JP4628066B2 - Measuring method by liquid chromatography using a filter for liquid chromatography - Google Patents

Description

The present invention relates to a measuring method using liquid chromatography using a filter over a liquid chromatography.

  In the fields of organic chemistry, biochemistry, medicine and the like, liquid chromatography is widely used for separation and fractionation of components in a sample. For example, as one of the measurements by liquid chromatography, glycated hemoglobin in blood, particularly hemoglobin A1c (hereinafter referred to as HbA1c), which is a stable glycated hemoglobin, is widely measured as an index for diagnosis of diabetes. Glycated hemoglobin is produced by combining sugar in blood with hemoglobin, and the ratio of HbA1c in the hemolyzed sample reflects the average sugar concentration in the blood for the past one to two months. It is used as an index for diagnosis of diabetes. For the measurement of HbA1c by the liquid chromatography method, an eluent having a pH of 5.0 to 9.0 is used as a separation column packed with a packing material for cation exchange liquid chromatography having a carboxyl group or a sulfonic acid group as a functional group. It is done using.

  In the liquid chromatography, a mobile phase such as an eluent is sent to a separation column by a liquid feed pump, a measurement sample is introduced from the middle of the flow path, and after introducing the measurement sample, the elution power of the eluent is changed. In this method, components in a measurement sample held in a separation column are separated and fractionated, and each component obtained is detected by a detector and processed as data, and then expressed as a chromatogram. In such liquid chromatography, a filter is often disposed in the middle of the flow path from the measurement sample introduction part to the separation column in order to prevent foreign matters in the sample from entering the separation column. . Further, in order to prevent the filler in the separation column from leaking out and entering the detector in addition to foreign matters, a filter may be disposed in the middle of the flow path from the separation column to the detector.

  Patent Document 1 discloses a high-performance liquid chromatography in which a stainless steel fiber is laminated and a sintered fiber filter is sintered integrally with the column main body at least on the outlet side of the column main body containing the packing material. A column is disclosed. However, if the column for liquid chromatography equipped with this filter is used to measure biological samples such as blood, serum, plasma, and urine containing proteins and lipids, the biological sample is easily adsorbed on the filter, and the measurement is repeated. The filter is clogged, and the filtration pressure of the filter rises, making it impossible to perform accurate measurement. Therefore, there is a problem that a large number of samples cannot be measured continuously. Even if the filter is subjected to a surface treatment to prevent the biological sample from being adsorbed, the fiber-sintered filter is likely to be clogged with foreign matter in the measurement sample. As the filtration pressure of the liquid crystal increases, accurate measurement cannot be performed. Therefore, there is a problem that a large number of samples cannot be measured continuously.

  Furthermore, Patent Document 2 is characterized in that a filter combining a filter paper and a membrane filter is disposed in a flow path leading to a column in liquid chromatography so that the filter paper is positioned on the side far from the column. A method for removing impurities and the like in a sample in a liquid chromatograph is disclosed. However, a filter consisting of filter paper and a membrane filter is likely to be clogged with foreign matter in the measurement sample. If continuous measurement is performed, the filter will be clogged in a short time, and the filtration pressure of the filter will increase, allowing accurate measurement. Therefore, there are problems that a large number of samples cannot be measured continuously, and the frequency of filter replacement increases.

JP-A-7-260763 JP-A-5-203634

  The present invention has been made in view of the above problems, and an object of the present invention is to provide a living body that can efficiently remove foreign matters and the like in a measurement sample without causing clogging, and contains hemoglobins and the like. Even when used for sample measurement, the biological sample is hardly adsorbed on the filter, clogging is unlikely to occur, and a liquid chromatography filter that does not adversely affect the separation performance and the filter are integrally provided in the separation column. An object of the present invention is to provide a liquid chromatography column.

Further, purpose of this onset Ming, said by using a filter for liquid chromatography, can be efficiently removed without causing any clogging of the foreign matter in the measurement sample, and the measurement of biological samples Even if it is used, there is little adsorption of biological samples to the filter, clogging is unlikely to occur, and many samples can be measured continuously with high accuracy. It is an object of the present invention to provide a measuring method capable of continuously separating and quantifying hemoglobins with high accuracy.

  The filter for liquid chromatography (hereinafter referred to as “filter”) of the present invention is a filter installed in a liquid flow path leading to a separation column in liquid chromatography, and a nonwoven fabric and a filter paper filter are laminated. The nonwoven fabric side is located on the side far from the column, the nonwoven fabric has a thickness of 0.05 mm or more and a porosity of 40% to 90%, and the filter paper filter has a retained particle diameter of 5 μm. It is characterized by the following.

