CN111562339B

CN111562339B - Method and equipment for detecting hemoglobin substances

Info

Publication number: CN111562339B
Application number: CN202010556522.5A
Authority: CN
Inventors: 徐岩; 刘先成; 曾映; 胡明龙; 胡文雍; 张波
Original assignee: Lifotronic Technology Co ltd
Current assignee: Lifotronic Technology Co ltd
Priority date: 2020-06-17
Filing date: 2020-06-17
Publication date: 2022-11-15
Anticipated expiration: 2040-06-17
Also published as: CN111562339A

Abstract

The invention relates to a detection method and detection equipment for hemoglobin substances. The detection method of the hemoglobin substances comprises the following steps: performing cation exchange liquid chromatography detection on a sample to be detected to obtain a detection signal of the sample to be detected, wherein a mobile phase comprises succinate, and the pH value of the mobile phase is 4 to 8; and analyzing the detection signal to judge whether the sample to be detected contains hemoglobin substances, wherein the hemoglobin substances comprise hemoglobin variants, and the hemoglobin variants comprise Hb New York. The method for detecting the hemoglobin substances can effectively detect the Hb New York, is beneficial to avoiding the interference of the detection result of the hemoglobin substances of the Hb New York, and solves the technical problem that people are eagerly to solve but can not succeed all the time.

Description

Method and equipment for detecting hemoglobin substances

Technical Field

The invention relates to the technical field of detection, in particular to a detection method and detection equipment for hemoglobin substances.

Background

Hemoglobin (Hb), a specific protein for transporting oxygen in erythrocytes, is a protein that gives blood a red color and is composed of globin and heme. Hemoglobin consists of HbA (hemoglobin a), hbA2 (hemoglobin A2), and HbF (hemoglobin F). Among them, hbA is mainly present in an adult, and a globin peptide chain thereof is composed of two α chains and two β chains. HbA is classified into HbA0 and HbA1, hbA0 is non-glycosylated HbA, and HbA1 is glycosylated HbA. The polypeptides of the HbA2 globin subunit are linked by two alpha chains and two delta chains. HbF is predominantly present in fetal phase, with globin peptide chains comprising two alpha and two gamma chains.

The hemoglobin variant is a substance generated by changing the structure of a globin molecule due to single or multiple amino acid substitutions or deletions of a peptide chain of the hemoglobin caused by the genetic mutation of the globin. Currently, more than 1173 hemoglobin variants have been found. Wherein Hb New York is abnormal hemoglobin of globin beta chain, and is abnormal hemoglobin formed by replacing Val (valine) at position 113 of the globin beta chain with Glu (glutamic acid).

Generally, methods for detecting hemoglobin include capillary electrophoresis and liquid chromatography. The capillary electrophoresis method has good precision and accuracy, and is not interfered by common hemoglobin variants. The capillary electrophoresis method is not easy to detect quickly because of its low detection speed due to its automation degree. The liquid chromatography has high automation degree and high detection speed, and can be used for quickly detecting hemoglobin. However, since the isoelectric points of hemoglobin variant and hemoglobin are relatively close, the detection by liquid chromatography is easily interfered by hemoglobin variant, and the accuracy of the detection result is affected. Particularly, the isoelectric points and the charge quantities of Hb New York and hemoglobin are relatively close, and the accuracy of the detection result of the liquid chromatography is greatly influenced. Then, the existing liquid chromatography cannot achieve the level of effectively detecting Hb New York because of the separation degree, so that the Hb New York is difficult to identify, and the interference of the Hb New York on the detection result is avoided. How to detect Hb New York through liquid chromatography so as to avoid the interference of Hb New York on the detection result becomes a technical problem in the technical field and cannot be overcome all the time.

Disclosure of Invention

Accordingly, it is necessary to provide a method for detecting hemoglobin-based substances with high separation degree. The detection method can effectively detect Hb New York.

A method for detecting hemoglobin substances comprises the following steps:

carrying out cation exchange liquid chromatography detection on a sample to be detected to obtain a detection signal of the sample to be detected, wherein a mobile phase comprises succinate, and the pH value of the mobile phase is 4-8; and

analyzing the detection signal to judge whether the sample to be detected contains hemoglobin substances, wherein the hemoglobin substances comprise hemoglobin variants, and the hemoglobin variants comprise Hb New York.

The research finds that the isoelectric points and the charge quantities of Hb New York and HbA0 are very close, and Hb New York is very difficult to detect and separate. At present, no report on the effective detection of Hb New York by liquid chromatography is available on a global scale. Through a great deal of research, the research creatively discovers that the cation exchange liquid chromatography detection of a sample to be detected is carried out by taking a solution containing succinate with the pH value of 4-8 as a mobile phase, so that the separation degree of Hb New York and HbA0 can be improved, hb New York can be effectively detected, the interference of the Hb New York on the detection result of hemoglobin substances can be avoided, and the technical problem that people are eagerly solved but fail to succeed all the time is solved. Tests prove that when the detection method of the hemoglobin substances is used for detecting a sample to be detected, the separation degree of Hb New York and HbA0 is greater than 80%, the separation degree of Hb New York and HbA0 is higher, and Hb New York can be effectively detected.

In one embodiment, the hemoglobin variant further comprises at least one of HbE, hbD, hbS, hbC, hbJ-Bangkok, hb G-Taipei, and Hb Q-Thailand.

In one embodiment, the hemoglobin-based substance further comprises at least one of glycated hemoglobin, non-glycated hemoglobin, and hemoglobin degrader, the glycated hemoglobin comprises at least one of HbA1a, hbA1b, hbA1c, and LA1c, the non-glycated hemoglobin comprises at least one of HbA0, hbA2, and HbF, and the hemoglobin degrader comprises at least one of P3 and P4.

In one embodiment, the concentration of the succinate in the mobile phase is 30 mmol/L-900 mmol/L.

In one embodiment, the succinate salt is selected from at least one of sodium succinate or potassium succinate.

In one embodiment, in the step of performing cation exchange liquid chromatography detection on the sample to be detected, the elution mode is three-phase isocratic elution, the mobile phase includes a first phase, a second phase and a third phase, the first phase includes 30mmol/L to 60mmol/L of the succinate, the second phase includes 80mmol/L to 120mmol/L of the succinate, and the third phase includes 150mmol/L to 900mmol/L of the succinate.

