JPH0774799B2 - Method for measuring blood components - Google Patents

Description

TECHNICAL FIELD The present invention relates to a method for accurately and easily measuring components in blood, particularly components contained in blood and blood cells, using high performance liquid chromatography. The present invention relates to an automatable measuring method.

(Prior Art) Various components contained in blood are measured for pathological research, diagnosis, treatment, and the like. For example, the drug dosage is determined by monitoring the drug concentration in blood. Examples of such drugs include:
Cyclosporin A (CyA) is mentioned.
CyA is a cyclic peptide and exhibits a strong immunosuppressive action. The digestion and absorption of this CyA varies greatly among individuals, and the absorption efficiency varies depending on the administration period, etc., so it is necessary to monitor the blood concentration. This CyA is usually obtained by the following method ("Ayumi of Medicine", Volume 129, No. 9, 623-627 (19
74)). First, blood is passed through a deproteinization column that has been appropriately hydrophilically treated in advance to remove CyA while adsorbing CyA and allowing protein to pass without adsorbing it. Methanol is passed through this column to elute CyA and the eluate is used as a solvent. After volatilization, CyA is measured by high performance liquid chromatography (HPLC) using acetonitrile-water (70:30) as an eluent.

When measuring components in blood as in the above CyA measurement method, plasma or serum obtained by removing blood cell components from whole blood is usually used. This is because when red blood cells containing hemoglobin are contained in the sample, large absorption appears throughout the ultraviolet and visible regions, making it impossible to perform measurement by a spectroscopic method. However, it has been pointed out that the method using plasma or serum is inaccurate because the target component to be measured may be contained in blood cells. For example, the CyA is also taken up by blood cells,
Moreover, the ratio of the drug concentration between blood cells and plasma gradually changes from the time when blood is collected.

This method cannot be used in the above-mentioned measurement method using high performance liquid chromatography when measuring components to be measured contained in both plasma and blood cells, because the deproteinization column causes clogging by blood cells. In order to measure the components to be measured in plasma and blood cells, it is necessary to hemolyze and destroy the blood cell components. For example, Journal of Chromatography, 341 (1
985), pp. 411-419, a method for measuring CyA after hemolysis using acetonitrile is described. According to this, first, acetonitrile is added to the collected whole blood to cause hemolysis, and a protein component is precipitated, followed by centrifugation to collect the supernatant. The supernatant is subjected to reverse-phase chromatography using silica gel to adsorb hydrophobic components other than CyA and CyA to remove other components, and then subjected to ODS silica gel column for separation and measurement.

As described above, a method capable of measuring a component to be measured in whole blood has been developed, but it cannot be applied to automatic measurement because the processing steps such as centrifugation are complicated.

(Problems to be Solved by the Invention) The present invention is to solve the above-mentioned conventional drawbacks, and an object of the present invention is to accurately and easily measure a substance to be measured contained in plasma and blood cells. To provide a method. Another object of the present invention is to provide a method capable of automatically measuring a component to be measured in blood, for example, using whole blood as a sample in a short time.

(Means and Actions for Solving the Problems) The method for measuring blood components of the present invention is a method for measuring a substance to be measured in a sample containing blood or blood cell components by high performance liquid chromatography, and has an HLB value A solution containing a nonionic surfactant of 10 to 17 is mixed with the sample to destroy blood cell components in the sample, and the mixed sample solution is introduced into a pretreatment column to remove the substance to be measured. A step of adsorbing and passing a protein component in the sample solution without adsorbing, and a step of flowing an eluent through the pretreatment column to desorb a substance to be measured and guiding it to a separation column, and measuring it. The above object is achieved thereby.

