JP6841503B2 - Column packing material for measuring hemoglobin and method for measuring hemoglobin - Google Patents

Description

The present invention relates to a column packing material for measuring hemoglobins used in high performance liquid chromatography, and a method for measuring hemoglobins using the column packing material.

Hemoglobin A1c (HbA1c) remains present in the blood for about 120 days, which is the lifespan of red blood cells. Therefore, by measuring hemoglobin A1c, the blood glucose status for the past 1 to 2 months can be estimated. Therefore, unlike other diabetes markers such as glycoalbumin, the transition of blood glucose level can be confirmed over a relatively long period of time by measuring hemoglobin A1c.

In the measurement of hemoglobin A1c, the measurement by high performance liquid chromatography is widely performed because the accuracy of the measured value is good. In high performance liquid chromatography, the measurement time is as important as the accuracy of the measured value. In order to reduce the burden on inspection engineers and to build an efficient inspection system, shortening the measurement time was a major issue along with improving the accuracy of measured values.

In Patent Document 1 below, there are two or more peaks of the frequency distribution obtained when the particle size distribution is measured, and among the peaks of the frequency distribution, the particle size of the peak showing the maximum particle size is the smallest grain. A column packing material for measuring hemoglobins, which is 1.1 to 3.0 times the particle size of the peak indicating the diameter, is disclosed.

Japanese Unexamined Patent Publication No. 2011-209040

An increase in the flow velocity greatly contributes to shortening the measurement time of high performance liquid chromatography. By the way, when hemoglobin A1c is measured by high performance liquid chromatography, if the other peaks generated adjacent to the peaks of hemoglobins such as hemoglobin A1c are large, the sharpness of the peaks of hemoglobins is impaired. Therefore, there is a risk that the separation performance will deteriorate.

In high performance liquid chromatography, sensitizing the peak of hemoglobins is an important issue in improving the separation performance. As one of the methods for determining the sensitivity of the hemoglobin peak, the bottom of the hemoglobin peak can be focused on. As shown in the schematic chromatogram diagram shown in FIG. 1, a downward protruding portion (hereinafter referred to as a “valley portion”) generated on the left side of the peak of hemoglobin A1c and an upward protruding portion (hereinafter referred to as “valley portion”) generated on the left side thereof. Both of the "mountain part") occur. In the chromatogram of hemoglobin A1c measurement, the ratio of the height from the baseline of the valley portion to the height from the baseline of the peak portion (hereinafter, referred to as "flatness on the left side of the A1c peak") is measured. It tends to increase as the flow velocity of time is increased. Therefore, the sharpness of the peak of hemoglobin A1c may be lost, and the separation performance of hemoglobin A1c may deteriorate.

When the column packing material described in Patent Document 1 is used, the column life can be extended. However, in the column packing material described in Patent Document 1, when the flow velocity is increased in order to speed up the measurement, the flatness on the left side of the peak tends to increase at the peak of hemoglobins such as hemoglobin A1c. Therefore, there is a risk that the separation performance of hemoglobins may deteriorate.

An object of the present invention is to provide a column packing material for measuring hemoglobins and a method for measuring the hemoglobins, which are excellent in the separation performance of the hemoglobins and can shorten the measurement time when measuring the hemoglobins by liquid chromatography. There is.

The present invention is a column packing material used for measuring hemoglobins by high-speed liquid chromatography, and has two or more peaks of frequency distribution obtained when measuring particle size distribution, and among the peaks of frequency distribution. the particle size of the peak indicating the maximum particle diameter is 1.1 to 3.0 times the particle size of the peak indicating the minimum particle size, the particle size of the peak (maximum peak) power in the frequency distribution is maximum , The particle size in the frequency distribution is smaller than the particle size showing the second largest peak (second peak), and the small particle size ratio represented by the following formula (1) is 0.91 to 0.99, and the particle size is the same. A column filler for measuring hemoglobins, wherein the average particle size of the column filler is in the range of 3.0 to 6.5 μm.

