Electrochemical Determination of Xanthine Oxidase and Inhibitors GEORGE G. GUILBAULT, DAVID N. KRAMER, and PAUL L. CANNON, Jr. Defensive Research Division,
U. S.
Army Chemical Research and Development laboratories, Edgewood Arsenal, Md.
b A recently developed electrochemical method i s extended to the determination of xanthine oxidase and sulfhydryl-attacking compounds, such as o-iodosobenzoic acid and p-chloromercuribenzoic acid. The method i s based on the catalyzed aerobic oxidation of hypoxanthine b y xanthine oxidase at pH 7.4 in tris(hydroxymethyl)aminomethane. A small, constant current of 3.8 pa. i s applied across two platinum thimble electrodes, and the change in potential of the anode with time during the reaction i s recorded. This small current, establishes a steady, reproducible potential with an electroinactive substrate, hypoxanthine, caused by oxidation of some component of the buffer solution, probably water. Thus the need of adding a potential poiser to establish the initial potential in this analysis i s eliminated. By this procedure, 0.0002 to 0.0040 unit per ml. of xanthine oxidase from milk and 0.12 to 1.2 pg. per ml. of pchloromercuribenzoic acid or o-iodosobenzoic acid may be determined with standard deviations of 1.5, 2.7,and 2.2, respectively. Various metal ions such as Ag(l) and Hg(ll) which bind the enzyme, may be determined in overall concentration of 0.5394 to 3.236 pg. and 1.003 to 6.018 pg. per ml., respectively (2.5 X lo-’ to 1.5 X 10-6M) with a standard deviation of about 2.0. Anodic polarography i s used to study the significance of the potentials observed in the described method, and a miniaturized apparatus i s described for the determination of micro amounts of xanthine oxidase and inhibitors of this enzyme.
I
a new electrochemical method was described for the determination of cholinesterase (8), highly toxic organophosphorus compounds (4), and the kinetics of fast enzymatic reactions ( 3 ) . The method consists of measuring the change with time of the potential of a platinum anode, through which a small current is passed, during the enzymatic hydrolysis of the thiocholine iodide esters. Recently, the general applicability of the method was shown in following C D, any reactions of the type: -4 N PREVIOUS PAPERS,
+
606
ANALYTICAL CHEMISTRY
where the substrate undergoes enzymolysis or simple catalysis by B to form products C and D,one or both of which must be electroactive, and have an oxidation potential lower than that of A . I n particular, the glucoseglucose oxidase reaction was studied, in which a potential poiser, diphenylamine sulfonic acid (DPASA), was added to set the initial potential (6), since the original substrate A was electroinactive. I n many instances, it is desirable to eliminate the addition of such a potential poiser, which might produce extraneous results in the reaction mixture under study or adversely affect the electrode system. Preliminary observations indicated that a reduction in the applied current causes a lowering of the initial potential, even in a solution of a n electroinactive substrate, such as hypoxanthine, to a constant reproducible value, an effect similar to that achieved with addition of a potential poiser, such as DPAS.4, in the glucose oxidase system. This potential is apparently due to the oxidation of a component in the buffer solution, probably water. Anodic polarographic studies were undertaken to establish the actual oxidation potentials, as indicated by the half-wave potentials, which occurred with hypoxanthine, and the products of its oxidation catalyzed by xanthine oxida-e. Xanthine oxidase catalyzes the oxidation of purines to uric acid, an important step in the metabolism of this class of compounds in mammalian cells. Since this enzyme is inactivated by a number of sulfhydryl-attacking compounds, a simple, rapid instrumentable method for its determination i s desirable. Some previous methods for the determination of this enzyme involve its catalysis of the oxidation of a substrate, such as hypoxanthine : 2 H20
