JP4814204B2 - Method for measuring glycated hemoglobin - Google Patents

Description

  The present invention relates to a method for measuring glycated hemoglobin in a liquid sample, for example, blood, and more particularly to a method for measuring glycated hemoglobin using liquid chromatography.

  There are various hemoglobins in human blood. About 90% of hemoglobin in adult blood is hemoglobin A0 and about 7% is glycated hemoglobin A1. The glycated hemoglobin A1 is divided into hemoglobin A1a1, hemoglobin A1a, hemoglobin A1b, hemoglobin A1c, and the like depending on the type of sugar bound to the β chain of hemoglobin. However, hemoglobin A1c accounts for about 60% of glycated hemoglobin A1.

  In hemoglobin A1c, glucose in blood is chemically bound to the β-chain N-terminus of hemoglobin. Since the ratio of hemoglobin A1c to the total hemoglobin depends on the blood glucose level, the ratio of hemoglobin A1c to the total of hemoglobin A1c and non-glycated hemoglobin is said to reflect the average blood glucose level of the patient over a period of 1 to 2 months. ing. For this reason, the hemoglobin A1c value (%), which is the ratio of hemoglobin A1c to the entire hemoglobin, is not widely changed unlike the blood glucose level, and is therefore widely used as an index for diagnosis of diabetes.

  In recent years, the hemoglobin A1c value has become one of the essential test items before diagnosis, and it is desired that the test results be obtained more quickly.

  In general, liquid chromatography is used to measure the hemoglobin A1c value. In order to increase the inspection speed by liquid chromatography, 1) downsizing of the column, 2) a method of increasing the flow rate, and 3) a method of reducing the particle size of the packing material are used. However, when the flow rate is increased or the particle size of the packing material is reduced, the pressure in the column increases. This places restrictions on the design of devices that include columns. In addition, expensive parts are required to increase the pressure resistance of the apparatus. Therefore, the cost of the apparatus becomes high, and expensive columns cannot be used for general-purpose clinical tests.

When measuring hemoglobin by liquid chromatography, a cation exchange column is used as described in Patent Document 1 below, and hemoglobins are separated by changing the elution power of the eluent. . Usually, when hemolyzed blood samples are supplied to cation exchange liquid chromatography and an eluent is flowed to separate them, hemoglobins are hemoglobin A1a, hemoglobin A1b, hemoglobin F, unstable hemoglobin A1c, stable hemoglobin A1c, and hemoglobin. Elute in A0 order.
Japanese Patent Publication No. 8-7197

  As described above, conventionally, the speed of measurement of hemoglobin has been increased by reducing the size of the column, improving the flow rate, or reducing the particle size of the packing material. However, an expensive device is required, and these high-speed methods are difficult to use for general-purpose clinical examinations. In addition, there was a limit to downsizing the column.

  An object of the present invention is to provide a method for measuring glycated hemoglobin capable of greatly reducing the measurement time without reducing the measurement accuracy of hemoglobin A1c in view of the above-described state of the prior art.

  The method for measuring glycated hemoglobin according to the present invention is a method for measuring glycated hemoglobin in a liquid sample by liquid chromatography, and has the following configuration.

That is, in the measuring method of the present invention, a column having an average particle diameter in the range of 5 μm to 20 μm is accommodated, the inner diameter is in the range of 3.0 mm to 5.0 mm, and the length is in the range of 10 mm to 20 mm. Next, a liquid sample is passed through, and then the eluent is passed through the column at a rate of 1.6 ml / min to 2.5 ml / min with different elution powers, and hemoglobin A0 and glycated hemoglobin are passed through. And a step of eluting a plurality of types of hemoglobin, wherein the eluent contains a nonionic surfactant when eluting the hemoglobin A0.

  The liquid sample supplied to the column in the method for measuring glycated hemoglobin according to the present invention is not particularly limited, but preferably a hemolyzed blood sample is used, whereby hemoglobin A1c, which is glycated hemoglobin in blood, is obtained. According to the invention, it is possible to measure with high accuracy in a short time.

  Details of the present invention will be described below.

  When measuring hemoglobins in blood by liquid chromatography, hemoglobins are eluted in the order of hemoglobin A1a, hemoglobin A1b, hemoglobin F, unstable hemoglobin A1c, stable hemoglobin A1c, and hemoglobin A0. And hemoglobin A0 occupies most of hemoglobin, and in a normal person, it accounts for about 90% of the whole hemoglobin. Therefore, in order to end the measurement, it is important to elute hemoglobin A0 in a shorter time. In the present invention, the column having the above-mentioned specific inside diameter and length, which is packed with the above-mentioned filler having the specific average particle diameter, is used, the flow rate of the eluent is set to 1.6 mm / min to 2.5 mm / min, and hemoglobin is used. By incorporating a nonionic surfactant into the eluent when eluting A0, the separation rate of each hemoglobin is increased, and the elution time of hemoglobin A0, which accounts for the majority, is reduced without degrading the separation performance of hemoglobin A1c. Characterized by shortening. Thereby, the measurement time can be greatly shortened without reducing the measurement accuracy of hemoglobin A1c.

