462 FRANT AND WESTENDORP: THE DETERMINATION OF [Vol. 75 The Determination of Trichloroacetic Acid in Urine Bx7 R. FRANT AND J. WESTENDORP SYNOPSIS-TWO methods are described for the estimation of trichloroacetic acid in urine, a semi-quantitative method with a possible error of about 25 per cent., and a more quantitative method with an average deviation of 5 per cent. Both methods are based on the well known Fujiwara reaction. Attention is drawn to the effect of carbon dioxide on the coloured solution. The final method proposed is suitable for samples having a minimum concentration of 10 pg. of trichloroacetic acid per ml. IN view of the widespread use of trichloroethylene, a siniple method for following its ingestion and excretion by industrial workers is of value. Studies of the metabolism of trichloroethylenel 7 2 have shown that it is converted into trichloroacetic acid.Any scheme of analysis must therefore determine trichloroethylene in air, blood and urine, and also trichloroacetic acid in blood and urine. Several excellent methods3 v4 j5 y 6 97 exist for the determination of trichloroethylene in the various media; we have, therefore, restricted our work to the determination of trichloroacetic acid in urine. EXPERIMENTAL Direct application of the well-known Fujiwara reaction3 9 4 gave orange colorations which were visually compared with a range of coloured papers. By this means standards were established equivalent to the range 1.5 mg. to 6 mg. of trichloroacetic acid per litre. This range proved insufficient to accommodate all the specimens received and it was found necessary to dilute the samples with urine free from trichloroethylene and trichloroacetic acid.It was noticed that, on standing, the pyridine layer changed colour from orange to yellow and this has been shown to be due to reaction with the carbon dioxide in the air; if this is excluded, the layer is orange. DIRECT METHOD- Pour the mixture on to 4.0 ml. of pyridine contained in a test tube. Close the mouth of the tube with a rubber bung with a tube leading to an absorption apparatus containing potassium hydroxide. Compare the red- orange coloration of the pyridine layer with a previously standardised series of coloured papers. Traces of trichloroacetic acid too small for determination by this means may be detected by passing a stream of carbon dioxide through the pyridine layer.A colour change from orange to yellow may then be perceived at a concentration as low as 1 mg. of trichloroacetic acid per litre. Table I shows the results obtained when this method was applied to a series of artificial standards. Apart from poor reproducibility this method suffers from the need for dilution with normal urine of samples containing large amounts of trichloroacetic acid. It was, therefore, decided to adapt the method to photometric determination. PHOTOMETRIC METHOD- The existence of two phases was considered a hindrance to successful photometric determination and attention was turned to the possibility of obtaining a single phase a5 in the process developed by Rogers and Kay7 for the estimation of carbon tetrachloride in air.The difficulty in applying this method to the determination of trichloroacetic acid in urine lay in the necessity of obtaining the chloro-compounds in acetone solution. Fortunately, acetone is immiscible with strongly alkaline aqueous solutions and we were thus able to obtain the required acetone solution by extracting alkaline urine with a measured volume of acetone. The use of ether instead of acetone was suggested, but although this would enable us to dispense with the addition of alkali, it was felt that the high vapour pressure of ether and its partial miscibility with water made acetone the more satisfactory solvent. The method finally adopted was as follows. Procedure-To 20 mi. of urine add 10 mi. of 12.5 K sodium hlvdroxide and mix.Heat to 70" C. for exactly 5 minutes, and cool rapidly.Sept., 19501 TRICHLOROACETIC ACID rx URINE 463 During the investigation into the suitability of this extraction for photometric work the following procedure was employed. TABLE 1 RESULTS OF DIRECT METHOD APPLIED TO KNOWN SOLUTIONS OF TRICHLOROACETIC ACID -%mount added in 20 ml., _\mount found, Error, Pba. PLg' '0 40 45 - 12.5 40 45 t 12.5 40 45 -y 12.5 50 45 - 10 50 45 - 10 70 60 - 14 90 90 0 90 