0.00 to 0.08 pg. vanadium calibration curve was 1 0 . 2 for samples 1 and 2 which contained, respectively, 0.8 and 6.8 pg. of vanadium per liter. Using a 0.0 to 1.0 pg. vanadium calibra- tion curve, the standard deviations for samples 3 (18 pg. per liter) and 4 (92 pg. per liter) were 1 0 . 6 and 1 0 . 8 , respectively.

LITERATURE CITED

(1) Haffty, J., U . S. Geol. Survey Water- Supply Paper 1540-A, 1 (1960).

(2) Jarabin, Z . , Szarvas, P., Acta b'niv. Debrecen 7, 131 (1961); ( ' . A . 57, 9192e (1962).

(3 ) Naito, H., Sugawara, K. , Bull. Chem. Sac. Japan 30, 799 (1957); C . A . 52, 5706i (1958).

(4) Silvey, FV. D., Brennan, R., ANAL. CHEM. 34,784 (1962).

( 5 ) Sugawara, K., Tanaka, M., Saito, H., Bull. Chem. Sac. Japan 26, 417 (1953); C.,4.49. (3842b (1958).

RECEIVED for review Rlarch 9, 1964. Accepted April 27, 1964. Division of Water and Waste Chemistry, 147th Meet- ing, ACS, Philadelphia, Pa., April 1964. Publication approved by the Director, L. S. Geological Survey.

Fluorometric Method for the Determination of Urea in Blood

JOSEF E. McCLESKEY

Clinical Chemistry Branch, U . S. Naval Medical School, National Naval Medical Center, Bethesda, Md.

b It has been observed that the compound produced by the reaction of urea with diacetyl monoxime ex- hibits fluorescence. A study of this fluorescent property has resulted in the development of a quantitative procedure for the determination of urea. The variables studied include the method of deproteinization of blood and serum, the time of heating, the concentration of diacetyl monoxime and the effect of pH. Comparison studies with the AutoAnalyzer and recovery studies show the method to be valid.

HE NEED fop a simple and accurate T method for quantitatively deter- mining urea nitrogen in blood has pro- vided impetus for the reevaluation of several urea nitrogen procedures. A variety of methods have been developed utilizing enzyme reactions to form am- monia with subsequent titration (3). Several objectionable features to these methods are evident, via., loss of am- monia and time consumed.

Several methods have also been pub- lished which utilize the reaction of urea with diacetyl ( 4 , 6), diacetyl monoxime (5 , 7-10)] or a-isonitrosopropio- phenone (1) in acid media to form a colored compound. Color intensities are then measured to yield urea concen- tration. The methods using diacetyl or diacetyl derivatives usually suffer from the fact that the color produced does not conform to Beer's Law and is photo- sensitive.

Experimentation oriented toward de- veloliment of a blood urea nitrogen method with more desirable characteris- tics led to the investigation, in our lab- oratory, of the method of Richter and Lapointe (9). During the course of this work a fluorescent property of the urea- alpha diketone compound was dis-

covered. Subsequent work indicated that this fluorescence could be adapted to a quantitative procedure for urea. A more complete study of reagents and reagent concentrations provided an ac- curate, simple, and reproducible pro- cedure for urea nitrogen in blood.

EXPERIMENTAL

The fluorescent properties of the com- pound formed by the reaction of urea and diacetyl monoxime were studied with a n Aminco-Bowman spectrophoto- fluorometer. The fluorescent peak was observed a t a wavelength of 415 mp when the compound was activated a t an optimal wavelength of 380 millimicrons.

Reagents and Solutions. VREA

PER ML. Weigh out 2.14 grams of urea (c.P., A.c.s.) and transfer to a 100-ml. volumetric flask. Add 50 ml. of 0.Ol.V sulfuric acid and swirl to dissolve. Then dilute to 100 ml. with 0.01,V sulfuric acid.

UREA NITROGEN WORKING STAXD- ARD, 40 MG. PER 100 ML. Dilute 4 ml. of the stock standard to 100 ml. with 0.01N sulfuric acid solution.

TRICHLOROACETIC ACID REAGENT, 30%. Dissolve 30 grams of reagent grade trichloroacetic acid in a few

NITROGENS TOCK S T A N D A R D . 1 MG.

Table I . Typical Calibration Data for Determination of Urea in Blood and

Blood Serum Urea nitrogen Fluorometer

concn. mg./100 ml. reading 0 . 0

10.0 15 .0 20.0 25.0 30.0 35.0 40.0

0 0 9 0

18 0 26 0 37 0 51 0 64 5 80 0

Each sample contained 2.0 ml. of dia- cetyl monoxime and 0.30 ml. of concen- trated sulfuric acid.

millilit,ers of water. Dilut'e to 100 ml. This reagent is stable for several months at' room temperature.

