JP2007057526A - Method for analyzing low-molecular-weight compound in sample containing water-soluble polymer and low-molecular-weight compound - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an analytical method which enables the execution of satisfactory separation between water-soluble polymers and low-molecular-weight compounds and speedily analyzing low-molecular-weight compounds in samples containing water-soluble polymers and the low-molecular-weight compounds under isocratic conditions without being affected by proteins etc. <P>SOLUTION: A filler material is made of a crosslinking organic polymer acquired by using a compound (glycerol dimethacrylate or the like) of ≥90 mass% having two ethylenic carbon-carbon double bonds and one hydroxyl group as a raw material monomer, has an exclusion limit at a molecular weight between 30,000-3,000 on a pullulan basis, and a mass mean particle diameter between 0.1-100 μm. A column using the filler material is used for analysis by high-performance liquid chromatography in the method for analyzing low-molecular-weight compounds in samples containing water-soluble polymers and the low-molecular-weight compounds. A column for liquid chromatography for the analysis of low-molecular-weight compound in samples containing water-soluble polymers and the low-molecular-weight compounds uses the filler material used for the analytical method. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to a method for analyzing a low molecular compound in a sample containing a water-soluble polymer and a low molecular compound by high performance liquid chromatography (hereinafter sometimes referred to as HPLC). Specifically, the present invention relates to a method for separating and analyzing a small amount of a low molecular compound (particularly a polar low molecular compound) in a sample containing a biopolymer compound (protein or the like) contained in a biological sample. More specifically, a mass spectrometer (hereinafter sometimes referred to as “MS (mass spectrograph)”) was used as a detector in a sample containing a protein such as serum albumin contained in a large amount of biological components. Even in this case, the present invention relates to an analysis method capable of analyzing a low molecular weight compound such as a drug metabolite contained therein without being affected by ion suppression or the like.

  When analyzing water-soluble polymers, especially proteins such as serum albumin and low-molecular compounds in samples containing biological metabolites of drugs, for example, addition of an organic solvent, solid phase Analysis is carried out after removing most of the proteins that are interfering components by extraction or the like. However, at this time, it is difficult to remove all the proteins, and a trace amount of the protein may not be removed and may remain.

  When these samples are analyzed with conventional ODS (silica gel packing with octadecyl group), a minute amount of protein is irreversibly adsorbed to the column, which accelerates deterioration of the column or changes the separation pattern. There was a problem such as. As a method for avoiding these problems, a column switching method and a method using a column using a packing called a permeation limiting packing have been developed.

  In order to compensate for the shortcomings of these silica gel-based fillers, those using a porous organic polymer as a base material for the filler have been developed. For example, since a column using a polyvinyl alcohol-based filler is hydrophilic, there is little adsorption of proteins and the like, and the protein is eluted first from the column without being adsorbed. However, since the substrate itself has a slightly hydrophobic portion, the low molecular weight compound can be held by hydrophobic interaction. For this reason, low molecular weight compounds can be separated from proteins and analyzed.

  Japanese Patent Application Laid-Open No. 2003-93801 (Patent Document 1) describes porous polymer particles having a feature of pore volume and surface area and having a hydrophilic layer on the surface. JP 2001-66295 A (Patent Document 2) and JP 2003-194793 A (Patent Document 3) describe fillers synthesized using a compound containing a polyethylene glycol skeleton as a crosslinkable monomer. This packing material has been introduced for its performance as a column for concentration in the column switching method.

  Columns using these organic polymers as packing materials have a property such that adsorption of hydrophilic polymers (especially serum albumin contained in a large amount in a biological sample in this case) does not occur. However, when analyzing a sample containing a water-soluble polymer and a low molecular weight compound using these columns, the problem is when analyzing a low molecular weight compound having a high polarity. Since these compounds have low hydrophobicity, ordinary hydrophobic fillers have not been sufficiently separated from water-soluble polymers. On the other hand, in the case of a filler that holds a highly polar low molecular weight substance well, the hydrophobicity is too strong, the retention of the low molecular weight compound is too large as a whole, and isocratic conditions (conditions where the eluent composition is constant) ) Has a problem that it takes too much time to analyze and is inconvenient as an analytical column.

  These problems may be avoided by using gradient conditions in the eluent, but there are other problems with gradient conditions. In other words, when water-soluble polymers such as albumin adsorbed on pipes and column housings elute little by little due to changes in the eluent composition accompanying the gradient and pressure fluctuations due to pump switching, low molecules are analyzed by MS and other means. In this case, ion suppression of the low molecular weight drug is prevented, and ion suppression that lowers the detection sensitivity occurs, which hinders quantification.

  Against this backdrop, the retention of high-polarity low-molecular compounds is strong, giving good separation of water-soluble polymers and low-molecular compounds, while allowing rapid analysis and being used under isocratic conditions. There has been a demand for a filler that can be used and an analysis method using the same.

  A porous separating agent using an organic polymer obtained by using 90% or more of glycerin dimethacrylate suitably used in the present invention is described in JP-A No. 58-32164 (Patent Document 4). However, there is no description of a method for analyzing a low molecular compound in a sample containing a water-soluble polymer and a low molecular compound using this filler.

JP 2003-93801 A JP 2001-66295 A JP 2003-194793 A JP 58-32164

  An object of the present invention is to solve the above-described problems of the prior art. That is, separation of water-soluble polymer and low molecular weight compound is good, and the sample containing water-soluble polymer and low molecular weight compound under isocratic conditions where the composition of the eluent is constant without being affected by protein. An object of the present invention is to provide an analysis method capable of quickly analyzing a low molecular weight compound.