The filter of the present invention is described in detail below.
Nonwoven fabric materials used in the filter of the present invention include, for example, polyolefin fibers such as polyethylene and polypropylene, polyester fibers such as polyethylene terephthalate, nylon fibers, rayon fibers, acrylic fibers, coconut fibers, vinylidene chloride fibers, cotton fibers, wool , Hemp fibers, glass fibers, etc., and the above materials may be used alone or in combination of two or more.

  The thickness of the non-woven fabric is 0.05 mm or more, and the upper limit may be appropriately determined, but is usually preferably 10 mm or less, and more preferably in the range of 0.1 mm to 5 mm. When the thickness of the nonwoven fabric is less than 0.05 mm, clogging is likely to occur due to foreign matter in the measurement sample, and when measuring a biological sample, clogging is likely to occur when the biological sample is adsorbed. Therefore, when continuous measurement is performed, the filtration pressure of the filter increases due to clogging, and accurate measurement cannot be performed. Moreover, when it exceeds 10 mm, a measurement sample will diffuse in a nonwoven fabric, and an accurate measurement may become impossible.

The porosity of the nonwoven fabric is in the range of 40% to 90%. When the porosity of the nonwoven fabric is less than 40%, clogging is likely to occur due to foreign matter in the measurement sample, and when measuring a biological sample, clogging is likely to occur when the biological sample is adsorbed. When continuous measurement is performed, the clogging of these filters increases the filtration pressure of the filter, making accurate measurement impossible. If the porosity of the nonwoven fabric exceeds 90%, the measurement sample diffuses into the nonwoven fabric, making it impossible to perform accurate measurements. In addition, the filter strength is weakened, and the liquid mobile phase during filter handling and measurement It may be damaged by pressure.
The porosity is a value measured by a fully automatic pore size distribution measuring device (mercury porosimeter).

  Although the said nonwoven fabric may be comprised from one nonwoven fabric, what combined several nonwoven fabrics may be sufficient. For example, the non-woven fabric on the sample introduction side that is the column side and the non-woven fabric on the sample outlet side that is far from the separation column can be laminated and used in such a combination. The method of lamination is not particularly limited. For example, the nonwoven fabrics may be overlapped, the nonwoven fabrics may be bonded together, or the nonwoven fabrics may be compressed and molded. The number of laminated nonwoven fabrics is usually in the range of 2 to 3 layers, but is not particularly limited, and may be increased as necessary.

  The filter paper filter used in the filter of the present invention includes paper made of cellulose fibers usually referred to as filter paper, and filters in which various fibers such as organic fibers and inorganic fibers are formed into filter paper. The material of the filter paper filter is not particularly limited, and specific examples include cellulose fiber, glass fiber, fluororesin fiber, silica fiber, titanium fiber, stainless steel fiber, etc., and these materials are used alone. It can also be used in combination of two or more. Further, a filter paper filter that has been subjected to a special treatment for reinforcing strength, suppressing adsorption of a biological sample, or the like can also be used. As a filter paper filter subjected to such a special treatment, for example, a filter paper (Advantech Toyo Co., Ltd .: trade name “Wet Strength Filter Paper”) with increased wet strength is commercially available.

  The retained particle diameter of the filter paper filter is 5 μm or less, preferably 3 μm or less, and more preferably 1.5 μm or less in order to reliably remove foreign matter in the measurement sample. When the retained particle size of the filter paper filter exceeds 5 μm, foreign matters in the measurement sample cannot be filtered, and these foreign matters are likely to flow into the separation column, increasing the liquid permeation pressure of the separation column. Accurate measurement may not be possible, and the separation performance of the separation column may be adversely affected. Here, the retained particle diameter of the filter paper filter is the maximum value of the leakage particle diameter when a test solution such as barium sulfate defined by JIS P3801 is naturally filtered.

  The filter paper filter may be composed of a single filter paper filter, but may be a combination of a plurality of filter paper filters. For example, it can be used by being laminated in such a combination that the retained particle diameter of the filter paper filter on the sample outlet side which is the column side is smaller than the filter paper filter on the nonwoven fabric side. The method of laminating is not particularly limited. For example, the filter paper filters may be simply stacked, the filter paper filters may be bonded together, or the filter paper filters may be compressed and molded. Also good. The number of filter paper filters is usually in the range of 2 to 3 layers, but is not particularly limited, and may be increased as necessary.

  The filter of the present invention is formed by laminating the nonwoven fabric and the filter paper filter, and may be silicone-coated as necessary in order to reliably prevent the biological sample from being adsorbed, and may be blocked with a blocking reagent. May be. Surface treatments such as silicone coating and blocking treatment may be applied only to either the nonwoven fabric or the filter paper filter, or may be applied to the entire filter.