+ hypoxanthine + uric Acid
xanthine
0 2
+2
H202
(1)
the assay being performed either manometrically as 02 uptake ( I ) , or spectrophotometrically as uric acid formation a t 290 mp ( 7 ) , or as cytochrome c reduction at 550 mp (6). The new electrochemical method, with the use of low current, provides a simple, fast sensitive method for the
determination of xanthine oxidase, and various sulfhydryl-attacking inhibitors, such as p-chloromercuribenzoic acid or o-iodosobenzoic acid, which inactivate this enzyme. Reaction requires only 3 to 5 minutes, and the accuracy is as good as with previous procedures. EXPERIMENTAL
Reagents. All solutions were prepared from reagent grade chemicals and triply distilled water. TRISBUFFER. Tris(hvdroxymethv1)aminomethane buffer, O . l J i , p H 7.40, mas prepared by dissolving the appropriate amount of Sigma 7 to 9 buffer (Sigma Chemical Co.) in distilled water. Hvdrochloric acid (0.l.W) was used to adjust the p H in all buffers except the one used in assay of &(I) and Hg(I1) concentrations. I n the latter case, nitric acid was used. MACILVAINEBUFFERS (pH 5.70, 6.76, and 7.58) were prepared by dissolving the appropriate amounts of disodium hvdrogen phosphate, citric acid, and potassium chloride in triply distilled water ( 2 ) . HYPOXdPvTTHINE. .k stock SOlUtiOn, l O - 3 M , was prepared by dissolving hypoxanthine (Sigma Chemical Co.) in 1 ml. of 1M sodium hydroxide, and diluting with tris buffer, p H 7.40. XaNTHINE OXIDASE. -411 standard solutions were prepared by diluting xanthine oxidase, prepared from milk b y the method of Ball (1) (Worthington Chemical Co.) in tris buffer, p H 7.40. The enzyme was assayed using the colorimetric procedure of Kalckar (7) and was found to contain 2.50 enzyme units per ml. of preparation. 0-IODOSOBENZOIC ACID. The salt (K & K Laboratories) was dissolved in 1 ml. of sodium hydroxide (1111), and the resulting solution was diluted with tris buffer, p H 7.40. METALIom. Solutions of the various perchlorate or nitrate salts of &(I), Cu(II), Bi(III), Hg(II), Ni(II), Fe(II), Mn(II), Zn(II), and Mg(I1) were prepared by dissolving C.P. material in triply distilled water. Apparatus. T h e equipment used t o apply a constant current, and t o measure t h e rate of depolarization was described in a previous paper (8). Beckman thimble type electrodes were used, and the applied current was 3.8 pa. -411 determinations were performed at a constant temperature, within % l oC., and all solutions were magnetically stirred.
Alternatively, a miniaturized apparatus was used for the determination of micro quantities of xanthine oxidase and inhibitors of this enzyme. It consisted of a 5-ml. be,$ker, in opposite ends of which were sealed near the bottom two pieces OF No. 20 B & S gauge platinum wire. Around this beaker, another larger beaker was sealed, with inlet and outlet holes, similar to those on a standard condenser, to allow for the circulation of water. A constant current of 0.85 &a. was applied across the platinum electrodes by means of a 11/2-volt battery with standard resistances. The potential observed was measured with a vacuum tube voltmetchr and recorder. A11 polarograms were recorded with a Sargent Model XXI I'olarograph, using a 600-r.p.m. Sargent synchronous rotator for rotating the platinum electrode ( 5 ) . The