  The indispensable condition as a filler that can solve the above-mentioned problems is that the present inventors do not cause problems such as adsorption of serum albumin that is likely to interfere with analysis because it is abundantly adsorbed as a water-soluble polymer. In order to separate a polar low molecular weight compound contained in the same sample from a water-soluble polymer, it must have hydrogen bonding properties at the same time. As a result, the inventors have intensively studied and succeeded in solving the above-mentioned problems, thereby completing the present invention. That is, the present invention relates to a method for analyzing a low molecular compound in a sample containing the following 1 to 10 water-soluble polymers and low molecular compounds, 11 analytical liquid chromatography fillers, and 12 water-soluble polymers The present invention relates to a liquid chromatography column for analyzing a low molecular compound in a sample containing the low molecular compound.

1. A high-speed liquid using a column using a filler composed of a crosslinked organic polymer obtained by using 90% by mass or more of a compound having two ethylenic carbon-carbon double bonds and one hydroxyl group as a raw material monomer A method for analyzing a low-molecular compound in a sample containing a water-soluble polymer and a low-molecular compound, wherein the analysis is performed by chromatography.
2. The analysis method according to 1 above, wherein the compound having two ethylenic carbon-carbon double bonds and one hydroxyl group is glycerin dimethacrylate.
3. 3. The analysis method according to 1 or 2 above, wherein the filler has an exclusion limit molecular weight by pullulan of 30000 or less.
4). Any one of the above 1-3, wherein high performance liquid chromatography analysis is performed under isocratic conditions using an eluent containing 15-40% by weight of an organic solvent compatible with water and 85-60% by weight of an aqueous buffer. The analysis method described in 1.
5. 5. The analysis method according to 4 above, wherein the organic solvent compatible with water is methanol and / or acetonitrile.
6). 6. The analysis method according to 5 above, wherein the organic solvent compatible with water is acetonitrile.
7). 7. The analysis method according to any one of 1 to 6, wherein the water-soluble polymer is a biologically derived water-soluble polymer that is contained in a biological component.
8). 7. The analysis method according to any one of 1 to 6, wherein the water-soluble polymer is serum albumin.
9. 9. The analysis method according to any one of 1 to 8 above, wherein the filler used in the analysis method is porous spherical particles having a mass average particle diameter of 0.1 to 100 μm.
10. 10. The analysis method according to any one of 1 to 9, wherein a low molecular weight compound separated by high performance liquid chromatography is analyzed with a mass spectrometer.
11. It consists of a crosslinked organic polymer obtained by using 90% by mass or more of glycerin dimethacrylate as a raw material monomer, the exclusion limit molecular weight by pullulan is 30000 or less and 3000 or more, and the mass average particle diameter is 0.1 to 100 μm. A packing material for liquid chromatography for analyzing low molecular weight compounds in a sample containing a water-soluble polymer and low molecular weight compounds.
12 12. A column for liquid chromatography for analyzing a low molecular compound in a sample containing a water-soluble polymer and a low molecular compound using the filler according to 11 above.

  By using the method according to the present invention, a low molecular compound in a sample containing a water-soluble polymer and a low molecular compound can be rapidly measured under isocratic conditions. In particular, even when a mass spectrometer (MS) is used as a detector, measurement can be performed without encountering interference such as ion suppression due to a protein component eluted in a trace amount.

filler:
The filler of the present invention is a crosslinked organic polymer obtained by polymerizing a raw material monomer mixed solution having 90% by mass or more of a compound having two ethylenic carbon-carbon double bonds and one hydroxyl group. If the monomer mixture is subjected to suspension polymerization, a particulate filler can be obtained.

  The sample to be analyzed of the present invention contains a water-soluble polymer and a low-molecular compound, but the water-soluble polymer itself is not the object of analysis, so it only needs to be separated from the low-molecular compound and eluted quickly. . Since the sample of the present invention is mainly derived from living organisms, many low-molecular compounds to be analyzed are highly polar. The degree of retention of highly polar low molecular weight compounds in the filler is determined by the sum of both electrostatic interactions (hydrogen bonding and dipolar interactions) and hydrophobic interactions with the filler. When the retention is increased by the hydrophobic interaction, the elution of other low molecular weight compounds having strong hydrophobicity coexisting in the sample is further delayed, which hinders rapid analysis. For this reason, as a filler suitable for rapid analysis and capable of separating a low-molecular compound having a high polarity from a water-soluble polymer, a filler that retains the low-molecular compound having a high polarity mainly by hydrogen bonding is preferable. In order to separate these low-molecular substances, it is preferable to have an appropriate hydrogen bonding property. Therefore, in the present invention, a hydroxyl group is introduced. If the number of hydroxyl groups increases too much, as a result, the polarity of the filler becomes too high and the hydrophobic interaction becomes too weak. The filler of the present invention is required to have a very delicate balance between hydrophobicity and hydrogen bonding.

Raw material monomer:
Examples of the filler satisfying such conditions include a crosslinked organic polymer obtained by using 90% by mass or more of a compound having two ethylenic carbon-carbon double bonds and one hydroxyl group as a raw material monomer. . Two ethylenic carbon-carbon double bonds are necessary to introduce a crosslinked structure during polymerization. An appropriate interval is required between two ethylenic carbon-carbon double bonds, but if it is too long, crosslinking of the filler becomes loose, swelling shrinkage increases, and the strength of the filler decreases. Is not preferable. The number of covalent bonds between carbon-carbon double bonds is preferably 6-10. Hydroxyl groups impart hydrogen bonding properties to the filler, but if it is too much, the hydrophobicity of the filler becomes small and it cannot be used for analysis. Therefore, one hydroxyl group is suitable for the raw material monomer.

