JPS6252434A - Absorption photometric analytic method - Google Patents

Abstract

PURPOSE:To make it possible to simply discriminate a measured value wherein analytical abnormality due to a photometry obstructing component is appearing, in a two- wavelength absorption photometry, by setting the absorbancy ratio of a standard reaction liquid to a standard and comparing the same with that of a measuring specimen reaction liquid. CONSTITUTION:The absorption spectrum curve (d) of a specimen reaction liquid is formed by adding the spectrum curve (c) of an obstructing component to the spectrum curve (a) of a standard reaction liquid having the same concn. as the specimen reaction liquid. The absorbancies of the obstructing component at the absorption max. wavelength lambda1 and non-absorption max. wavelength lambda2 of the reaction product are set to A1lambda1 and A1lambda2 (A1lambda2=0 in the embodiment of a drawing) and the absorbances of the standard reaction liquid and a reagent blank at a wavelength are respectively set to Astlambda and Ablambda. When the ratio of the absorbancy ratio Vsp of the specimen reaction liquid to the absorbancy ratio Vst of the standard reaction liquid is taken, said ratio is represented by formula. Therefore, when the absorbancy A1lambda1 at the wavelength lambda1 of the obstructing component in a specimen increases to reach a non- negligible value, the value of the ratio Vsp/Vst comes to a value larger than 1 and the abnormality of the measured value can be known.

Description

DETAILED DESCRIPTION OF THE INVENTION (a) Industrial Application Field The present invention relates to spectrophotometric analysis, particularly for blood, plasma,
This book relates to spectrophotometric analysis of specimens such as serum, urine, other body fluids, secretions, and other biochemical samples.

The present invention also relates to a spectrophotometric analysis method for specimens and other biochemical samples in an automatic chemical analyzer.

(b) Conventional technology Spectrophotometric analysis is one of the most commonly used analytical techniques, and with the development of highly sensitive and highly selective reagents,
It is used in various automatic analyzers.

Automated chemical analyzers that employ such spectrophotometric analysis are used, for example, to analyze each component of samples such as plasma, serum, urine, other body fluids, and secretions, and the analytical values are used for diagnostic purposes. Since it is used for treatment guidelines, etc., accuracy and speed are always required.

However, spectrophotometric measurements on specimens such as plasma, serum, urine, and other body fluids and secretions are based on chromogens, e.g.
It may be affected by bilirubin in the yellow stain, hemoglobin due to hemolysis, chyle, etc. In such cases, it is not possible to measure absorbance accurately, and therefore all samples can always be measured with a certain degree of accuracy. This was a problem because it could not be analyzed.

In addition to such interference from coexisting components in the sample, dirt or scratches on the measurement cell, or interference from air bubbles or solid matter mixed in the reaction solution can cause errors in photometric values. There is.

Conventionally, in order to avoid such errors, a sample blank solution is prepared separately and the measurement sample of the sample reaction solution is corrected from the measurement results obtained under the same measurement conditions, or the so-called dual wavelength method is used.

(c) H target point to be solved by the invention However, providing a sample blank solution for each sample or for each measurement cell is not preferable as it reduces the amount of sample throughput, and it also changes the analysis procedure. Problems will remain as it becomes complicated. In addition, in the two-wavelength method, the two wavelengths of light used in the measurement have an apparent absorbance that can be considered to be roughly equivalent, such as 1. It has a corrective effect on solids in the sample liquid, microbubbles, and scratches on the surface of the measurement cell. However, with regard to chromogens such as bilirubin, hemoglobin, and chylomicron, which is the cause of chyle, the influence of these two wavelengths is different, so it is difficult to obtain accurate analytical values even with the two-wavelength method.

The present invention is intended to eliminate the decrease in analytical precision and accuracy due to abnormalities in measured values due to interfering components and substances in the conventional spectrophotometric analysis method.

(d) Six ways to solve the problem The present invention detects when absorbance measurement is affected by interfering components in the sample, particularly in the specimen, and performs re-analysis.
Alternatively, it provides an absorption photometric analysis method that notifies or instructs to perform correction.

In the present invention, the ratio between the absorbances of two waves on the absorption spectrum of the substance to be measured is always constant unless there is any interference. This was done by focusing on α.

That is, the present invention measures the absorbance of transmitted light at two different wavelengths on the absorption spectrum of the photometric target substance for a sample reaction liquid having an arbitrary liquid layer length, and compares the two measured absorbances. On the other hand, light having the same wavelength as the two types of light described in iq is transmitted through a standard solution reaction solution having the same wL/l as the sample solution, and the standard solution reaction for each wavelength of light is measured. Measure the absorbance of the solution, compare the two measured absorbances, compare the ratio between the absorbances of the sample reaction solution and the absorbance of the standard solution reaction solution, and determine the difference in the measured value of the sample reaction solution. This spectrophotometric analysis method is characterized by detecting the presence or absence of the influence of interfering substances.