sample was contained in one side of a n H-cdl, the other half of which contained a saturated calomel electrode. Procedures. DE?ERMINATION OF XANTHINEOXIDASX. Twenty-five milliliters of a 1 x l O - + M hypoxanthine I solution in MacTlvain: buffer, p H 6.76, I I I I I was placed in a 50-ml. beaker, and the 0 0.30 0.40 0.50 0.60 0.70 o'80 resulting solution was automatically stirred. The two phtinum electrodes E vp. S . C . E . , VOLTS and the standard ealomel electrode Figure 1. Voltage-time curve for enzymatic oxidation of hypoxanthine (SCE) were immersed into the soluby xanthine oxidase tion, and a constant w r r e n t of 3.8 pa. was applied across the platinum electrodes. The recorder was switched on, of sulfhydryl inhibitor originally present silver(1) or 20.0 to 120 pg. of merand the resulting potential of the cury(II), add 0.5 ml. of a 0.1-unitmay be determined. platinum anode us. SCE was autoDETERMINATION OF MERCURY (11) per-ml. solution of xanthine oxidase, matically recorded. At zero time, a 1.0AND SILVER(I). To 0.5 ml. of a soluand let the resulting solution sit for ml. solution of the xanthine oxidaqe to 1 minute. At the end of this incubation containing 10.7 to 50.4 pg. of be determined (coni aining 0.005 to 0.10 unit of enzyme) was added to effect the enzymatic oxidation of substrate (Figure 1). Table 1. Determination of Xanthine Oxidase and Sulfhydryl-Binding Compounds Addition of xanthine oxidase to the Xanthine oxidase magnetically stirred solution of hypoxunit/ml. of total solution Rel. error, anthine causes an oxidation to peroxide Added Found" 4. /U and uric acid, according to Equation 1. 0.000200 0.000205 f2.5 This peroxide and uric acid produced 0,000400 0.000404 +1.0 causes a reduction in the potential of 0.000800 0.000788 -1.5 the system (Figure 1). I n all de0.00120 0.00121 $0.8 terminations, the slorie of the electro0.00200 0.00201 $0.5 chemical curve was determined a t the -1.0 0.00400 0,00396 first major inflection point, as illusStd. dev. 1.5 trated in Figure 1. iifter each use, the electrodes were cleaned by rinsing wChloromerciiribenzoic acid o-Iodosobenzoic acid with distilled water. pg./ml. _ _ Of total SOlUtiOn Rei. error, pg./ml. of total soln. Rel. error, From calibration plots of A E / A t us. Added Founda 70 Added Founda % xanthine oxidase concentration, the + 3 . 3 0.120 0.120 0.124 0.122 + 1.7 activitv of the unknoan enzyme may be +0.8 0.240 0.240 0.240 0.242 0.0 calculated. 0.360 0.354 -1.6 0.360 0.351 -2.5 DETERMINATION ~ C H L O R O M E R +2.0 0.600 0.600 0.612 0.610 +1.7 0147
CURIBENZOIC A C I D AIqD 0-IODOSORENZOIC ID. To 24 ml. of a 1 X 10-6-V
solution of hvpoxanthjne in MacIlvaine buffer, pH 6.76, is added 1.0 ml. of the sulfhvdrvl-binding inhibitor to be determined (containing 2.0 to 30 pg. of material). The solution is stirred, the electrodes are immersed into the solution, and the resulting potential is recorded. -4t zero tiine, 1.0 ml. of a 0.1-unit-per-ml. solution of xanthine oxidase inhibited by the agent is added to effect the oxidation of the substrate. By means of calibration plots of A E / A t vs. inhibitor concentration, the amount
0.960 1.20
0.989 1.16
+3.0 -3.0 Std. dev. 2 . 7
Silver(I ) pg./ml. of total solution Added 0.5394 1.078 1,502 2.150 3.236
0
Founds 0.5505 1.060 1.522 2.110 3,310
~
~error, 1 . %
0.840 1.08
0.867 1.06
+3.0 -2.0 Std. dev. 2 . 2
Mercury(11) pg./ml. __ of total solution Added 1.003 2.006 3.001 4.012 6.018
~
FGG1,020 2.046 3.004 3.942 6.138
~error, 1 .
%
+2.1 +1.7 -1.7 f2.0 fl.3 fl.O -1.8 -1.8 +2.2 f2.0 Std. dev. 2 . 0 Std. dev. 1 . 9 An average of 3 or more determinations, performed by the micro and macro procedures.
VOL. 36, NO. 3, MARCH 1964
607
Table
II.
