US 3753863 A
Description (OCR text may contain errors)
United States Patent O m 3,753,863 REAGENT FOR MEDICAL TESTING WHICH CON- TAINS A BENZIDlNE-LIKE COMPOUND Roy E. Speck, Indianapolis, Ind., assignor to Bio-Dynamics, Inc., Indianapolis, Ind. No Drawing. Filed Nov. 22, 1971, Ser. No. 200,842 Int. Cl. G01n 31/14 US. Cl. 195-103.5 C 11 Claims ABSTRACT OF THE DISCLOSURE A liquid reagent is disclosed which has improved stability. 'It has been found that reagents containing benzidine or benzidine-like compounds are made stable if they contain at least about 50% of a compound selected from the group consisting of 1,2-ethanediol, 1,2-propanediol, 1,3-propanediol, 1,4-butanediol, 2,3-butanediol and 1,2,3- propanetriol.
BACKGROUND OF THE INVENTION This invention relates to the field of medical testing. In particular, it relates to tests which use reagents containing benzidine or benzidine-like compounds such as o-dianisidine and o-tolidine.
In the prior art, reagents containing benzidine-like compounds had to be prepared shortly prior to use if they contained significant quantities of water. Even reagents containing only absolute ethanol or anhydrous methyl alcohol (with the benzidine-like compounds) could only be kept for a few months, and they had to be refrigerated to obtain such stability.
This problem of stability caused many suppliers who sell predispensed medical testing kits to freeze dry the dispensed reagent to impart increased stability. This was somewhat successful in increasing stability, but it greatly increased the cost.
SUMMARY OF THE INVENTION The invention relates to reagents which are used in medical tests and which contain benzidine or benzidinelike compounds. It has been found that such reagents are made stable if they contain at least about 50% of a compound selected from the group consisting of 1,2-ethanediol, 1,2-propanediol, 1,3-propanediol, 1,4-butanediol, 2,3- butanediol, and 1,2,3-propanetriol.
The invention provides stability even at elevated temperatures for at least a year. Through the use of high purity compounds, there is no interference with readings taken of optical density; and due to the fact that the compounds are completely miscible with Water, there is no dilficulty in mixing the reagent With other aqueous reagents, serum, plasma, or urine. The invention is significantly more economical than the freeze drying used in the prior art.
DESCRIPTION OF THE PREFERRED EMBODIMENTS The most important medical test for which the invention is useful is the test for glucose. But it is to be understood that the novelty of this invention relates to a method of increasing the stability of a reagent used in medical testing and not in any particular medical test itself. For purposes of illustration of the invention, the examples will refer to the use of the improved reagent in testing for glucose in serum.
Reagents containing at least .001% benzidine or benzidine-like compounds, such as o-dianisidine and o-tolidine, are commonly used in medical testing procedures. 'It has been found that if such reagents contain at least about 50% of a stabilizing compound selected from the group consisting of 1,2-ethanediol, 1,2-propanediol, 1,3-propane- Patented Aug. 21, 1973 diol, 1,4-butanediol, 2,3 butanediol, and 1,2,3 propanetriol, that they will be extremely stable for long periods of time without the need for refrigeration. Preferably the reagents should contain at least of the stabilizing compound, and ideally the reagents would contain only the stabilizing compound and benzidine or a benzidinelike compound. The stabilizing compound should be of high purity to provide optical clarity so that there is no interference with optical density determinations made in the medical test.
Example 1 In obtaining blood sugar level, the following reagents are used: (a) glucose oxidase-peroxidase solution, (b) buffer solution, (c) o-dianisidine solution, (b) buffer-dye mixture, (e) sulfuric acid solution, and (f) glucose stock standard.
The glucose oxidase-peroxidase solution is obtained from Fermco Laboratories, Chicago 9, Ill. and is a standard commercially available substance.
The 0.15 molar acetate buffer solution, pH 5.0, is prepared by adding 14.98 gm. of sodium acetate trihydrate to 500 ml. of distilled Water in a 1000 ml. volumetric flask. The flask is shaken in order to dissolve the contents. 2.3 ml. of acetic acid is added to the contents of the flask and distilled water is added thereto to provide 1000 ml. of liquid. The solution is then adjusted potentiometrically.
The o-dianisidine reagent is prepared by placing 2 grams of o-dianisidine hydrochloride (Fermco Lot No. 97-966) in a blender. 500 ml. of chromatoquality 1,2- propanediol (99.37% pure, manufactured by Matheson, Coleman & Bell: Cincinnati, Ohio) is added to the blender, and the mixture is blended at high speed for five minutes. The blended mixture is then transferred to centrifuge bottles and centrifuged at 2,500 r.p.m. for 10 minutes. After centrifugation, the supernatant is poured into a 500 ml. flask and stoppered. Care is taken to protect the mixture from atmospheric moisture. The mixture is then stored at 22 C. for a year. After the year, the test is run using the year old o-dianisidine reagent.
The buffer-dye mixture is prepared by adding 0.5 ml. of o-dianisidine filtrate to 99.5 ml. of acetate buffer solution, pH 5.0.
The sulfuric acid solution is prepared by adding 2 parts of concentrated sulfuric acid to 3 parts of distilled Water. The acid should be added cautiously to the Water in order to prevent spattering and overheating. After such addition has been performed the resulting substance should be allowed to cool.