The two wavelengths of light used in the present invention can be any of the two wavelengths that fall within the range where the absorbance of the substance to be measured can be measured.
Among the two wavelengths of light, select the light with the maximum absorption wavelength in the absorption vector of the substance to be measured as the light of one wavelength, and select the light with the non-absorption maximum wavelength as the light of the other wavelength. This is preferable because there are fewer types of light. Here, the light with the maximum absorption wavelength refers to the light with the maximum absorption wavelength in the absorption spectrum of the substance to be measured, and is unique to the substance under certain measurement conditions. In addition, light with a non-absorbing maximum wavelength refers to light with an absorption wavelength other than the maximum absorption wavelength in the absorption spectrum of the photometric target substance, but it also refers to light with an absorption wavelength other than the maximum absorption wavelength in the absorption spectrum of the substance to be measured. Light is preferred. For example, for a photometric target substance, the ratio of the absorbance at the maximum absorption wavelength to the absorbance at the maximum non-absorption wavelength is constant as long as the same wavelength is always selected as the maximum non-absorption wavelength.
It is preferable to determine the maximum non-absorption wavelength for each analysis item in advance. Of course, this absorbance ratio is unique to the photometric NflL substance, so it is sufficient to find the absorbance ratio for one non-absorbing maximum wavelength, but it is sufficient to calculate the absorbance ratio for multiple, for example, two non-absorbing maximum wavelengths. can be used.

In the present invention, a solution of a substance to be measured in the analysis is used as the standard solution. Therefore, for example, a standard solution used for creating a calibration curve can be used. Furthermore, in the present invention, reagent blank values are determined for, for example, light at the absorption maximum wavelength of the photometric target substance and light at the non-absorption maximum wavelength used, and used as reagent blank values for the respective wavelengths.

(E) Function In the present invention, the ratio of the absorbance at the absorption maximum wavelength and the absorbance at the non-absorption maximum wavelength of the photometric target substance in the standard solution reaction solution, that is, the absorbance ratio of the standard solution reaction solution, is used as the standard,
The ratio of the absorbance at the absorption maximum wavelength to the absorbance at the non-absorption maximum wavelength for the photometric N-parallel in the photometric sample liquid reaction liquid, that is, the absorbance ratio of the photometric sample liquid reaction liquid, is compared with the standard, and the photometric sample is determined. Detects abnormalities in measured values for liquid reaction liquids.

It is something that Therefore, in the present invention, the absorbance ratio of the photometric sample liquid reaction liquid is compared with the absorbance ratio of the standard liquid reaction liquid for each measured value of the sample. In this ↑ ratio, the absorbance ratio of the photometric sample solution and the mark i? ! When the ratio or difference with the absorbance ratio of the reaction solution exceeds a threshold value, it indicates that an influence or abnormality due to an interfering substance appears in the measured value, and it means that the analytical value concerned will be inaccurate. .

(F) Example Hereinafter, one embodiment of the present invention will be described with reference to the accompanying drawings, but the present invention is not limited in any way by this description.

The figure is an absorption spectrum diagram for explaining the principle of the absorption photometric analysis method of the present invention.

A predetermined amount of a standard solution containing only the component to be measured is placed in a photometric reaction tube, a predetermined amount of a reaction reagent is added to it, and the reaction is allowed to occur under predetermined conditions for a predetermined time to generate the desired reaction product, which is the photometric target substance. generate things.

Next, the reaction tube for photometry after this reaction is sent to the photometry section, where the standard solution reaction solution absorbance A is measured for each of the maximum absorption wavelength and non-absorption maximum wavelength λ2 of the reaction product.
Measure stλ1 and Astλ2. Then, instead of the standard solution, the same amount of pure water is placed in another photometric reaction tube, a predetermined amount of the reagent is dispensed therein, and the photometric reaction tube containing this reagent is sent to the photometric section. Therefore, for the photometric reaction tube, the reagent blank absorbance AI) λ is determined for each of the wavelengths of absorption maximum wavelength λ1 and non-absorption maximum wavelength λ2.

and vAbλ2 are measured.

Therefore, the standard solution absorbance ratio Vst for the target reaction product in the absence of interfering factors is determined by the following equation.

formula Further, the concentration conversion coefficient is determined by the following formula.

