JP2018179505A - Column packing material for measuring hemoglobins, and measuring method of hemoglobins - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a column packing material for measuring hemoglobins, capable of maintaining separation performance even when a flow rate is increased.SOLUTION: A column packing material used for measuring hemoglobins by high-performance liquid chromatography has two or more peaks of the frequency distribution obtained when the particle size distribution is measured. Among the frequency distribution peaks, the particle diameter of the peak showing the maximum particle diameter (maximum peak) is 1.1-3.0 times the particle diameter of the peak showing the minimum particle diameter. The particle diameter of the maximum peak is smaller than the particle diameter showing the second largest peak (second peak). A small particle diameter ratio represented by the following formula (1) is 0.91-0.99, and the average particle diameter of the column packing material is 3.0-6.5 μm. Small particle diameter ratio=(the maximum peak height from the baseline)/(the maximum peak height from the baseline + second peak height from the baseline) . . . (1))SELECTED DRAWING: None

Description

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

  Hemoglobin A1c (HbA1c) continues to be present in the blood for about 120 days, which is the life of red blood cells. Therefore, by measuring hemoglobin A1c, it is possible to estimate the state of blood glucose in the past one to two months. Therefore, unlike other diabetes markers such as glycoalbumin, the measurement of hemoglobin A1c can confirm the transition of the blood glucose level over a relatively long period.

  In the measurement of hemoglobin A1c, measurement by high performance liquid chromatography is widely performed because the accuracy of the measurement value is good. In high performance liquid chromatography, measurement time is one of the important indexes as well as measurement accuracy. In order to reduce the burden on the laboratory technicians and to construct an efficient inspection system, shortening the measurement time has been a major issue as well as improving the accuracy of the measurement values.

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

JP, 2011-209040, A

  A major contribution to shortening the measurement time of high performance liquid chromatography is the increase in flow velocity. By the way, when measuring hemoglobin A1c 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 lost. Therefore, the separation performance may be degraded.

  In high performance liquid chromatography, sharpening the peaks of hemoglobins is an important issue in the improvement of separation performance. As a method of determining the sharpness of the hemoglobin species peak, attention can be paid to the bottom of the hemoglobin species peak. As the chromatogram schematic diagram shown in FIG. 1 shows, a downward projecting portion (hereinafter referred to as "valley portion") generated on the left side of the peak of hemoglobin A1c and an upward projecting portion (hereinafter referred to as " Both occur). In the chromatogram of hemoglobin A1c measurement, the ratio of the height from the baseline of the valley to the height from the baseline of the peak (hereinafter referred to as “flatness on the left side of the A1c peak”) is measured It tends to increase when the flow rate of time is increased. Therefore, the sensitivity of the peak of hemoglobin A1c is lost, and the separation performance of hemoglobin A1c may be reduced.

  When the column packing described in Patent Document 1 is used, the column life can be extended. However, in the column packing described in Patent Document 1, when the flow rate is increased in order to speed up the measurement, the flatness of the left side of the peak tends to increase at the peak of hemoglobins such as hemoglobin A1 c. Therefore, there is a possibility that the separation performance of hemoglobins may be reduced.

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

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

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

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

  In the column packing 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 filler for measuring hemoglobins which is constituted according to the present invention.

  The method of measuring hemoglobins by liquid chromatography according to the present invention is a method of measuring hemoglobins by high performance liquid chromatography, wherein two or more peaks of frequency distribution obtained when particle size distribution is measured, frequency Among the distribution peaks, the particle size of the peak showing the largest particle size (maximum peak) is 1.1 to 3.0 times the particle size of the peak showing the smallest particle size, and the particle size of the largest peak is the second Smaller than the particle diameter showing a large peak (second peak), the small particle diameter ratio represented by the following formula (1) is 0.91 to 0.99, and the average particle diameter of the column filler is It is characterized by using a column packing characterized by satisfying 3.0 to 6.5 μm, and setting the flow rate to 2.5 to 3.7 mL / min.

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

  According to the column packing for measuring hemoglobins according to the present invention and the method for measuring hemoglobins, even when the flow rate is increased to increase the speed, the sensitivity of the peaks of hemoglobins can be maintained. Therefore, it is possible to achieve both shortening of the measurement time and improvement of the separation performance of hemoglobins.

