STABILIZATION OF DIAZONIUM SALT SOLUTIONS

Abstract: The stability of diazonium salt solutions useful as colorimetric reagents in clinical laboratory determinations of various component levels in body fluids has been found to be enhanced by the inclusion of certain surfactants.

Description: STABΪLIZATION OF DIAZONIUM SALT SOLUTIONS The stability of diazonium salt solutions useful as colorimetric reagents in clinical laboratory determina- tions of various component levels in body fluids has been found to be enhanced by the inclusion of certain surfactants. This invention relates to a method for enhancing the stability of certain diazonium salt solutions used in clinical laboratory determinations of enzyme levels in various body fluids. Diazotized aromatic amines are conventionally added to clinical tests as an indicator means to determine the concentrations of various components in body fluids. Acid phosphatase is present in the prostate gland of humans and, in cases of cancer to the prostate, the enzyme is released into the blood stream. Cancer of the prostate, therefore, can be diagnosed by tests, such as that disclosed in United States Patent No. 3,002,893, which measure serum acid phosphatase. This is determined by a process in which serum is incubated with a buffered phenolic phosphate ester substrate, and after incubation, an azonium salt of an aromatic amine containing from one to two a ino groups per molecule is added. This azonium salt reacts with the enzyme-liberated phenol, and the resultant color of the test solution is proportional to the prostatic acid phosphatase activity in the serum being tested. Gluta ic-oxalacetic transaminase enzyme levels in blood serum or other body fluids is indicative of certain types of cellular destruction as found in, for example, yocar- dial infarction, intrahepatic lymphoma or carcinoma, and UTE SHEET OMPI the like. A method for determining glutamic-oxalacetic transa inase levels in body fluids is disclosed in United States Patent No. 3,069,330. As discussed in United States Patent No. 3,069,330, the level of glutamic- oxalacetic transaminase enzyme is determined by a process in which a body fluid is incubated with a buffered mixture of L-aspartic acid and o-ketoglutaric acid. After incuba¬ tion, an azonium salt capable of coupling with the oxal¬ acetic acid formed by enzymatic action on the substrate is added, and the resultant color of the test solution is proportional to the glutamic-oxalacetic transaminase level in the fluid being tested. In addition to the use of diazonium salts as indicators for enzyme determinations, they are also conventionally used as indicators for the detection and quantitation of 2-napthol (and other phenols) and for bilirubin. Although the diazonium salts for these and other clinical tests using colorimetric determinations can be stabilized as solids in the form of double salts of zinc chloride and the like, they are normally hydroscopic and, therefore, if not kept in tightly sealed containers they will absorb moisture from the air and become unstable. The diazotized aromatic amines used as colorimetric indi¬ cators are also unstable in solutions. While stability of these diazonium salt solutions can be enhanced by keeping the solution cold, dark, and at an acid pH, even under these conditions significant deterioration as evidenced by discoloration, precipitate formation, and loss of reactivity is seen in a relatively short time. I have now discovered that the addition of relatively small amounts of certain surfactants significantly improves the stability of diazonium salt solutions. ET - Although the mechanism for this unexpected improvement in stability is not clear, the addition of these surfactants also surprisingly prevents the discoloration and precipitate formation noted above. Among those surfactants that can be added to diazonium salt solutions to increase stability are various non-ionic surfactants (such as those disclosed in Kirk-Othmer, Encyclopedia of Chemical Technology, 3rd Edition, volume 8, pages 912 et seq) . This classification of surfactants includes the polyethoxylated fatty alcohol ethers such as polyoxyethylene 23 lauryl ether sold under the tradename of Brij 35 by ICI Americas, Inc. of Wilmington, Delaware; polyethylene glycol p-nonylphenyl ethers such as that sold under the tradename of