Examples of the compound having two ethylenic carbon-carbon double bonds and one hydroxyl group include di (ethylenically unsaturated carboxylic acid) esters of polyhydric alcohols having three or more hydroxyl groups, or ester bonds of such diesters. Are substituted with ether bonds or single bonds, such as glycerin di-1,3- (meth) acrylate, glycerin di-1,2- (meth) acrylate, 2-hydroxy-1,3-diaryloxy. Examples include propane and 2-hydroxy-1,3-divinyloxypropane. Here, “(meth) acrylate” means “methacrylate” or “acrylate”. Of these, glycerol dimethacrylate (2-hydroxy-1,3-dimethacryloxypropane) is particularly preferable. Hereinafter, glycerol dimethacrylate will be described as an example.
Use of a monomer having a lower hydrophobicity than glycerin dimethacrylate, such as acrylamide and a crosslinking agent (polyfunctional monomer), is not preferable because the hydrophobicity of the filler becomes too low. Also, the use of a highly hydrophobic monomer such as divinylbenzene is not preferable because the hydrophobicity increases and the elution of the hydrophobic low molecular weight compound is greatly delayed and the analysis time is prolonged.

  Since glycerin dimethacrylate is a crosslinkable monomer, the resulting filler has a high degree of crosslinking and high strength. For this reason, since the diameter of the filler particles can be reduced, a high-performance filler for liquid chromatography can be obtained. On the other hand, if a non-crosslinkable monomer other than glycerin dimethacrylate is used, the strength of the filler is lost so much, which is not preferable. A monomer that is a cross-linking agent and has a similar degree of hydrophobicity, such as polyethylene glycol dimethacrylate, has a long molecular chain at the cross-linking part. This is not preferable because the strength is lowered.

  The concentration of glycerin dimethacrylate in the raw material monomer mixture must be 90% by mass or more. More preferably, it is 95% by mass, more preferably 99% by mass or more. If it is less than 90% by mass, the hydrogen bonding property becomes small, and the separation of a low-molecular compound having a high polarity is worsened. When it is used in an amount of 90% by mass or more, a filler having sufficient strength, high hydrogen bonding properties and low hydrophobicity can be obtained.

  The separation performance and characteristics of the filler of the present invention can be finely adjusted by mixing other monomers within a range where glycerin dimethacrylate does not become less than 90% by mass. As the monomer that can be added in addition to glycerin dimethacrylate, most of the radical polymerizable monomers used for producing ordinary fillers can be used. For example, styrene, divinylbenzene, methyl acrylate, bis (meth) acrylamide, ethyl (meth) acrylate, hydroxyethyl (meth) acrylate, glycidyl (meth) acrylate, ethylene glycol di (meth) acrylate, (meth) acrylamide, glycerin mono (Meth) acrylate etc. are mentioned.

polymerization:
The polymerization can be carried out by usual radical polymerization such as solution polymerization, bulk polymerization, suspension polymerization and emulsion polymerization. The case of producing spherical particles by aqueous suspension polymerization will be described below as a representative example, but is not limited to this method.
The oil phase used for aqueous suspension polymerization is prepared by adding a polymerization initiator to a mixture of a raw material monomer mixture and a diluent (used as a solvent or dispersion medium or a monomer diluent).

  The diluent is added to the monomer mixture for the purpose of making the resulting spherical crosslinked organic polymer particles (filler) porous. The type is not particularly limited when water is not used as a medium such as bulk polymerization, but when water is used as a medium such as aqueous suspension polymerization, a water-insoluble organic compound is preferable. . Specific examples include toluene, xylene, diethylbenzene, heptane, octane, dodecane, butyl acetate, dibutyl phthalate, isoamyl alcohol, 1-hexanol, cyclohexanol, 2-ethylhexanol, 1-dodecanol, uncrosslinked polystyrene, and the like. . These solvents or dispersion media can be used alone or as a mixture of two or more thereof.

  Moreover, the addition amount of these diluents is 10-90 mass% on the basis of the total amount of a raw material monomer and a diluent, Preferably it is 20-80 mass%, More preferably, it is 25-60 mass%. If the addition amount is less than 10% by mass, the porosity of the filler becomes insufficient, such being undesirable. When a large amount of diluent is used, the pore volume of the filler becomes large, which is preferable. However, when it exceeds 90% by mass, the physical strength of the filler is insufficient and the pressure resistance as a column is lowered.

  Examples of the polymerization initiator include azo compounds such as 2,2′-azobis (isobutyronitrile) and 2,2′-azobis (2,4-dimethylvaleronitrile); benzoyl peroxide, dicumyl peroxide And generally used polymerization initiators such as di-t-butyl peroxide, tert-butyl perbenzoate, and organic peroxides such as methyl ethyl ketone peroxide. These are used alone or in combination of two or more. The concentration of the polymerization initiator to be used is appropriately determined depending on the type of the monomer and cannot be defined unconditionally, but is preferably 0.1 to 5 parts by mass with respect to 100 parts by mass of the total amount of monomers.

  An oil phase dispersion stabilizer is added to the aqueous phase. Examples of the dispersion stabilizer used include water-soluble polymer compounds such as polyvinyl alcohol, alkyl cellulose, hydroxyalkyl cellulose, carboxyalkyl cellulose, sodium polyacrylate, and gelatin. Although the density | concentration of a dispersion stabilizer does not have limitation in particular, 0.1-5 mass parts is preferable with respect to 100 mass parts of water. In order to prevent a part of the monomer from dissolving in the aqueous phase, it is preferable to add salts to the aqueous phase. Examples of salts to be added include sodium chloride, calcium chloride, sodium sulfate and the like, and these salts are used alone or in combination of two or more. The concentration of the salt to be used is not particularly limited, but is preferably as high as possible within the range allowed by solubility. For example, with respect to water, it is 1-15 mass parts for sodium chloride, and 1-40 mass parts for calcium chloride.

