US3733177A - Methods and compositions for the determination of uric acid in blood - Google Patents

Abstract

COLORIMETRIC METHODS AND COMPOSITIONS FOR THE QUANTITATIVE DETERMINATION OF THE URIC ACID CONTENT OF BLOOD BY TREATING SERUM AT AMBIENT TEMPERATURES WITH AN ALKALINE FERRICYANIDE SOLUTION, FOLLOWED BY THE ADDITION OF FERRIC IONS AND A 5-(2-PYRIDYL)-2H-1,4-BENZODI-

AZEPINE OR WATER SOLUBLE SALE THEREOF TO PRODUCE A BRILLIANT PURPLE COLORED SOLUTION WHICH IS THEN QUANTITATIVELY MEASURED BY STANDARD COLORIMETRIC MEANS.

Description

May15,1973 B. KLEIN 7 3,733,777

METHODS AND COMPOSITIONS FOR THE DETERMINATION OF URIC ACID IN BLOOD Filed Jan. 1972 2 Sheets-Sheet 2 40 SPECIMENS/HOURQII WASH RATIO) 60 SPECIMENS/HOUR (2=| WASH RATIO) 0.6-

o l l l 2 4 s 8 IO l2 I416 mg URIC ACID/I00 ml FIG. 2

L0: we F m p n n 7 f f 0.6-

7 4o 30 so 40 L 0- SPECIMENS/HOUR (2=l WASH RATIO) FIG. 3

United States Patent- 3,733,177 I METHODS AND COMPOSITIONS FOR THE DETERMINATION OF URIC ACID IN BLOOD Bernard Klein, New Hyde Park, N.Y., assignor to HolImann-La Roche Inc., Nutley, NJ. Filed Jan. 3, 1972, Ser. No. 214,770

Int. Cl. Gllln 31/22 US. Cl. 23-230 B 11 Claims ABSTRACT OF THE DISCLOSURE Colorimetric methods and compositionsfor the quantitative determination of the uric acid content of blood by treating blood serum at ambient temperatures with an alkaline ferricyanidesolution, followed by the addition of ferric ions and a -(2-pyridyl)-2H-1,4-benzodiazepine or water soluble salt thereof to produce a brilliant purple colored solution which is then quantitatively measured by standard colorimetric means.

BACKGROUND OF THE INVENTION The need for an accurate quantitative method for determining the uric acid content in blood which utilizes a small amount of sample, does not require a large degree of technical skill, thus being readily adaptable to clinical use, and is sufficiently economical for mass screening has long been felt. Additionally, it has been considered most desirable that such a method be readily adaptableto an automated sequential or continuous flow system inorder that a great many samples may be processed rapidly and with the highest possible accuracy. Thereis a need-for such an automated sequential or continuous flow system which is capable of highly accurate results for the .diagnostic testing of large numbers of persons for the incidence of abnormal amounts of uric acid in their blood.

Uric acid is normally present in some quantities in the fluids of mammals, particularly humans, as a result of normal body functions. In a given warm blooded animal, the uric acid content of body fluids must be held within carefully prescribed limits in order to preventundesirabl e consequences. v

For example, ingestion of certain purine-containing foods which normally have no effect on blood uric acid levels, may unpredictably elevate uric acid blood levels.

3,733,177 Patented May 15, 1973 methods, although improving over the uricase and alkaline phosphotungstate processes, also do not afford the degree of sensitivity as'well as adaptability to automated procedures which are realized with the methods of the present invention.

The diagnostic compositions and methods of the present invention provide a reliable, convenient test for the quantitating of uric acid in the blood as well as affording a method whereby the quantitative determination may be carried out in a sequential or continuous flow system. Further, the diagnostic compositions and methods of the I present invention overcome many of the disadvantages of the prior art methods of determining uric acid in blood by not requiring a high degree of laboratory skill and technology, using a small specimen volume, and being highly accurate in the clinical situation.

BRIEF SUMMARY OF THE INVENTION In accordance with the invention, a 5-(2-pyridyl)-2H- 1,4-benzodiazepine, or a water-soluble salt thereof, preferably in combination with a buffer, is added with an aqueous solution of ferric chloride to deproteinized serum to which has been treated with an aqueous alkaline ferricyanide solution, whereby a purple solution is obtained which can be quantitated as to its uric acid content by standard colorimetric means.-

7 DETAILED DESCRIPTION OF THE INVENTION In accordance with the invention a compound selected from the group consisting of compounds of the formula wherein A is selected from the group consisting of Such elevation may be indicative of renal insufficiency.

Uric acid, in abnormally high concentrations in the blood, tends to crystallize out in the body joints causing a very painful inflammatory condition known as gOutJHighairic acid blood levels are also known to be associated'with such conditions as uremia and those characterized-by whiteblood cells,

an excessive destruction of the nuclei'of e.g., leukemia and pneumonia. 1 a I It is therefore important to provide a' test for uric acid which is both simple and accurate and which can be used as an adjunct to routine screening'operations in clinics and for periodic screening of patients in hospitals, nursing homes andthe like.