Igepal CO 630 by GAF of Wayne, New Jersey; and ethoxylated tridecyl alcohol sold under the tradename of Lipal 610 by Drew Chemical Corporation of Boonton, New Jersey. Polyethoxylated fatty acid esters such as that sold under the tradename of Myrj by ICI Americas, Inc., and polyethoxylated sorbitan fatty acid esters such as that sold under the tradename of Tween by ICI Americas, Inc. , also show the ability to enhance diazonium salt solution stability. The Myrj and Tween surfactants are not very stable in an acid environment, however, and should be avoided if the solution is to be kept at an acidic pH. Other surfactants which may be used in the practice of the present invention are those classified as cationic surfactants such as Monaquat PTD sold by Mona Industries of Paterson, New Jersey, and described in United States Patent No. 4,209,449. Amphoteric surfactants such as Miranol H2M sold by Hiranol Chemical Company, Inc. of Irvington, New Jersey may also be used in the practice of the present invention. Surfactants tested which are - TRSX .UBSTITUTE SHEET ( . - j ≥S. classified as being anionic have not shown the ability to increase stability of diazonium salt solutions. Table 1 shows the enhanced recovery of diazotized 5-nitro- 2-aminomethoxybenzene (Fast Red B, one of the many conven¬ tional diazonium salt solutions used as colorimetric clinical reagents) from solutions prepared by adding Fast Red B to a 0.3N hydrochloric acid solution in a 1 mg to 1 ml proportion. In all instances shown, the experimental solutions were kept at 37°C. Table 1 shows the enhanced stability obtained with the addition of amphoteric, cationic, and nonionic surfactants to diazonium salt solutions. The effect noted for the sodium lauryl sulfate surfactant listed in the table carries to all those anionic surfactants tested - although an initial enhancement may occur, this rapidly decreases to an effect which differs little from that found in those solutions to which no surfactant has been added. The data contained in Table 1 indicates that differing concentrations of the same surfactant have differing effects upon the recovery of the diazonium salt from solution. This concentration phenomena was studied, therefor, for a number of different surfactants. The results of a number of these studies are tabulated in the following tables. SUBSTITUTE SHEET TABLE 1 EFFECT OF SURFACTANT CLASSES ON THE RECOVERY OF FAST RED B FROM SOLUTIONS (percentage recovery) Surfactant Percent of In Surfactant Class Concentr. 3 Days 6 Days 8 Days 10 Days tlonaquat P-TD Cationic 0.33% 97 79 69 59 X 3.3% 87 75 71 pi 61 Lipal 610 Nonionic 0.67% 83 58 41 27 2.5% 78 50 27 12 Igepal CO 630 Nonionic 2.5% 88 80 72 58 Brij 35 Nonionic 1.25% 92 85 71 59 TABLE 2 STABILITY OF DIAZONIUM SALT (percentage recovery) Brij 35 (%) DAYS; 2 4 6 8 0.0 80 49 26 12 0.04 84 64 46 30 0.1 89 76 60 48 0.2 93 81 67 57 0..4 95 83 68 61 1.0 97 82 64 55 2.0 95 78 58 44 4.0 92 67 38 23 SUBSTITUTE SHEET OMPI TABLE 3 STABILITY OF DIAZONIUM SALT (percentage recovery) Monaquat P-TD (%) DAYS: 2 4 6 8 0.0 75 44 24 11 0.1 92 73 64 45 0.2 95 82 76 50 0.4 95 86 83 70 1.0 94 87 82 70 2.0 89 84 79 66 4.0 83 77 72 61 SUBSTITUTE SHEET ( ._ OMPI _ TABLE 4 STABILITY OF DIAZONIUM SALT (percentage recovery) Igepal CO 630 (%) DAYS: 0.0 75 44 24 11 0.1 78 60 42 20 0.2 86 73 64 41 0.4 92 83 75 57 1.0 92 85 78 63 2.0 89 84 77 64 4.0 87 81 73 59 As represented in Tables 2, 3, and 4, the effect of the surfactant concentrations on the stability of diazotized 5-nitro-2-aminomethoxybenzene shows a marked improvement with as little as 0.04% surfactant added to the diazonium salt solution; as increasing amounts of surfactant are added, the stability of the salt solution increases to a maximum point of stability, and then begins to decline. In Table 2, for example, the two day maximum point of stability is reached with the addition of 0.4 to 2.0% Brij 35; increasing the concentration of Brij to 4.0% results in a decrease in stability of the salt solution. This discovery of a maximum point of stability (a concentration range resulting in maximum stability of the solution) SUBSTITUTE SHEET OMPI appears consistent for other non-ionic, amphoteric, and cationic surfactants tested. Although stability for diazonium salt solutions is enhanced over a broad concen¬ tration of surfactant, because of the maximum