  If the ratio of the aqueous phase to the oil phase is too large, the amount of a part of the monomer dissolved in the aqueous phase increases, and if it is too small, coalescence of oil droplets is likely to occur. Therefore, the mass of water to be used is preferably 200 to 1000 parts by mass with respect to 100 parts by mass of the total amount of monomer and diluent.

  Before starting the aqueous suspension polymerization, the oil phase and the aqueous phase are mixed and dispersed so that the oil droplets have a target particle diameter (particle diameter). For the dispersion, a stirrer with a stirring blade for atomization or a high-speed disperser (homogenizer) can be used. To make an adsorbent with a relatively large particle size (for example, for solid-phase extraction), an agitation device with a stirring blade for atomization is used, and an adsorbent with a small particle size (for example, an adsorbent for liquid chromatography). It is better to use a high-speed disperser (homogenizer).

The polymerization reaction is carried out at a temperature range of 40 to 100 ° C. for 5 to 16 hours under normal stirring.
The filler fine particles thus obtained are sufficiently washed with hot water, an organic solvent or the like to remove the dispersion stabilizer, solvent, residual monomer, diluent or the like contained in or adhering to the particles. Furthermore, if necessary, the particles are classified to obtain a chromatographic filler.

Filler particle size:
In HPLC, it is necessary to use fine particles so as to have a sufficient number of theoretical plates. The mass average particle diameter of the filler is preferably from 0.1 to 100 μm, more preferably from 1 to 10 μm, still more preferably from 3 to 5 μm. When the particle diameter is less than 0.1 μm, the column pressure becomes too high, which causes a problem that a burden is imposed on the apparatus. If it exceeds 100 μm, the performance as an analytical column is lowered, which is not convenient. In order to obtain a desired particle size, the amount of dispersing agent and the stirring speed in the polymerization are adjusted. Optimum conditions can be determined by obtaining the correlation between the size of the finally obtained filler and these conditions. Further, the polymerization particles may be classified. Classification operations include classification with a sieve, air classification, and the like.

The mass average particle diameter can be measured with a Coulter Counter (registered trademark), an optical microscope or the like.
Although the case where the filler is a porous particle has been described above, an integral filler such as a monolith may be used as long as the filler is porous.

Exclusion limit molecular weight of filler:
The exclusion limit molecular weight of the filler of the present invention is preferably 30000 or less. If the exclusion limit molecular weight exceeds 30000, elution of a water-soluble polymer such as serum albumin is delayed, resulting in poor separation from low molecular weight compounds. Further, if the exclusion limit molecular weight is less than 3000, the retention of the low molecular compound itself to be analyzed is lowered or the separation is deteriorated, which is not preferable.

  The exclusion limit molecular weight is packed in a stainless steel column with an inner diameter of 4.6 mm and a length of 150 mm, pure water is used as an eluent, pullulan (manufactured by Showa Denko KK) with a known molecular weight as a standard sample, Create and ask. When the exclusion limit molecular weight is 30000 or less, most of the water-soluble proteins contained in a large amount in the living body and the like are eluted near the exclusion limit (Vo). On the other hand, since the low molecular weight compounds are sequentially eluted according to their hydrophobicity after the permeation limit (Vt), the separation between the water-soluble polymer and the low molecular weight compound is improved. For this reason, the value of the exclusion limit molecular weight is important for improving the separation of the water-soluble polymer and the low-molecular compound.

  When the filler of the present invention is produced by usual suspension polymerization or the like, the exclusion limit molecular weight of the filler can be controlled by the amount and type of diluent added together with the monomer. In general, by increasing the amount of diluent, the pore size of the filler particles becomes larger and the exclusion limit molecular weight becomes larger. If a poor solvent for the polymer produced by polymerization of the monomer is used as the diluent, the exclusion limit molecular weight increases, and if a good solvent is used, the exclusion limit molecular weight decreases.

Substances to be analyzed:
The water-soluble polymer refers to a protein, an organic polymer, or a natural polymer having a molecular weight of about 10,000 or more. In particular, examples include biologically derived water-soluble polymers, and serum albumin, albumin dimer, and the like, which are compounds that are generally included in biological samples and are likely to interfere with analysis.

ｋ_caffeine：カフェインの保持係数，ｋ_toluene：トルエンの保持係数，ｋ_r：（ｔ_r−ｔ₀）／ｔ₀，ｔ_r：サンプルの保持時間（ピーク位置の時間，ｒはカフェインまたはトルエンを示す。），ｔ₀：非保持時間。
が、０．０５〜１．０が好ましく、０．０８〜０．５がより好ましい。αが下限値未満では水溶性高分子と低分子化合物の分離が悪くなり、上限値を超えると低分子化合物同士の分離が悪くなる。 In this specification, the low molecular weight compound represents an organic compound having a molecular weight of 4000 or less. In particular, when the biological sample is a sample to be analyzed, a drug or a metabolite of the drug contained in the sample is included. Specific examples include caffeine, theobromine, theophylline and barbitals.
Although the packing material of the present invention has low hydrophobicity, the hydrogen bonding property is remarkably higher than that of a reverse phase analytical column that has been used so far. For this reason, it has the characteristic that retention of strong low molecular weight compounds, such as caffeine, is large. This property is one factor that enables the analysis method of the present invention. The reason for this high hydrogen bonding property is presumed to be the influence of hydroxyl groups scattered not only on the particle surface but also on the inner surface of the pore, but it is not known in detail.
The column of the present invention is represented by the following formula:

k _caffeine: retention factor of caffeine, k _toluene: retention coefficient of _{toluene, k r: (t r -t} 0) / t 0, t r: sample of retention time (time of peak position, r is the caffeine or toluene ), T ₀ : non-holding time.
However, 0.05-1.0 is preferable and 0.08-0.5 is more preferable. When α is less than the lower limit, the separation of the water-soluble polymer and the low molecular compound is poor, and when the value exceeds the upper limit, the separation of the low molecular compounds is poor.