In one embodiment, the pH of the first phase is 5.2 to 5.5.

In one embodiment, the pH of the second phase is 5.2 to 5.5.

In one embodiment, the pH value of the third phase is 5.2 to 5.5.

In one embodiment, in the step of performing cation exchange liquid chromatography detection on the sample to be detected, the step of eluting includes: eluting with the first phase within 0min to 1.5min, eluting with the second phase within 1.5min to 3.2min, and eluting with the third phase within 3.2min to 5.0 min.

In one embodiment, the step of eluting further comprises the steps of: eluting with the first phase for 5min to 6 min.

In one embodiment, in the step of performing cation exchange liquid chromatography detection on the sample to be detected, the particle size of the filler of the chromatographic column is 3-6 μm.

In one embodiment, in the step of performing cation exchange liquid chromatography detection on the sample to be detected, the column pressure of the chromatographic column is above 4 MPa.

In one embodiment, in the step of performing cation exchange liquid chromatography detection on the sample to be detected, the chromatographic column includes a housing, and the material of the housing includes at least one of stainless steel or polyetheretherketone.

In one embodiment, in the step of performing cation exchange liquid chromatography detection on the sample to be detected, the inner diameter of the chromatographic column is 4.0mm to 4.6mm, and the column length of the chromatographic column is 20mm to 50mm.

In one embodiment, in the step of performing cation exchange liquid chromatography detection on the sample to be detected, the flow rate is 0.5mL/min to 6.0mL/min.

In one embodiment, in the step of performing cation exchange liquid chromatography detection on the sample to be detected, the column temperature is 25 ℃ to 40 ℃.

In one embodiment, in the step of performing cation exchange liquid chromatography detection on the sample to be detected, the sample injection amount is 1 μ L-100 μ L.

In one embodiment, in the step of performing cation exchange liquid chromatography detection on the sample to be detected, the detection wavelength is 200nm to 500nm.

In one embodiment, before the step of performing cation exchange liquid chromatography detection on the sample to be detected, the method further comprises the following steps: and diluting the sample to be detected by adopting a diluent.

In addition, this research still provides a check out test set of hemoglobin class material, includes:

the detection module can perform cation exchange liquid chromatography detection on a sample to be detected by taking a solution containing succinate with the pH of 4-8 as a flowing phase to obtain a detection signal of the sample to be detected;

and the analysis module can analyze the detection signal to judge whether the sample to be detected contains hemoglobin substances, wherein the hemoglobin substances comprise hemoglobin variants, and the hemoglobin variants comprise Hb New York.

Drawings

One or more embodiments are illustrated by corresponding figures in the drawings, which are not to be construed as limiting the embodiments, unless expressly stated otherwise, and the drawings are not to scale.

FIG. 1 is a liquid chromatogram of a sample to be tested in example 1;

FIG. 2 is a liquid chromatogram of a sample to be tested in example 2;

FIG. 3 is a liquid chromatogram of a sample to be tested in example 3;

FIG. 4 is a liquid chromatogram of the sample to be measured obtained in example 3, plotted after the maximum value of the ordinate is reduced;

FIG. 5 is a liquid chromatogram of four different samples to be tested in example 4;

FIG. 6 is a liquid chromatogram of a sample to be tested in example 5;

FIG. 7 is a liquid chromatogram of a sample to be tested in example 6;

FIG. 8 is a liquid chromatogram of a sample to be tested in example 7;

FIG. 9 is a liquid chromatogram of a sample to be tested in example 8;

FIG. 10 is a liquid chromatogram of a sample to be tested in example 9;

FIG. 11 is a liquid chromatogram of a sample to be tested in example 10;

FIG. 12 is a liquid chromatogram of the sample to be tested in example 11;

FIG. 13 is a liquid chromatogram of a sample to be tested in comparative example 1;

FIG. 14 is a liquid chromatogram of a sample to be tested in comparative example 2;

fig. 15 is a liquid chromatogram of the sample to be measured in comparative example 3.

Detailed Description

The present invention will now be described more fully hereinafter for purposes of facilitating an understanding thereof, and may be embodied in many different forms and are not limited to the embodiments described herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete. It should be noted that the terms "first," "second," and the like are used for descriptive purposes only and are not to be construed as indicating or implying relative importance.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.

Unless otherwise specified, in the following description, hb New York (New York Variant Hemoglobin) is abnormal Hemoglobin of the globin β chain, and is abnormal Hemoglobin formed by replacing Val (valine) at position 113 of the globin β chain with Glu (glutamic acid).

HbE (HbE Variant Hemoglobin) is an abnormal Hemoglobin formed by replacing Glu (glutamic acid) at position 26 of the beta chain of globin by Lys (lysine). HbE is the most common one of globin β chain aberrant hemoglobins. HbE is mainly distributed in southeast asian countries. In China, hbE mainly exists in the south of Yangtze river and in coastal areas.

HbS (HbS Variant Hemoglobin) is an abnormal Hemoglobin formed by replacement of Glu (glutamic acid) at position 6 of the globin β chain by Val (valine). Homozygous HbS is called sickle cell disease, hbS most commonly found in north africa and north america.

HbC (HbC Variant Hemoglobin) is abnormal Hemoglobin formed by replacing Glu (glutamic acid) at position 6 of a beta chain of globin by Lys (lysine), and is mainly distributed in Western Africa.

HbD (HbD Variant Hemoglobin) is distributed in northwest and north-south America of India. Among them, there are many people carrying HbD in Panjab region of India and Indian.

HbJ-Bangkok (J-Bangkok Variant Hemoglobin) is abnormal Hemoglobin formed by replacing Gly (glycine) at the 56 th site of a globin beta chain with Asp (aspartic acid), and is highly developed in southern areas of China.

Hb G-Taipei (G-Taipei Variant Hemoglobin) is abnormal Hemoglobin formed by replacing 22 th Glu (glutamic acid) of a globin beta chain with Gly (glycine), and is highly prevalent in northern areas of China.

Hb Q-Thailand (Q-Thailand Variant Hemoglobin) is abnormal Hemoglobin formed by replacing 74 th Asp (aspartic acid) of globin alpha chain with His (histidine), and is highly prevalent in northern areas of China.