90 0 100 90 - 11 110 60 - 45 120 120 0 120 90 - 25 190 90 - 25 140 120 - 14 oc Procedure-To 26ml. of urine add 10ml. of 12-5A7 sodium hydroxide and 10ml. of acetone. Shake vigorously for 5 minutes, and transfer 2 ml. of the acetone layer by means of a pipette into a test tube. Add 4 ml. of alkaline pyridine solution (100 ml. of pyridine, 40 ml.of water, 0.48 ml. of 15 per cent. sodium hydroxide) and heat the tube a t 70" C. for exactly 5 minutes, and then cool with running water. Immediately after cooling,- transfer the coloured solution to a 5-mm. cell and measure its extinction at 5400 A. with a Bleeker photo-electric colorirneter. Visual comparison of repeated acetone extracts from a single sample of urine led us to believe that almost complete separation of trichloroacetic acid and trichloroethylene was achieved with only a single extraction. The results obtained by single extraction are shown in Table 11; they suggest that Beer's law is followed by the coloured solution. TABLE I1 RESULTS OF SINGLE EXTRACTIONS OF SAMPLES OF URINE CONTAINING T7.-2RIOUS AMOUNTS OF TRICHLOROACETIC ACID Trichloroacetic acid, x 104 /*g-/ml.E I.: - Eo E - Eo (= C) c 0 0 g;;} Eo 10 0.122 0,076 '76 10 0.123 0.077 77 30 0.204 0.158 79 20 0.198 0.1qj2 76 A study was next made of the effect of variation in the time of heating the pyridine - It is clear from this table that the acetone mixture; the results are shown in Table 111. TABLE 111 EFFECT OF VARIATION IN TIME OF HEATING Trichloroacetic Heated for 5 minutes Heated for 10 minutes A acid, r 1 -- x 104 E - Eo E - E, (= C ) E C E E-Eo ____ E-EO ~ x 104 /*.g-/ml- 0 0.040 0.040 0 0.043 0.043 30 0.226 0.184 61 0.259 0.2 17 72 30 0.223 0.181 60 0.258 0.216 72 40 0.295 0-253 63 0.326 0-284 71 40 0.893 0.25 1 63 0.334 0.292 73464 FRANT AND WESTENDORPI THE DETERMINATIOS OF [Vol.75 time of heating is an important factor. The fact that the values (E - E,)/C x 1W are lower in Table I11 than in Table I1 is attributed to variation in the alkali content of the pyridine - sodium hydroxide mixture and we suggest that more consistent results might be obtained by mixing 100 ml. of pyridine with 40 ml. of 0.18 per cent. sodium hydroxide. The application of the method to urines containing large amounts of trichloroacetic acid with heating for 7 minutes gave the results shown in Table IV. RESULTS OBTAINED ON URINES CONTAINING LARGE AMOUNTS OF TRICHLORO*lCETIC ACID Heating time = 7 minutes Trichloroacetic acid, 0 0 30 30 60 60 90 90 120 150 150 PfT /ml- E 0.034 0.043 0.213 0.217 0-387 0.409 0.61 1 0-639 0,841 1.020 1.020 E - E, x 104 E - E, C 0.174 0-178 0.348 0.370 0.572 0.600 0.802 0.980 0.980 55 59 58 62 04 6 i 67 6.5 05 APPLICATION TO HIGH CONCENTRATIONS- An attempt to apply the method to higher concentrations by diluting the acetone before taking an aliquot proved unsatisfactory as the extract became turbid on the addition of pure acetone.This difficulty was overcome by diluting the acetone extract with the acetone extract from a trichloroacetic acid- and trichloroethylene-free urine. The results given in Table V were obtained with a urine containing lZOpg. of trichloroacetic acid per ml. TABLE V RESULTS OBTAINED AFTER DILUTION WITH ACETONE EXTRACT FROM TRICHLOROXCETIC ACID- AND TRICHLOROETHYLENE-FREE URINE Acetone extract, ml. 2 2 1.6 1-6 1.2 1.2 0.8 0.8 0.4 0 0 (= 4 Blank extract, ml. 0 0 0 4 0 4 0.8 0.8 1.2 1.2 1.6 2.0 2.0 E 1.010 1.020 0.716 0.773 0-60 1 0.608 0.423 0-420 0.238 0,030 0.036 E - E, 0.977 0.987 0.683 0.740 0.568 0.575 0.390 0-387 0*20.3 E - E, :: s 104 82 82 71 $7 79 so 81 81 85 120 a These results suggested that analysis of a urine sample containing an unknown amount of trichloroacetic acid might be successfully carried out by this method, provided that a urine containing a known amount of trichloroacetic acid were examined concurrently to serve as a standard.The method was found to be improved by closing the mouth of the test tube with a plug of cotton wool containing solid potassium hydroxide to remove carbon dioxide from the atmosphere during the colour development, and by adding 1 ml. of boiled water to prevent turbidity. We thus had two methods applicable to urine samples containing large amounts of trichloroacetic acid, viz., (a) to dilute the sample with trichloroacetic acid-free urine, and (b) to take an aliquot from the acetone extract.A sample was taken, which a semi-quantitative examination by the method already outlined had shown to contain about 600 pg. of trichloroacetic acid per ml., and was examined by both methods. The results are shown in Tables VI and VII.Sept., 19601 TRICHLOROACETIC ACID I N URINE TABLE VI 466 Sample, ml. 0 0 1 1 2 2 3 3 4 4 5 5 (= P ) RESULTS ON Trichloroacetic acid-free urine, rnl. E 25 0.009 25 0.009 24 0-152 24 0.155 23 0.261 23 0.261 22 0.383 22 0.386 21 0.533 21 0.500 20 0.627 20 0-6J 1 DILUTED SAMPLE E - E, x 103 P E - EO 0.143 0.146 0-252 0-252 0.374 0.377 0.524 0-49 1 0.618 0.642 Sample containing 60 pg./ml.. . .. .. 