point of stability, a preferred concentration range of surfactant would be 0.04% to 4.0%. More preferred ranges, however, would be 0.2% to 2.0%, or 0.4% to 2.0%. As discussed previously, the stability of these salts in solution can be enhanced if the solution is kept cold. This stability is even more enhanced by the addition of a small amount of surfactant. Table 5 shows the effect of adding 0.8% Brij 35 to a 0.05% diazotized 5-nitro-2- aminomethoxybenzene solution in 0.1N hydrochloric acid. _-.. _Eι-Et_. TT f OMPI TABLE 5 STABILITY OF DIAZONIUM SALT (percentage recovery) Days of 37°C Room Temp. 4°C Storage +Brij -Brij +Brij -Brij +Brij -Brij 3 85 18 80 7 43 0 100 79 16 99 41 24 93 7 38 82 Θ 100 87 50 76 0 99 73 80 55 0 100 52 143 94 21 205 95 0 Diazotized sulfanilic acid solutions, that is the indica- tor means for clinical determination of bilirubin, was also tested to determine the stability of this reagent with the addition of surfactant. The surfactant chosen was Brij 35 at 1.0%, and stability was examined at both room temperature and at 4°C. As shown in Table 6, stability is also improved by the addition of surfactant to this test reagent. O PI TABLE 6 STABILITY OF DIAZOTIZED SULFANILIC ACID (percentage recovery) Days of Room Temperature 4°C. Storage +1% Brij -Brij +1% Brij -Brij 2 95 90 100 100 4 69 37 100 96 39 92 81 38 54 13 In all instances of times and temperatures, the effect of adding the surfactant enhanced the stability of the test diazonium salt solutions. As discussed previously, the present invention is directed to a means of stabilizing diazonium salt solutions, especially those solutions or reagents which are used in clinical determinations. Although each reagent may vary in accordance with the determination to be made, the addi¬ tion of a relatively small amount (that is from about 0.01 to 4.0%) of an amphoteric, cationic, or nonionic surfac¬ tant has been found to greatly enhance the stability of the reagent. A particularly stable reagent useful in the quanitification of alpha-naphthol released from alpha- naphthylphosphate in an assay for the enzyme acid phospha¬ tase, for example, consists of a diazonium salt solution containing 0.05 grams of diazotized 5-nitro-o-anisidine and 0.8 grams of Brij 35 in 100 ml. of 0.3N hydrochloric acid. E SH EE T From the foregoing description, one skilled in the art can easily ascertain the essential characteristics of the present invention and without departing from the spirit and scope thereof can make various changes and/or modifi- cations to the invention for adapting it to various usages and conditions. Accordingly, such changes and modifica¬ tions are properly intended to be within the full range of equivalents of the following claims. Having thus described my invention and manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same. SUBSTITUTE S-H

Claims: I Claim ; 1. A liquid composition of matter for the determination of components found in body fluids which composition includes a diazonium salt, the improvement which comprises said composition further comprising a surfactant selected from nonionic, amphoteric and cationic surfactants in a concentration of from 0.04% to 4.0%. 2. The liquid composition of Claim 1 wherein the surfactant is in a concentration of from 0.2% to 2.0%. 3. The liquid composition of Claim 2 wherein the surfactant is in a concentration of from 0.4% to 2.0%. 4. The liquid composition of Claim 1 wherein the surfactant is a non-ionic surfactant. 5. The liquid composition of Claim 4 wherein the surfactant is selected from the group consisting of polyethoxylated fatty alcohol ethers, polyethylene glycol p-nonylphenyl ethers, ethoxylated tridecyl alcohol, polyethoxylated fatty acid esters, and polyethoxylated sorbitan fatty acid esters. 6. The liquid composition of Claim 4 wherein the surfactant is polyoxyethylene 23 lauryl ether. 7. The liquid composition of Claim 1 which comprises 5- nitro-2-aminomethoxybenzene. 8. The liquid composition of Claim 1 which comprises diazotized sulfanilic acid. SUBSTITUTE SHEET f OMPI 9. The liquid composition of Claim 1 which is a colorimetric reagent for the determination of a component within a body fluid. O Π

Inventors: BABSON ARTHUR, BABSON, ARTHUR
Applicants: BABSON ARTHUR, BABSON, ARTHUR
Application year: 1983
Main classification: C09K3
IPC codes: C12Q1, G01N33