United States Patent [191 [11] 3,807,956 Morin [45] Apr. 30, 1974 [54] MODIFIED BIURET REAGENT FOR GLASS STORAGE AND METHOD Primary Examiner—Morris O. Wolk [75] Inventor: Leo G. Morin, Miami, Fla. Assistant Examiner—-R. E. Serwin Attorney, Agent, or Firm-Fisher, Christen & Sabol [73] Assignee: The First National Bank of Miami, Miami, Fla. [22] Filed: Aug. 30, 1972 [21} Appl. No.: 284,880 [57] CT [52] US. Cl. ......... .. 23/230 B, 23/230 R, 23/253 R, A modi?cation of the biuret reagent with zirconium 252/408 and reduced alkalinity to make the reagent suitable [51] Int. Cl. ......................................... .. G0ln 33/16 for storage in glass is disclosed. The performance of [58] Field of Search .................. .. 23/230 B; 252/408 the modi?ed reagent in measuring protein is not re duced. [56] References Cited UNITED STATES PATENTS 11 Claims, No Drawings 3,310,382 3/1967 Kingsley .......................... .. 23/230 B 3,807,956 , 1 2 MODIFIED BIURET REAGENT FOR GLASS Such biuret reagents often contain other non STORAGE AND METHOD essential ingredients, and this invention covers such bi— uret reagents. The biuret reagents often contain copper chelating agents,~such' as sodium potassium tartrate, ci BACKGROUND OF THIS INVENTION trates and ethylenediamine tetracetic acid, and ingredi 1. Field of 'this Invention ents, such as potassium iodide. The biuret reagent also This invention relates to a modi?cation of the biuret contains surfactants, such as the Tritons, e.g., Triton reagent so that it may be stored in glass. X-lOO and Triton X-l55, sodium salts of high molecu 2. Prior Art lar weight, alkyl sulfates or sulfonates, Merpol SE, The determination of protein in biological ?uids with 0 Zonyl A, Merpol HCS, and the Igepal CA’s, such as the biuret reagent is a useful and frequently performed lgepal CA 630. test in clinical laboratories. The biuret reagent is stable Examples of useful cupric compounds are cupric per at room temperature for several years. It must, how chlorate, cupric sulfate, cupric acetate, cupric buty ever, be stored in a plastic or other non-glass container, rate, cupric bromate, cupric chlorate, cupric bromide, since it will slowly react with glass. This is a problem for 5 cupric chloride, cupric fluoride, cupric dichromate, cu commercial suppliers, since biuret reagent supplied in pric formate, cupric iodate, cupric lactate, cupric or glass tubes or ampules that are used as both the reac thophosphate, cupric laurate, cupric salicylate, cupric tion tube and the cuvette for measurement in colorime nitrate, cupric tartrate and cupric oxalate. ters has a shelf life of about 3 months only. There is a The term alkali hydroxide includes the hydroxides of genuine commercial need for a modi?ed biuret reagent 20 alkali metals, such as potassium hydroxide, sodium hy that will not react with glass for a minimum of a year. droxide, lithium hydroxide, rubidium hydroxide, ce sium hydroxide, and francium hydroxide; of the alka line earth metals, such as barium hydroxide,- calcium BROAD DESCRIPTION OF THIS INVENTION hydroxide, strontium hydroxide, and radium hydrox The primary object of this invention is to provide a ide, and of the ammonium radical (ammonium hydrox biuret reagent that will not react with glass for at least ide). The preferred alkali hydroxide is sodium hydrox a year. ide. Other useful hydroxides include such metal hy Another object is to provide a biuret reagent that droxides such as lead hydroxide, silver hydroxide, zinc may be stored in glass tubes or ampules. hydroxide, iron hydroxide and beryllium hydroxide, Still another object is to provide a biuret reagent that and organic hydroxides such as hydroxylamine. A will not have diminished capacity as a protein indica strong hydroxide should be used. This invention in tor. volves lowering the hydroxide content to below a cer It has been found that with the addition of zirconium tain level. ions and the reduction of alkalinity, the biuret reagent Examples of useful water-soluble zirconium salts are will maintain its capacity to indicate proteins, yet will 35 zirconium perchlorate (preferred), zirconium nitrate, not interact with glass and becomes suitable for storage zirconium chlorate, zirconium sulfate, zirconium chlo in glass tubes and ampules. Broadly speaking, this in rite, zirconium phosphate, zirconium borate, zirconium vention is practised by reducing the hydroxide content bromate, zirconium acetate, zirconium sul?te, zirco of any conventional biuret reagent to 0.1 to 2 gram per 'nium tetrachloride, zirconium lactate, zirconium per cent and adding 0.01 to 0.5 gram percent of a water sulfate, zirconium nitrate, zirconium perborate, zirco soluble zirconium salt. , nium oleate, zirconiumoxalate, zirconium citrate, zir The biuret