Prior art" methods of determining uric acidican ibe boardly classified as enzymatic, alkaline phosphotungstate and miscellaneous chemical colorimetric. The-enzymatic method utilizing the enzyme uricase suifers the-disadvam tages of requiring a prolonged incubation'gperiod =with verycareful control of the variables-in.theconditions thus requiring highly skilled laboratory personnel ergpensive equipment and a great deal of timeqThe alkaline phosphotungstate method is, disadvantageous in that ;it

lacks sensitivity which necessitated theuse of arr-excessively large amount of sample-andhas e hijbitedadyerse side reactions which create turbidity,whichngives false high readings. vThe morerecently developed chemical hydrogen, lower alkyl; and

is selected-from the C=N and 4 R4 0 B is selected from and CH R is selected from the group consisting of halogen, hydrogen, trifluoromethyl, nitro and amino; R is selected from the group consisting of and GEN; and R and R where taken together with their attached nitrogen atom form a radical selected from the group consisting of piperazinyl, lower alkyl substituted plperazinyh. pyrrolidinyl, lower alkyl substituted pyrrolidinyl, piperidinyl and lower'alkyl substituted piperidinyl;

.isiloweralkyl; and R, is selected from the group consisting of lower alkyl and hydrogen and water-soluble salts thereof, preferably in combination with a buffer, is added with an aqueous solution of ferric chloride to deproteinized serum which has been treated with an aqueous alkaline ferricyanide solution, whereby a purple solution is obtained which can be quantitated by standard colorimetric means.

Examples of benzodiazepine compounds of Formula I above which are particularly suitable as the color-forming reagent in the process of this invention include the following:

7-bromo-1,3-dihydro-1- [4- (4-methyl-l-piperazinyl) butyl]--(2-pyridyl)-2H-1,4-benzodiazepin-2-one;

7-amino-1,3-dihydro-5-(2-pyridyl)-2H-,1,4-

benzodiazepin-2-one;

1-methyl-1-[3-(7-bromo 5 (2pyridyl)-1,3-dihydro-2- oxo 2H 1,4-benzodiazepine-1-yl)propyl]urea Whose preparation is disclosed in US. Pat. No. 3,464,978, issued Sept. 2, 1969;

7-bromo-1,3,-dihydro-S- (2-pyridyl)-2H 1,4-

benzodiazepine;

7-amino-1,3-dihydro-1-methyl-5-(2-pyridyl)-1H- 1,4-benzodiazepine;

7-bromo-1,3-dihydro-(3-dimethylaminopropyl)-5-(2- pyridyl)-2H-1,4-benzodiazepin-2-one;

7-bromo-1,3-dihydxo-5-(2-pyridyl)-2H-1,4-benzodiazepin- 2-one-4-oxide',

7-bromo-1,3-dihydro-5-(2-pyridyl) -2H-1,4-benzo diazepin-Z-one;

7-bromo-1,3-dihydro-1-(fl-hydroxypropyl)-2-(2-pyridyl)- 2H-1,4-benzodiazepin-2-one; and

7-bromo-5-(2-pyridyl) 1,3 dihydro 1 [3-(N-cyanomethylamino)propyl] 2H 1,4 benzodiazepin-Z-one whose preparation is also disclosed in U.S. Pat. No. 3,464,978.

The term lower alkyl as used throughout this specification includes both straight and branched chain alkyl groups having from 1 to 7 carbon atoms such as methyl, ethyl, propyl, isopropyl and the like. The term lower alkanoyloxy refers to both straight chain and branched chain aliphatic carboxylic acid moieties such as acetoxy, propionyloxy, butyryloxy and the like. The term halo gen includes bromine, chlorine, fluorine and iodine. Also included within the purview of the present invention are the water soluble acid addition salts of the compounds of Formula I above. Any conventional Water soluble acid addition salts of the compounds of Formula I above may be utilized in the process of this invention to quantitatively determine the iron content of aqueous solutions. Among the acid addition salts which can be utilized in accordance with this invention, includes salts of compounds of the Formula I with organic or inorganic acids such as hydrochloric acid, hydrobromic acid, nitric acid, sulfuric acid, acetic acid, formic acid, succinic acid, maleic acid, p-toluenesulfonic acid and the like.

The color differentiation with varying concentrations of ferrous ions produced by the compound of Formula I above is such that the concentration of ferrous ions pro duced by the instant diagnostic reagent compositions in situ can easily be determined by standard colorimetric instruments. Furthermore, the compounds of Formula I are not sensitive to extraneous sources and therefore are not affected by trace contaminants. The method of this invention provides a simple colorimetric means for quantitatively determining the uric acid content of serum.