The glucose stock standard solution is prepared by diluting 1.0 gm. of dextrose to 100 ml. with 0.25% aqueous benzoic acid solution. The resulting diluted solution should be allowed to stand for 24 hours to mutarotate.
In order to obtain the blood sugar level of serum or plasma, 0.02 ml. of glucose oxidase-peroxidase solution is added to an optically selected tube. The contents of the tube are lyophilized. The tube is then plugged with a polyethylene plug. The contents of the tube, the enzyme, is stable for months in this state under refrigeration.
2.0 ml. of the buffer-dye mixture is added to the enzyme prepared in the above tube in order to reconstitute the enzyme. The tube is inverted two or three times to dissolve. 0.013 ml. of the serum or plasma to be tested is added to the above tube. The plug is replaced and the tube is inverted to mix. The tube is incubated at 37 C. for 30 minutes inn a heating block or water bath. 2.0 mls. of the sulfuric acid mixture are added to the tube and the plug replaced. The tube is inverted to mix. The tube is then placed in the colorimeter with a 525 m filter and the reading taken.
In order to calibrate the colorimeter, 0.01 ml. of each of the above-mentioned 25 mg., 50 mg., 100 mg., 300 mg., 400 mg., and 500 mg./ 100 ml. working standards are added to respective tubes. Each of the tubes is treated exactly as an unknown serum or plasma as in the above procedure.
Results obtained using the year old reagent are at least as accurate as results obtained using freshly prepared odianisidine reagent. Results at high glucose concentrations are found to be more accurate using the reagent with 1,2- propanediol than using a reagent which uses only methanol or ethanol.
Example 2 The procedure in Example 1 is followed with the mixture of l,2-propanediol and odianisidine being stored for 6 months at 50 C. Identical results were obtained.
Example 3 To determine the effect of moisture in the o-dianisidine reagent, additional tests are run using the procedure of Examples 1 and 2 except that water is added to the odianisidine reagent in amounts of 5%, and Essentially no difference is noted in the results of tests run using 5%, 10%, and 20% water. Accuracy is reduced at 30% and 40% water, and 50% water results in only slightly better results than are obtained without any 1,2-propanediol.
Examples 4-6 The procedures of Examples 1-3 are followed using 1,2-eth'anediol, 1,3-propanediol, ll,4-butanediol, 2,3-butanediol, and 1,2,3-propanetriol, each individually in place of the 1,2-propanediol used. All of these compounds produce essentially the same effect, although 2,3-butanediol requires the test to be run at above its melting point (34 C.), thus presenting somewhat of a ditficulty.
Examples 7-12 The procedures of Examples 1-6 are followed using 0- tolidine, o-dianisidine, and benzidine each individually in place of o-dianisidine, hydrochloride. In each instance it is noted that these compounds have increased stability in reagents of the examples in comparison to reagents using ethanol, methanol, or simply water.
It is understood that the foregoing detailed description is given merely by way of illustration and that many variations may be made therein Without departing from the spirit of my invention.
Having described my invention, What I desire to secure by Letters Patent is the following and equivalents thereof:
1. A reagent for use in medical testing which comprises:
(a) at least 50% of a stabilizing compound selected from the group consisting of 1,2-ethanediol, 1,2- propanediol, 1,3-propanediol, l1,4-butanediol, 2,3-butanediol, and 1,2,3-propanetriol, and
(b) at least 001% of a second compound selected from the group consisting of benzidine, o-dianisidine, o-dianisidine hydrochloride and o-tolidine.
2. The reagent of claim 1 in which the stabilizing compound is of such a purity so as to provide optical clarity, whereby there is no interference with optical density determinations made in a medical test in which said reagent is used.
3. The reagent of claim 1 in which the stabilizing compound is 1,2-propanediol.
4. The reagent of claim 1 in which said second compound is o-dianisidine hydrochloride.
5. The reagent of claim 1 in which said reagent contains at least of said stabilizing compound.
6. The reagent of claim 1 in which said reagent contains only said stabilizing compound and said second compound.
7. In a method for determining the concentration of glucose in a biological fluid, in which glucose oxidase is used to produce hydrogen peroxide and in which peroxidase is used to convert an indicating compound in the presence of the hydrogen peroxide to a colored compound, the improvement which comprises:
having the indicating compound be a compound selected from the group consisting of o-dianisidine, odianisidine hydrochloride, o-tolidine and benzidine and providing said compound in reagent form in which the reagent contains at least 001% of said compound and at least 50% of a stabilizing compound selected from the group consisting of 1,2- ethanediol, 1,2-propanediol, 1,3-propanediol, 1,4-butanediol, 2,3 butanediol, and 1,2,3 propanetriol, whereby said reagent is stable over long periods of time even at relatively high temperatures.
8. The improved method of claim 7 in which the stabilizing compound is of such a purity so as to provide optical clarity, whereby there is no interference with any optical density determinations made.
9. The improved method of claim 7 in which said stabilizing compound is 1,2-propanediol.
10. The improved method of claim 7 in which said indicating compound is o-dianisidine hydrochloride.
11. The improved method of claim 7 in which said reagent contains at least 80% of said stabilizing compound.
References Cited Analytical Abstracts 1923220 (October 1970).
ALVIN E. TANENHOLTZ, Primary Examiner M. D. HENSLEY, Assistant Examiner US. Cl. X.R. 252-407; 23-230; 99