HbA1a (Hemoglobin-A1 a) is lactosylated Hemoglobin. HbA1b (Hemoglobin-A1 b) is a fructosylated Hemoglobin. HbA1c (Stable Glycated Hemoglobin) is Stable Glycated Hemoglobin, and the content of HbA1c can characterize the mean blood glucose level from 2 to 3 months in the past. LA1c (Labile Glycated Hemoglobin) is unstable Glycated Hemoglobin, and Schiff base, an intermediate product in the glycation process, is degraded into glucose and Hemoglobin.

HbA0 (Non-glycated Hemoglobin) is unglycated Hemoglobin, and its content is more than 80% of that of normal persons. HbF (total Hemoglobin) is Fetal Hemoglobin. HbA2 (A2 Hemoglobin), hemoglobin HbA2, is an increased level of Hemoglobin, which is mainly seen in mild β -globin aplastic anemia, a β -anemic factor.

P3 (Peak 3) and P4 (Peak 4) are the degradation peaks of hemoglobin HbA0, typically at less than 5%. If the blood sample is not fresh or has other variant hemoglobin, the P3 and P4 content will increase.

The method for detecting a hemoglobin-based substance according to an embodiment can effectively detect Hb New York, and can be used for detection and analysis of a hemoglobin-based substance for the purpose of diagnosis and treatment of a non-disease. Wherein the hemoglobin-based substance comprises a hemoglobin variant. The hemoglobin variant comprises Hb New York.

In one embodiment, the hemoglobin variant further comprises at least one of HbE, hbD, hbS, hbC, hbJ-Bangkok, hb G-Taipei, and Hb Q-Thailand. Further, the hemoglobin-based substance may further include at least one of glycated hemoglobin, non-glycated hemoglobin, and hemoglobin degradation products. The glycated hemoglobin includes at least one of HbA1a, hbA1b, hbA1c, and LA1 c. The non-glycated hemoglobin includes at least one of HbA0, hbA2, and HbF. The hemoglobin degradation product comprises at least one of P3 and P4.

Specifically, the detection method of hemoglobin substances comprises the following steps S110-S120:

s110, carrying out cation exchange liquid chromatography detection on a sample to be detected to obtain a detection signal of the sample to be detected, wherein the mobile phase comprises succinate, and the pH value of the mobile phase is 4-8.

Research shows that the isoelectric points and the charge quantities of Hb New York and HbA0 are very close, and Hb New York is very difficult to detect and separate. Currently, chloride is mainly used as an eluent for elution. However, chloride cannot elute and separate hemoglobin variants such as Hb New York. There is no report on the effective detection of Hb New York by liquid chromatography on a global scale. Through a great deal of research, the research unexpectedly discovers that the cation exchange liquid chromatography detection of a sample to be detected is carried out by taking a succinate-containing solution with the pH value of 4-8 as a mobile phase, so that the separation degree of Hb New York and HbA0 can be improved, hb New York can be effectively detected, the interference of the detection result of hemoglobin substances of Hb New York can be avoided, and the technical problem that people are eagerly to solve but are not successful all the time is solved.

In one embodiment, the succinate salt is sodium succinate or potassium succinate. The two succinate raw materials are easy to obtain, and are beneficial to detecting and separating various hemoglobin substances, in particular to detecting and separating Hb New York. It should be noted that the succinate salt is not limited to the succinate salt indicated above, but may be another succinate salt in the art.

In one embodiment, the concentration of succinate in the mobile phase is 30 mmol/L-900 mmol/L. Research shows that when the concentration of succinate is less than 30mmol/L, the elution effect cannot be achieved, so that hemoglobin substances cannot be separated; if the concentration of succinate exceeds 900mmol/L, salts may be separated out, which affects the liquid flow rate and elution process of the pipeline. The concentration of succinate in the mobile phase is set to be 30 mmol/L-900 mmol/L, so that detection and separation of various hemoglobin substances are facilitated, and particularly Hb New York is detected and separated.

Further, in the step of performing cation exchange liquid chromatography detection on the sample to be detected, the elution mode is three-phase isocratic elution. The mobile phase includes a first phase, a second phase, and a third phase. The first phase comprises 30 mmol/L-60 mmol/L succinate. The second phase comprises succinate of 80 mmol/L-120 mmol/L. The third phase comprises 150 mmol/L-900 mmol/L succinate.

Wherein the pH value of the first phase is 5.2-5.5. The arrangement ensures that various hemoglobin substances have higher separation degree, better peak type and high detection result accuracy.

The pH of the second phase is from 5.2 to 5.5. The arrangement ensures that various hemoglobin substances have higher separation degree, better peak type and high detection result accuracy.

The pH value of the third phase is 5.2 to 5.5. The arrangement ensures that various hemoglobin substances have higher separation degree, better peak type and high detection result accuracy.

Further, the first phase is a succinate aqueous solution with the pH value of 5.2 to 5.5 and the concentration of 30 to 60 mmol/L. The second phase is a succinate aqueous solution with the pH value of 5.2 to 5.5 and the concentration of 80mmol/L to 120 mmol/L. The third phase is a succinate aqueous solution with the pH value of 5.2 to 5.5 and the concentration of 150mmol/L to 900mmol/L. The separation degree of various hemoglobin substances can be improved by the arrangement, particularly the separation degree of Hb New York and HbA0 is improved, and the separation and detection of various hemoglobin substances are facilitated.

In one embodiment, in the step of performing cation exchange liquid chromatography detection on the sample to be detected, the step of eluting includes: eluting with the first phase within 0min to 1.5min, eluting with the second phase within 1.5min to 3.2min, and eluting with the third phase within 3.2min to 5.0 min. The arrangement is favorable for detecting and separating various hemoglobin substances such as Hb New York, hbA1c, hb J-Bangkok, hb E, hb G-Taipei and the like. It should be noted that "elution is performed at 0min to 1.5min with the first phase" means that elution is performed with the first phase as a mobile phase from the start of elution (i.e., 0 min) to 1.5min after elution. And so on.

Further, the step of eluting further comprises the steps of: eluting with the first phase for 5min to 6 min. The arrangement is favorable for separating various hemoglobin substances such as Hb Q-Thailand and the like.