  As the filler used in the present invention, various fillers conventionally used for separating hemoglobin in blood can be used. Examples of such fillers include polymer-based and silica-based materials.

  The average particle system of the filler needs to be in the range of 5 μm to 20 μm. When the average particle size is less than 5 μm, the pressure in the column becomes too high. More preferably, the average particle diameter is 6 μm or more, and the increase in the internal pressure of the column can be more effectively suppressed.

  If the average particle size exceeds 20 μm, the separation performance of hemoglobin A1c may be reduced. More preferably, the average particle size is 12 μm or less, whereby the separation performance of hemoglobin A1c can be further enhanced.

  As the column filled with the above packing material, a known column for ion exchange liquid chromatography can be appropriately used. The column material is not particularly limited, and examples thereof include metals such as stainless steel, synthetic resins such as polyetheretherketone and polytetrafluoroethylene, and glass.

  The inner diameter of the column is in the range of 3.0 mm to 5.0 mm. If the inner diameter is less than 3.0 mm, the linear velocity becomes too high and the pressure in the column rises too much. If the inner diameter exceeds 5.0 mm, diffusion in the column will occur too much, and the separation performance of hemoglobin A1c will deteriorate.

The length of the column is in the range of 10 mm to 20 mm. If it is less than 10 mm, the separation performance of hemoglobin A1c decreases as the number of theoretical plates decreases. More preferably, the length of the column is 15 mm or more, whereby the separation performance of hemoglobin A1c can be further enhanced.

If the column length exceeds 20 mm, it takes time to elute the hemoglobin fraction, and the measurement time cannot be shortened .

  In the present invention, the flow rate at the time of elution of hemoglobin by varying the elution power of the eluent is set in the range of 1.6 ml / min to 2.5 ml / min. When the flow rate is less than 1.6 ml / min, it takes time to elute the hemoglobin fraction, and the measurement time cannot be shortened. More preferably, measurement time can be further shortened by setting it as 1.7 ml / min or more.

  When the flow rate exceeds 2.5 ml / min, the pressure in the column rises and a column with excellent pressure resistance is required. More preferably, it is desirably 2.0 ml / min or less, whereby a cheaper column can be used.

  Further, in the present invention, when eluting various hemoglobin fractions sequentially by flowing an eluent with different elution powers, a nonionic surfactant is contained in the eluent when hemoglobin A0 is eluted. Accordingly, hemoglobin A0 that is firmly adsorbed to the filler by hydrophobic interaction can be quickly eluted. Such a nonionic surfactant is not particularly limited, and any known nonionic surfactant can be used. Typical examples of such nonionic surfactants include polyoxyethylene alkyl ether, polyoxyethylene alkyl phenyl ether, polyoxyethylene polyoxypropylene glycol, polyoxyethylene fatty acid ester, polyoxyethylene sorbitan fatty acid. Examples include esters, sorbitan fatty acid esters, glycerin fatty acid monoesters, and sucrose fatty acid esters. Among them, polyoxyethylene (20) sorbitan laurate (Twenn 20) which is a polyoxyethylene sorbitan fatty acid ester type is particularly preferable.

  The nonionic surfactant only needs to be contained in the eluent when eluting hemoglobin A0. Even if the nonionic surfactant is added only to the eluent when hemoglobin A0 is eluted. Of course, a nonionic surfactant may or may not be added to the eluent used to elute other hemoglobin fractions. However, preferably, the addition of a nonionic surfactant to the eluent when eluting other Hb fractions may affect the separation, so that only non-existent eluent when eluting hemoglobin A0 is used. It is preferred that an ionic surfactant is present.

  Therefore, preferably, when the eluent is supplied to the column, the nonionic surfactant is not present in the eluent until the stage before elution of hemoglobin A0, and the nonionic interface is eluted when hemoglobin A0 is eluted. It is desirable to add an activator to the eluent. In that case, when different elution outputs are used, plural types of eluents may be switched and used. When supplying eluents to the column, the elution liquid may be changed while changing the composition of the supplied eluents. Good.

  Further, in order to elute hemoglobin A0 in a short time, a known elution efficiency improving method as described in JP-A No. 2003-14719 or JP-A No. 2005-128030 may be further used in combination.

  As the liquid sample supplied to the column in the present invention, a hemolyzed blood sample is used to determine the hemoglobin A1c value as an index of diabetes of the patient. However, the method for measuring glycated hemoglobin of the present invention may be used to measure glycated hemoglobin in a liquid sample other than a hemolyzed blood sample.