Analysis
of Xanthine Oxidase Solutions
Reported activity units /ml. Worthineton. lot 3.70 11.7
8.0
...
a
Activity found units/ml. Colorimetrically Electrochemicallya (7) 2.50
2.50
11.5
11.5
7.50 0,501
7.52 0.505
An average of 3 runs.
tion time, add the resulting solution to 20 ml. of a 1 X 10-4M solution of hypoxanthine in tris nitrate buffer, p H 7.40. Proceed as described above for assay of xanthine oxidase. From calibration plots of A E / A t us. Ag(1) and Hg(I1) concentration, the amount of these materials originally present may be determined. MICROPROCEDURE. Two milliliters of a 1 X 10-5Jf solution of hypoxanthine in LlacIlvaine buffer, p H 6.76, is placed into the miniaturized apparatus, and the resulting solution automatically stirred. A constant current of 0.85 Ma. is applied across the electrodes, the recorder is switched on, and the potential of the platinum anode us. cathode is automatically recorded. - i t zero time, a 0.1-ml. solution of xanthine oxidase (containing to 0.01 unit of enzyme) is 4 X added, and the amount of xanthine oxidase present is determined as described previously. If o-iodosobenzoic acid or p-chloromercuribenzoic acid is to be determined, 0.1 ml. of these materials (containing 0.2 to 3.0 pg. of compound) is added to the hypoxanthine solution, and 0.1 ml. of a 0.1-unit-per-ml. xanthine oxidase solution is then added. The amount of these inhibitors present is determined as described previously. If mercury(I1) or silver(1) is to be analyzed, incubate for 1 minute 0.1 ml. of 2 to 10 pg. of silver(1) or 4 to 24 p g . of mercury(I1) with 0.1 ml. of a 0.1-unit-per-ml. solution of xanthine oxidase. Add 0.1 ml. of the resulting solution to 2.0 ml. of hypoxanthine, and proceed as described previously.
and 1.9. -411 results represent an average of 3 separate determinations, a t least one of which was by the micro procedure. To test the reliability of the proposed method, a number of samples of xanthine oxidase, separated from milk by the method of Ball ( I ) , were tested and these results are given in Table 11. The results obtained electrochemically agree with those obtained colorimetrically (following the production of uric acid a t 290 mp) within 0.3%. DISCUSSION
Effect of Substrate and pH. At constant concentrations of xanthine oxidase, t h e rate of hydrolysis of hypoxanthine increases with decreasing substrate concentration from 1 X
10-3 to 1 x 10-5M, then decreases (Figure 2). At constant substrate concentrations of 10-3 to 10-5M, it was found that the enzyme activity is a maximum a t approximately pH 6.76 in MacIlvaine buffer (Table 111). Also, the rate is considerably higher in MacIlvaine buffer, pH 7.58, than in tris buffer, pH 7.40. For maximum sensitivity in assay of enzymic activity, a 1 X 10-5Alfhypoxanthine solution in PIlacIlvaine buffer, p H 6.76, was used. By this procedure, 0.005 to 0.10 unit of enzyme per ml. to 4 X unit added (or 2 X per ml. of total solution used) may be determined with a standard deviation of 1.5. Effect of Enzyme. T h e experimental curves of A E / A t obtained by addition of xanthine oxidase to hypoxanthine possess 2 slopes. Addition of enzyme causes a sharp initial decrease in the potential of approximately 30 mv. (Figure l ) , believed to be caused by a n electroactive metal ion present in the enzyme [probably Fe(II)]. Following this, a smooth, straight line is observed, $i, followed by a steeper slope, s2. These two slopes, the second steeper than the first, might be expected, based on the equations of aerobic oxidation of hypoxanthine : Hypoxanthine
+ 0: xanthine oxidase xanthine
+ Hz02
RESULTS
The results of the determination of samples of enzyme and inhibitors are indicated in Table I. I n general, samples of xanthine oxidase, containing 2 X to 4 X unit per ml. of total solution, were analyzed with a standard deviation of 1.5. o-Iodosobenzoic acid and p-chloromercuribenzoic acid, in concentrations of 0.12 to 1.2 pg. per ml. of solution were analyzed with standard deviations of 2.2 and 2.7, respectively. Finally, silver(1) and mercury(II), from 0.5394 to 3.236 and 1.003 to 6.018 fig. per ml. were determined with standard deviations of 2.0 608
ANALYTICAL CHEMISTRY
1~10-3
~ o - 4
iX10-4
5xio-5
1~10-5
5xio-6
HYPOXANTHINE, M
Figure 2.