The time control of the elution step is beneficial to eluting and separating various variant hemoglobins, improves the resolution and the anti-interference performance of detection, and particularly can thoroughly elute Hb New York variant hemoglobins.

The elution method is not limited to three-phase isocratic elution, and may be other elution methods, for example, two-phase gradient elution, four-phase isocratic elution, or four-phase gradient elution.

In one specific example, the off-peak time of Hb E is 230.7 seconds ± 0.30 seconds. The peak off time of Hb New York was 118.8 seconds. + -. 0.30 seconds. The peak-off time of Hb G-Taipei was 226.8 seconds. + -. 0.50 seconds. The peak off time of Hb J-Bangkok was 105.0 seconds. + -. 0.30 seconds. The peak time of Hb Q-Thailand was 300.1 sec. + -. 0.30 sec.

Wherein the filler particle size of the chromatographic column is 3-6 μm. Researches show that when the particle size of the filler is less than 3 mu m, the pressure of a chromatographic column can be more than 20MPa, and the common pipeline is easy to have liquid leakage. When the particle size of the filler is larger than 6 μm, the column efficiency and the resolution of the chromatographic column can be reduced, the separation of hemoglobin substances is incomplete, and part of critical variant hemoglobin such as Hb New York cannot be separated. The filler particle size of the chromatographic column is set to be 3-6 microns, so that various hemoglobin substances can be separated. The particle diameter of the filler is equal to the average particle diameter of the filler.

Further, cation exchange groups are bonded to the filler. The cation exchange group is used for adsorbing and separating hemoglobin substances. Further, the cation exchange group includes at least one of a sulfonic acid group and a carboxyl group.

The column pressure of the chromatographic column is above 4 MPa. The arrangement is favorable for improving the column efficiency, the separation degree and the resolution of the chromatographic column, is favorable for separating and detecting various hemoglobin substances, and is particularly favorable for separating and detecting Hb New York. Further, the column pressure of the chromatographic column is from 4MPa to 20MPa. The arrangement is not only beneficial to the separation and detection of various hemoglobin substances, but also can reduce the liquid leakage probability of the detection pipeline.

The chromatography column comprises a housing. The material of the housing comprises at least one of stainless steel or polyetheretherketone. The two materials have the characteristics of high insulation stability, hydrolysis resistance, compression resistance, corrosion resistance and the like, can not react with hemoglobin and microspheres, and are favorable for ensuring the separation and detection of various hemoglobin substances. Further, the housing includes a vial and an end fitting. The material of both the vial and the end fitting comprises at least one of stainless steel or polyetheretherketone.

The inner diameter of the chromatographic column is 4.0mm to 4.6mm. The column length of the chromatographic column is from 20mm to 50mm.

The chromatographic column is an SX type cation exchange chromatographic column independently produced by Shenzhen Pumen science and technology Limited.

In one embodiment, in the step of performing cation exchange liquid chromatography detection on the sample to be detected, the flow rate is 0.5 mL/min-6.0 mL/min. When the flow rate is lower than 0.5mL/min, the total testing time is far longer than 6 minutes, and the market demand is difficult to meet; when the flow rate is higher than 6.0mL/min, the pressure is over 20MPa, and the liquid leakage of the pipeline is easy to occur. Further, the flow rate is 1.5mL/min to 3.0mL/min.

In one embodiment, in the step of performing cation exchange liquid chromatography detection on the sample to be detected, the column temperature is 25 ℃ to 40 ℃. Researches find that when the column temperature is lower than 25 ℃, the instrument can generate serious condensed water in a high-temperature and high-humidity environment to influence the normal test of the colorimetric module; when the temperature of the column is higher than 40 ℃, the instrument can be recovered after waiting for a long time (more than half an hour) in a low-temperature environment (lower than 10 ℃) after being started. The column temperature is controlled to be 25-40 ℃, so that the normal operation of detection equipment can be ensured, and the separation and detection of hemoglobin substances can be ensured.

In one embodiment, in the step of performing cation exchange liquid chromatography detection on the sample to be detected, the sample injection amount is 1-100 μ L. When the sample introduction amount is less than 1uL, the total peak output area is far lower than the sensitivity lower limit of the instrument, the system is difficult to identify peaks of various hemoglobin substances, and meanwhile, the repeatability is very poor; when the sample introduction amount is more than 100uL, the total peak-out area is more than the highest limit of the instrument, the resolution is poor, the chromatographic column residue is serious, and the service life is shortened. Furthermore, the sample injection amount is 1-10 mu L.

In one embodiment, in the step of performing cation exchange liquid chromatography detection on the sample to be detected, the detection wavelength is 200nm to 500nm. The hemoglobin substance has an absorption spectrum at the detection wavelength of 200nm to 500nm. Further, the detection wavelength was 415nm.

In one embodiment, the sample to be tested is a blood sample. The sample to be measured is not limited to a blood sample, and may be an experimental sample requiring detection of hemoglobin-based substances or an experimental sample containing hemoglobin-based substances.

Further, before the step of performing cation exchange liquid chromatography detection on the sample to be detected, the method also comprises the following steps: and diluting the sample to be detected by using a diluent. The diluent may be deionized water or a hemolytic agent, for example. Furthermore, the sample to be tested is a blood sample, and the step of diluting the sample to be tested by using the diluent comprises the following steps: and diluting 5-10 mu L of sample to be detected by adopting 1500 mu L of diluent.

It should be noted that, if the concentration of the sample to be measured can meet the actual requirement, the step of diluting the sample to be measured with the diluent is omitted.

In one embodiment, the detection signal is a response signal of the substance to be detected. The detection signal may be, for example, an electrical signal of each component in the substance to be detected.

And S120, analyzing the detection signal to judge whether the sample to be detected contains hemoglobin substances, wherein the hemoglobin substances comprise hemoglobin variants, and the hemoglobin variants comprise Hb New York.

In one embodiment, the hemoglobin variant further comprises at least one of HbE, hbD, hbS, hbC, hbJ-Bangkok, hb G-Taipei, and Hb Q-Thailand. The method for detecting hemoglobin can separate and detect not only Hb New York but also at least one of HbE, hbD, hbS, hbC, hbJ-Bangkok, hb G-Taipei and Hb Q-Thailand.