Analysis conditions by high performance liquid chromatography:
<Eluent>
In the analysis method of the present invention, it is preferable to use an HPLC eluent containing 15 to 40% by mass of an organic solvent compatible with water and 85 to 60% by mass of an aqueous buffer.

  The organic solvent that is compatible with water is an organic solvent that can be dissolved in water (including an aqueous buffer solution) in an amount of 25% by mass or more in the range of normal temperature to HPLC analysis temperature. Such an organic solvent is not particularly limited as long as it is a solvent that can be used in ordinary liquid chromatography. Examples thereof include methanol, ethanol, acetonitrile, and acetonitrile is particularly preferable. These organic solvents can be used in combination of several kinds.

  As the aqueous buffer, various buffers used for stabilizing the pH at the time of analysis can be used. Further, pure water can be used without using a buffer solution. When MS is used as a detector, a volatile buffer is desired. For this, ammonium formate and ammonium acetate are preferred. Commonly used is a 5-10 mM aqueous ammonium acetate solution.

The mixing ratio of the organic solvent compatible with water and the aqueous buffer is preferably 15 to 40% by mass: 85 to 60% by mass, more preferably 20 to 35% by mass: 80 to 65% by mass, and more preferably 20 to 30% by mass. : 80-70 mass% is the most preferable.
The filler of the present invention is not completely free of adsorption of proteins such as serum albumin. However, the hydrophobic interaction with proteins such as serum albumin is minimized and hardly causes adsorption under the eluent conditions in which the organic solvent is 15 to 40% by mass where the present filler can be suitably used. Therefore, when the analysis is performed in this eluent range, the recovery rate of albumin is 90% or more, and at this time, the retention of the hydrophilic low molecular compound is sufficiently large, and the strongly hydrophobic compound is not delayed. Elute. For this reason, HPLC analysis can be suitably performed under isocratic conditions. When the organic solvent concentration is less than 15% by mass, not only protein such as serum albumin is adsorbed but also elution of a highly hydrophobic compound becomes too late. Furthermore, under conditions where the organic solvent concentration exceeds 40% by mass, the retention of low-molecular compounds with high polarity becomes small, and the separation becomes poor because it approaches the permeation limit. Further, under these conditions, proteins such as serum albumin are not preferable because they have poor solubility and risk of precipitation.

  In the HPLC analysis of the present invention, it is preferable to carry out the eluent under isocratic conditions. This is to minimize the elution of the hydrophilic polymer adsorbed on the column housing, piping, etc., and to perform MS detection and quantification of low molecular weight compounds stably. There is no problem even if the gradient conditions are applied when the observation is not performed by MS or when the column or analyzer is washed.

  EXAMPLES Hereinafter, although an Example and a comparative example are given and this invention is demonstrated, this invention is not limited to these examples.

Example 1:
1. Synthesis of glycerin dimethacrylate filler A mixture of glycerin dimethacrylate (NK ester 701, Shin-Nakamura Chemical Co., Ltd.) 2000 g and cyclohexanol (Pure Chemical Co., Ltd.) 1340 g was mixed with 2,2′-azobis (isobutyro). 30 g of nitrile (Wako Pure Chemical Industries, Ltd.) was dissolved to prepare an oil phase. On the other hand, 120 g of polyvinyl alcohol (Kuraray Co., Ltd., Kuraray Poval PVA-224) is dissolved in 3 liters of water, 7 liters of water and then 240 g of sodium chloride (2 liters) in water (2 liters) are added and mixed, and the aqueous phase is mixed. Was prepared. Mix the oil phase and the water phase in a 20L stainless steel container, apply it to a high-speed disperser (homogenizer), and adjust the rotation speed and dispersion time to adjust the maximum particle size of the oil droplets to 5μm. did.

Subsequently, reaction was performed at 60 degreeC for 7 hours, stirring at 150 rpm using a normal stirring blade. The resulting crosslinked polymer particles were centrifuged (2000 rpm, 10 minutes), the supernatant was discarded, and the precipitate was dispersed in 12 liters of warm water at 70 ° C. (using an ultrasonic cleaner) and then stirred at 70 ° C. for 3 hours. This was subjected to suction filtration, and the cake on the funnel was washed with 60 liters of hot water at 70 ° C. and then with 18 liters of acetone, spread on a stainless steel vat and air-dried, and further dried under reduced pressure at 60 ° C. for 24 hours. This was classified with a wind classifier. When measured with a Coulter counter (Beckman Coulter Multisizer 3), 1300 g of crosslinked polymer particles having a mass average particle diameter of 5.1 μm were obtained.
After adding 500 ml of pure water to 50 g of the crosslinked polymer particles obtained above and heating and stirring at 60 ° C. for 5 hours, the particles were collected by filtration and washed successively with 2000 ml of 70 ° C. hot water and 300 ml of methanol. This was spread on a stainless steel bat and air-dried, and further dried under reduced pressure at 70 ° C. for 24 hours to obtain 48 g of a filler.