Small particle size ratio = (height from the baseline of the maximum peak) / (height from the baseline of the maximum peak + height from the baseline of the second peak) ... (1)

In the column packing material for measuring hemoglobins according to the present invention, preferably, the column packing material contains an acrylic monomer as a main component.

In the column packing material for measuring hemoglobins according to the present invention, preferably, there are two peaks of the frequency distribution obtained when the particle size distribution is measured.

In the liquid chromatography column for measuring hemoglobins according to the present invention, the column is filled with the above-mentioned column packing material for measuring hemoglobins, which is configured according to the present invention.

The method for measuring hemoglobins by liquid chromatography according to the present invention is a method for measuring hemoglobins by high-speed liquid chromatography, in which two or more peaks of the frequency distribution obtained when measuring the particle size distribution are present and the frequencies are present. of distribution peak, the particle size of the peak indicating the maximum particle diameter is 1.1 to 3.0 times the particle size of the peak indicating the minimum particle size, peak power in the frequency distribution is maximum (maximum peak) The particle size of is smaller than the particle size showing the second largest peak (second peak) in the frequency distribution , and the small particle size ratio represented by the following formula (1) is 0.91 to 0.99. A column filler is used, and the average particle size of the column filler is 3.0 to 6.5 μm, and the flow velocity is 2.5 to 3.7 mL / min. And.

Small particle size ratio = (height from the baseline of the maximum peak) / (height from the baseline of the maximum peak + height from the baseline of the second peak) ... (1)

According to the column packing material for measuring hemoglobins and the method for measuring hemoglobins according to the present invention, the sharpness of the peak of hemoglobins can be maintained even when the flow velocity is increased to increase the speed. Therefore, it is possible to achieve both a reduction in the measurement time and an improvement in the separation performance of hemoglobins.

FIG. 1 is a schematic diagram of a general chromatogram when hemoglobin A1c is measured in the technical field of the present invention. FIG. 2 shows the flatness on the left side of the peak of hemoglobin A1c at the peak of hemoglobin A1c when the flow velocity is changed stepwise in the measurement of the blood sample using the column packed with the column packing material according to the embodiment of the present invention. It is a figure which shows the change of the measured value of.

The details of the present invention will be described below.

(Diameter of filler particles)
The column packing material for measuring hemoglobins of the present invention (hereinafter, also simply referred to as a packing material) is composed of a particulate packing material.

The lower limit of the average particle size of the filler of the present invention is 3.0 μm, and the upper limit is 6.5 μm. If the average particle size of the filler is less than 3.0 μm, the pressure required to flow the eluent through the column increases, and the liquid chromatography device requires special parts for imparting pressure resistance. Become. If the average particle size of the filler exceeds 6.5 μm, the porosity in the column increases, which may affect the separation performance when the flow velocity is increased. The preferable lower limit of the average particle size of the filler of the present invention is 6.0 μm, and the preferable upper limit is 6.4 μm.

In this specification, the average particle size and the particle size distribution are based on the measurement results obtained by the particle size distribution measuring device based on the principle of the number counting method. However, even if the measuring device is based on another measurement principle, it can be used as long as the measurement result has a known correlation with the measurement result based on the principle of the number counting method or has been calibrated. Examples of such an apparatus include an apparatus based on a known principle such as a centrifugal sedimentation method, a dynamic light scattering method, a laser diffracted light scattering method, an ultrasonic attenuation method, a capillary method, and a Coulter method.

(Peak of frequency distribution)
In the filler of the present invention, there are two or more peaks of the frequency distribution (hereinafter, also simply referred to as peaks of the particle size distribution) obtained when the particle size distribution is measured. The filler of the present invention preferably has two to four peaks of the particle size distribution, and more preferably has two peaks.

In the present specification, the peak means a measurement point having a height higher than the baseline than the adjacent left and right measurement points. That is, the peak of the particle size distribution is the apex of each mountain in the graph showing the particle size distribution.

