JPS60178361A - Diazo reagent for measuring bilirubin - Google Patents

Abstract

PURPOSE:To enhance the stability of a measuring reagent without changing the fundamental composition thereof and to eliminate preparation trouble at the time of use, by allowing alkali halide to coexist along with diazonium salt formed through the reaction of sodium nitrite and sulfanilic acid. CONSTITUTION:Alkali halide such as sodium chloride is allowed to coexist along with diazonium salt formed through the reaction of sulfanilic acid and sodium nitrite as a stabilizer in an amount 10-20 times that of diazonium salt in a mol ratio.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a highly stable diazo reagent used for the determination of BiIJ) Levin.

Measuring the total bilirubin and direct bilirubin levels in serum is one of the essential test items for diagnosing hepatic filament diseases. Quantification of bilirubin is carried out by using a diazonium salt of an aromatic primary amine as a measuring reagent and measuring azobilirubin produced by a diazo coupling reaction of bilirubin. The typical method is Malloy-F+vell.
yn) method and Michaellso
n) method, the former measures azobilirubin, and the latter shifts azobilirubin from red to blue under alkaline conditions and measures it as alkaline azobilirubin. The diazonium salt used for the determination of bilirubin is prepared immediately before use with sulfanilic acid and sodium nitrite. From the preparation of diazonium salts, azobilirubin (red color,
The process up to the generation of characteristic absorption: 580 nm is as follows.

SO2F SO2F 'SO2F [S/L/7 Anipic acid] [Diazonium salt] 11 (X)011 (S00H 1Hylylbin) 1 (Red) 0tJH The diazonium salt used in the above reaction is extremely unstable. Since it cannot be stored, it must be prepared each time bilirubin is measured.Recently, in order to eliminate the inconvenience of one-time preparation, a stabilized diazonium salt method has been developed to stabilize the diazo reagent. A method for measuring bilirubin has also been developed.However, in this method,
) Due to the difference in the type of Levin from that of conventional methods, and due to the roughness of Levin in vivo, there are differences in data from conventional methods in specimens of newborns, dialysis patients, kidney transplant patients, etc. is beginning to be pointed out.

The present invention has been made in view of the above, and it is possible to stabilize the conventional measurement reagent without changing its basic composition.
To provide a diazo test solution for bilirubin determination, which can be stored for a long time by increasing the temperature, eliminates the trouble of one-time preparation, and provides 311I quantitative data exactly the same as conventional ones.

The diazo reagent of the present invention comprises sulfanilic acid and nitrous acid.
It is made by coexisting an alkali metal halide salt as a stabilizer with the diazonium salt produced by the reaction with lithium.

In the diazonium reagent of the present invention, the unstable diazonium salts are stabilized by the presence of the alkali metal halide.

As the alkali metal halide salt, especially chloride (J-)
! J UJ-(N:, +Cl) E, 1: Bichloride/'
J, u ram (KO4)y)s preferred.

The coexisting amount of the alkali metal halide salt may be 10 to 20 times the amount of the diazonium salt in terms of mo/I/number.

The diazo reagent of the present invention comprises nulfanilic acid, nitrite, and alkali metal halide in a molar ratio of 1:2.
It is manufactured by blending at a ratio of ~3:10-20. There are no separate restrictions on other manufacturing conditions, and normal IL
l, l manufacturing conditions at that size! -It's fine as long as it's valid. For example, 90-1205J of thorium chloride as a stabilizer in purified water.
/l, 4-6 g/l of nurphanilic acid and 6-8 ml/IJ of 11° hydrochloric acid dissolved in test solution C (hereinafter referred to as "diazo test solution Δ"), and 6-8 g/4 of 1-lium nitrite in purified water. It is prepared by mixing the dissolved test solution (S1, Diazo Test Solution T-3j) in an appropriate ratio.

Next, the preparation of the diazo reagent of the present invention and the quantification of BiIJ) Levin using the reagent will be specifically explained with reference to Examples.