D I ~ C E T Y L ~ ~ O N O X I M E . Dissolve 5.0 grams of diacetyl monoxime (Eastman Organic Chemicals) in 500 ml. of dis- tilled water. Add 150 grams of sodium chloride, 100 ml. of distilled water and shake well until dissolved. Dilut'e to 1000 ml. an3 filter. If stored in an amber bottle a t room temperature this reagent is stable for about six weeks.

Procedure. The recommended pro- cedure for the fluorometric determina- tion of blood urea nitrogen is as follows:

Add 0.2 ml. of whole blood or serum to 4.0 ml. of distilled water. Mix well and allow to stand for 10 to 15 minutes. Add 5.0 ml. of 30Y0 trichloroacetic acid to precipkate the prot,ein. Mix well and centrifuge at 2500 r.p.m. for 15 minutes or, alt,ernat'ively, filter t'hrough Whatman #40 filter paper. Pipet 2.0 ml. of the protein-free filtrate into a screw cap test t'ube (16 X 125 mm.) . .4dd 2.0 ml. of diacetyl monoxime and 0.30 ml. of concentrated sulfuric acid. Screw the cap down tightly and mix well. Heat the tube in a boiling water bath for 15 minutes. After heat'ing, release the pressure by loosening t'he cap, mix well, and allow to cool to room temperature. Run a standard (40 mg. per 100 ml.) in the same manner by using 0.2 ml. of the working standard in place of blood or serum. Measure the fluorescence in the spectrophoto- fluorometer a t a wavelength of 415 mp with an activation wavelength of 380 mp, Use the 40 mg. per 100 ml. standard to adjust the instrument to a predetermined fluorescence. An 8070 adjustment is used in t8his laboratory. Read t,he results from a previously prerared calibration curve.

RESULTS A N D DISCUSSION

Table I shows a typical calibration data using the procedure described above, but substituting standard urea nitrogen solutions for blood and serum

1646 ANALYTICAL CHEMISTRY

Table II. Comparison of the Fluores- cent Method with the AutoAndyzer

55- 1

I , , , , , , , , , , , , , , , 1 .M .lo . I5 2 0 .25 .30 -35 .LO 4 5 .50 .55 .60 .65 .70 *75

CONCD. HzSOI ADDED, MI.

Figure 1 . Effect of sulfuric acid concentration on fluorescence

samples. These solutions are prepared by making appropriate dilutions of the working standard. The points on the calibration curve can be reproduced from day to day within experimental error (*I%) when the same reagents are used. However, when new reagents or standards are used a new calibration curve has to be prepared.

The concentration of urea in blood found by this procedure was compared with the results of the AutoAnaEyzer (Technicon Instrument Co., Chauncey, N. Y.) under rout'ine conditions. De- scription and operat'ion of the Auto- Analyzer are discussed in reference (11). The agreement between the two meth- ods was satisfactory and showed an average relative error of 0.617, when the ;iutoAnalyzer results were taken as valid. The data from the comparison studies are shown in Table 11.

The reproducibility of this procedure is shown in Table 111. Each sample was run a minimum of four times with mean errors and standard deviations being calculated. The standard devia- tions for the series' shown range from 0.11 to 0.18 with an average of 0.13.

Recovery studies were made by adding known amounts of urea to blood samples which had previolisly been analyzed by this method and by the duto.4nalyzer. These determinations were carried out in quadruplicate. Re- sults are shown in Table IV.

The sensitivity of this method is much greater than the colorimetric proce- dures. Using a 0.2-ml. sample the concentration of urea nitrogen is 1-5 pg./ml. of test solution (1.7 X 10-5N). However, a sample as small as 0.025 ml. may be used which yields a concentra- tion in the final volume of test solution of 3 X 10-6.\f.

Selection of Method of Deprotein- ization. Several methods of depro- teinization of biological samples were tried. .\fter considering simplicity and t ime needed, the trichloroacetic

acid method was selected. I t was found tha t by laking 0.2 ml. of blood in 4.0 ml. of water, followed by de- proteinization with 5 ml. of 3070 trichloroacetic acid, a satisfactory filtrate could be obtained.

Effect of Time of Heating at 100' C. The formation of the urea-alphadi- ketone compound increases with time while heating a t 100" C. (2,4). The time required to produce maximum forma- tion, as reported by other workers, varies from 20 to 35 minutes. I t has been observed tha t by heating a t 100' C. under a positive pressure the opti- mum heating time for maximum fluo- rescence is from 14 to 16 min.