  The silicone coating material is not particularly limited as long as it can form a silicone film, and examples thereof include curable silicone rubber, oil, varnish, rubber, silane, and the like, preferably curable silicone rubber. .

  Examples of the blocking reagent include proteins such as bovine serum albumin, casein, gelatin, hemoglobin and myoglobin, polar lipids such as phospholipid, and surfactants such as polyethylene glycol mono-4-octylphenyl ether (Triton X-100). Etc. Examples of the blocking method include a method of passing a 0.01% to 5% by weight blocking reagent solution through the nonwoven fabric, filter paper filter, or filter and then drying.

  Furthermore, in order to reinforce the strength of the filter of the present invention and increase the effective filtration area, a support may be used as necessary on both sides of the filter or on the side close to the column side, that is, on the filter paper filter side. Can do. The support is not particularly limited as long as it can reinforce the filter strength and increase the effective filtration area, and the retained particle diameter is larger than that of the filter paper filter. Is preferred. Specific examples include a mesh sheet made of a synthetic resin such as polypropylene, polyethylene, nylon, and polyethylene terephthalate, and a mesh sheet made of a metal such as stainless steel and titanium. In the case of a mesh sheet made of a material that easily adsorbs a biological sample, such as stainless steel, it is preferable to apply a silicone coating to the surface or a blocking treatment with a blocking reagent, as described above.

  The shape of the filter of the present invention is not particularly limited as long as it has the required strength and effective filtration area, and may be a desired shape. For example, the shape viewed from the flow direction of the liquid flow path includes circular, elliptical, polygonal, etc. Among them, a circular shape is preferable because it is easy to manufacture and secures an effective filtration area. .

  The size of the filter of the present invention is preferably 1 mm to 100 mm in diameter, and more preferably 2 mm to 50 mm. When the filter diameter is less than 1 mm, the effective filtration area of the filter becomes insufficient, and the filter is likely to be clogged with foreign matter in the sample. Also, when measuring a biological sample, the biological sample is adsorbed. In this case, clogging is likely to occur, so that the number of continuous measurements can be reduced and the frequency of filter replacement is increased. If the diameter exceeds 100 mm, the measurement sample diffuses into the filter, making accurate measurement impossible, and the filter becomes larger than necessary. The above diameter is the diameter when the filter shape is circular, but in the other shapes, it is the diameter of a virtual circle when a circle that is in contact with the filter is assumed.

  The thickness of the filter of the present invention, that is, the length of the liquid flow path in the flow direction is preferably in the range of 0.1 mm to 50 mm, and more preferably in the range of 0.2 mm to 10 mm. When the thickness of the filter is less than 0.1 mm, the filter is likely to be clogged with foreign matter in the measurement sample, and when measuring a biological sample, the filter is clogged when the biological sample is adsorbed. If continuous measurement is performed, clogging may increase the filtration pressure of the filter, which may prevent accurate measurement. If the thickness of the filter exceeds 50 mm, the measurement sample may diffuse into the filter and accurate measurement may not be possible. Here, the thickness of the filter is the total thickness of the nonwoven fabric and the filter paper filter, and does not include the thickness of the support when the support is laminated, for example.

  The ability to collect foreign matters in the measurement sample by the filter of the present invention can be defined by the retained particle diameter of the filter filter because the filter of the present invention is composed of a nonwoven fabric and a filter paper filter having a relatively high porosity. . For this reason, the retained particle diameter of the filter of the present invention is preferably 5 μm or less, more preferably 3 μm or less, and more preferably 1.5 μm or less in order to reliably remove foreign matter or the like from the measurement sample. Further preferred. When the retained particle diameter of the filter of the present invention exceeds 5 μm, foreign substances in the measurement sample cannot be filtered, and the foreign substances easily flow into the separation column, and the liquid permeation pressure of the separation column is reduced. This may increase the accuracy of measurement and may adversely affect the separation performance of the separation column. Here, the retained particle diameter of the filter of the present invention is the maximum value of the leaked particle diameter when a test solution such as barium sulfate defined in JIS P3801 is naturally filtered, as is the retained particle diameter of the filter paper filter.

  The filter of the present invention is installed in the order of the nonwoven fabric and the filter paper filter from the side away from the separation column in the liquid flow path leading to the separation column of liquid chromatography. The filter of the present invention may be installed at least in the liquid flow path leading to the separation column, and may be installed at other positions, for example, on the outlet side of the separation column. When it is also installed on the outlet side of the separation column, these can be collected when the packing material leaks from the separation column. In this case, the filter paper filter of the filter is preferably on the separation column side.