Further, the hemoglobin-based substance may further include at least one of glycated hemoglobin, non-glycated hemoglobin, and hemoglobin degradation products. The glycated hemoglobin includes at least one of HbA1a, hbA1b, hbA1c, and LA1 c. The non-glycated hemoglobin includes at least one of HbA0, hbA2, and HbF. The hemoglobin degradation product comprises at least one of P3 and P4. The method for detecting a hemoglobin substance can separate and detect not only a hemoglobin variant but also at least one of glycated hemoglobin, non-glycated hemoglobin, and a hemoglobin degradation product.

In one embodiment, the step of analyzing the detection signal to determine whether the sample contains hemoglobin includes analyzing the detection signal by using data analysis software to obtain a chromatogram of absorbance and peak-off time of each component of the substance. And judging whether the sample to be detected contains hemoglobin substances and the types of the contained hemoglobin substances according to the absorbance and the peak-off time.

Some studies have analytically detected hemoglobin variants by high performance liquid chromatography, which has focused on four common hemoglobin variants of HbE, hbD, hbS and HbC. Hb New York and HbA0 have very close isoelectric points and charge amounts, so that Hb New York is very difficult to detect and separate. At present, the effective detection of Hb New York by liquid chromatography has not been reported on a global scale. Through a large amount of researches, the inventor unexpectedly discovers that the method can improve the separation degree of Hb New York and HbA0 and effectively detect Hb New York by taking a succinate-containing solution with the pH value of 4-8 as a mobile phase and performing cation exchange liquid chromatography detection on a sample to be detected, is beneficial to avoiding the interference of a detection result of a hemoglobin substance of the Hb New York, and solves the technical problem that people are eagerly solved but are not successful all the time. Tests prove that when the detection method of the hemoglobin substances is used for detecting a sample to be detected, the separation degree of Hb New York and HbA0 is greater than 80%, the separation degree of Hb New York and HbA0 is higher, and Hb New York can be effectively detected.

In the method for detecting hemoglobin substances, the elution mode is three-phase isocratic elution, the mobile phase comprises a first phase, a second phase and a third phase, the first phase comprises 30-60 mmol/L succinate, the second phase comprises 80-120 mmol/L succinate, and the third phase comprises 150-900 mmol/L succinate, so that the method is favorable for separating and detecting at least one hemoglobin variant in Hb New York, hbE, hbD, hbS, hbC, hbJ-Bangkok, hbG-Taipei and Q-Thialand, can separate and detect at least one glycated hemoglobin in HbA1a, hbA1b, hbA1c and LA1c, can separate and detect at least one non-glycated hemoglobin in HbA0, hbA2 and HbF, can improve the separation and detection results of at least one hemoglobin degradation product in P3 and P4, can improve the separation and detection results of various hemoglobin substances, and is particularly favorable for detecting various hemoglobin A and Hb, and improving the accuracy of the separation and detection results of hemoglobin and Hb, and the hemoglobin degradation products.

In the method for detecting a hemoglobin-based substance, the step of eluting includes: elution is carried out at 0min to 1.5min by using a first phase, elution is carried out at 1.5min to 3.2min by using a second phase, elution is carried out at 3.2min to 5.0min by using a third phase, and elution is carried out at 5min to 6min by using the first phase, so that separation and detection of at least one of Hb New York, hbE, hbD, hbS, hbC, hbJ-Bangkok, hb G-Taipei, hbQ-Thailand, hbA1a, hbA1b, hbA1c, LA1c, hbA0, hbA2, hbF, P3 and P4 are facilitated, separation and detection of at least one of Hb New York, hbE, hbD, hbS, hbC, hbJ-Bangkok, hbG-Taipei, hbQ-Thland, hbA1a, hbA1b, hbA1c, LA1c, hbJ-Bangkok, hb, hbF, hbP 3 and P4 are also simultaneously achieved, and the separation and the interference of Hb protein can be improved, and the variation of Hb can be more accurately detected, and the Hb protein variation can be more accurately detected.

The existing detection methods for detecting hemoglobin substances mainly comprise capillary electrophoresis, ion exchange high performance liquid chromatography and gene diagnosis. The capillary electrophoresis technology is mainly monopolized by foreign companies, the price is high, the price of a single instrument is more than several times of that of an HPLC instrument, and the capillary electrophoresis technology cannot be applied in batches; the sample analysis needs to be carried out in batches, the speed is slow, and the real-time detection cannot be carried out; the degree of automation is low, and the functions of emergency insertion, automatic maintenance and the like are relatively deficient; the multi-channel capillary has channel difference, the calibration procedure is more complex, and the difference between instruments is larger. However, the price of gene diagnosis is high, the operation is complex, and the method is not suitable for large-scale screening and the like. In the method for detecting hemoglobin substances, the mobile phase and the elution time sequence are optimized on the basis of ion exchange high performance liquid chromatography, so that on the basis of successfully separating and detecting HbA1a, hbA1b, hbF, LA1c, hbA1c, P3, P4, hbA0 and HbA2 and four common variants HbE, hbD, hbS and HbC in the world, hemoglobin variants such as Hb New York, hbJ-Bangkok, hbG-Taipei and HbQ-Thieland in China can be separated and detected, the peak of the hemoglobin variant in a chromatogram is obviously different from the peak of normal hemoglobin, the peak type of each peak is good, the resolution is high, and the performance of the detection method adopting the hemoglobin substances and the capillary electrophoresis method are equivalent through a large number of experimental verification and clinical feedback. Moreover, the hemoglobin substance detection method has the advantages of low cost, high automation degree, convenience in operation and maintenance, high repeatability and accuracy and the like, can be used for batch detection, is favorable for automatically screening and identifying various hemoglobin variants in the screening process of the thalassemia, finds interference possibly existing in the hemoglobin variants in advance, reduces artificial judgment errors and risks, and avoids reporting inaccurate results.

In addition, the present inventors have also provided a hemoglobin-based substance detection apparatus according to an embodiment, which can effectively detect Hb New York and can be used for detection and analysis for the purpose of diagnosis and treatment of non-diseases of hemoglobin-based substances.

Further, the detection device of the hemoglobin substances detects the sample to be detected according to the detection method of the hemoglobin substances.