In accordance with the present invention, the uric acid content of serum is determined by treating a deproteinized sample of serum with an aqueous alkaline ferricyanide reagent and allowing the mixture to stand for a reasonable time, e.g., about 5 minutes, 'at ambient, i.e. room temperatures, thus forming in solution ferrocyanide ions and allantoin. An aqueous solution containing ferric ions in the form of a water soluble ferric salt and a compound of Formula I is then added wherein ferricyanide ions and ferrous ions are produced. The ferrous ions thus produced react with the compound of Formula I, preferably in the presence of a buffer, to produce a brillian deep purple color which is read colorimetrically thus affording a rapid, simple quantitative determination of the uric acid content of the sample which is ideally suited for routine diagnostic use.

In accordance with the present invention, the serum sample to betested is initially treated with a conventional deproteinizing agent. Acidic deproteinizing agents such as, for example, trichloroacetic acid or tungstic acid, are preferred. The specimen is well mixed with the deprotein izing agent in a ratio of 1:10 and centrifuged at high speed to obtain a clear supernate. A 1.0 ml. aliquot of the clear supernate containing 0.1 ml. of specimen is treated at ambient temperature with about 2.0 ml. of an aqueous solution of an alkaline ferricyanide reagent and allowed to stand for about 5 minutes. The mixture is then treated with about 1.0 ml. of an aqueous solution of a water soluble ferric salt such as, for example, ferric chloride and 2.0 ml. of an aqueous solution of a compound of Formula I. The solutions are mixed and the absorbance of the violet blue color which develops over about 10 minutes is measured at 580 nm. against both a standard uric acid solution similarly treated and a reagent blank.

The solution containing ferricyanide ions can be made from any water soluble ferricyanide salt which does not otherwise interfere with the reaction such as, for example, potassium ferricyanide and sodium ferricyanide. Potassium ferricyanide is preferred in the practice of the present invention. This reagent may be made in quantity if so desired and used as needed. The appropriate amount of potassium ferricyanide is dissolved in an aqueous alkaline medium such as, for example, a 2% sodium carbonate solution. The quantity of ferricyanide salt utilized in preparing the reagent is variable. However, a sufficient quantity must be utilized to react with all the uric acid present in the specimen to furnish a positive indication of elevated uric acid blood levels when the diagonstic method of the present invention is being utilized as a diagnostic or a mass screening tool.

Generally, it is preferred that for each 0.1 ml. of serum to be tested, the reagent solution contains from about 0.18 ,umoles to about 1.5 11110165, most preferably from about 0.35 moles to about 0.7 ,umoles of ferricyanide salt. It is preferred that the ferricyanide reagent solution to be at a pH of from about 9 to about 11. This is readily accomplished by the addition thereto of a sufficient amount of an alkali metal hydroxide or carbonate. Most preferred for this purpose is sodium carbonate.

The quantity of ferric ions added to the sampleferricyanide ion mixture is again variable However, it is preferred to utilize a quantity of ferric ion slightly in excess of the molar quantity of ferricyanide ions added to the sample. The utilization of such an excess that there will be sufficient ferric ions present to react with the ferrocyanide ions generated by the initial reaction between the ferricyanide ions and the uric acid in the sample. The ferric ions may be supplied as any water soluble ferric salt which does not interfere with the diagnostic determination such as, for example, ferric chloride, ferric nitrate, ferric sulfate and the like. Of these, ferric chloride is preferred.

The quantity of the compound of Formula I which is addedto the aqueous-reaction mixture is variable. In all instances, however, there must be a sufiicient quantity of the compound of Formula I present to react with all of the ferrous ions generated by the reaction between the ferric ions and the ferrocyanide ions. This quantity is most conveniently determined by equating the quantity of the compound of Formula I with that of the ferric ions to insure the stoichiometry of the chelation reaction.

It is preferred to maintain the test medium ata pH of about 4.0 to about 5.0, preferably about 4.5. This can most easily be accomplished by adding suitable buffers to'the ferric ion reagent and the reagent containing the compound of Formula I. Buffering these reagents also makes them stable in aqueous solution when they are made up in quantity for large scale laboratory testing.

In general, any recognized butler pair suitable for the maintenance of such a pH rangeas described above can be utilized. Preferably, there can be utilized as a buffer pair a water soluble salt of acetic acid and acetic acid. Of the Water soluble salts of acetic acid sodiu'rri acetate is preferred. However, ammonium acetate, potassium acetate or other water soluble salt of acetic acid can be used, if desired. Although the quantities of the buffer pair comprising awater soluble acetic acid salt and acetic acid are variable, the present invention contemplates the use of a sufiicient quantity of the acid component, e.g., acetic acid, to provide a final test sample having a pH in the range of from about 4.0 to about 5.0; preferably 4.5.-By fiinal test sample is meant a solution containing the ferricyanide ions, the ferric ions and the benz'odiazepine color reagent. In general, there is contemplated the preparation of a solution of both the ferric ions and the benzodiazepine color former which contains per liter about 1.0 mole of a water soluble salt of acetic acid toabout 1.0 to about 2.0 moles of acetic acid.