Next, a liquid chromatography system using the filter of the present invention will be described.
The filter of the present invention only needs to be positioned in the middle of the liquid flow path leading to the separation column. Even if the filter of the present invention is installed in a holder separate from the separation column, it is in the same container as the separation column, that is, the separation column main body. May be housed and mounted. Furthermore, it may be installed separately from the separation column or may be installed integrally with the separation column. However, in consideration of filter replacement work, filter handleability, and the like, it is preferable that the filter is mounted on a holder separate from the separation column and installed separately from the separation column. On the other hand, if the filter is integrally provided in the separation column and these are provided as a column for liquid chromatography, the number of members can be reduced and workability is improved.

  The shape of the holder to which the filter of the present invention is attached is not particularly limited, and the filter can be held, and the diffusion of the measurement sample hardly occurs, and the airtightness can be maintained between the filter of the present invention and the holder. Any known holder can be used. Examples of the material of the holder include metals such as stainless steel, polyethylene, polyetheretherketone, fluororesin, a mixture of polyetheretherketone and fluororesin, and particularly polyetheretherketone and fluorophore that hardly absorb biological samples. Resins are preferred. In addition, a material such as stainless steel that is easily adsorbed by a biological sample is preferably coated with a silicone coating or a material that suppresses the adsorption of the biological sample such as a fluororesin or polyetheretherketone.

  The material of the separation column body is not particularly limited as long as it has strength as the separation column body. For example, metals such as stainless steel and titanium, plastics such as fluororesin and polyetheretherketone, glass, etc. Can be mentioned. When using a metal such as stainless steel, the biological sample is easily adsorbed, so the inner surface of the separation column body is coated with silicone or coated with a material that suppresses the adsorption of the biological sample such as fluororesin or polyetheretherketone. Is preferred.

  The thickness of the silicone coating in the separation column body is preferably in the range of 0.1 nm to 100 μm, more preferably in the range of 0.1 nm to 10 μm, and even more preferably in the range of 0.7 nm to 2 μm. If the thickness of the silicone coating is less than 0.1 nm, it is difficult to coat the silicone itself, and if it exceeds 100 μm, the separation performance of the separation column may be affected.

  The coating ratio inside the separation column body is preferably 10% or more, more preferably 30% or more, and even more preferably 60% or more with respect to the total surface area inside the separation column body. When the coating ratio is less than 10%, it is difficult to suppress the biological sample and the like from being adsorbed inside the separation column main body.

  Moreover, the separation column main body may be subjected to a blocking treatment with a blocking reagent as necessary. The blocking reagent and the blocking treatment method are the same as those described above as the blocking treatment used in the liquid chromatography filter of the present invention.

  In the liquid chromatography system of the present invention, the same one as the conventional liquid chromatography system is used except that the filter is provided.

  As a method for measuring a sample using a liquid chromatography equipped with the filter of the present invention, the measurement sample may be introduced upstream of the filter of the present invention, and the rest is performed in the same manner as in the conventional liquid chromatography. Can do. Moreover, it is preferable to measure by high performance liquid chromatography among liquid chromatography.

  The measurement sample is not particularly limited as long as it can be conventionally measured by liquid chromatography, and examples thereof include biological samples such as blood, serum, plasma, urine, and particularly hemoglobins in blood, preferably It is preferable to separate and quantify HbA1c. The measurement of hemoglobins, particularly HbA1c, requires accurate continuous measurement in a short time. On the other hand, when a filter is used, clogging is likely to occur. By using the filter of the present invention in high performance liquid chromatography, Accurate continuous measurement in time is possible.

  The filter for liquid chromatography of the present invention is formed by laminating a nonwoven fabric having a thickness of 0.05 mm or more and a porosity of 40% to 90% and a filter paper filter having a retained particle diameter of 5 μm or less. Therefore, it is difficult for clogging to occur due to foreign matter in the measurement sample, and even when used for measurement of biological samples containing hemoglobins, the biological sample to the filter Adsorption is unlikely to occur, and therefore, even when a large number of samples are measured continuously, the increase in the filtration pressure of the filter is very small, and accurate measurement can be performed. In addition, since the filter performance life is very long, the replacement frequency of the filter can be reduced, and the measurement efficiency can be increased.

  In the liquid chromatography column of the present invention, since the filter is provided integrally with the separation column, in addition to the above effects, the number of members can be reduced and workability is improved.

  Since the liquid chromatography system of the present invention is equipped with the above-mentioned filter, clogging hardly occurs due to foreign matters in the measurement sample, and the liquid chromatography system is used when measuring a biological sample containing hemoglobins. However, it is difficult for the biological sample to be adsorbed to the filter. Therefore, even when a large number of samples are continuously measured, the increase in the filtration pressure of the filter is very small, and an accurate measurement can be performed. In addition, since the filter performance life is very long, the replacement frequency of the filter can be reduced, and the measurement efficiency can be increased. Furthermore, when the filter is installed away from the separation column, the filter can be exchanged efficiently.