Variation of AE/At
with hypoxanthine concentration
A.
First slope
B.
Second slope
(1)
Xanthine
+
xanthinc.
0 2
-+ oxidase
uric tcid
+ HzOz
Table 111.
(2)
I n the first step one electroactive species (H202) is produced, and in the second step peroxide and uric acid are formed, both of which are electroactive (Figure 3). I t was f o m d by repeated evaluation, that the initial slope, sl, gave the most reproducible values and showed a linear dependence of rate upon enzyme concentration. To eqtablish the eig,nificance of the initial and final potentials observed, anodic polarograrns w r e run, using a rotating platinum eleci rode, which had a n area calculated fr3m the 13 & S gauge number of 0.0052 sq. em. When a current of approximitely 40 pa. was applied across the platinum electrodes in a 1 X 10- 3.11solutior of hypoxanthine in 0.131 tris, p H 7.40, he thimble electrode very slon ly assumed a potential of about 0.97 volt E S . SCE,which tended to drift off scale. However, a lowering of the current to 3 8 pa. produced a steady initial potential a t 0 75 volt. With the smaller electrode used in polarography, this potential occurs a t a current of 0 080 pa. (Figure 3, C). Table ISs h o w the good agreement of potentials in various solutions for the two methods a h e n the constant cur-ent through the thimble anode is 3 8 pa, and the polarographic current is 0 080 pa. The polarographic curve C in Figure 3 obqerved for hypoxanthine is identical to the polarogram ibtained for a 1 X 10-3.1f xanthinci solution or a
Effect of p H and Substrate Concentration on Rate of Oxidation of Hypoxanthine by Xanthine Oxidase
[Hypoxanthine]
AE/At,
~
91
PH 5 . 7oa
1x 1x 1x 1x 1x 1x I x 1x I x 1x
6 . 76=
7.40b 7.58. 8.0b a
10-3 10-3 10-4 10-5 10-3 10-4 10-5 10-4 10-5 10-3
... ...
1.65 2.60 4.90 5.95 0.65 1 90 3 10 3 0 4 5 0.650
2.76 2.96 0.325 0 . 700 1.50 1.70 2.50
...
glucose solution ( 5 ) , and is identical to that of a tris buffer, p H 7.40, thus showing that hypoxanthine like glucose is electroinactive. By using lower current, 3 8 pa., a steady initial potential, 0.75 volt, is observed--apparently because of the oxidation of a component in the buffer solution, probably water. The final potential, 0 25 volt, is due to the peroxide produced upon oxidation of hypoxanthine. It was found experimentally that the most linrar variation of rate with enzyme concentration is obtained at a current of 3.8 pa., nonreproducible results being obtained at 5.6, 10.0, and 25.0 pa. Stability of Reagents. T h e enzyme and substrate solutions are normally stored a t 8" C. in a refrigerator when
5.0 ...
... 1.40
not in use. Xanthine oxitlast> solutions were stable for s e w r a l days under quch treatment. and lost 7570 of their activity only after 2 neeks. .Ifter 6 to 8 hours without rrfrigeration, some decrease in t h e activity of the enzyme solutions results. Solutions of hypouanthine were stable for weeks when kept on ice. Effect of Temperature. The rate of hydrolysis of substrate dc.pends on the temperature used. Hence, for best results, all determinations and
Table IV.
Potentials of Anodes in Tris Buffer, pH 7.40
1
5.0-
Potentials 1's. SCE, volt Constant current voltaniPolarmetry, ography, 3 8 pa.