reagent of this invention will not react conium iodate, zirconium bromide, zirconium fluorate, with glass for at least one year, can be stored in glass zirconium chloride, Zirconium fluoride and zirconium containers and does not have diminished capacity as a iodide. protein indicator after having been stored in glass con 45 Any suitable colorimeter or spectrophotometer can tainers for at least one year. be used to measure the absorbance. Examples of useful This invention further includes the process-of using colorimeters are: Coleman, Model 44; Perkin-Elmer, the novel biuret reagent of this invention to determine . Model 124; the colorimeter disclosed in US. Ser. No. the protein content of biological ?uids, such as blood, 224,457, applicants: Raymond W. Kiess and Peter H. 50 by colorimetric means or similar means. Stewart, tiled: Feb. 8, 1972, assignee: Kiess Instru This biological ?uid can be those of man or animal. ments, Inc., 8768 S.W. 131st Street, Miami, Florida, Examples of such biological ?uids are serum, plasma, 33156; and the direct reading colorimeter disclosed in urine and spinal fluid. US. Pat. No. 3,561,878, inventor: R. W. Kiess. The following examples are included to further illus DETAILED DESCRIPTION OF THIS INVENTION trate the invention, but .they do not limit it. A large number of biuret reagents are known. All‘ of EXAMPLE 1 them (within the scope of this invention) involve the reaction of copper (cupric form) in the presence of To one liter of deionized water is added 2.16 grams of cupric perchlorate, 6 grams of sodium potassium tar large amounts of a strong base, such as sodium hydrox 60 ide, with protein to produce a violet color. This inven trate, 1 gram of zirconium perchlorate, 8 grams of so tion involves the modification of any biuret reagent dium hydroxide, 1 gram of potassium iodide, and 2 ml which contains cupric ions and a high pH (due to the of Triton X-l00 (the trade name for a synthetic organic presence of an amount of a hydroxide base). So when surface active agent which is a nonionic, water soluble, the phrase “a biuret reagent” or “the biuret reagent” alkyl aryl polyether alcohol). The solution is dispensed is used herein, it generally refers to the above formula in 2.5 ml lots to a set of ampules and the ampules are tion (unless it is obvious that the modi?ed biuret rea sealed. The ampules are stored for one year. On exami gent of this invention is being referred to). nation, after one year, there is no apparent action on 3,807,956 3 4 the glass or any signes of degradation. A set of stan droxide is an alkali hydroxide. dards made to contain 1, 2, 4, 6, 8 and 10 gram-percent 4. A reagent as described in claim 1 wherein said al of protein are prepared. To a number of tubes of modi kali hydroxide is sodium hydroxide. ?ed reagent, 50A of speci?ed levels of protein stan 5. A reagent as claimed in claim 1 wherein said hy dards, are added. The absorbance is measured using a droxide is present in an amount between 0.1 and about Coleman Model 44. There is a linear proportion be 2 gram percent. tween absorbance and protein concentration. 6. A method of preparing a mixture of a biological ?uid and a biuret reagent, the absorbance of said mix EXAMPLE 2 ture being capable of being colorimetrically deter A reagent is prepared as in Example 1 and is tested mined in order to ascertain the protein content of the fresh with standards as in Example 1. The linear pro biological ?uid, which comprises admixing a sample of portion is the same as in Example 1. biological ?uid and a biuret reagent, which contains cu pric ions in an alkali medium that contains about 2 EXAMPLE 3 gram percent or less of an hydroxide and between A reagent is prepared as in Example 1, except that about 0.01 to 0.5 gram percent of a zirconium salt, the the sodium hydroxide is raised to 6 gram percent and ratio of the biological ?uid to the reagent being be the zirconium is omitted. The reagent is tested with tween 1:100 and 1:25. standards as in Example 1. The linear proportion is the 7. A method as described in claim 6 wherein said zir same as in Example 1. conium salt is zirconium perchlorate. 20 8. A method as described in claim 6 wherein said hy EXAMPLE 4 droxide is an alkali hydroxide. A reagent is prepared as in Example 3 and is stored 9. A method as described in claim 6 wherein said al for 6 months in glass ampules. There is a translucent kali hydroxide is sodium hydroxide. clouding of the glass and serious signs of degradation. 10. A method wherein the absorbance of a mixture The reagent is tested as in Example 1. The reagent 25 of biological ?uid and a biuret reagent, which contains functions very poorly with a loss of about 60 percent cupric ions in an alkali medium that contains about 2 capacity to measure proteins.