  Since the measurement method of the present invention uses liquid chromatography equipped with the filter, clogging hardly occurs due to foreign matters in the measurement sample, and even when used for measurement of biological samples, It is difficult for the biological sample to be adsorbed to the filter. Therefore, even when a large number of samples are measured continuously, the increase in the filtration pressure of the filter is very small, and accurate measurement can be performed. In particular, it can be suitably used for measuring hemoglobins in blood, preferably HbA1c, which requires accurate continuous measurement in a short time.

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

Example 1
Non-woven fabric (made by Asahi Kasei Kogyo Co., Ltd .: trade name “Microweb”, material polyester fiber, thickness 0.1 mm, porosity 70%), filter paper filter (made by Advantech Toyo Co., Ltd .: trade name “High Purity Filter Paper No. 5C”, material Cellulose fibers, retained particle diameter 1 μm) and support (Advantech Toyo Co., Ltd .: mesh sheet, polyester material) are stacked in this order, then punched into a circle with a diameter of 8 mm, and the filter of the present invention is manufactured as an integrally molded product. did. The thickness of the obtained filter excluding the support was 0.3 mm, and the retained particle diameter was 1 μm.
This filter was fitted into a stainless steel holder, and the holder was fixed with a screw to produce a filter held by the holder. In addition, this filter is arrange | positioned so that the nonwoven fabric side may become a flow path upstream.

(Example 2)
The present invention was carried out in the same manner as in Example 1 except that a filter paper filter (manufactured by Advantech Toyo Co., Ltd .: trade name “wet strength filter paper No. 412”, material cellulose fiber, retained particle diameter 1.5 μm) was used as the filter paper filter. A filter was prepared. The thickness of the obtained filter excluding the support was 0.4 mm, and the retained particle diameter was 1 μm.
This filter was fitted into a stainless steel holder, and the holder was fixed with a screw to produce a filter held by the holder. In addition, this filter is arrange | positioned so that the nonwoven fabric side may become a flow path upstream.

(Example 3)
A filter paper filter (manufactured by Advantech Toyo Co., Ltd .: trade name “filter paper for standard use, No. 131”, material cellulose fiber, retained particle diameter 3 μm) was used in the same manner as in Example 1 except that a filter paper filter was used. A filter was produced. The thickness of the obtained filter excluding the support was 0.4 mm, and the retained particle diameter was 3 μm.
This filter was fitted into a stainless steel holder, and the holder was fixed with a screw to produce a filter held by the holder. In addition, this filter is arrange | positioned so that the nonwoven fabric side may become a flow path upstream.

Example 4
A nonwoven fabric (made by Asahi Kasei Co., Ltd .: trade name “ELTAS”, material polyester fiber, porosity of 60%) was used as a nonwoven fabric in the same manner as in Example 1 except that it was laminated to a thickness of 0.8 mm. A filter was produced. The thickness of the obtained filter excluding the support was 1 mm, and the retained particle diameter was 1 μm.
This filter was fitted into a stainless steel holder, and the holder was fixed with a screw to produce a filter held by the holder. In addition, this filter is arrange | positioned so that the nonwoven fabric side may become a flow path upstream.

(Example 5)
Support (made by Advantech Toyo Co., Ltd .: mesh sheet, polyester material), non-woven fabric (made by Asahi Kasei Co., Ltd .: trade name “ELTAS”, material polypropylene fiber, thickness 0.8 mm, porosity 55%), filter paper filter (made by Advantech Toyo Co., Ltd.) : Product name “High-purity filter paper No. 5C”, raw material cellulose fiber, retention particle diameter 1 μm) and support (Advantech Toyo Co., Ltd .: mesh sheet, raw material polyester) are superposed in this order, and then circular with a diameter of 8 mm. The filter of the present invention was manufactured as an integrally molded product. The thickness of the obtained filter excluding the support was 1 mm, and the retained particle diameter was 1 μm.
This filter was fitted into a stainless steel holder, and the holder was fixed with a screw to produce a filter held by the holder. In addition, this filter is arrange | positioned so that the support body side by which the nonwoven fabric was laminated | stacked may become a flow path upstream.

(Example 6)
The filter of the present invention is the same as in Example 5 except that a filter paper filter (manufactured by Advantech Toyo Co., Ltd .: trade name “standard filter paper No. 2”, material cellulose fiber, retained particle diameter 5 μm) is used as the filter paper filter. Was made. The thickness of the obtained filter excluding the support was 1 mm, and the retained particle diameter was 5 μm.
This filter was fitted into a stainless steel holder, and the holder was fixed with a screw to produce a filter held by the holder. In addition, this filter is arrange | positioned so that the support body side by which the nonwoven fabric was laminated | stacked may become a flow path upstream.