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

  Hereinafter, the present invention will be described in detail.

(Particle size of filler particles)
The column filler for measuring hemoglobins of the present invention (hereinafter, also simply referred to as a filler) comprises a particulate filler.

  The lower limit of the average particle diameter 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 becomes high, and special parts for imparting pressure resistance to the liquid chromatography apparatus are required. Become. When the average particle size of the filler exceeds 6.5 μm, the porosity in the column may increase, and the separation performance may be affected when the flow rate is increased. The preferable lower limit of the average particle diameter of the filler of the present invention is 6.0 μm, and the preferable upper limit is 6.4 μm.

  In the present specification, the above-mentioned average particle diameter and particle size distribution are based on measurement results obtained by a particle size distribution measuring device based on the principle of the number counting method. However, even if it is a measurement device based on other measurement principles, it should be used if the measurement result has a known correlation with the measurement result according to the principle of the number counting method, or if the device is calibrated. Such devices include, for example, devices based on known principles such as centrifugal sedimentation method, dynamic light scattering method, laser diffraction light scattering method, ultrasonic attenuation method, capillary method, Coulter method and the like.

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

  In the present specification, the above peak means a measurement point whose height from the baseline is larger than that of the adjacent left and right measurement points. That is, the peak of the above particle size distribution is the top 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 exhibiting the maximum particle size is 1.1 to 3.0 times the particle size of the peak exhibiting the minimum particle size, and the peak exhibits 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 baseline of maximum peak) / (height from baseline of maximum peak + height from baseline of second peak) (1)

  When the small particle size ratio is in the above-mentioned range, it is possible to achieve both the packing of the filler particles in the interior of the column with high density and the control of the optimum specific surface area of the filler particles. Therefore, it is possible to provide a column which has both pressure resistance and high analysis ability. The preferred range of 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 in which two or more fillers having a single particle size distribution are mixed so as to meet the above-mentioned conditions. And a method of obtaining a filler having a plurality of peaks by polymerization.

(Material of filler)
As the material of the filler of the present invention, a known material such as an inorganic material such as silica, ceramic or glass, or an organic synthetic material such as acrylic polymer or styrene polymer may be used. it can. Among them, 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 of producing the filler in the case of using the above-mentioned acrylic polymer as a raw material, a method of polymerizing an acrylic monomer by a known polymerization method can be used.

  Examples of the acrylic monomer include, for example, at least two (meth) acryloyloxy groups in the molecule, such as polyethylene glycol di (meth) acrylates, polypropylene glycol di (meth) acrylates, and alkylene glycol di (meth) acrylates. Examples thereof include crosslinkable acrylic monomers having a group.

  The above acrylic polymer is a method of polymerizing the above crosslinkable acrylic monomer, or the above crosslinkable acrylic monomer, methyl (meth) acrylate, ethyl (meth) acrylate, polyethylene glycol mono (meth) acrylate It can be prepared by a method of copolymerizing with a non-crosslinkable acrylic monomer such as 2-hydroxyethyl (meth) acrylate.

(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. The filler having the ion exchange group, for example, when the main component of the filler is the acrylic polymer, a) an acrylic polymer is prepared by polymerizing an acrylic monomer and a monomer having an ion exchange group. And a method of introducing an ion exchange group after the preparation of the acrylic polymer and b).

  In the present specification, fillers and filler particles are used as the same term unless otherwise specified.

(Object of measurement)
Using the filler of the present invention, various hemoglobins can be measured by high performance liquid chromatography. Specifically, hemoglobin A0, hemoglobin A1c, hemoglobin F (fetal hemoglobin), hemoglobin A2, hemoglobin S, etc. can be measured by liquid chromatography using the column material for measuring hemoglobins of the present invention. .

  In addition, by using the filler of the present invention, it is also possible to perform measurement under conditions where the flow rate is higher than in the prior art. In order to shorten the measurement time, the method of measuring hemoglobins by high performance liquid chromatography using the column packing for measuring hemoglobins of the present invention under the condition of a flow rate of 2.5 to 3.7 mL / min Moreover, it is one of the present invention.