In the filler of the present invention, among the frequency distribution peaks, the particle size of the peak showing the maximum particle size is 1.1 to 3.0 times the particle size of the peak showing the minimum particle size, and the peak shows the maximum. The particle size is smaller than the particle size showing the second largest peak, and the small particle size ratio defined by the following formula (1) is 0.91 to 0.99.

Small particle size ratio = (height from the baseline of the maximum peak) / (height from the baseline of the maximum peak + height from the baseline of the second peak) ... (1)

When the small particle size ratio is in the above range, it is possible to both fill the column with the filler particles at a high density and control the optimum specific surface area of the filler particles. Therefore, it is possible to provide a column having both pressure resistance and high analytical ability. The preferred range for the small particle size ratio is 0.94 to 0.99.

(Production method)
As a method for producing the filler of the present invention, a known method can be used. For example, a method of mixing two or more kinds of fillers having a single peak in particle size distribution so as to meet the above-mentioned conditions. , A method of obtaining a filler having a plurality of peaks by polymerization and the like.

(Material of filler)
As the material of the filler of the present invention, a known material such as an inorganic material such as silica-based, ceramic-based or glass-based material, or an organic synthetic material such as an acrylic polymer or a styrene-based polymer can be used. it can. Of these, it is preferable to use an organic synthetic material, and it is more preferable to use an acrylic polymer as a main component.

In the present specification, "acrylic" means having an acryloyloxy group or a methacryloyloxy group, and "(meth) acrylic" means "acrylic or methacrylic".

As a method for producing a filler when the acrylic polymer is used as a material, a method of polymerizing an acrylic monomer by a known polymerization method can be used.

Examples of the acrylic monomer include polyethylene glycol di (meth) acrylates, polypropylene glycol di (meth) acrylates, alkylene glycol di (meth) acrylates, and the like, and at least two (meth) acryloyloxys in the molecule. Examples thereof include a crosslinkable acrylic monomer having a group.

The acrylic polymer is a method for polymerizing the crosslinkable acrylic monomer, the crosslinkable acrylic monomer, methyl (meth) acrylate, ethyl (meth) acrylate, polyethylene glycol mono (meth) acrylates. , 2-Hydroxyethyl (meth) acrylate and the like can be prepared by a method of copolymerizing with a non-crosslinkable acrylic monomer or the like.

(Surface condition)
The filler of the present invention preferably has an ion exchange group.

The ion exchange group is preferably a cation exchange group, more preferably a sulfonic acid group. For the filler having an ion exchange group, for example, when the main component of the filler is the acrylic polymer, a) an acrylic monomer and a monomer having an ion exchange group are polymerized to prepare an acrylic polymer. It can be prepared by a known method such as a method of preparing an acrylic polymer and then introducing an ion exchange group.

In addition, in this specification, a filler and a filler particle are used as synonymous terms unless otherwise specified.

(Measurement target)
By using the filler of the present invention, various hemoglobins can be measured by high performance liquid chromatography. Specifically, by using the column packing material for measuring hemoglobins of the present invention, hemoglobin A0, hemoglobin A1c, hemoglobin F (fetal hemoglobin), hemoglobin A2, hemoglobin S and the like can be measured by liquid chromatography. ..

Further, by using the filler of the present invention, it is possible to perform measurement under conditions where the flow velocity is higher than before. In order to shorten the measurement time, a method for measuring hemoglobins by high performance liquid chromatography using the column packing material for measuring hemoglobins of the present invention under the condition that the flow rate is 2.5 to 3.7 mL / min is also available. It is also one of the present inventions.

When measuring hemoglobins such as hemoglobin A1c by high performance liquid chromatography using the column packing material for measuring hemoglobins of the present invention, it is known to be equipped with a pump, sampler, detector, etc. for eluent delivery. A column filled with the column packing material for measuring hemoglobins of the present invention can be connected to a high performance liquid chromatography system to measure hemoglobins in a blood sample.