From the foregoing description it is evident that the compositions of the present inventionmay be utilized or handled as prepared aqueous stock solutions, aqueous concentrates or in a dry powder formLIn 'either the con- 'centrate or the power form, sufficient buifering agents are added to stabilize the compositions when the working dilutions are made and maintain the pH ofthe reaction mixture at between 4.0 and 5.0, preferably about 4.5.

In another aspect of the. invention, the diagnostic compositions of the invention may be packaged in a dry state as a diagnostic kit or reagent system. In such; a reagent system, the reagents may be packaged in amounts such that stock solutions can be formed therewith which are suitable for large scale testing either manually'o'r by continuous flow procedure as contemplated herein; Alternatively, reagent systems maybe prepared which are suitable for single diagnostic determination. A typical reagent system would also include uric acid from which -an aqueous solution would be prepared to be utilized as a standard for the colorimetric determinations. The amount of reagents utilized in a given reagent system may be easily calculated in relation to the specimen being tested from the molar quantities given herein. These calculations are considered to be Well within the purview of a person skilled in the art. A representative reagent system utilizing specific compounds would be as follows.

The above quantities represent sufficientfreag e nts for 1000 tests. ThusfReagents. A. andfiarehd sso vs 1n 2 liters of deionized water and 2 liters of a pH 4.5 acetate buffer, respectively. Reagent B is dissolved in 1 liter of a 1 normal hydrochloric acid. The uric acid standard may be prepared as a 100ml. stock solution as.described by Caraway in f sta'ndar d Methods of Clinical Chemistry (D. Seligson, eel), v01. Apps, ass-gagAsadsmis Press, NewYork (196 3). This stock: solution canthen be conveniently diluted to form standards for comparative purposes. It is also within the purview of the invention to prepare a reagent system for a single test utilizing quantities representing one thousandth of those given above. Further, the uric acid standard can be packaged in dry form or as an aqueous solution prepared as above.

In utilizing the compositions of the present invention, the addition of the compound of Formula I to the test system immediately produces the desired purple coloration. The color deepens as the reaction proceeds to completion. Accordingly, in order to insure uniform coloring, the aqueous solution should be allowed to stand until its color appears to have become constant. *In general, it has been found that the full development of the purple color will occur over a period of from about 5 to 15 minutes after the addition of the compound of Formula I. In most cases 10 minutes is a sufficient period of time to allow for full color development.

The quantitation of the uric acid in the colored sample can be carried out by any conventional colorimetric method utilizing standard spectrophotometers such as Beckman Spectrophotometer, Coleman Spectrophotometer and the like.

The principle of the diagnostic method according to the present invention is based on a series of coupled reactions. Initially, uric acid present in the sample undergoing analysis reduces the ferricyanide ion in the added first reagent to ferrocyanide ions, which in turn form ferricyanide ions and ferrous ions with the addition of the second reagent which comprises a source of ferric ions such as, for example, ferric chloride, a buffer and a com pound of the Formula I. The ferrous ions thus generated react with the compound of the Formula I to produce a brilliant deep purple color. The purple color is thereafter colorimetrically measured and the uric acid content of the sample quantitatively determined.

The quantitative determination of the uric acid content in a specimen is carried out as follows: the absorbance of the purple color developed in the sample by the method of the present invention is measured against a reagent blank at 580 nm. utilizing a standard spectrophotometer such as, for example, a Beckman DBG Spectrophotometer, employing a cuvette with a 10 mm. light path. The quantity of uric acid in the specimen is determined in the conventional manner from the absorbance of the specimen with reference to the absorbance of the color produced by a uric acid standard similarly treated. The uric acid content of the specimen is calculated in accordance with the following formula:

Uric acid content of specimen (mg. ml.)

Uric acid content of standard (mg/100 ml.)

Absorbance of specimen 'Absorbance of standard apparatus. The latter method consists essentially of mixing specimens in continuous sequential flow with normal saline, dialyzing the mixture to produce an aqueous protein-free solution containing the uric acid, mixing the aqueous solution with an aqueous alkaline solution containing ferricyanide ions, mixing the aqueous solution with an aqueous solution of a ferric salt and a compound of the Formula I at a constant pH of from about 4.5 to about 5.5 and passing the resulting solution through an apparatus which quantitatively determines the glucose content thereof photometrically.

FIG. 1 is a schematic flow diagram illustrating a continuous flow automated system for analyzing uric acid in biological fluids utilizing the diagnostic composition of the present invention.

FIG. 2 is a recording of the photometric response obtained when utilizing the automated system of FIG. 1.

FIG. 3 is a plot in terms of absorbance of the photometric response illustrated in FIG. 2.