0.320 Acetone extract sample, ml. (= 4 ) 0 0 0.2 0.2 0.4 0.4 143 146 126 126 125 126 131 123 124 128 TABLE VII RESULTS ON ACETOSE EXTRACT OF SAME s h m x Blank extract, E - E, x 10' 4 ml. E E - Eo 2.0 0.009 2.0 0.009 1.8 0.29' 0.283 142 1.8 0.293 0.284 142 1-6 0-578 0.569 142 1.6 0.580 0.57 1 143 Sample containing 60 pg./ml. . . .. .. 0.330 pg./mf. 67 1 684 59 1 59 1 586 59 1 615 57 7 582 600 609 - pg./ml. 533 533 533 536 534 - A series of urines was then examined to determine the accuracy of the method; the results These results were considered satisfactory of this examination are shown in Table VIII. and the final method is given in detail below. Trichloroacetic acid added, pg. /ml. PO 40 80 80 133 133 120 120 173 173 211: 214 213 213 254 234 '267 267 Test 2 2 2 2 2 2 1 1 1 1 1 1 0.5 0.5 0.5 0.5 0.5 0 .5 T-4BLE VIII EXAMINATION OF ACCURACY Blank E 0 0 0 0 0 0 1 1 1 1 1 1 1.5 1.5 1.5 1.5 1.5 1.5 0.206 0.218 0.429 0.405 0.736 0.717 0.330 0-318 0.486 0.484 0-605 0.555 0.318 0-286 0.365 0.32 1 0.357 0.35." Blank 0 2-0 0.007 Blank 0 2.0 0.005 OF METHOD E - E, acid found, Trichloroacetic CLg. /ml. 0.200 364 0.212 38.5 0.423 76.6 0.399 724 0.730 132.5 0.71 1 129.0 0-324 117-5 0.312 113.1 0.480 174 0.478 173.5 0.599 217.5 0.549 199 0.312 216 0.280 203 0.359 260.2 0.3 15 228.5 0.35 1 254 0.346 25 1 Standard 2 Standard 2 0.336 0.326 ) 0 0.342 0 0.332 Average deviation . . .. Error, - 9.0 - 3.6 - 4.0 - 9-5 - 0-4 - 3-2 - 2.0 - 6.6 +- 0.6 + 0.3 + 1.7 - 7.0 +- 1.4 - 4.7 $.2.9 - 9.6 - 4.5 - 6-0 % - 4.3466 FRANT AND \YESTENDORP [Vol. 75 METHOD REAGENT- and 0-48 ml. of 15 per cent. sodium hydroxide. Alkaline pyridine reagent-A solution containing 100 ml. of pyridine, 40 ml. of water PROCEDURE- To 25 ml. of the specimen, add 10ml. of 12.5 N sodium hydroxide and 10-Oml. of acetone. Also carry out this procedure on a trichloroacetic acid-free urine and on 24ml. of trichloroacetic acid-free urine to which has been added 1 ml. of 0.15 per cent. trichloroacetic acid solution. Transfer 2-ml. portions of the acetone extract into test tubes by means of a pipette. If the amount of trichloroacetic acid is high, take a smaller quantity from the sample extract and dilute to 2ml. with acetone from the blank determination. Add 4 rnl.of alkaline pyridine reagent and close the tubes with a plug of cotton wool containing a little solid potassium hydroxide. Heat the tubes at 70” C. in a water-bath for 7 minutes, remove them from the bath and rapidly cool to room temperature under running water. Lift the cotton wool plugs, add 1.0 ml. of boiled water from a burette and replace the plugs. Finally transfer the coloured solutions to 5-mm. cells and record their extinction by means of a Bleeker photo-electric colorimeter and using a wavelength of 5400 A. The trichloroacetic acid in the sample (or samples) may be determined by comparison of the extinction of the sample with that of the standard. This method is suitable for samples having a minimum concentration of 1Opg. of tri- chloroacetic acid per ml. Agitate the whole vigorously for exactly 5 minutes. REFERENCES 1. 2 . 3. 4. 6. 6. 7. Barrett, H. M., and Johnston, J . H., J . Biol. Chent., 1939, 127, 765. Powell, J . F., Brit. J . I n d . Med., 1945, 2, 142. Forssman, S., Svenska Lakartidn., 1945, 29, 1. Fujiwara, K., S.B. Natuyf. Ges. Rostock., 1914, 6, 33. Jacobs, M. B. , “Analytical Chemistry of Industrial Poisons, Hazards and Solvents,” Interscience Burgen, A. S. V., Brit. Med. J . , 1948, 1, 1238. Rogers, G. W., and Kay, K. K., J . I n d . Hyg. Toxicol., 1947, 29, 229. Publishers, Inc, New York, 1941. THE MEDICAL DEPARTMENT AND ANALYTICAL LABORATORY N.V. PHILIPS GLOEILAMPENFABRIEKEN EINDHOVEN, HOLLAND December, 1949