0 080 pa.
0 25
0 24
uric acid
...
0 32
hypoxanthine
0 75
0 73
1 X 10-354 H20? 1 x 10-3.11
x 10-3.11
4.0-
9
Table V.
8d
Effect of Ions on Depolarization Curves
[Hypoxanthine] = 1 X 10-4.1f, [xanthine oxidase] = 0 004 unit/nil of total eolution, pH = 7.40, T = 25' (2.
3.0
r' M
5
...
... 1.30 2.90
MacIlvaine buffer.
Solutions
8
1.66 4.45
Tris buffer.
~~
sg
mv./sec.
0.002 unit/ml. 0.004 unit/nil. 0.008 unit/ml.
ci
Ingbimv. /secx. tion AE/At,
2.0
u
Ion added None 1.0
0 0.2
0.4
0.6
0.8
1.0
A P P L I E D P O T E N T I A L y-s, S . C . E . ,
1:2
VOLTS
Figure 3. Poliirograms of Hz02,hypoxanthine, and uric acid solutions described in Table IV
1 o 10 10 1 o 10 10 10 10 1o 1o 10 1o
x X X
x
1 0 - 5 ~-4g+ 10-5M Cu+2 lO-5M Bi+3 10-55f~g+* 10-531 Xi+Z 10-5LlfFe'l
X X X l O - 5 N Mn+2 X lO-5M Zn+2 x 10-5.11 ;\Ig+*
x x x
1 0 - 3 ~csIO-'M cs-
po4-2
i o - 3 ~ 1
1 45 0 0 0 76 1 45
oo
1 45 0 78 1 45 0 64 o 50
0 100 47 6 0 100 0 46 2 0 55 9 59 3
1 0
0 3 1 45
~
VOL. 36, NO. 3, MARCH 1964
609
Table VI. Effect of Time of Incubation on Determination of Inhibitors ( A ) 1 x lO-5M Ag(1) or Hg(I1) Time of
incubation, min. Blank 0 0
1.o 2.0 5.0
AElAt
mv./sec. 1.50 1.40
0 0 0
(B) 1.2 pg./ml. p-Chloromercuribenzoic Acid or o-Iodosobenzoic Acid 1.50 Blank 0.0 2.0 5.0 10.0 20.0
0 0 0 0 0
standard curves must be run a t the same temperature. A constant temperature bath was used to regulate t h e temperature t o within + l o C. Effect of Ions. T h e effect of various ions on the slope of the depolarization curves is shown in Table V. Cations t h a t bind to sulfhydryl groups greatly inhibit t h e enzyme, and thus must be absent in enzyme assays. Concentrations 1 X 10-5V of silver(1) and mercury(I1) completely inhibit the enzymatic oxidation, and 10-5N concentrations of copper(II),
iron(II), zinc(II), and magnesium(I1) cause approximately a 50% inhibition of the enzyme. A method of analysis for these cations by enzymatic inhibition is thus possible. After incubation times of 1 minute, silver(1) and mercury(I1) may be quantitatively determined in concentrations as low as 2.5 X 10-7.U (Table VI), Phosphate ion, in concentrations up to 10-3MJ does not affect the results, but cyanide which is electrochemically active [and is a known inhibitor of xanthine oxidase ( I ) ] does, and should be absent. Effect of Inhibitors. Since xanthine oxidase has a n active -SH group, a n y compounds t h a t tie up this group will inhibit its enzymatic action on hypoxanthine, and hence may be sensitively determined. Two such sulfhydryl-binding inhibitors studied mere o-iodosobenzoic acid and p-chloromercuribenzoic acid. Studies on the effect of substrate, pH, and time of incubation upon the determination of these compounds, revealed that a 1.0 x 10-5Ji solution of hypoxanthine in MacIlvaine buffer, pH 6.76, using a 0.1-unit-per-ml. solution of xanthine oxidase in 25 ml. of solution, yieldad best results. Under these conditions, incubation times of 2, 5 , 10, and 20 minutes yielded no advantage over nonincubation, and none of the compounds need be
preincubated with enzyme for good sensitivity (Table VI), The proposed electrochemical method for xanthine oxidase offers a rapid, specific, sensitive method for the determination of this enzyme and for the determination of inhibitors of this enzyme, provided the identity of these materials is known. Since only a very limited number of compounds and ions inhibit xanthine oxidase, the method is specific and sensitive for the determination of these materials. LITERATURE CITED