In FIG. 1 a continuous flow automated testing system is shown schematically wherein a specimen sample to be tested, i.e., serum, is drawn up in sequence from separate sample cups in the sample plate which rotates at a constant speed to provide the system with 20-60 specimen samples with a 2:1 wash ratio per hour. A sample, so drawn, is mixed in flow with normal saline and passed through a glass mixing coil of conventional design. After the mixture has passed through the mixing coil, it is next pumped through a dialyzer module that is provided with a cellophane membrane or the like through which the uric acid passes in aqueous solution by dialysis. The dialyzer module is maintained at a constant temperature of 37 C. The residual, non-diffusable portion of the sample is discarded. As the aqueous uric acid solution passes through the dialyzer module membrane it is admixed with an aqueous alkaline solution containing ferricyanide ions, preferably in the form of potassium ferricyanide which react to form allantoin and ferrocyanide ions. The aqueous stream is then mixed in continuous flow with an aqueous solution containing ferric ions, preferably in the form of ferric chloride, and a reagent stream comprising the 5-(2-pyridyl) 2H 1,4- benzodiazepine color reagent of Formula I. The color reagent, preferably 7- bromo 1,3 dihydro-1-(3-dimethylaminopropyl) 5 (Z-pyridyl) 2H 1,4 benzodiazepin-Z-one, is maintained at a pH of about 4.5 to 5.5, preferably at about 5.0. The mixture is then passed through a second mixing coil. As the mixture is in transit through this coil, the ferric ions and ferrocyanide ions react to form ferricyanide ions and ferrous ions which in turn react with the benzodiazepine color reagent to form a brilliant purple coloration. Photometric measurements are then performed at 580 nm. in a mm. flowcell colorimeter, i.e., the absorbance of the solution to be tested in measured at 580 nm. in a flow-cell colorimeter using a 580 nm. filter. The results of the colormetric readings are recorded on a conventional recording mechanism.

The continuous flow system illustrated in FIG. 1 aspirates at a rate of to 60 specimens/hour. The rate of flow in ml./min. of the materials entering the system according to a preferred technique is illustrated in FIG. 1. The materials entering the system are pumped into it by any suitable pumping means adjusted to maintain the rate of flow illustrated in FIG. 1. The mechanism for the system of the present invention can be conveniently provided by a manifold assembly prepared in accordance with the system illustrated in FIG. 1 adaptable to the Technicon Autoanalyzer.

In FIG. 2 the absorbance of solutions containing graduated amounts of uric acid, e.g., 2 rug/100 ml., 4 mg./ 100 ml., 8 mg./ 100 ml., etc. are plotted as a graph against concentration.

In FIG. 3 the photometric response of solutions containing dilferent concentrations of uric acid is demonstrated. The drawing illustrates four separate experiments, each of which represents passage through the automated system of FIG. 1 of a sequence of at least three solutions having uric acid concentrations in the order of low to high to low, such as, for example, 2 mg. per

8 100 ml. to 16 mg. per 100 ml. to 2 mg. per 100 ml. These experiments were conducted to illustrate the sensitivity of the automated system. The difference in the response curve for similar concentration sequences represents a variance in the speed with which they were passed through the system.

The reagents utilized in connection with the automated procedure of uric acid determination comprise aqueous solutions of a ferricyanide reagent, a ferric ion containing reagent and the buffered color forming reagent. The ferricyanide reagent comprises sufiicient ferricyanide to react with all the uric acid in the sample, for example, 0.115 g. potassium ferricyanide dissolved in 1 liter of 2.0% sodium carbonate and 0.9% sodium chloride. The ferric ion containing solutions comprises sufiicient ferric ions to react with all the ferrocyanide ions formed in the initial reaction, for example, 0.27 g. ferric chloride dissolved in 1 liter of 1 N hydrochloric acid. The colorforming reagent comprises sufiicient color-forming compound to react with the ferrous in the ions formed by the reaction of the ferric ions and the ferrocyanide ions, for example, 1.88 g; of a compound of Formula I, 82.0 g. of anhydrous sodium acetate and approximately 40.0 ml. of glacial acetic acid in a liter of distilled water. The pH of the solution is maintained between about 4.4 and 4.6.

In the practice of the invention according to the automated procedure, iron-free distilled water is pumped through the system for 10 minutes. The system is then switched to reagent and the pumping is continued until a steady base line is obtained on the recorder chart. The base line is set to -0.0lA percent transmission).

The standards in the sample tray are aspirated at a rate of 20 to 60 (2:1 wash ratio) samples per hour. The specimens to be analyzed are then sampled, with a standard uric acid specimen which is aspirated intermittently to insure qualitative control.

The uric acid content of each specimen is determined by reference to a calibration curve prepared by plotting the corrected absorbances of the uric acid standards against concentrations in mg./ ml. Table I sets forth a comparison of results obtained when 10 randomly selected plasma specimens were analyzed utilizing the automated and manual uric acid procedures of the present invention and a prior art method of determining uric acid utilizing uric acid oxidase. The uric acid oxidase method utilized was that described by Remp in Standard Methods of Clinical Chemistry (R. P. MacDonald, ed.,) vol. 6, pp. 1-12, Academic Press, New York, New York 15 75.