2. Column performance evaluation About 3 g of this packing material was packed into a stainless steel column having an inner diameter of 4.6 mm and a length of 150 mm by a wet packing method.
Eluent: pure water,
Flow rate: 0.33 ml / min,
Standard sample: 0.1% by weight of pullulan,
Molecular weight of pullulan: 758000, 338000, 194000, 95400, 46700, 20800, 12000, 5300 (all are Shodex STANDARD P-82, Showa Denko KK),
Molecular weight: 2930 (manufactured by Showa Denko KK)
Molecular weight: 1330 (manufactured by Showa Denko KK)
Detector: RI,
Injection volume: 100 μl.
The elution position of pullulan of each molecular weight was measured, and from the retention time, the elution volume was calculated and a calibration curve was created. That is, each point was plotted on a graph with the logarithmic value of the molecular weight on the vertical axis and the elution volume on the horizontal axis (FIG. 1). When the value of the vertical axis at the point where the extension of the inclined straight line and the extension of the line parallel to the vertical axis intersect in FIG. 1 was determined as the exclusion limit molecular weight, the exclusion limit molecular weight of this filler was 20000.

3. Evaluation of recovery rate of BSA (bovine serum albumin) The packing material was packed in a column having a diameter of 4.6 mm and a length of 50 mm.
The recovery rate of BSA when bovine serum albumin (manufactured by SIGMA, hereinafter sometimes referred to as BSA) was injected into this column was measured when the column was not attached (a tube made of polytetrafluoroethylene (instead of the column) The measurement was performed with an inner diameter of 0.5 mm and a length of 10 m).
Eluent: 10 mM ammonium acetate / acetonitrile = 850 g / 150 g,
Flow rate: 1 ml / min
Column temperature: 30 ° C.
Sample: BSA 7 mg / ml,
Injection volume: 10 μl,
Detector: UV (220 nm).
BSA was observed as one peak at the exclusion limit, and it was observed that 98% of BSA was eluted from the peak area.

ｋ_caffeine：カフェインの保持係数，ｋ_toluene：トルエンの保持係数，ｋ_r：（ｔ_r−ｔ₀）／ｔ₀，ｔ_r：サンプルの保持時間（ピーク位置の時間），ｔ₀：非保持時間。
この値は、疎水性相互作用と静電的相互作用の比を表しており、この値が大きいほど、極性の高い低分子化合物の保持が大きく、かつ、疎水性の低分子化合物の溶出も遅くなりすぎないということを示す。つまり、この値が（ある程度までは）大きいほど、始めに溶出する親水性高分子と極性の高い低分子化合物の分離が好調であり、かつ疎水性の強い低分子化合物の溶出に長い時間待つ必要がないカラムを与えることを示す。 4). Evaluation of the separation performance of the column In order to compare the electrostatic interaction of each packing material, considering the hydrophobicity, the relative electrostatic interaction strength was calculated by the following equation. .

k _caffeine: retention factor of caffeine, k _toluene: retention coefficient of _{toluene, k r: (t r -t} 0) / t 0, t r: sample of retention time (time of peak position), t _0: non-holding time.
This value represents the ratio of hydrophobic interaction to electrostatic interaction. The higher this value, the greater the retention of highly polar low molecular compounds and the slower the elution of hydrophobic low molecular compounds. Indicates that it will not be too much. In other words, the higher this value is (to a certain extent), the better the separation of the hydrophilic polymer that elutes first from the low-molecular compound with high polarity, and the longer it is necessary to wait for the elution of the low-molecular compound with strong hydrophobicity. Indicates that a column without is given.

Analysis conditions:
Eluent 10 mM ammonium acetate / acetonitrile = 750 g / 250 g,
Flow rate: 1 ml / min
Column temperature: 40 ° C
Detector: UV (254 nm),
Injection volume: 10 μl,
Sample: BSA 700 mg / L, caffeine 10 mg / L, toluene 150 mg / L,
Result: α = 0.086.

  FIG. 2 shows a chromatogram. Peak 1: BSA, peak 2: caffeine, peak 3: toluene. After BSA eluted at the exclusion limit (0.39 minutes), it eluted at 0.97 minutes for caffeine and 4.45 minutes for toluene. Since the separation of BSA and caffeine was good and the elution of toluene was not slow, rapid analysis under isocratic conditions was possible.

Example 2 (copolymerization type filler)
Polymerization and air classification were performed in the same manner as in Example 1 except that 1880 g of glycerin dimethacrylate and 120 g of glycidyl methacrylate were used instead of 2000 g of glycerin dimethacrylate to obtain 1200 g of crosslinked polymer particles. The mass average particle diameter of the crosslinked polymer particles was 5.1 μm.
To 30 g of the crosslinked polymer particles obtained above, 500 ml of 0.3M aqueous formic acid solution (adjusted to pH 3.0 with 1N aqueous sodium hydroxide solution) was added, and the mixture was heated and stirred at 60 ° C. for 5 hours. Then, it was washed successively with 2000 ml of hot water at 70 ° C. and 300 ml of methanol. This was spread on a stainless steel bat and air-dried, and further dried under reduced pressure at 70 ° C. for 24 hours to obtain 30 g of a filler.
2. The exclusion limit molecular weight (analysis conditions are the same as in Example 1) was 20000.
3. Evaluation of BSA recovery rate (analysis conditions are the same as in Example 1):
93% BSA was recovered.
4). Column separation performance (analysis conditions are the same as in Example 1):
α = 0.087
A filler similar to that in Example 1 was obtained.