Effect of Varying Diacetyl Monoxime Concentration. Varying the amount of diacetyl monoxime used in a sample produced significant changes in fluorescence intensity. The fluorescence increased with addition of diacetyl monoxime up to 2.0 ml. With addition of more than 2.0 ml., the fluorescence intensity decreased slightly. This can probably be a t - tributed t o a quenching effect of the reagent itself. Based on this finding it was decided to use 2.0 ml. of diacetyl monoxime in the procedure.

Effect of Varying Acid Concentra- tion. Varying amounts of concen- trated sulfuric acid were added t o

Rela- tive

Fluorescent duto- Mean error, method Analyzer error yo

Mg./100 ml.

24.1 f 0 . 1 24 18 .1 f 0 . 1 18 26.8 f 0 .2 27 22.3 f 0 . 0 22 33.6 f 0 . 1 34 10.1 f 0 . 1 10 26.8 f 0 . 2 27

7 . 9 rt 0 . 1 8 16 .9 f 0 . 1 17 20.2 f 0 . 2 20 24 l r t 0 1 24 2 0 9 f O l 21 7 0 f 0 0 7

1 3 1 f O l 13 1 4 2 f O 2 14 18.0 f 0 .2 18

+ o . 1 + o . 1 -0.2 + 0 . 3 -0.4 +O. l -0.2 -0.1 -0.1 +0 .2 $0.1 -0 .1

0 . 0 +o . 1 +0 .2

0 . 0

0 41 0 55 0 74 1 36 1 17 1 00 0 74 1 25 0 58 1 00 0 41 0 47 0 00 0 76 1 42 o on

2 3 O f O 2 23 0 0 0 0 0 2 9 2 5 0 3 29 - 0 2 0 6 9 2 9 2 f 0 2 29 - 0 2 0 6 9 1 3 O f O 2 13 0 0 0 0 0

Average mean error = 0.13 Average relative error = 0.657'

Table 111. Reproducibility of Determinations

Mg./100 ml. Sam- Urea Standard ple nitro- LZIean Mean devia-

# gen value error tion

53 8 . 0 7 9 + O 1 0 11 -53 8 n +n 1 _ _ 53 7 . 8 53 7 . 8

, - ~

-0 .1 -0 .1

53 7 . 8 -0 .1 65 20.8 20.8 0 . 0 0.12 66 21 . o +0 .2 ~~

65 65 65 82 82 82 82 82 83 83 83 83 83

20 8 20 8 20 8 29 2 29 0 29 2 29 2 29 1 13 0 12 7 13 0 13 2 13 1

29.1

13 .0

0 . 0 0 . 0 0 . 0

+o. 1 -0 .1 +0 .1 +o . 1

0 . 0 0 . 0

+0 .3 0 . 0

+0 .2 +o . 1

0.11

0 .18

Average Standard Deviation = 0.13

Table IV. Recovery Studies

Mg./100 ml. Urea Urea Added Recovered,

nitrogen Theoretical nitrogen urea N 7c Sample value added value found recovered

13 0 10 0 23 0 22 8 9 8 98 0 13 0 20 0 33 0 33 1 20 1 100 5 18 1 10 0 28 1 28 1 10 0 100 0 9 2 10 0 19 2 19 0 9 8 98 0 9 2 20 n 29 2 29 0 19 8 90 0 9 2 18 1 27 3 27 2 18 0 99 5

These data are the average of at least four separate determinations on each sample

VOL. 36, NO. 8, JULY 1964 1647

samples of a constant concentration of urea nitrogen, and the fluorescence was measured. As seen in Figure 1, the fluorescence increased as the amount of sulfuric acid increased. The maxi- mum fluorescence intensity occurred after the addition of 0.3 ml. Further addition caused a decrease in the intensity of fluorescence. This was the amount of acid selected for use in the procedure.

The specificity of this reaction for urea has been discussed in prev ioa publica- tions (2, 10, 18). These authors show that contribution by similar compounds such as uric acid, et'c., is relatively in- significant, due to their minute presence in these samples.

;1 preliminary effort to adapt this procedure to the Coleman Model 12C

photofluorometer has thus far proved unsuccessful. This may be the result of poor resolution of the filter system which we have employed. Kork is continuing on this modification in instru- mentation.

A C K N O W L E D G M E N T

The author thanks T. E. Wheeler and R. I. Morgan for their interest and assistance during the course of this in- vest igat ion.

LITERATURE CtTED

(l! Archibald, R. M., J . Bzol. Chem. 167, 007 (1945).

(2 ) Beale, R. N., Croft, D., J . Clin.

(3 ) Day, H. G., Bernstorf, E., Hill, R.

(4 ) Dickenman, R. C., Crafts, B., Zak,

Path. 14, 418 (1961).

T., ANAL. CHEM. 21, 1290 (1949).