,...I n Tabl e II, the recovery of uric acid added to pooled serum aliquots as well as standard uric acid solutions and serum standard combinations is given. An average recovery of 99.97% (98.7%-l01.0%) was realized.

Total uric Found uric L acid, mg./100 acid, 1ng./100 Recovery,

7 ml... m

Specimens combined in 1:1 ratio (mg/100 ml.) I percent Serum Pool #1, 6.4 plus Serum Pool #2, 5,5 j 6. 6. 0 100.0 Serum Pool #1, 6.4 plus Serum Pool #3, 6.3 6. 4 6. 5 101. 5 Serum Pool #1, 6.4 plus Serum Pool #4, 7.4 6.9 7.1 Y 102. 8 Serum Pool #2, 5.5 plus Serum Pool #3, 6.3-. 5.9 6.0 101. 7 Serum Pool #2, 5.5 plus Serum Pool #4, 7 .4 0. 5 6. 6 101. 5 Serum Pool #3, 6.3 plus Serum Pool #4, 7.4 6. 8 6. 7 98. 5

Mean I 101.0

Standard, 2.0 plus Standard, 4.0 3. 0 2. 9 96. 6 Standard, 4.0 plus Standard, 16.0 10. 0 10. 1 101. 0 Standard, 2.0 plus Standard, 16.0 9. 0 9. 0 100. 0 Standard, 8.0 plus Standard, 16.0 12.0 12. 4 103. 3

Mean. 100.2 Serum Pool #1, 6.4 plus Standard, 2.0 4. 2 "4.1 07.? Serum Pool # 1, 6. 4 plus Standard, 4.0 5. 2 5. 1 98. 0 Serum Pool #1, 6.4. plus Standard, 8.0 7.2 7.1 98.6 Serum Pool #1, 6.4. plus Standard, 12.0.. 9. 2 9. 5 103.2 Serum Pool #1, 6.4 plus Standard, 16.0 11.2 11.2 100.0 Serum Pool #1, 6.4. plus water 3. 2 3. 1 96.8 Serum Pool #1, 6.4. plus LiCOa solution 3.2 3.1 96. 8

Mean. 98.7

Composite mean 99. 9

The following examples further illustrate the inven- 2.0 ml. of a color reagent prepared by dissolving 1.88 tion. All temperatures are in degrees centigrade. g. of 7br0m0-l,3-dihydro-1-(3-dimethylaminopropyl)-5- EXAMPLE 1 V (2 pyridyl)-2 L I -1,4-benzodiazepin-2-one dihydrochloride I 1 in one liter of a pH 4.5 acetate buffer. The acetate buffer To a stirred solutlon of 22.0 g. of 7-bromo1,3-d1hydr0- was prepared by dissolving 272.0 g. sodium acetate tri- 5-(2-py11dyl)-2H1,4-benzod1azep1n-2-one 1n. 55.0 ml. of hydrate and 80.0 ml. glacial acetic acid in 500 ml. of dry N,N-dimethylformam1de Was treated wlth 11.0 m deionized water, adjusting the pH if necessary and dilut- Of a methanolic sOlutiOn of sodium methoxide (0.0835 ing to one liter, The solutions were, thoroughly mixed mole of NaOCH and stirred for 30 minutes. After 30 and the absorbance of the violet blue color that developed minutes, 15.0 ml. of a toluene solution containing 0.0174 was measured after about 10 minutes against a reagent mole of 'y-dimethylaminopropyl chloride 'Was thereafter blank at 580 nm. in a Beckman DBG Spectrophotometer added, and the mixture stirred at 75 for 5.5 hours. S0lusinga cuvette with a 10 mm. light path. vents were removed under reduced pressure. and the resi- The uric acid content of the specimens was obtained dual oil was dissolved in 100 ml. of dichlorornethane The by reference to a calibration curve prepared by plotting resultant solution Was washed with water, dried and evapthe absorbances (A) given by standard uric acid solutions orated. The oil was next dissolved in .100 ml. of ethyl 40 treated in the same manner against concentration or by acetate and filtered over 100 g. of activated neutral alu-' 1 the Beer-Lambert formula. Utilizing 10 mg. uric acid/100 mina (Grade I); Using ethyl acetate as the eluant, 7- ml. as a standard, the concentration of the specimen was bromo-1,3-dihydro 1 (3-dirnethylaminopropyl). 5 (2- calculated according to the formula: pyridyl)-2H-1,4-benzodiazepin-2-one was recovered from the column. 7 A580 um. (specimen) EXAMPLE 2 mm (Standard) X10=mg. uric acid/100 ml. The 7-bromo-1,3-dihydro-l-(3 dimethylaminopropyl)- 5 2. 1 4 2 f d in For comparatlve purposes, ur c acid analyses were also ample 1 was dissolved in sufficient methanol to provide conducted on a llke number of Samples utilizing a a 10 percent solution. This solution was then saturated bonate'phPsphomngstat-e Procedure described y with hydrogen chloride. A sufficient amount of ether was cafaway 111 Standard Methods of Cllnical Chemistry added to cause the solution toturn turbid. The resultant sellgson, L PP. 239-247 Academic Press, New mixture was allowed to cool for several hours. 7-bromo- York 1,3-dihydro 1 (3,-dimethylaminopropyl)-5-(2- id 1)- The results obta ned ut1l1z1ng the aforesaid two tech- 2H-1,4-benzodiazepin-2-one dihydrochloride precipitated mques are Set forth In the fQllowlng table! out on standing and 'was separated by filtration. The salt i was recrystallized from a methanol-ether mixture as pale yellow prisms, M.P. 181-183 dec. acid/10 Benzo- Pho pho- EXAMPLE V M m 0 s m N dlazepine tungstate Difference, This example demonstrates theapplicability of the test 0 pee en test i Difierence percent vmethod to the uric acid content of blood serum. 2 -3 0.1 1.6 In the method, 0.5 ml. of serum is deproteinized by the 2:? a? 8}, addition of 4.5 ml. of 5% trichloroacetic acid, allowing 1 +011 t ifs the mixture to stand at least minutes and centrifuged at 2:8 2:3 is; high speed, e.g., 2,500 r.p.m. to obtain a clear, super- 7-0. 6.8 +012 +2I9 natant fluid. A 1.0 ml. aliquot of the clear supernatant 3 3 $1 3 582, was treated with 2.0 ml. of a ferricyanide solution .which 9.1 1 +011 1 :1 had been prepared by dissolving 0.115 g. of potassium 1 2 18% i' ferric anide in one liter of 27 b ht 6:4 y a 0 y werg aqueous 1 6.0 +0.4 +6.3 solution of sodium carbonate and allowed to stand at f? 3:3 g8 f g-Z 13% room temperature for about 5 minutes. The mixture was -3 i; 713-, f 030 then treated sequentially with 1.0 ml. of a ferric chloride 2:8 2'; $8} solution prepared by. dissolving 0.27.0..g: ferric chloride -11 :9 +01 +20 5.6 -0. 2 3.7