Specifically, the detection device for hemoglobin substances comprises a detection module and an analysis module. The detection module can perform cation exchange liquid chromatography detection on a sample to be detected by taking a solution containing succinate with the pH of 4-8 as a flowing phase to obtain a detection signal of the sample to be detected. The analysis module can analyze the detection signal to judge whether the sample to be detected contains hemoglobin substances, wherein the hemoglobin substances comprise hemoglobin variants, and the hemoglobin variants comprise Hb New York.

Wherein, the detection module is a high performance liquid chromatography device or a hemoglobin analyzer. The analysis module includes data analysis software. The detection equipment of the hemoglobin substances is a high performance liquid chromatography device with an analysis module or a hemoglobin meter with an analysis module. Specifically, the detection module comprises an automatic sampler, a high-pressure pump for delivering eluent, a detector for detecting hemoglobin substances and the like. The analysis module includes hemoglobin analysis system software. It should be noted that the high performance liquid chromatography device is a common high performance liquid chromatography device in the art, the hemoglobin analyzer is a common hemoglobin analyzer in the art, and the analysis module is common data analysis software in the art, which is not described herein again.

The hemoglobin-based detection apparatus further comprises a reservoir for storing a mobile phase. The storage pool is in communication with the detection module. The storage pool can convey a solution containing succinate with the pH of 4-8 to the detection module to be used as a mobile phase for detection.

The detection equipment for the hemoglobin substances can effectively detect Hb New York, is beneficial to avoiding the interference of the detection result of the hemoglobin substances of the Hb New York, and solves the technical problem that people are eagerly to solve but can not succeed all the time.

DETAILED DESCRIPTION OF EMBODIMENT (S) OF INVENTION

The following detailed description is given with reference to specific examples. The following examples are not specifically described, and other components except inevitable impurities are not included. The examples, which are not specifically illustrated, employ drugs and equipment, all of which are conventional in the art. The experimental procedures without specifying the specific conditions in the examples were carried out under the conventional conditions such as those described in the literature, in books, or as recommended by the manufacturer.

Unless otherwise stated, the samples to be tested in the following examples are all blood samples taken from Shenzhen hospital of Beijing university. The detection equipment of the hemoglobin substances is a commercial high performance liquid chromatograph. The chromatographic column is an SX type cation exchange chromatographic column (cargo number: 04-I02B 000-00035) which is independently produced by Shenzhen Pumen science and technology corporation. The diluent is deionized water.

Examples 1 to 3

The procedure for detecting hemoglobin in examples 1 to 3 was as follows:

v2. Mu.L of the sample to be tested was diluted with V1. Mu.L of the diluent. And carrying out cation exchange high performance liquid chromatography detection on the diluted sample to be detected to obtain a detection signal of the sample to be detected. And analyzing the detection signal to obtain a chromatogram of the detection sample. Wherein, the control parameters of the liquid chromatogram are as follows: the elution mode is three-phase isocratic elution, the mobile phase comprises a first phase, a second phase and a third phase, the first phase is an aqueous solution of succinate with the concentration of C1, the second phase is an aqueous solution of succinate with the concentration of C2, and the third phase is an aqueous solution of succinate with the concentration of C3. The pH of the first phase is pH1. The pH of the second phase is pH2. The pH of the third phase is pH3. The step of eluting includes: eluting with the first phase in 0min to T1min, eluting with the second phase in T1min to T2min, eluting with the third phase in T2min to T3min, and eluting with the first phase in T3min to T4 min. The flow rate is v1. The column pressure was f1MPa. The column temperature was T ℃. The amount of sample was V3. Mu.L. The detection wavelength is lambda nm. The specific control parameters are detailed in table 1. In Table 1, mM means mmol/L. The samples to be tested in examples 1 to 3 were all blood samples clinically determined to contain HbA1a, hbA1b, hbF, hbA1c, P3, P4, hbA0, hbA2, and Hb New York. The measurement results are shown in FIGS. 1 to 3. FIG. 1 is a liquid chromatogram of a sample to be tested in example 1. FIG. 2 is a liquid chromatogram of a sample to be tested in example 2. FIG. 3 is a liquid chromatogram of a sample to be tested in example 3. Fig. 4 is a liquid chromatogram of the sample to be measured obtained in example 3 plotted after the maximum value of the ordinate is reduced. Since the maximum value of the ordinate is reduced in fig. 4, the bifurcation of the peak of HbA0 and the peak of Hb New York is not shown. The black filled portion in fig. 4 is the filling of its corresponding peak, for example: the filling at HbF is the filling of its corresponding peak of HbF. By analogy, the black fills in other figures (for example, fig. 6 to fig. 9) in the following description are all similar meanings, and are not described in detail later.

Table 1 control parameters for hemoglobin-based detection in examples 1 to 3

As can be seen from FIGS. 1-3, hb New York variant hemoglobin showed a peak position before HbA 0. The calculated separation degree of Hb New York and HbA0 in the figures 1-3 is greater than 80%, which shows that the separation degree of Hb New York and HbA0 is higher by the detection method for hemoglobin substances, hb New York can be effectively detected and separated, and the interference of Hb New York on HbA0 detection is avoided.

As can be seen from fig. 3 and 4, the above-described method for detecting hemoglobin can simultaneously detect and separate HbA1a, hbA1b, hbF, hbA1c, P3, P4, hbA0, hbA2, and Hb New York, and the separation degree between the peaks is high.

Example 4

The procedure for detecting a hemoglobin-based substance and the control parameters in example 4 were substantially the same as those in example 3, except that in example 4, four different samples to be detected were detected. The four different samples to be detected are respectively numbered as samples 1-4.

Sample 1 is a blood sample clinically determined to contain HbA1a, hbA1b, hbF, LA1c, hbA1c, P3, P4, hbA0, hbA2, and HbE.

Sample 2 is a blood sample clinically determined to contain HbA1a, hbA1b, hbF, LA1c, hbA1c, P3, P4, hbA0, hbA2, and HbD.

Sample 3 is a clinically determined blood sample containing HbA1a, hbA1b, hbF, LA1c, hbA1c, P3, P4, hbA0, hbA2, hbS.

Sample 4 is a clinically determined blood sample containing HbA1a, hbA1b, hbF, LA1c, hbA1c, P3, P4, hbA0, hbA2, hbC.