  In the method for measuring glycated hemoglobin according to the present invention, a column having the above-mentioned specific average particle size range is packed, the inner diameter and the length are in the above-mentioned specific range, and the hemoglobin fraction is eluted. Since the eluent flow rate is in the range of 1.6 ml / min to 2.5 ml / min and a nonionic surfactant is added to the eluent when hemoglobin A0 is eluted, the measurement accuracy of hemoglobin A1c Various hemoglobin fractions can be promptly eluted without lowering the pH, and hemoglobin A0 occupying most of the hemoglobin fraction can be promptly eluted. Therefore, the measurement time can be greatly shortened, and the hemoglobin A1c value can be measured quickly.

  Further, when the measurement speed is increased by the conventional method of increasing the flow rate or the method of reducing the average particle diameter of the packing material, a column having excellent pressure resistance is required, and expensive parts must be used. On the other hand, in the present invention, it is not necessary to increase the flow rate so much and it is not necessary to reduce the average particle size of the filler so much, so that the measurement of hemoglobin A1c can be accelerated without increasing the cost of the apparatus including the column. It becomes possible to plan. Therefore, a method for measuring glycated hemoglobin suitable for general-purpose clinical tests can be provided.

  Hereinafter, the present invention will be described in more detail by giving specific examples and comparative examples of the present invention. However, the present invention is not limited to the following examples.

(1) Preparation of filler In a reactor equipped with a stirrer, an aqueous solution of 3% by weight polyvinyl alcohol (manufactured by Nippon Synthetic Chemical Co., Ltd.), 300 g of tetraethylene glycol dimethacrylate (manufactured by Shin Nakamura Chemical Co., Ltd.), triethylene glycol dimethacrylate (Shin Nakamura) A mixture of 100 g) and 1.0 g benzoyl peroxide was added. The mixture was sized while stirring, and then polymerized at 80 ° C. for 1 hour under a nitrogen atmosphere. Separately, as a monomer having an ion exchange group, 100 g of 2-methacrylamide-2-methylpropanesulfonic acid (manufactured by Toagosei Co., Ltd.) and 100 g of polyethylene glycol methacrylate (manufactured by NOF Corporation, ethylene glycol chain n = 4) Was dissolved in ion-exchanged water. The solution obtained by dissolving the monomer in ion-exchanged water was added to the reactor, and polymerization was performed at 80 ° C. for 2 hours in a nitrogen atmosphere while stirring. The obtained polymer-containing composition was washed with water and acetone to obtain hydrophilic coated polymer particles having an ion exchange group.

  10 g of the obtained coated polymer particles were immersed in 300 ml of ozone water having a dissolved ozone gas concentration of 100 ppm and stirred for 30 minutes. After stirring, the mixture was centrifuged using a centrifuge (Hitac CR20G manufactured by Hitachi, Ltd.), and the supernatant was removed. This operation was repeated twice to give a hydrophilic treatment to obtain a packing material for ion exchange liquid chromatography.

  The ozone water is a hollow tubular ozone gas permeable membrane made of perfluoroalkoxy resin having an inner diameter of 0.5 mm, a thickness of 0.04 mm and a length of 350 cm in an outer tube having a cylindrical shape with an inner diameter of 15 cm and a length of 20 cm. It was prepared using an ozone water production system (manufactured by Sekisui Chemical Co., Ltd.) including the ozone dissolution module accommodated.

  In the preparation of the filler for ion exchange liquid chromatography, the average particle size was measured using a laser diffraction flow distribution measuring device at the stage where the above-mentioned hydrophilic coated polymer particles having an ion exchange group were obtained. Further, in the production of the coated polymer particles, the following three types of fillers A to C having different average particle diameters were prepared by varying the number of revolutions of stirring in the granulation step.

Filler A: average particle size 9 μm, CV value 15%
Filler B: average particle diameter 3 μm, CV value 14%
Filler C: average particle size 25 μm, CV value 12%
(2) Columns Columns A to H having the following five dimensions were prepared as liquid chromatography columns.

Column D: inner diameter 4.6 mm, length 20 mm
Column E: inner diameter 4.6 mm, length 9 mm
Column F: inner diameter 4.6 mm, length 35 mm
Column G: inner diameter 2.0 mm, length 20 mm
Column H: inner diameter 6.0 mm, length 20 mm
(3) Experimental example 1
About each column apparatus 1-15 shown in following Table 1 obtained combining the said fillers A-C and columns D-H, the liquid which elutes Hb fractions other than A0 is a feed pump (made by Shimadzu Corporation) LC-10AS), and the pressure in the column was measured when the solution was fed at a rate of 1.7 ml / min. The results are shown in Table 1 below. For those having a pressure in the column of 5 MPa or less, the pressure was not excessively high, and therefore, ○ was given, and for those having a pressure exceeding 5 MPa, × was given.