hexahydrate in one liter; of 1: N, hydrochloric acid and 11 EXAMPLE 4 In an analogous manner to that employed in Example 3, tests were conducted wherein uric acid was added to aliquots of serum pools. Over a 20-fold range of uric acid additions, a mean recovery of 99.5% (range 93.7 to 105.7%) was realized. The results of these tests are summarized in the following table.

TABLE Recovery of uric acid added to serum pools Total uric Uric acid present; Uric acid acid, g, Uric acid Recovery, in sample, pg. added, pg. calculated found, pg. percent What is claimed is:

wherein A is selected from the group consisting of -C=N and -C=N- B is selected from the group of o H C and CH R is selected from the group consisting of halogen, hydrogen, trifluoromethyl, nitro and amino; R is selected from the group consisting of H -R1(|]-R5 hydrogen, lower alkyl and 'CI1H211N/ n is an integer from 2 to 7; R is selected from the group consisting of hydrogen, hydroxy, lower alkyl,

lower alkoxy and lower alkanoyloxy; R is 2-pyridyl; R is selected from the group consisting of lower alkyl, hydrogen,

12 substituted piperidinyl; R is lower alkyl; and R is selected from the group consisting of lower alkyl and hydrogen, and water soluble acid addition salts thereof; and

(d) colorimetrically quantitating the uric acid present by means of said color.

2. The process in accordance with claim 1 wherein said source of ferric ions consists essentially of an aqueous solution of ferric chloride buffered to a pH of between from about 4.0 to about 5.0.

3. The process in accordance with claim 1 wherein said source of ferric iron ions is an aqueous solution of ferric chloride and said source of ferricyanide ions is potassium ferricyanide.

4. The process in accordance with claim 1 wherein said color-forming benzodiazepine compound is added as an aqueous solution buffered to a pH of from about 4.0 to about 5.0 with a buffer pair comprising a watersoluble salt of acetic acid and acetic acid.

5. The process in accordance with claim 4 wherein said color-forming benzodiazepine compound is selected from the group consisting of 7-bromo-1,3-dihydro-l-(3-dimethylaminopfopyl) 5 (Z-pyridyl)-2H-l,4-benzodiazepin-Z-one and water soluble acid addition salts thereof.