Comparative Example 1:
1. Production of Filler A filler having a mass average particle diameter of 4.9 μm was obtained in the same manner as in Example 1 except that 2000 g of ethylene dimethacrylate was used instead of 2000 g of glycerin dimethacrylate.
2. The exclusion limit molecular weight (analysis conditions are the same as 2 in Example 1) was 30000.
3. Evaluation of recovery rate of BSA (analysis conditions are the same as 3 in Example 1):
90% BSA was recovered.
4). Evaluation of separation performance of the column (analysis conditions are the same as 4 in Example 1):
α = 0.028.
The value of α was smaller than that of the filler of Example 1. FIG. 3 shows the chromatogram. Peak 1: BSA, peak 2: caffeine, peak 3: toluene. In the chromatogram, for comparison with FIG. 2, the time axis is reduced so that the distance between the BSA peak and the toluene peak is substantially the same. It can be seen that the elution time of toluene is about 12 minutes, which is considerably slower than about 5 minutes of Example 1, and is not suitable for rapid analysis under isocratic conditions.

Comparative Example 2:
1. Production of Filler A filler having a mass average particle diameter of 5.1 μm was obtained in the same manner as in Example 1 except that 1000 g of ethylene dimethacrylate and 1000 g of glycerin dimethacrylate were used instead of 2000 g of glycerin dimethacrylate.
2. The exclusion limit molecular weight (analysis conditions are the same as Example 1) was 50000.
3. Evaluation of BSA recovery rate (analysis conditions are the same as in Example 1):
93% BSA was recovered.
4). Evaluation of column separation performance (analysis conditions are the same as in Example 1):
α = 0.044.
The value of α was smaller than that of the filler of Example 1. FIG. 4 shows a chromatogram. Peak 1: BSA, peak 2: caffeine, peak 3: toluene. In the chromatogram, for comparison with FIG. 2, the time axis is enlarged so that the distance between the BSA peak and the toluene peak is substantially the same.
The elution time of toluene was not too slow and was in an appropriate position, but the separation of caffeine was inferior to Example 1.

Comparative Example 3:
1. As the filler, a commercially available column: GF-310 4B (manufactured by Showa Denko KK, polyvinyl alcohol-based filler, mass average particle diameter 5 μm) was used.
2. Exclusion limit molecular weight:
Catalog value 40000.
3. Evaluation of BSA recovery rate (analysis conditions are the same as in Example 1):
98% BSA was recovered.
4). Evaluation of column separation performance (analysis conditions are the same as in Example 1):
α = 0.019.
The value of α was smaller than that of the filler of Example 1. FIG. 5 shows a chromatogram. Peak 1: BSA, peak 2: caffeine, peak 3: toluene. In the chromatogram, for comparison with FIG. 2, the time axis is reduced so that the distance between the BSA peak and the toluene peak is substantially the same. The separation of BSA and caffeine is inferior.

水溶性高分子マトリックスとして牛血清アルブミン（ＢＳＡ、分子量：約６７，０００）を選んだ。ＬＣ−ＭＳシステムとしては、Agilent 1100シリーズＨＰＬＣシステム（アジレントテクノロジー製）にLCQ Advantage （サーモエレクトロン社製）を連結し、エレクトロスプレーイオン化（ＥＳＩ）法で使用した。カラム温度３０℃、（１０ｍＭ酢酸アンモニウム）／アセトニトリル＝７０ｍｌ／３０ｍｌを移動相とする５分間のイソクラティック溶出（０.２０ｍｌ／ｍｉｎ、スプリットなし）で分析を行った。
まず、薬物（各５μｇ／ｍｌ）のみをイオン交換水に溶かした試料１０μｌを注入し、ＵＶ検出（２２０ｎｍ）およびＥＳＩ−負イオンモードによる選択イオンモニタリング（ＳＩＭ）にて測定した（イオン化電圧：５ｋＶ）。次に、３種の薬物（各５μｇ／ｍｌ）とＢＳＡ（０.７ｍｇ／ｍｌ）をイオン交換水に溶かした試料１０μｌを注入し、同様に測定した。ただし、後者ではＢＳＡが高濃度で溶出している部分（１.２ｍｉｎまで）の溶出液はＭＳへ導入しなかった。
結果：
薬物試料の分析結果を図６に、ＢＳＡ含有薬物試料の分析結果を図７に示す。ＵＶ検出クロマトグラムの縦軸最大値は、ＢＳＡのピークトップに合わせてある。また、ＳＩＭクロマトグラムの縦軸最大値は、同じｍ／ｚにおける測定では合わせてある。
初めに行った薬物試料の分析結果から、各薬物は脱プロトン化した負の分子イオン［Ｍ−Ｈ］_-として感度よく検出され、バルビタール（ｍ／ｚ １８３.２）、フェノバルビタール（ｍ／ｚ ２３１.１）、ヘキソバルビタール（ｍ／ｚ ２３５.２）が保持時間１.３〜４.５ｍｉｎの間によく分離して溶出することがわかった。
次に行ったＢＳＡ含有薬物試料の分析では、ＢＳＡが保持時間０.４〜０.９ｍｉｎに集中して溶出したので、バルビタール溶出の直前で流路を切り替える瞬間（１.２ｍｉｎ）まで充分な余裕があった。得られた各薬物のＳＩＭクロマトグラムは、ピークの形状・高さともに、ＢＳＡを含まない場合の結果と非常によく一致した。フェノバルビタールの感度は他の２種に比べて低かったが、ＢＳＡの有無による差は認められなかった。
充填剤の排除限界より大きいＢＳＡの場合は、サイズ排除モードが働くため逆相的な相互作用は無視できるほど小さくなり、その結果、先端にまとまって溶出されたと考えられる。それに対し、低分子のバルビタール類は細孔内に入って逆相モードで分離されるが、細孔内面に点在する水酸基との水素結合により保持が若干増加するので、ＢＳＡとの分離がさらに促進されたと推定される。 Example 3: Analysis using MS as a detector A column packed with the packing material of Example 1 in a stainless steel column having a diameter of 2.0 mm and a length of 50 mm by wet packing was used. Three kinds of drugs as samples of low molecular weight compounds, namely, barbital, phenobarbital, hexobarbital represented by the following formula,