(Comparative Example 1)
Non-woven fabric as the non-woven fabric (manufactured by Asahi Kasei Corporation: trade name "Eltas", material polypropylene fiber, a void 60% rate), and used in laminated so that the Thickness 0.8mm, filter paper-like filter (Advantec Toyo as filter paper-like filter Manufactured: A filter was produced in the same manner as in Example 5 except that the product name “High-purity filter paper No. 5A”, raw material cellulose fiber, retained particle diameter 7 μm) was used. The thickness of the obtained filter excluding the support was 1 mm, and the retained particle diameter was 7 μm.
This filter was fitted into a stainless steel holder, and the holder was fixed with a screw to produce a filter held by the holder. In addition, this filter is arrange | positioned so that the support body side by which the nonwoven fabric was laminated | stacked may become a flow path upstream.

(Comparative Example 2)
A filter was produced in the same manner as in Example 1 except that a membrane filter (raw material cellulose acetate, pore diameter 0.8 μm) was used instead of the nonwoven fabric. The thickness of the obtained filter excluding the support was 0.4 mm, and the retained particle diameter was 0.8 μm.
This filter was fitted into a stainless steel holder, and the holder was fixed with a screw to produce a filter held by the holder. This filter is arranged so that the filter paper filter side is upstream of the flow path.

(Comparative Example 3)
Filter paper filter (manufactured by Advantech Toyo Co., Ltd .: trade name “filter paper for standard No. 2”, material cellulose fiber, retained particle diameter 5 μm), filter paper filter (manufactured by Advantech Toyo Co., Ltd .: trade name “high purity filter paper No. 5C”, Material cellulose fibers, retention particle diameter 1 μm), membrane filter (material cellulose acetate, pore diameter 0.8 μm), and support similar to Example 1 were superposed in this order, then punched into a circle with a diameter of 8 mm, and the filter was removed. Manufactured as an integral molded product. The thickness of the obtained filter excluding the support was 0.7 mm, and the retained particle diameter was 1 μm.
This filter was fitted into a stainless steel holder, and the holder was fixed with a screw to produce a filter held by the holder. This filter is arranged so that the filter paper filter side is upstream of the flow path.

(Comparative Example 4)
A filter was produced in the same manner as in Example 1 except that no filter paper was used. The thickness of the obtained filter excluding the support was 0.1 mm, and the retained particle diameter was in the range of 10 μm to 100 μm.
This filter was fitted into a stainless steel holder, and the holder was fixed with a screw to produce a filter held by the holder. In addition, this filter is arrange | positioned so that the nonwoven fabric side may become a flow path upstream.

  Using the filters held in the holders produced in Examples 1 to 6 and Comparative Examples 1 to 4, the following was performed: (1) degree of pressure increase during measurement of biological sample and (2) adsorption of biological sample And the effect on measured values.

(1) Degree of pressure increase during measurement of biological sample In the liquid chromatography under the measurement conditions shown below, the filters held in the holders prepared in Examples 1 to 6 and Comparative Examples 1 to 4 were introduced into the separation column as samples. The pressure increase test at the time of biological sample measurement was performed. The filters of Examples 1 to 4 and Comparative Example 4 are so that the nonwoven fabric side is far from the separation column. The filters of Examples 5 and 6 and Comparative Example 1 are far from the separation column on the support side on which the nonwoven fabric is laminated. The filters of Comparative Examples 2 and 3 were connected so that the filter paper filter side was far from the separation column.
Measurement condition 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: Eluent A: 100 mmol / l phosphate buffer (pH 5.5)
Eluent B: 300 mmol / l phosphate buffer (pH 6.8)
Flow rate: 2.0 ml / min Detection wavelength: 415 nm
Sample injection volume: 10 μl
Eluent A was allowed to flow from 0 to 3 minutes from the start of measurement, eluent B was allowed to flow from 3 to 5 minutes, and eluent A was allowed to flow from 5 to 10 minutes.

Healthy human blood collected using sodium fluoride as a blood anticoagulant was diluted 100-fold with a hemolysis diluent (0.1 wt% Triton X-100 phosphate buffer, pH 7.0) did. The measurement sample was continuously injected up to 800 samples from the sample inlet upstream of the liquid chromatography filter, and the increase in the filtration pressure of the filter was measured for every 100 samples. The measurement results are shown in Table 1.
Increase value of filtration pressure of filter (N / mm ² ) = filter filtration pressure at measurement (N / mm ² ) −filter filtration pressure before sample injection (N / mm ² )
Further, after the injection of 800 specimens, the increase value of the liquid permeation pressure of the separation column was measured and shown in Table 2.
Increase value of liquid permeation pressure of separation column (N / mm ² ) = liquid permeation pressure of separation column at measurement (N / mm ² ) −liquid permeation pressure of separation column before sample injection (N / mm ² )