6. A method for the quantitative analysis of the uric acid content of blood serum consisting esesntially of providing in continuous flow the sequential steps comprising:

(a) combining in continuous flow a measured specimen of plasma with an isotonic solution of sodium chloride;

(b) passing said mixture through a dialysis zone, thereby separating from said mixture a clear aqueous solution;

(c) mixing said clear aqueous solution with a measured amount of an alkaline aqueous solution of a water-soluble ferricyanide salt;

((1) mixing the aqueous solution produced in step (c) by concurrent flow with a first reagent comprising a measured amount of a buffered aqueous solution of a ferric iron salt and a second reagent comprising a buffered, aqueous solution of a color-forming benzodiazepine compound selected from the group of compounds of the formula wherein A is selected from the group consisting of -C=N and C=N l 4 l 0 B is selected from the group consisting of and CH R is selected from the group consisting of halogen, hydrogen, trifluoromethyl, nitro, and amino; R is selected from the group consisting of H R72JRg hydrogen, lower alkyl and CnH2I1N n is an integer from 2 to 7; R is selected from the group consisting of hydrogen, hydroxy, lower alkyl, lower alkoxy and lower alkanoyloxy; R is 2-pyridyl;

R is selected from the group consisting of lower alkyl; hydrogen,

and CEN; and R and R where taken together with their attached nitrogen atom, form a radical selected from the group consisting of piperazinyl, lower alkyl substituted piperazinyl, pyrolidinyl, lower alkyl substituted pyrrolidinyl, piperidinyl, and lower alkyl substituted piperidinyl; R is lower alkyl; and R is selected from the group consisting of lower alkyl and hydrogen thereof and water soluble acid addition salts thereof thereby forming a colored so lution; and

(e) flowing said colored solution to an analyzing zone and photometrically determining quantitatively during the flow of said colored solution through said analyzing zone the concentration of uric acid present in said sample.

7. The method in accordance with claim 6 wherein said first reagent and said second reagent are buffered to a pH of from about 4.0 to about 5.0 with a bulfer pair comprising a Water-soluble salt of acetic acid and acetic acid.

8. The method in accordance with claim 6 wherein said color-forming benzodiazepine compound is selected from the group consisting of 7-bron1o-1,3-dihydro-l-(3-dimethylaminopropyl)-5-(2-pyridyl) 2H 1,4 benzodiazepin- 2-one and water-soluble acid addition salts thereof.

9. The method in accordance with claim 6 wherein said water-soluble ferricyanide salt is potassium ferricyanide and said Water-soluble ferric iron salt is ferric chloride.

10. A reagent system for the determination of the uric acid content of blood serum samples consisting essentially of:

(a) a first container containing for each 0.1 ml. of serum to be tested from about 0.18 to about 1.5 moles of a water-soluble ferricyanide and a sufficient amount of an alkalinizing substance selected from the group consisting of alkali metal hydroxide and carbonate to render the pH of an aqueous solution thereof between from about 9 and 11;

(b) a second container containing at least an equimolar amount of a water-soluble ferric iron salt based on the moles of ferricyanide present in said reagent (a);

(c) a third container containing at least an equimolar amount of a color-forming compound selected from the group of compounds represented by the formula wherein A is selected from the group consisting of B is selected from the group of and CH R is selected from the group consisting of halogen, hydrogen, trifluoromethyl, nitro and amino; R is selected from the group consisting of H -R7-Ru hydrogen, lower alkyl and R5 -Cu'lIznN 12 is an integer from 2 to 7; R is selected from the group consisting of hydrogen, hydroxy, lower alkyl, lower alkoxy and lower alkanoyloxy; R is Z-pyridyl;

R is selected from the group consisting of lower alkyl, hydrogen,

and -CEN; and R and R where taken together with their attached nitrogen atom, form a radical selected from the group consisting of piperazinyl, lower alkyl substituted piperazinyl, pyrrolidinyl, lower alkyl substituted pyrrolidinyl, piperidinyl and lower alkyl substituted piperidinyl; R is lower alkyl; and R is selected from the group consisting of lower alkyl and hydrogen, and water soluble acid addition salts thereof based on the amount of said water-soluble ferric iron salt present in reagent (b); and

(d) a fourth container containing, as a standard, a substance selected from the group consisting of uric acid and aqueous solutions thereof.

11. A reagent system in accordance with claim 10 wherein said water-soluble ferricyanide salt in said first container is potassium ferricyanide, salt alkalinizing substance is sodium carbonate, said water-soluble ferric iron salt in said second container is ferric chloride, said colorforming compound in said third container is selected from the group consisting of 7-bromo-1,3-dihydro-1-(3-dimethylaminopropyl) 5 (2-pyridyl)-2H-1,4-benzodiazepin-2- one and water-soluble acid addition salts thereof and said first container contains from about 0.35 to about 0.7a moles of potassium ferricyanide.

References Cited UNITED STATES PATENTS 3,449,081 6/1969 Hughes 23---253 R 3,506,404 4/1970 Evans et a1. 260-239.3 D X MORRIS O. WOLK, Primary Examiner R. M. REESE, Assistant Examiner US. Cl. X.R.

260-2393 D, 239 BD

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