, Scandia Laboratories, Albuquerque, 1967. Grisper, R., and Griffini, M. A., Medna Lav., 1970, 61, 509. Boillat, A., Praeventivmedizin, 1970, 15, 447. Seto, A., and Schulze, M., Analyt. Chem., 1956, 28, 1625. Friedman, P., and Cooper, J., Analyt. Chem., 1958, 30, 1675. Weichart, H., and Bardodej, Z., Zentbl. ArbMed. ArbSchutz, 1970, 20, 219. Linder, J., Zentbl. ArbMed. ArbSchutz, 1973, 23, 161. Fujiwara, K., Sber. Abh. Naturf. Ges. Rostock, 1916, 6, 33. Frant, R., and Westendrop, J., Archs Ind. Hyg., 1950, 1, 308. Moss, M. S., and Kenjon, M. W., Analyst, 1964, 89, 802. Leibmann, K., and Hindman, J., Analyt. Chem., 1964, 32, 348. Tanaka, S., and Ikeda, M., BY. J . Ind. Med., 1968, 25, 214. Linder, J., and Weichardt, H., 2. Analyt. Chem., 1973, 267, 267. Berry, D., J. Chromat., 1975, 107, 107. Breiner, D., Ketelaars, H., and Van Rossum, J., J . Chromat., 1974, 88, 55. Mantel, M., Molco, M., and Stiller, M., Analyt. Chem., 1963, 35, 1737. Mantel, M., in Research Laboratories Annual Report, No. IA-984, Israel Atomic Energy Commission,

Reith, J . F., van Ditmarsch, W. C., and de Ruiter, Th., Analyst, 1974, 99, 652. Analyt. Chem., 1968, 40, 618R. Jackson, S., Hltk Phys., 1966, 12, 843.

Yavne, 1964, p. 112.

Received December 22nd, 1976 Accepted March 21st, 1977

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