As shown in Table 1, in the filters obtained in Examples 1 to 6, the filtration pressure of the filters did not change even after 400 samples were measured, and these filters are less likely to be clogged. Recognize. Further, as shown in Table 2, when the filters obtained in Examples 1 to 6 were used, the liquid permeation pressure of the separation column located downstream of the filter was measured even after 800 samples were measured. It can be seen that there is no increase, foreign matter in the measurement sample is removed by the filter, and it is not mixed in the separation column. In the filters obtained in Comparative Examples 1 and 4, there was no increase in the filtration pressure of the filter, but the increase in the liquid permeation pressure in the separation column located downstream of the filter was 10 N / mm ² . This is presumably because the foreign matter trapping properties of the filters obtained in Comparative Examples 1 and 4 were poor and foreign matters were mixed in the separation column. In Comparative Examples 2 and 3, there was no increase in the liquid permeation pressure of the separation column located downstream of the filter, but the increase in the filtration pressure of the filter was very high. If the filtration pressure of the filter increases, the measurement cannot be performed accurately, or the measurement itself becomes difficult when a measuring device with low pressure resistance is used, so it is necessary to frequently replace the filter.

  From the above, the filter of the present invention does not increase the filtration pressure of the filter even when continuous measurement is performed, and the use of the filter of the present invention prevents foreign matters in the measurement sample from entering the separation column. This can be reliably prevented, and the liquid permeation pressure of the separation column does not increase.

(2) Adsorption of biological sample and influence on measurement values In the liquid chromatography under the measurement conditions shown below, using the filters held in the holders prepared in Examples 1 to 6 and Comparative Examples 1 to 4, The effect of the biological sample on the adsorptivity and the measured value was evaluated.
Measurement condition 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: Eluent A: 50 mmol / l sodium perchlorate added to 50 mmol / l phosphate buffer (pH 5.4)
Eluent B: 300 mmol / l sodium perchlorate added to 50 mmol / l phosphate buffer (pH 5.4)
Flow rate: 2.0 ml / min Detection wavelength: 415 nm
Sample injection volume: 10 μl
Eluent A was allowed to flow from 0 to 3 minutes from the start of measurement, eluent B was allowed to flow from 3 to 5 minutes, and eluent A was allowed to flow from 5 to 10 minutes.

A commercially available control blood level 2 (Glyco Hb Control, International Reagents Co., Ltd.) was dissolved and diluted as described in the attached document, and used as measurement sample 1. In addition, healthy human blood added with blood anticoagulant heparin Na ((1) hemolyzed diluted 100-fold by the same method as the pressure increase test at the time of biological sample measurement) was used as measurement sample 2. For the measurement samples 1 and 2, the HbA1c value was measured alternately. The measurement was repeated 10 times after measuring 1 sample of control blood level 2 and then 5 samples of healthy human blood. That is, control blood level 2 was measured 10 times in this order.
Table 3 shows the HbA1c value in each measurement of level 2 of the obtained control blood. Furthermore, the average value, standard deviation, and CV value of these HbA1c values were calculated and shown in Table 3.

  From the results in Tables 1 and 2, it is considered that the filters obtained in Comparative Examples 1 and 4 have poor foreign matter trapping properties, and it is considered that foreign matters are mixed into the separation column. Then, it turns out that a stable and accurate measured value cannot be obtained.

(Examples 7 to 11)
A filter of the present invention was produced in the same manner as in Example 1 except that the thickness of the nonwoven fabric was as shown in Table 4. The thickness of the obtained filter excluding the support was a value obtained by adding 0.2 mm, which is the thickness of the filter paper filter, to the thickness of the nonwoven fabric shown in Table 4, and the retained particle diameter was 1 μm.
This filter was fitted into a stainless steel holder, and the holder was fixed with a screw to produce a filter held by the holder. In addition, this filter is arrange | positioned so that the nonwoven fabric side may become a flow path upstream.

(Comparative Example 5 )
A filter was produced in the same manner as in Example 1 except that the non-woven fabric was not used. The thickness of the obtained filter excluding the support was 0.2 mm, and the retained particle diameter was 1 μm.
This filter was fitted into a stainless steel holder, and the holder was fixed with a screw to produce a filter held by the holder. This filter is arranged so that the filter paper filter side is upstream of the flow path.

(Comparative Example 6 )
A filter was produced in the same manner as in Example 1 except that the thickness of the nonwoven fabric was 0.01 mm. The thickness of the obtained filter excluding the support was 0.2 mm, and the retained particle diameter was 1 μm.
This filter was fitted into a stainless steel holder, and the holder was fixed with a screw to produce a filter held by the holder. In addition, this filter is arrange | positioned so that the nonwoven fabric side may become a flow path upstream.

