METHOD FOR THE DETERMINATION OF BILIRUBIN

Abstract: The present invention provides a method for the deter­ mination of bilirubin which comprises reacting the bilirubin with a compound represented by the formula (I) and determining the pigment thus formed.

Description: Background of the Invention The present invention relates to a method for the determination of bilirubin and a composition suitable therefor. Bilirubin is present in serum and the determination of bilirubin in serum is useful for the diagnosis of liver function. With regard to the determination of bilirubin, a method using diazonium salt of sulfanilic acid is widely used. In this method, bilirubin is determined by coupling the diazonium salt of sulfanilic acid with bilirubin and determining a formed pigment colorimetrically. However, the method has the following disadvantages. (1) It is necessary to carry out the diazotization of sulfanilic acid using a strong acid each time just before use, because the diazonium salt of sulfanilic acid is unstable. Therefore, it is somewhat troublesome to perform the determination. (2) The reaction rate of bilirubin with diazonium salt of sulfanilic acid is slow and it takes about 15 to 30 minutes to complete the reaction. (3) It is difficult to apply the method to the determination with an automatic analyzer because a strong acid must be utilized and metals are readily corroded in the presence of the strong acid. Thus, development of a method for the determination of bilirubin which can easily be performed without utilization of strong acid in a short period of time has been desired. Prior Art EP-A-0 098 562 discloses a method and reagents for the determination of direct and total bilirubin. The reagents used include a diazonium salt, although the particular diazonium salts of the present invention are not specifically disclosed. Summary of the Invention In accordance with the present invention, bilirubin can be determined by reacting bilirubin with a diazonium compound [hereinafter referred to as Compound (I)] represented by the formula wherein X represents halogen atom and Y represents Cl ⊖ or a double salt anion consisting of zinc chloride, stannous chloride, mercuric chloride or aluminium sulphate, to form a pigment and by determining the pigment thus formed. Halogen atom includes chlorine, fluorine, bromine and iodine. The present invention is based on the principle that the reaction of bilirubin in a sample with the Compound (I) proceeds stoichiometrically to form a pigment and that the amount of the formed pigment is proportional to the amount of bilirubin in the sample. Detailed Description of the Invention In carrying out the present invention, Compound (I) is dissolved in a buffer solution having a pH in a range of 1 to 11 to prepare a reagent solution. A sample containing bilirubin is added thereto and the mixture is incubated at a temperature of 5 to 50°C, preferably 15 to 40°C for 2 to 20 minutes. After completion of the reaction, the absorption of the reaction solution colored by the pigment thus formed is measured at the maximum absorption wavelength of the pigment in the visible ray region on the basis of a reagent blank as a control. Bilirubin in the sample is determined from a calibration curve obtained in advance by tests on known amounts of bilirubin. As the buffer, hydrochloride-potassium chloride, phosphate, Tris-hydrochloride, succinate, oxalate, acetate and Good are mentioned. A surfactant such as Triton®X-100 a polyethyleneglycol mono-p-iso-octylphenyl ether of Sigma Co. is used, if required, to clear the turbid solution. Compound (I) is a known compound as an intermediate for dye synthesis which is prepared by diazotizing the compound represented by the formula (II) wherein X has the same meaning as defined above, which is also a known compound as an intermediate for dye synthesis. It is preferred to prepare Compound (I) at the time when the determination is conducted. The preparation of Compound (I) is carried out by adding Compound (II) to a mixture of hydrochloric acid and sodium nitrite. In the reaction, Compound (II), hydrochloric acid and sodium nitrite are used in concentrations of 0.01 - 2.0 mg/ml, 0.1 - 5.0% and 0.1 - 5.0%, respectively. The buffer solution is used in a concentration of 0.005 - 2 mM. Compound (I) is used in a concentration of equimolar amount or more, usually 10 to 1000 times molar to that of bilirubin in the sample. Preferable concentration is about 0.01 - 2.0 mg/ml. The maximum absorption wavelength of the pigment is about 535 nm. A composition for the determination of bilirubin in the present invention comprises Compound (I) and a buffer. Certain specific embodiments of the present invention are illustrated in the following representative examples. Example 1 In 100 ml of 0.1 M phosphate buffer (pH 5.0) were dissolved 500 mg of Triton®X-100 and 40 mg of Compound (I) (X = Cl) to prepare a reagent solution. Bilirubin standard solution (Daiichi Kagaku Yakuhin Co., Ltd.) was added to the reagent solution which was warmed at 37°C in advance. After incubation at 37°C for 5 minutes, the absorbance of the reaction solution at λ max of Compound (I) of 535 nm was measured in contrast to a reagent blank test as a control. Table 1 shows the relation between the concentration of bilirubin and the absorbance. It is evident from Table 1 that the concentration of bilirubin and the absorbance are correlated by a single straight line. Example 2 In 100 ml of 0.1 M phosphate buffer (pH 5.0) were dissolved 500 mg of Triton®X-100 and 40 mg of Compound (I) (X = Cl) to prepare a reagent solution. Five serum samples were added to (1) the reagent solution immediately after preparation (2) the reagent solution which was stored at 30°C for 48 hours (3) the reagent solution which was stored at 37°C for 4 hours and (4) the reagent solution which was stored at 5°C for a week. The mixtures were subjected to the same incubation and measurement as in Example 1. The concentrations of bilirubin in the serum samples were calculated by a calibration curve which was prepared in advance using the bilirubin standard solution. Table 2 shows the results together with the result obtained by measuring the concentration of bilirubin in the serum samples using Bilirubin B test "Wako" (Wako Junyaku, Co.) which is a reagent for bilirubin determination.

Claims: Method for the determination of bilirubin in a sample by reacting the bilirubin in the presence of a buffer solution and, if required, a surfactant with a diazonium compound and measuring the change in absorption of the resulting reaction solution characterized in that the diazonium compound conforms to formula (I) wherein X represents halogen atom and Y represents Cl ⊖ or a double salt anion consisting of zinc chloride, stannous chloride, mercuric chloride or aluminium sulfate. A method according to claim 1, wherein said halogen atom is a member selected from chlorine, bromine, iodine and fluorine. A method according to claim 1 or 2, wherein said absorption is measured in the visible ray region. A method according to claim 1-3, wherein said buffer is hydrochloride-potassium chloride, phosphate, Tris-hydrochloride, succinate, oxalate, acetate or a Good buffer. A method according to claim 1-4, wherein said surfactant is polyethyleneglycol mono-p-iso-octylphenyl ether. A method according to claim 1-5, wherein said Compound (I) is used in a double salt form. A composition for the determination of bilirubin which comprises Compound (I) defined in claim 1 and a buffer.

Inventors: AOYAMA, NORIHITO, MIIKE, AKIRA, OKANO, MIKIO, TATANO, TOSHIO
Applicants: KYOWA MEDEX CO. LTD.
Application year: 1986
Main classification: G01N33
IPC codes: G01N33