Example 1 [(] Diazo reagent Prepare the following buffer solution and diazo test solutions Δ and B.

Buffer Sotrium benzoate 569 Caffeine anhydride 879 Sotrium acetate 94g Bridge-35709 Purified water 1e Diazo test armpit A Storichium chloride 11.7fl Sulfanilic acid 0.52g Concentrated hydrochloric acid 0.72 ml Purified water 100m (! Diazo test B Sodium nitrite 076g fi'j ''-L water 100ml! [■] Add 2.5 me of buffer solution to 4111 constant blood Wi 20 till of bilirubin amount,
Measure the absorbance at 580 nm and take this absorbance as 19.

Next, diaso 5-shiki music (giant armpit A: 100me
Diazo test solution f3: l me added to) 0.
After adding 1 me and leaving it for 1 minute, 53 Q nm
The absorbance is measured and the absorbance is defined as 1.

Bi-IJ 7 Perform the same procedure as above for standard 1ff-serum with known Levin concentration, add diazo reagent [) 5 after IJ
Calculate the absorbance So and Sl at 30nrrl.

The total amount of bilirubin in the serum is expressed in the following manner.

(C is the total bilirubin value of the standard serum) Table 1 shows the results obtained on the day of diazo reagent preparation and subsequent full determination. As a comparison, oral oxidation was investigated using the same 4111 test using a reagent that did not contain the stabilizer 1-lium chloride, but in this case, only the liter of preparation was used, as was conventionally said. That's all I could do.

Table 1 (Unit: mg/de> Example 2 The total amount of bilirubin in serum was automatically measured using a Beckman Ametora-8 automatic analyzer. The test solutions used (slow solution, diazo test armpit A and B) were It is the same as that in Example 1.

In this automatic analyzer, the absorbance of sample serum and standard serum before and after addition of the diazo reagent is determined by
, So , 8 + are automatically measured, and the reaction time of the diazo coupling is as short as 25 seconds (1 minute in Example 1). In addition, this device stores the general absorbance of predetermined standard serum, and the measured absorbance is
If the absorbance deviates from the stored absorbance by more than a certain value, the measurement data cannot be measured because it is unreliable.

Using the above diazo reagent, the total amount of bilirubin was automatically measured on the day of preparation and thereafter. The sample serum was the same as that used in Example 1. 7IllI determination results are shown in Table 2.The total bilirubin value was determined by the above automatic analyzer using the conventional method [manual method]. Diazo reagent (stabilized side does not combine) was compared with the measured value by one-time preparation] to find a correlation, and the results shown in Figure 1 were obtained. However, n=30°3'=1.013X-0,
086, γ-0,9937.

As shown in Table 2 onwards, the diazo reagent of the present invention has excellent stability and can be used after long-term storage, whereas conventional diazo reagents can only be used on the day of preparation. Moreover, its stability is achieved by simply adding an alkali metal halide salt to the conventional diazo reagent, and there is no change in the basic composition of the reagent.
/Levin measurement operation is exactly the same as before, and can be easily applied to measurement by an automatic analyzer, and measurement data exactly the same as before can be obtained.

[Brief explanation of drawings]

FIG. 1 is a graph showing the correlation between the BiIJ/Levin measurement value using the diazo reagent of the present invention (by automatic analyzer) and the BiIJ/Levin measurement value using the conventional diazo reagent (manual method). Agent Patent Attorney Miyazaki Shinhachi Department 1st TM

Claims (2)

[Claims] (1) A highly stable diazo reagent for measuring bilirubin, which is made by coexisting an alkali metal halide salt with a diazonium salt produced from thorium nitrite and nurphanil. (2) Item (1) above, wherein the amount of the alkali metal halide salt added is 10 to 20 times the amount of the diazonium salt in terms of number of moles.
BiIJ) Diazo reagent for measuring Levin as described in Section 1.