(1) . . Ball, E. G., J . B i d . Chem. 128, 51 (1939 j. (2) Elving, P., Olson, E. C., J . Bm. Chem. SOC.79, 2697 (1957). (3) Guilbault, G. G., Kramer, D. N., Cannon, P. L., Anal. Biochem. 5 , 208 (1963). (4) Guilbault, G. G., Kramer, D. N., Cannon, P. L., .%NAL. CHEM.34, 1437 (1962). ( 5 ) Guilbault, G. G., Tyson, B. C., Kramer, D. S., Cannon, P. L., Ibid., 35. 582 (1963). (6) Horecker, B. L., Heppel, L. A., J. Riol. Chem. 178,683 (1949). ( 7 ) Kalckar, H. M., Ibid., 167, 429 (1947). (8) Kramer, D. N., Cannon, P. L.,
Guilbault. G. G.. ANAL. CHEW 34,
RECEIVEDfor review August 6, 1963. Accepted December 18, 1963.
Brucine Method for Determination of Nitrate in Ocean, Estuarine, and Fresh Waters DAVID JENKINS and LLOYD L. MEDSKER Sanitary Engineering Research laboratory, University of California, Berkeley, Calif.
b A technique which uses brucine for the determination of nitrate in natural waters and employs controlled heating and chloride masking is described. It is not significantly influenced by variations in chloride concentration between 0 and 20 grams per liter of chloride. The method gives highly reproducible results, and in the range 0.05 to 0.8 mg. per liter nitrate nitrogen, the color produced bears an essentially linear relationship to the nitrate concentration. The standard curve can b e reproduced from day to day using the same reagents and test conditions. Recoveries of added nitrate from natural waters are quantitative but the method is not recommended for untreated sewages from which recoveries are poor.
N
although often present in concentrations of less than 0.1 mg. per liter, is the principal form of nitro610
ITRATE,
ANALYTICAL CHEMISTRY
gen in most natural bodies of water. I n quality surveys of estuarine and offshore waters it is necessary to use a nitrate determination which is unaffected either by high concentrations of chloride or by wide fluctuations in the chloride concentration. The 11th edition of Standard Jiethods (3) lists two procedures for nitrate determination in water-the phenol disulfonic acid method and tentatively the brucine method. I n the former, chloride exerts a strong interference while the latter method is not recommended for nitrate concentrations of less than 1 mg. per liter. The work presented in this paper describes an attempt to adapt the brucine method to concentrations of nitrate below 1 mg. per liter and to control the conditions of the reaction so that reproducibility and linearity may be obtained in samples containing from 0 to 20 grams per liter of chloride. The reactions of brucine and strychnine compounds with nitrate had
been recorded by the turn of the century (6, 6), but it is only recently that the quantitative potential of the reaction has been recognized. HOWever, past experience in this laboratory had shown that the brucine method as described in Standard Methods (3) gave erratic, unreproducible results with standards as well as samples. I t was therefore necessary to run a large number of standards with each group of samples. I n an attempt to increase the sensitivity of the method to nitrate concentrations below 1 mg. per liter, the sample volume was increased from 2 to 5 ml. while keeping the volumes of the other solutions the same. h typical standard curve obtained using this method is shown in Figure 1 (curve A ) . There is still a strong curvature below 1 mg. per liter of nitrate and the erratic nature persists. By close control of the reaction conditions and by use of a high concentration of chloride to mask chloride variations