  In all the columns filled with the combination of the packing material A and the column G and the packing material B, the pressure exceeded 5 MPa and the pressure standard was not satisfied.

(4) Experimental example 2
For the column apparatus marked with ◯ in Experimental Example 1, HbA1c was measured in the following manner, and chromatograms were compared. However, the column packed with the packing material C did not elute the HbA1c peak, or was broadened even if it was eluted, so the measured value of HbA1c could not be calculated. Therefore, chromatography was performed using packing A and columns D, E, F or H.

Measurement of HbA1c:
Glyco Hb control level 1 (manufactured by Kokusai Reagent Co., Ltd., reference value 5.8 ± 0.5%) was dissolved in 200 μL of water for injection and then diluted (phosphate buffer containing 0.1% Triton X-100). (PH 7.0)) was diluted 100 times and used as a measurement sample.
The amount of hemoglobin A1c and the amount of non-glycated hemoglobin in the measurement sample were measured, and the ratio of hemoglobin A1c to the total of hemoglobin A1c and non-glycated hemoglobin (hemoglobin A1c value (%)) was determined.

System: Liquid feed pump LC-9A (manufactured by Shimadzu Corporation)
Autosampler ASU-420 (manufactured by Sekisui Chemical Co., Ltd.)
Detector SPD-6AV (manufactured by Shimadzu Corporation)
Eluent: 1st liquid Phosphate buffer (pH 5.3)
Second liquid 0.05% Tween20-containing 250 mM phosphate buffer (pH 8.0)
* The concentration of the first solution is adjusted so that HbA1c has the same retention time in each column (combination of filler and column).

Measurement time: 50 seconds Flow rate: 1.7 mL / min Detection wavelength: 415 nm
Sample injection volume: 10 μL

  The results are shown in FIGS. Table 2 shows combinations of the packing material A and columns D, E, F, and H used to obtain the chromatograms of FIGS. 1 to 4, 1 to 5 indicate the following peaks.

1: Peak 1 HbA1a + HbA1b
2: Peak 2 HbF
3: Peak 3 unstable HbA1c
4: Peak 4 Stable HbA1c
5: Peak 5 HbA0

  Table 3 shows the measured values of HbA1c obtained from the chromatograms shown in FIGS.

  As is apparent from FIGS. 1 to 4, with the combination of the packing material A and the column D, HbA1c could be separated and quantified in a measurement time of 50 seconds. HbA0 could also be eluted within the measurement time.

  In the combination of the packing material A and the column E, the length of the column is too short, the peak is broadened, and there is a problem in the quantitativeness of HbA1c. HbA0 could be eluted within the measurement time.

  In the combination of the packing material A and the column F, the HbA1c value was within the reference value, but the column length was too long, so that the peak-to-peak was compressed, and there was a problem in peak separation. Further, HbA0 could not be completely eluted within the measurement time due to the influence of the column length being too long.

  In the combination of the packing material A and the column G, since the inner diameter of the column was too large, the peak was broadened, and there was a problem in the quantitativeness of HbA1c. Further, HbA0 could not be completely eluted within the measurement time.

It is a figure which shows the chromatogram at the time of using the packing material A and the column D in a 2nd experiment example. It is a figure which shows the chromatogram at the time of using the packing material A and the column E in a 2nd experiment example. It is a figure which shows the chromatogram at the time of using the packing material A and the column F in a 2nd experiment example. It is a figure which shows the chromatogram at the time of using the packing material A and the column H in a 2nd experiment example.

Explanation of symbols

1 ... Peak 1 (HbA1a + HbA1b)
2 ... Peak 2 (HbF)
3 ... Peak 3 (unstable HbA1c)
4 ... Peak 4 (stable HbA1c)
5 ... Peak 5 (HbA0)

Claims (2)

A method for measuring glycated hemoglobin in which glycated hemoglobin in a liquid sample is measured by liquid chromatography,
A step of passing a liquid sample through a column in which a filler having an average particle diameter of 5 μm to 20 μm is stored, an inner diameter of 3.0 to 5.0 mm, and a length of 10 to 20 mm. When,
Next, the eluent is passed through the column at a rate of 1.6 ml / min to 2.5 ml / min with different elution powers to elute a plurality of types of hemoglobin including hemoglobin A0 and glycated hemoglobin. And a method for measuring glycated hemoglobin, wherein an eluent containing a nonionic surfactant is used as an eluent when eluting the hemoglobin A0.   The method for measuring glycated hemoglobin according to claim 1, wherein the liquid sample is a hemolyzed blood sample.

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