K=Cst/(Astλ, As Lλ2)−(A
I:lλ, -All)λ2) [In the formula, Cst is the concentration of the standard solution of the substance to be measured. :Next, a predetermined amount of the sample is dispensed into a measurement reaction tube, and the predetermined amount of the reaction reagent is dispensed into this, and the reaction is allowed to occur under predetermined conditions for a predetermined period of time to perform the desired reaction. send to Specimen absorbances Aspλ1 and Aspλ2 are measured for the absorption maximum wavelength λ1 and non-absorption maximum wavelength λ2 of the reaction product in the reaction tube sent to the photometry section, respectively, and the analyte absorbance ratio Vsp is determined by the following formula.

Then, calculate the ratio of the absorbance ratio of the standard solution and the reaction solution, V s t t: to the absorbance ratio of the sample, Vsp, and calculate the ratio (Vsp/V
s t ) is within the threshold of 0.95 to 1.05, the concentration of the substance to be measured in the sample C3p=K[: (A
sp included I Aspλ2) (Ahλ1-Al)
λ2)]. However, the ratio V s p / V s
When t is outside the threshold of 0.95 to 1.05, it indicates an abnormality in the measured value and requires re-analysis or a caution mark or comment.

In this manner, by determining the ratio of the sample absorbance ratio Vsp to the standard solution absorbance ratio VSt, an abnormality in the sample measurement value can be easily detected. If no abnormality in the sample measurement value is detected, the measurement value is determined by normal spectrophotometry, that is, one-wavelength or two-wave absorption spectrophotometry.

The figure shows a case where the spectrum of the interfering component affects the measured value of the analyte. In this example, the absorption spectrum curve d of the sample reaction solution is formed by adding the spectrum curve C of the interfering component to the spectrum curve #ia of the standard solution reaction solution at the same concentration.

In this case, the maximum absorption wavelength λ1 and the maximum non-absorption wavelength in the absorption spectrum C of the interfering component.

The absorbance of the interfering component is A! λ1 and AIλ2, and in this example, At λ2 is zero. The absorbance ratio Vsp of the sample reaction solution is, On the other hand, the absorbance ratio Vst of the standard solution reaction solution is, therefore, the ratio of the absorbance ratio Vsp of the sample reaction solution to the absorbance ratio Vst of the standard solution reaction solution V sp / V st is as follows. The absorbance of the interference component at wavelength λ1 At λ
1 becomes thick (when it becomes a value that cannot be ignored, the ratio V s
The value of p/V s t becomes a value greater than 1, and an abnormality in the measured value can be known.

Also, contrary to the example in the figure, the absorbance AI of the wavelength λ1 of the interference component is
When λ1 is zero and the absorbance at wavelength λ2 is large, the light is ``b'' - 1 - 1 - J engineering university! Nijiro”-11-Vs
t Astλ2 +A Iλ2−Abλ2 and I
), the absorption magician of the wavelength λ2 of the interfering component in the sample. When λ2 increases to a value that cannot be ignored, the ratio V s p /
The value of V s t becomes a value smaller than 1, which makes it possible to know whether there is an abnormality in the measured value.

(g) Effects of the invention The present invention uses the absorbance ratio of a standard solution reaction solution as a standard and compares it with the absorbance ratio of a measurement sample reaction solution. It is now possible to easily identify measurement values in which analytical abnormalities due to photometric interference components or photometric interference substances have appeared, and it is therefore possible to detect inaccurate analysis values due to these photometry interference components or photometry interference substances. It is hoped that the reliability will improve.

[Brief explanation of the drawing]

The figure is an absorption spectrum diagram for explaining the principle of the spectrophotometric analysis method of the present invention. In the figure, a is the absorption spectrum of the standard solution reaction solution, b is the absorption spectrum of the reagent blank solution, C is the absorption spectrum of the interfering component in the sample reaction solution, d is the absorption spectrum of the sample reaction solution, λ1 is the absorption maximum wavelength, λ2 is the non-absorbing maximum boyfriend. Agent

Claims (1)

[Claims] The absorbance of transmitted light at two wavelengths on the absorption spectrum of the photometric target substance is measured in a sample reaction solution having an arbitrary liquid layer length, and the two measured absorbances are compared. The absorbance of the standard solution reaction solution having the same liquid layer length as that of the sample reaction solution is measured in the transmitted light of the two types of wavelengths, and the two measured absorbances are compared. A spectrophotometric analysis method, which is characterized in that the presence or absence of an influence of an interfering substance on the measured value of a sample reaction solution is detected by comparing the ratio between the absorbance of a standard solution and the absorbance of a reaction solution.