Next, using the filters held in the holders prepared in Examples 7 to 11 and Comparative Examples 5 and 6 , as in Example 1, (1) the degree of pressure increase during biological sample measurement was evaluated. The filters of Examples 7 to 11 and Comparative Example 6 were connected so that the nonwoven fabric side was the far side from the separation column, and the filter of Comparative Example 5 was connected so that the filter paper filter side was the far side from the separation column.
Table 5 shows the increase value of the filtration pressure of the obtained filter, and Table 6 shows the increase value of the liquid permeation pressure of the separation column.

As shown in Table 5, in the filters obtained in Examples 7 to 11, the filtration pressure of the filters did not change even after 400 samples were measured, and these filters are less likely to be clogged. Recognize. In addition, as shown in Table 6, when the filters obtained in Examples 7 to 11 were used, the liquid permeation pressure of the separation column located downstream of the filter was measured even after 800 samples were measured. It can be seen that there is no increase, foreign matter in the measurement sample is removed by the filter, and it is not mixed in the separation column. In Comparative Examples 5 and 6 , there was no increase in the liquid permeation pressure of the separation column located downstream of the filter, but the increase in the filtration pressure of the filter was very high. If the filtration pressure of the filter increases, the measurement cannot be performed accurately, or the measurement itself becomes difficult when a measuring device with low pressure resistance is used, so it is necessary to frequently replace the filter.

  From the above, it has been clarified that the filter of the present invention tends to increase the filtration pressure of the filter when continuous measurement is performed when the thickness of the nonwoven fabric is less than 0.05 mm.

(Examples 12 to 16)
A filter of the present invention was produced in the same manner as in Example 8 (nonwoven fabric thickness 0.2 mm) except that the porosity of the nonwoven fabric was as shown in Table 7. The thickness of the obtained filter excluding the support was 0.4 mm, and the retained particle diameter was 1 μm.
This filter was fitted into a stainless steel holder, and the holder was fixed with a screw to create a filter held by the holder. In addition, this filter is arrange | positioned so that the nonwoven fabric side may become a flow path upstream.

(Comparative Examples 7 and 8 )
A filter was produced in the same manner as in Example 8 (nonwoven fabric thickness 0.2 mm) except that the porosity of the nonwoven fabric was as shown in Table 7. The thickness of the obtained filter excluding the support was 0.4 mm, and the retained particle diameter was 1 μm.
This filter was fitted into a stainless steel holder, and the holder was fixed with a screw to create a filter held by the holder. In addition, this filter is arrange | positioned so that the nonwoven fabric side may become a flow path upstream.

Next, using the filters held in the holders produced in Examples 12 to 16 and Comparative Examples 7 and 8 , as in Example 1, (1) the degree of pressure increase during biological sample measurement was evaluated. The filters were all connected so that the nonwoven fabric side was far from the separation column.
The increase value of the filtration pressure of the obtained filter is shown in Table 8, and the increase value of the liquid permeation pressure of the separation column is shown in Table 9.

As shown in Table 8, in the filters obtained in Examples 12 to 16, the filtration pressure of the filters did not change even after 400 samples were measured, and these filters are less likely to be clogged. Recognize. Further, as shown in Table 9, when the filters obtained in Examples 12 to 16 were used, the liquid permeation pressure of the separation column located downstream from the filter was measured even after the 800 sample measurement was completed. It can be seen that there is no increase, foreign matter in the measurement sample is removed by the filter, and it is not mixed in the separation column. In Comparative Example 7 , there was no increase in the liquid permeation pressure of the separation column located on the downstream side of the filter, but the increase in the filtration pressure of the filter was very high. If the filtration pressure of the filter increases, the measurement cannot be performed accurately, or the measurement itself becomes difficult when a measuring device with low pressure resistance is used, so it is necessary to frequently replace the filter. In Comparative Example 8 , almost no increase in the filtration pressure of the filter was observed, but the HbA1c peak obtained by the measurement was broadened. This is probably because the non-woven fabric used in the filter has a large porosity of more than 90%, and thus the diffusion of the measurement sample in the non-woven fabric has become larger than necessary.

Claims (1)

A measurement method for measuring hemoglobin by liquid chromatography in which a liquid chromatography filter is attached to a liquid flow path leading to a separation column,
The filter for liquid chromatography is formed by laminating a nonwoven fabric and a filter paper filter, and is mounted so that the nonwoven fabric side is located on the side far from the separation column, and the nonwoven fabric has a thickness of 0.05 mm or more and a porosity. Is 40% to 90%, and the retention particle diameter of the filter paper filter is 5 μm or less.