(Eluent)
As the eluent used for liquid chromatography using the column packing material for measuring hemoglobins of the present invention, it is preferable to use buffers or organic solvents containing known salt compounds. Specific examples of the buffer solution include organic acids, inorganic acids, salts thereof, amino acids, Good's buffer solution and the like.

The organic acid is not particularly limited, and examples thereof include citric acid, succinic acid, tartaric acid, and malic acid.

The inorganic acid is not particularly limited, and examples thereof include hydrochloric acid, nitric acid, sulfuric acid, phosphoric acid, boric acid, and acetic acid.

The amino acids are not particularly limited, and examples thereof include glycine, taurine, and arginine.

Examples of the salts include sodium salt, potassium salt, calcium salt, magnesium salt and the like.

In addition, other commonly added substances such as surfactants, various polymers, and hydrophilic low molecular weight compounds may be appropriately added to the buffer solution.

The preferable lower limit of the salt concentration of the buffer solution when measuring hemoglobin A1c is 10 mmol / L, and the preferable upper limit is 1000 mmol / L. If the salt concentration of the buffer solution is less than 10 mmol / L, the ion exchange reaction may not occur and hemoglobins may not be separated. If the salt concentration of the buffer exceeds 1000 mmol / L, salt may precipitate and adversely affect the system.

(Peak of hemoglobins)
As shown in FIG. 1, in the chromatogram of hemoglobin A1c, a downward protruding portion (“valley portion”) generated on the left side of the peak of hemoglobin A1c and an upward protruding portion (“mountain portion”) generated on the left side thereof. ") Both occur.

The "flatness on the left side of the peak", which is an evaluation index of the present invention, is obtained as follows.

"Flatness of the left part of the peak" = (height from the baseline that is the apex of the valley part) / (height from the baseline that is the apex of the mountain part)

In general, this value tends to increase as the flow velocity is increased during measurement, and it is judged that by suppressing the increase, the peak of hemoglobin A1c can be sharply maintained and a high separation ability can be maintained.

(Example)
Examples for explaining the effects of the invention will be described below, but the present invention is not limited to the following examples.

[Manufacturing Example 1]
2-Hydroxy-3-acryloyloxypropyl methacrylate (manufactured by Shin-Nakamura Chemical Co., Ltd.) 40 g, triethylene glycol dimethacrylate (manufactured by Shin-Nakamura Chemical Co., Ltd.) 140 g, pentaerythritol tetraacrylate (manufactured by Shin-Nakamura Chemical Co., Ltd.) 5 g, and 1.0 g of benzoyl peroxide was dissolved in a monomer mixture containing 15 g of pentaerythritol triacrylate (triester 37%) (manufactured by Shin-Nakamura Chemical Co., Ltd.). The obtained mixture was dispersed in 2 L of a 4 wt% polyvinyl alcohol aqueous solution, and the polymerization reaction was carried out for 1 hour by heating to 80 ° C. in a nitrogen atmosphere while stirring at 390 rpm using a stirring blade having a blade length of 60 mm. It was. After 1 hour, 200 mL of an aqueous solution in which 80 g of acrylamide-tert-butylsulfonic acid was dissolved was added to the reaction system, and the polymerization reaction was further carried out at 80 ° C. for 1 hour.

The obtained polymer particles were washed to obtain filler particles. The average particle size and particle size distribution of the obtained filler particles were measured by a particle size distribution measuring device. In the measurement, a semiconductor laser was used as the light source and LE400-05 was used as the sensor, and the measurement was performed for 120 seconds to display the volume distribution and calculate the average particle size. As a result, the average particle size was 4.45 μm, and the particle size distribution was a single peak.

[Manufacturing Example 2]
Filler particles were obtained in the same manner as in Production Example 1 except that the rotation speed during stirring in Production Example 1 was changed from 390 rpm to 300 rpm. The average particle size and particle size distribution of the obtained filler particles were measured. As a result, the average particle size was 9.47 μm, and the particle size distribution was a single peak.

Table 1 shows the average particle size and the number of particle size distribution peaks of the filler particles obtained in Production Examples 1 and 2.