The results are shown in detail in FIG. 5. FIG. 5 is a liquid chromatogram of four different samples tested in example 4. The arrows (4-1), (4-2), (4-3) and (4-4) indicate the samples 1, 2, 3 and 4, respectively. a represents HbA1a, b represents HbA1b, f represents HbF, A0 represents HbA0, A2 represents HbA2, E represents HbE, D represents HbD, S represents HbS, and C represents HbC.

As can be seen from fig. 5, the above-mentioned method for detecting hemoglobin-based substances can simultaneously detect and separate HbA1a, hbA1b, hbF, LA1c, hbA1c, P3, P4, hbA0, hbA2, hbE, hbD, hbS, and HbC, the separation degree between the peaks is high, and it is possible to avoid interference of variant hemoglobin such as HbE, hbD, hbS, and HbC with detection of other hemoglobin-based substances (particularly, normal hemoglobin), and ensure the accuracy of the detection result.

Example 5

The procedure for detecting hemoglobin and the control parameters in example 5 were substantially the same as those in example 3, except that the sample to be measured in example 5 was a blood sample clinically determined to contain HbA1a, hbF, hbA1c, P4, hbA0, hbA2, and HbE. The results are shown in detail in FIG. 6. FIG. 6 is a liquid chromatogram of the sample to be tested in example 5.

As can be seen from fig. 6, the above method for detecting hemoglobin substances can simultaneously detect and separate HbA1a, hbF, hbA1c, P4, hbA0, hbA2, and HbE, and the separation degree between the peaks is high, particularly glycated hemoglobin HbA1c and β -thalassemia factor HbA2, which is advantageous for non-disease diagnosis and treatment purpose analysis of glycated hemoglobin HbA1c and β -thalassemia factors HbF and HbA2.

Example 6

The procedure for detecting a hemoglobin-based substance and the control parameters of example 6 were substantially the same as those of example 3, except that the sample to be measured of example 6 was a blood sample clinically determined to contain HbA1b, hbF, LA1c, hbA1c, P3, P4, hbA0, hbA2, and Hb J-Bangkok. The results are shown in detail in FIG. 7. FIG. 7 is a liquid chromatogram of the sample to be tested in example 6.

As can be seen from FIG. 7, the method for detecting hemoglobin can simultaneously detect and separate HbA1b, hbF, LA1c, hbA1c, P3, P4, hbA0, hbA2 and Hb J-Bangkok, has high separation degree between peaks, and is beneficial to non-disease diagnosis and treatment purpose analysis of Hb J-Bangkok.

Example 7

The procedure for detecting a hemoglobin-based substance and the control parameters of example 7 were substantially the same as those of example 3, except that the sample to be measured of example 7 was a blood sample clinically determined to contain HbA1a, hbF, hbA1c, P3, P4, hbA0, hbA2, hbE, and Hb G-Taipei. The results are shown in FIG. 8. FIG. 8 is a liquid chromatogram of the sample to be tested in example 7.

As can be seen from fig. 8, the above method for detecting hemoglobin can simultaneously detect and separate HbA1a, hbF, hbA1c, P3, P4, hbA0, hbA2, hbE, and Hb G-Taipei, and the separation degree between the peaks is high, which is advantageous for non-disease diagnosis and analysis of Hb G-Taipei for therapeutic purposes.

Example 8

The procedure for detecting hemoglobin and the control parameters in example 8 were substantially the same as those in example 3, except that the sample to be tested in example 8 was a blood sample containing HbA1a, hbA1b, hbF, hbA1c, P3, hbA0, hbA2, hbD, hbC, and HbQ-Thailand determined clinically. The results are shown in detail in FIG. 9. FIG. 9 is a liquid chromatogram of the sample to be tested in example 8.

As can be seen from fig. 9, the above-described method for detecting hemoglobin-based substances can simultaneously detect and separate HbA1a, hbA1b, hbF, hbA1c, P3, hbA0, hbA2, hbD, hbC, and Hb Q-Thailand, and the separation degree between the peaks is high, which is advantageous for non-disease diagnosis and analysis of Hb Q-Thailand for therapeutic purposes.

Example 9

The procedure for detecting a hemoglobin-based substance and the control parameters of example 9 were substantially the same as those of example 3, except that T1 was 1, T2 was 2.5, T3 was 4, and T4 was 5. The detection result is shown in detail in FIG. 10. FIG. 10 is a liquid chromatogram of the sample to be tested in example 9.

As can be seen from fig. 10, the separation degree between the peaks in example 9 is significantly lower than that in example 3, for example, the separation degree between HbF and HbA2, which indicates that shortening the time decreases the separation degree of the peaks of hemoglobin, particularly the separation degree of the main hemoglobin such as HbF and HbA2.

Example 10

The procedure for detecting a hemoglobin-based substance and the control parameters in example 10 were substantially the same as those in example 1, except that C1 was 20. The detection result is shown in detail in FIG. 11. FIG. 11 is a liquid chromatogram of the sample to be tested in example 10.

As can be seen from fig. 11, the separation degree between the peaks of example 10 is significantly lower than that of example 1, and a significant interference peak also appears in the chromatogram of example 10.

Example 11

The procedure for detecting a hemoglobin-based substance and the control parameters in example 11 were substantially the same as those in example 2, except that C3 was 1000. The detection result is shown in detail in FIG. 12. FIG. 12 is a liquid chromatogram of the sample to be tested in example 11.

As can be seen from FIG. 12, the peak appearance is forward and the kurtosis is not obvious due to the larger C3 in example 11.

Comparative example 1

The procedure for measuring hemoglobin and the control parameters of comparative example 1 were substantially the same as those of example 1, except that pH1 was 3, pH2 was 3, and pH3 was 3. The results are shown in FIG. 13. Fig. 13 is a liquid chromatogram of the sample to be tested in comparative example 1.

As can be seen from fig. 13, since pH1, pH2, and pH3 are all lower than 4, the peak of comparative example 1 becomes shorter and smaller, the column efficiency becomes significantly worse, and hemoglobin HbF and HbA2 cannot be completely separated.

Comparative example 2

The procedure for measuring hemoglobin and the control parameters of comparative example 2 were substantially the same as those of example 2, except that pH1 was 10, pH2 was 10, and pH3 was 10. The detection result is shown in detail in FIG. 14. Fig. 14 is a liquid chromatogram of the sample to be tested in comparative example 2.