B., '4m. J . Clin. Path. 24, 981 (1954). (5) Frieaman, H. S., ANAL. CHEM. 25,

662 (1953). ( 6 ) LeMar, R. L., Bootzin, D., Ibid. , 29,

1233 11957). ( 7 ) satelson,' S., Scott, M. L., Beffa, C.,

(8 ) Ormsby, A. A., J . Bzol. Chem. 146,

(9 ) Richter, H. J., Lapointe, Y. S., Clin.

(10) Rosenthal, H. L., AXAL. CHEM. 27,

(11) Skeggs, L. T., Am. J . Clin. Path.

(12) Wheatley, V. R., Biochem. J . 43,

Am. J . Clzn. Path. 21, 275 (1951).

595 (1942).

Chem. 5 , 617 (1959).

1980 (1955).

28, 311 (1957).

420 (1948).

RECEIVED for review January 21, 1964. Accepted April 21, 1964. The opinions and views expressed are those of the author and are not to be construed as official or necessarily reflecting those of the 3ledical Department of the 1Jnited States Savy or the Saval Service at large.

Polarographic Behavior of Silver in Cyanide Solutions

ROBERT F. LARGE and EDWIN P. PRZYBYLOWICZ

Research laboratories, Eastman Kodak Co., Rochester, N. Y. I4650

b The polarographic behavior of aqueous solutions of the argento- cyanide ion has been investigated. A combined anodic and cathodic wave with an E l l 2 of -0.18 volt vs. SCE was observed with a solution 1 .O X 1 OP3M in argentocyanide ion, buffered at a pH of 6.7 and containing no excess cyanide ion. The wave has been shown to be the result of the anodic dissolution of mercury in the presence of cyanide ion and the re- duction of mercuric cyanide, both species having been produced by a rapid chemical reaction between mer- cury and the argentocyanide ion:

Hg f 2 Ag(CN)2-$ Hg(CN)Z f 2 Ag + 2 CN-

The analytical utility of a previously reported method has been evaluated in the light of the mechanism presented in this work, with an extension to include the simultaneous polarographic determination of silver and mercury.

H E POLAROGRAPHY of silver ion has T been described by a number of authors (2-4, 9, 13). With a dropping mercury electrode and the usual solution conditions, silver does not give a well- defined reduction wave, but rather a wave which merges with the anodic dissolution curve of mercury. Although the diffusion current may be measured and related linearly t o the concentration of silver. no distinct step-wave is observed. This results from the fact that silver ion is reduced at a potential

which is more positive than that for the dissolution of mercury. The simul- taneous reduction of ions of other metals more noble than mercury, as well as species of mercury itself, constitutes a serious interference. As a result, polarography does not offer a selective measure of silver ion, and it3 application in this area has been limited.

Complex formation has not proved successful in efforts to shift the polaro- graphic wave away from the mercury dissolution curve. In such cases, the system usually contains an escess of the complesing agent, most of which form more stable complexes with mercury than with silver. Thus, the relative position of the polarographic wave for the silver complex, as compared with that of the anodic dissolution wave of mercury, cannot be altered and again no step-wave is observed.

Recently, Dagnall and R e s t (3) have reported a procedure whereby a step- wave is obtained with solutions of the argentocyanide ion in which the excess complexing agent is masked through the use of a metal ion which forms a slightly less stable complex with cyanide ion than does silver. Nickel(I1) was chosen as the masking agent for this purpose since it forms a relatively stable cyanide compleh, and the excess nickel (11) would not constitute an inter- ference polarographically. This use of masking reactions is novel in polaro- graphic work and could well find ap- plication in other similar systems.

However, our attempts to use the system described by Dagnall and

West led to unexpected results. Cn- like the results reported by these authors, a step-wave was observed for silver which was a composite anodic- cathodic curve, the ratio of anodic to cathodic currents being approximately 1 : 1. It thus appeared that the electro- chemical process was not simply the re- duction of the argentocyanide ion, but a process of a more complex nature. This combined wave was observed previously by Bowers and Kolthoff (1) in studies of the induced reduction of colloidal silver bromide by the argento- cyanide ion. However, the electrode processes were not completely char- acterized by these authors. An in- vestigation was undertaken to define the nature of the observed wave, and to evaluate the analytical utility of the Dagnall and West procedure in view of these new developments.

EXPERIMENTAL

Apparatus. All polarographic da ta exclusive of tha t associated with the tracer experiments were obtained with a Sargent Model XV Polaro- graph, equipped with a pen speed of 3.3 seconds for full-scale, deflection. The polarographic cells and auxiliary equipment used were those marketed by Metrohm Ltd. A silver-silver chloride reference electrode was em- ployed. Solution contact was made through a saturated potassium nitrate salt bridge. The capillary employed had a drop time of 4.73 seconds and a rate of mercury flow of 1.86 mg. per second at an applied potential of -0.10

1648 ANALYTICAL CHEMISTRY