The filler particles obtained in Production Examples 1 and 2 were mixed at the mixing ratios shown in Table 2 to obtain column fillers of Examples 1 and 2 and Comparative Examples 1 to 3. Table 2 shows various parameters of these column packing materials obtained.

The column packing materials obtained in Examples 1 and 2 and Comparative Examples 1 to 3 were filled in a stainless steel column having an inner diameter of 4.6 mm and a length of 20 mm to obtain a column for measuring hemoglobins. The obtained column was set in a general-purpose high performance liquid chromatography measuring device, and a blood sample was measured.

(Measurement of blood sample)
Control blood for quality control was used as a sample, and measurement was performed under the following conditions.

Measurement sample: Control blood for quality control (manufactured by Sysmex Corporation)
Eluent: 200 mmol / L phosphate buffer (pH 5.3) as eluent A, 400 mmol / L phosphate buffer (pH 8.0) as eluent B
Absorbance: 415 nm
Measuring device: LC20A (manufactured by Shimadzu Corporation)

In addition, at the time of measurement, the flow velocity was changed from 1.7 to 3.7 mL / min over 11 steps. FIG. 2 shows changes in the values of other peaks adjacent to the peak of hemoglobin A1c when the flow velocity is changed in this way.

In Examples 1 and 2, it can be seen that the flatness of the left side of the peak of hemoglobin A1c is not increased even under the measurement conditions in which the flow velocity is increased, and the separation ability is maintained.

On the other hand, in Comparative Examples 1 and 2, the flatness of the left side of the peak of hemoglobin A1c increased as the flow velocity increased. Therefore, under the measurement conditions in which the flow velocity is increased for the purpose of shortening the measurement time, the column packing materials of Comparative Examples 1 to 3 impair the sharpness of the peak and cannot maintain a sufficient separation ability.

Claims (5)

A column packing material used for measuring hemoglobins by high-speed liquid chromatography. There are two or more peaks of the frequency distribution obtained when measuring the particle size distribution, and the maximum particle size of the frequency distribution peaks. the particle size of the peaks indicating that a 1.1 to 3.0 times the particle size of the peak indicating the minimum particle size, the particle size of the peak (maximum peak) power in the frequency distribution is maximum, in the frequency distribution The particle size of the column filler is smaller than the particle size showing the second largest peak (second peak), and the small particle size ratio represented by the following formula (1) is 0.91 to 0.99. A column filler for measuring hemoglobins having an average particle size in the range of 3.0 to 6.5 μm.
Small particle size ratio = (height from the baseline of the maximum peak) / (height from the baseline of the maximum peak + height from the baseline of the second peak) ... (1) The column packing material for measuring hemoglobins according to claim 1, wherein the column packing material contains an acrylic monomer as a main component. The column filler for measuring hemoglobins according to claim 1 or 2, wherein the frequency distribution obtained when the particle size distribution is measured has two peaks. A liquid chromatography column for measuring hemoglobin, wherein the column is filled with the column packing material for measuring hemoglobin according to any one of claims 1 to 3. A method of measuring hemoglobin by high performance liquid chromatography, the peak of the frequency distribution obtained when measuring the particle size distribution is present two or more, of the frequency distribution peak, particle size of a peak indicating a maximum particle size , The particle size of the peak (maximum peak ) , which is 1.1 to 3.0 times the particle size of the peak showing the minimum particle size and has the maximum frequency in the frequency distribution, is the peak with the second largest frequency in the frequency distribution. It is smaller than the particle size showing (second peak), the small particle size ratio represented by the following formula (1) is 0.91 to 0.99, and the average particle size of the column packing material is 3.0 to. A method for measuring hemoglobins, which comprises using a column filler characterized by satisfying 6.5 μm and setting a flow velocity of 2.5 to 3.7 mL / min.
Small particle size ratio = (height from the baseline of the maximum peak) / (height from the baseline of the maximum peak + height from the baseline of the second peak) ... (1)

Images