As can be seen from fig. 14, since pH1, pH2, and pH3 are all higher than 8, all hemoglobin-based substances in the chromatogram of comparative example 2 peak early, and the types of hemoglobin-based substances cannot be distinguished.

Comparative example 3

The procedure for measuring hemoglobin and the control parameters of comparative example 3 were substantially the same as those of example 3, except that the first phase was an aqueous solution of phosphate having a concentration of C1, the second phase was an aqueous solution of phosphate having a concentration of C2, and the third phase was an aqueous solution of phosphate having a concentration of C3. The results are shown in detail in FIG. 15. Fig. 15 is a liquid chromatogram of the sample to be tested in comparative example 3.

As can be seen from fig. 15, the HbA2 separation degree in the chromatogram of comparative example 3 decreased, with the occurrence of an interference peak.

In summary, the method for detecting hemoglobin can efficiently detect variant hemoglobin in a sample to be detected, has a high separation degree between variant hemoglobin and normal hemoglobin, a good peak shape and a high resolution, and can be used for detection and analysis of non-disease diagnosis and treatment of hemoglobin substances.

The technical features of the embodiments described above may be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the embodiments described above are not described, but should be considered as being within the scope of the present specification as long as there is no contradiction between the combinations of the technical features.

The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent should be subject to the appended claims.

Claims

1. A method for detecting hemoglobin for non-disease diagnosis and treatment purposes, comprising the steps of:

performing cation exchange liquid chromatography detection on a sample to be detected to obtain a detection signal of the sample to be detected, wherein a mobile phase comprises succinate, the pH value of the mobile phase is 4-8, in the step of performing cation exchange liquid chromatography detection on the sample to be detected, the elution mode is three-phase isocratic elution, the mobile phase comprises a first phase, a second phase and a third phase, the first phase comprises 30-60 mmol/L of the succinate, the second phase comprises 80-120 mmol/L of the succinate, the third phase comprises 150-900 mmol/L of the succinate, and the step of elution comprises: eluting with the first phase for 0min to 1.5min, eluting with the second phase for 1.5min to 3.2min, and eluting with the third phase for 3.2min to 5.0 min; and

analyzing the detection signal to judge whether the sample to be detected contains hemoglobin substances, wherein the hemoglobin substances comprise hemoglobin variants, and the hemoglobin variants comprise Hb NewYork; wherein the hemoglobin variant further comprises at least one of HbE, hbD, hbS, hbC, hbJ-Bangkok, hb G-Taipei and Hb Q-Thailand; and/or the hemoglobin-based substance further comprises at least one of glycated hemoglobin, non-glycated hemoglobin and hemoglobin degradation products, wherein the glycated hemoglobin comprises at least one of HbA1a, hbA1b, hbA1c and LA1c, the non-glycated hemoglobin comprises at least one of HbA0, hbA2 and HbF, and the hemoglobin degradation products comprise at least one of P3 and P4.

2. The method of claim 1, wherein the succinate salt is at least one selected from sodium succinate and potassium succinate.

3. The method of claim 1, wherein the first phase has a pH of 5.2 to 5.5;

and/or the pH value of the second phase is 5.2-5.5;

and/or the pH value of the third phase is 5.2-5.5.

4. The method of claim 1, wherein the step of eluting further comprises the steps of: and eluting with the first phase for 5-6 min.

5. The method for detecting hemoglobin according to any one of claims 1-4, wherein the step of subjecting the sample to cation exchange liquid chromatography comprises packing the sample with a particle size of 3-6 μm;

and/or in the step of detecting the sample to be detected by cation exchange liquid chromatography, the column pressure of the chromatographic column is more than 4 MPa;

and/or in the step of performing cation exchange liquid chromatography detection on the sample to be detected, the chromatographic column comprises a shell, and the material of the shell comprises at least one of stainless steel or polyether-ether-ketone;

and/or in the step of detecting the sample to be detected by the cation exchange liquid chromatography, the inner diameter of the chromatographic column is 4.0 mm-4.6 mm, and the column length of the chromatographic column is 20 mm-50 mm;

and/or in the step of carrying out cation exchange liquid chromatography detection on the sample to be detected, the flow rate is 0.5 mL/min-6.0 mL/min;

and/or in the step of performing cation exchange liquid chromatography detection on the sample to be detected, the column temperature is 25-40 ℃;

and/or in the step of carrying out cation exchange liquid chromatography detection on the sample to be detected, the sample injection amount is 1-100 muL;

and/or in the step of carrying out cation exchange liquid chromatography detection on the sample to be detected, the detection wavelength is 200 nm-500 nm.

6. The method according to any one of claims 1 to 4, wherein the step of performing cation exchange liquid chromatography on the sample to be tested is preceded by the following steps: and diluting the sample to be detected by adopting a diluent.

7. An apparatus for detecting hemoglobin, comprising:

the detection module can perform cation exchange liquid chromatography detection on a sample to be detected by taking a solution containing succinate with the pH of 4-8 as a flowing phase, so as to obtain a detection signal of the sample to be detected, wherein the elution mode is three-phase isocratic elution in the cation exchange liquid chromatography detection on the sample to be detected, the flowing phase comprises a first phase, a second phase and a third phase, the first phase comprises 30-60 mmol/L of the succinate, the second phase comprises 80-120 mmol/L of the succinate, the third phase comprises 150-900 mmol/L of the succinate, and the elution step comprises: eluting with the first phase for 0min to 1.5min, eluting with the second phase for 1.5min to 3.2min, and eluting with the third phase for 3.2min to 5.0 min;

the analysis module can analyze the detection signal to judge whether the sample to be detected contains hemoglobin substances, wherein the hemoglobin substances comprise hemoglobin variants, and the hemoglobin variants comprise Hb NewYork; wherein the hemoglobin variant further comprises at least one of HbE, hbD, hbS, hbC, hbJ-Bangkok, hb G-Taipei and Hb Q-Thailand; and/or, the hemoglobin substance further comprises at least one of glycated hemoglobin, non-glycated hemoglobin and hemoglobin degradation products, wherein the glycated hemoglobin comprises at least one of HbA1a, hbA1b, hbA1c and LA1c, the non-glycated hemoglobin comprises at least one of HbA0, hbA2 and HbF, and the hemoglobin degradation products comprise at least one of P3 and P4.