  In the case of measuring hemoglobins such as hemoglobin A1c and the like by high performance liquid chromatography using the column packing for measuring hemoglobins of the present invention, a known pump, sampler, detector or the like for feeding an eluent is provided. A column packed with the column 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 an eluent used for liquid chromatography using the column packing for measuring hemoglobins of the present invention, it is preferable to use buffers containing known salt compounds and organic solvents. Specific buffers include, for example, organic acids, inorganic acids, and salts thereof, amino acids, buffers of Good and the like.

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

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

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

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

  In addition, substances generally added to the above buffer, for example, surfactants, various polymers, hydrophilic low molecular weight compounds and the like may be added as appropriate.

  The preferable lower limit of the salt concentration of the above buffer when performing measurement of hemoglobin A1c is 10 mmol / L, and the preferable upper limit is 1000 mmol / L. When the salt concentration of the buffer solution is less than 10 mmol / L, the ion exchange reaction may not be performed, and the hemoglobins may not be separated. When the salt concentration of the buffer solution exceeds 1000 mmol / L, salts may be precipitated to adversely affect the system.

(The 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 hemoglobin A1c peak and an upward protruding portion ("crest portion" generated on the further left side ') Both occur.

  The “peak left flatness”, which is an evaluation index of the present invention, is determined as follows.

  "Flatness of the peak left portion" = (height from the baseline at the top of the valley) / (height from the baseline at the top of the peak)

  Generally, this value tends to increase as the flow velocity increases during measurement, and it is judged that the peak of hemoglobin A1c can be kept sharp and the high separation ability can be maintained by suppressing the increase.

(Example)
Although the Example for demonstrating the effect of invention is described below, this invention is not limited to a following example.

Production Example 1
40 g of 2-hydroxy-3-acryloyloxypropyl methacrylate (manufactured by Shin-Nakamura Chemical Co., Ltd.), 140 g of triethylene glycol dimethacrylate (manufactured by Shin-Nakamura Chemical Co., Ltd.), 5 g of pentaerythritol tetraacrylate (manufactured by Shin-Nakamura Chemical Co., Ltd.) In a monomer mixture containing 15 g of pentaerythritol triacrylate (triester 37%, manufactured by Shin-Nakamura Chemical Co., Ltd.), 1.0 g of benzoyl peroxide was dissolved. The obtained mixture is dispersed in 2 L of a 4% by weight aqueous solution of polyvinyl alcohol, and while stirring at 390 rpm using a stirring blade with a blade length of 60 mm, the mixture is heated to 80 ° C. under a nitrogen atmosphere to carry out a polymerization reaction for 1 hour The 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 a polymerization reaction was further performed 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. At the time of measurement, measurement was carried out for 120 seconds using a semiconductor laser as a light source and LE 400-05 as a sensor, and volume distribution display was performed to calculate an average particle diameter. As a result, the average particle diameter was 4.45 μm, and the particle size distribution was in the form of a single peak.

Production Example 2
In the same manner as in Production Example 1, except that the number of revolutions during stirring in Production Example 1 was changed from 390 rpm to 300 rpm, filler particles were obtained. The average particle size and particle size distribution of the obtained filler particles were measured. As a result, the average particle diameter was 9.47 μm, and the particle size distribution was in the form of a single peak.

  The average particle diameter and the number of particle size distribution peaks of the filler particles obtained in Production Examples 1 and 2 are shown in Table 1.

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

  The column packings obtained in Examples 1 and 2 and Comparative Examples 1 to 3 were packed 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 apparatus, and the blood sample was measured.

(Measurement of blood sample)
The 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 (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 (made by Shimadzu Corporation)

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

  In Examples 1 and 2, no increase in flatness of the left side of the peak of the hemoglobin A1c is observed under measurement conditions in which the flow rate is increased, and it can be seen that the separation ability is maintained.

  On the other hand, in Comparative Examples 1 and 2, an increase in the flatness of the peak left portion of the hemoglobin A1c was observed as the flow velocity increased. Therefore, in the measurement conditions in which the flow rate is increased in order to shorten the measurement time, the column packings of Comparative Examples 1 to 3 lose the sharpness of the peak and can not maintain sufficient separation ability.

Claims (5)

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

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