Bovine serum albumin (BSA, molecular weight: about 67,000) was selected as the water-soluble polymer matrix. As the LC-MS system, an LCQ Advantage (manufactured by Thermo Electron) was connected to an Agilent 1100 series HPLC system (manufactured by Agilent Technologies), and used by electrospray ionization (ESI) method. The analysis was performed by isocratic elution (0.20 ml / min, no split) for 5 minutes using a column temperature of 30 ° C. and a mobile phase of (10 mM ammonium acetate) / acetonitrile = 70 ml / 30 ml.
First, 10 μl of a sample in which only drugs (5 μg / ml each) were dissolved in ion-exchanged water was injected and measured by UV detection (220 nm) and selective ion monitoring (SIM) in ESI-negative ion mode (ionization voltage: 5 kV). ). Next, 10 μl of a sample prepared by dissolving three kinds of drugs (each 5 μg / ml) and BSA (0.7 mg / ml) in ion-exchanged water was injected and measured in the same manner. However, in the latter, the eluate of the portion where BSA was eluted at a high concentration (up to 1.2 min) was not introduced into the MS.
result:
The analysis result of the drug sample is shown in FIG. 6, and the analysis result of the BSA-containing drug sample is shown in FIG. The maximum value of the vertical axis of the UV detection chromatogram is adjusted to the peak top of BSA. Further, the maximum value of the vertical axis of the SIM chromatogram is matched in the measurement at the same m / z.
From the results of the analysis of the first drug sample, each drug was detected with high sensitivity as a deprotonated negative molecular ion [MH] ₋ , and barbital (m / z 183.2), phenobarbital (m / z 231.1), hexobarbital (m / z 235.2) was well separated and eluted during a retention time of 1.3 to 4.5 min.
In the subsequent analysis of the BSA-containing drug sample, since BSA was concentrated and eluted at a retention time of 0.4 to 0.9 min, there was a sufficient margin until the moment of switching the flow path (1.2 min) immediately before barbital elution. was there. The SIM chromatograms of the obtained drugs were in good agreement with the results obtained when BSA was not included in both the peak shape and height. The sensitivity of phenobarbital was lower than the other two, but no difference was observed depending on the presence or absence of BSA.
In the case of BSA larger than the exclusion limit of the filler, since the size exclusion mode works, the reverse-phase interaction becomes negligibly small, and as a result, it is considered that the BSA is eluted together at the tip. In contrast, low molecular weight barbitals enter the pores and are separated in the reverse phase mode, but the retention is slightly increased due to hydrogen bonding with the hydroxyl groups scattered on the inner surface of the pores. Presumed to have been promoted.

The calibration curve showing the relationship between the molecular weight of the analyte in the size exclusion chromatography and the elution volume of the eluent, and the exclusion limit molecular weight are shown. The chromatogram of Example 1. Peak 1: BSA, peak 2: caffeine, peak 3: toluene. The chromatogram of Comparative Example 1. Peak 1: BSA, peak 2: caffeine, peak 3: toluene. The chromatogram of Comparative Example 2. Peak 1: BSA, peak 2: caffeine, peak 3: toluene. The chromatogram of Comparative Example 3. Peak 1: BSA, peak 2: caffeine, peak 3: toluene. The chromatogram of Example 3. UV detector (without BSA), MS (without BSA). The chromatogram of Example 3. UV detector (with BSA), MS (with BSA).

Claims (12)

  A high-speed liquid using a column using a filler composed of a crosslinked organic polymer obtained by using 90% by mass or more of a compound having two ethylenic carbon-carbon double bonds and one hydroxyl group as a raw material monomer A method for analyzing a low-molecular compound in a sample containing a water-soluble polymer and a low-molecular compound, wherein the analysis is performed by chromatography.   The analysis method according to claim 1, wherein the compound having two ethylenic carbon-carbon double bonds and one hydroxyl group is glycerin dimethacrylate.   The analysis method according to claim 1 or 2, wherein the filler has an exclusion limit molecular weight by pullulan of 30000 or less.   The high performance liquid chromatographic analysis is carried out under isocratic conditions using an eluent containing 15 to 40% by weight of an organic solvent compatible with water and 85 to 60% by weight of an aqueous buffer. The analysis method of crab.   The analysis method according to claim 4, wherein the organic solvent compatible with water is methanol and / or acetonitrile.   The analysis method according to claim 5, wherein the organic solvent compatible with water is acetonitrile.   The analysis method according to any one of claims 1 to 6, wherein the water-soluble polymer is a biologically derived water-soluble polymer contained in a biological component.   The analysis method according to any one of claims 1 to 6, wherein the water-soluble polymer is serum albumin.   The analysis method according to any one of claims 1 to 8, wherein the filler used in the analysis method is a porous spherical particle having a mass average particle diameter of 0.1 to 100 µm.   The analysis method according to any one of claims 1 to 9, wherein the low molecular weight compound separated by high performance liquid chromatography is analyzed by a mass spectrometer.   It consists of a crosslinked organic polymer obtained by using 90% by mass or more of glycerin dimethacrylate as a raw material monomer, the exclusion limit molecular weight by pullulan is 30000 or less and 3000 or more, and the mass average particle diameter is 0.1 to 100 μm. A packing material for liquid chromatography for analyzing low molecular weight compounds in a sample containing a water-soluble polymer and low molecular weight compounds. A liquid chromatography column for analyzing a low molecular weight compound in a sample containing a water-soluble polymer and a low molecular weight compound using the filler according to claim 11.

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