The surfactant used in the method of the present invention is used as a hemolytic agent that destroys blood cells. Nonionic surfactants are used and their HLB values range from 10 to 17. The HLB value is a parameter indicating the relative proportion of hydrophilic groups and lipophilic groups that the molecule of the surfactant has. When the HLB value is large, the proportion of hydrophilic groups is large, and therefore the degree of hydrophilicity is high, and when the HLB value is small, the proportion of hydrophobic groups is large,
Therefore, the degree of lipophilicity increases. The hydrophilic group of the nonionic surfactant is a polyoxyethylene group or a hydroxyl group, and the hydrophobic group is an aliphatic or aromatic hydrocarbon group. Examples of the surfactant used in the present invention include polyoxyethylene alkyl ether, polyoxyethylene alkylphenyl ether, polyoxyethylene aralkyl phenyl ether (for example, polyoxyethylene tribenzylphenyl ether) having an HLB value in the above range. To be When the HLB value is less than 10, the hydrophilicity becomes low, and micelles are formed in an aqueous solution to become opaque, so the measurement cannot be performed accurately, and if these are supplied to the column, the column or filter will be clogged. The problem arises. Conversely, when the HLB value exceeds 17, the cell membrane of blood cells is not completely destroyed.

The above-mentioned surfactant is usually used as an aqueous solution, but an organic solvent miscible with water may be added in order to enhance the solubility of the surfactant. Such organic solvents include methyl alcohol, ethyl alcohol, acetone,
Such as acetonitrile. These organic solvents are used as long as proteins in blood do not coagulate and cause precipitation. Its concentration is usually less than 30%. The amount of the above surfactant used is 10 to 500 mg per 1 ml of blood or the amount of blood cell components contained in 1 ml of blood. For example, when a solution containing the above-mentioned surfactant in a proportion of 0.01 to 2% by weight is used, it is added in a proportion of 2 to 100 ml per 1 ml of blood.

As a method of destroying blood cell components, a method of dissolving lipids in blood cell membranes using an organic solvent; and a method of adding water to destroy the above cell membranes due to an osmotic pressure difference are known. It is not suitable because the membrane components and other protein components precipitate.

As the packing material packed in the pretreatment column used in the present invention, a packing material for reverse phase chromatography is used. Of these, synthetic polymer-based fillers having no dissociative group are preferable. Generally, synthetic polymer fillers with strong hydrophobicity are
It strongly adsorbs proteins, but if it is made hydrophilic, it will not adsorb proteins. If the degree of hydrophilicity is further increased, the substance to be measured such as CyA will not be adsorbed.
As the filler of the present invention, a synthetic polymer type filler having a hydrophilic group such as a hydroxyl group and a hydrophobic group such as an alkyl group in a predetermined ratio is used. Such a filler adsorbs the substance to be measured, which is a non-protein substance, under certain conditions, for example, in water or a buffer solution having a particular pH, but does not adsorb the protein, and under other conditions, for example, It has the property of eluting the substance to be measured adsorbed to a buffer solution having a pH different from the above. As such a filler, for example, particles that are a (co) polymer having an acrylic monomer as a (co) polymerization component are used. Such monomers include polyethylene glycol mono (or di)-(meth) acrylate, polypropylene glycol mono (or di) -acrylate, tetramethylolmethane mono (or di, tri) acrylate. It is obtained by suspension polymerization of these monomers and has a particle size of 5-30.
Fine particles of about μm are particularly suitable. Generally, among the packing materials for reversed phase chromatography, the packing materials such as ODS silica gel (silica gel having an octadecylsilyl group bonded) and CN silica gel (silica gel having a nitrile group bonded) have silanol groups and adsorb proteins. Therefore, it is not suitable for the method of the present invention. Although packing materials for ion exchange chromatography can be used under certain limited conditions, they are, in most cases, unsuitable for use in the method of the present invention.

As the packing material to be packed in the analytical column, a packing material having a higher adsorptivity of the substance to be measured than the packing material to be packed in the pretreatment column may be appropriately used depending on the types of the substance to be measured and the packing material of the pretreatment column. To be selected. For example, ODS silica gel filler is used.

The method of the present invention is embodied by, for example, the apparatus shown in FIG. This device consists of a hexagonal valve 4 with a connection A-F,
It has a pretreatment column 5 connected via the connecting ends B and E of the hexagonal valve and an analytical column 8 connected to the connecting end C of the hexagonal valve. The pretreatment column 5 is packed with the above-mentioned packing material for reverse phase chromatography, and the analytical column is
An appropriate filler (for example, ODS silica gel when measuring CyA) is filled depending on the type of the substance to be measured.

For example, when measuring the substance to be measured in whole blood,
The surfactant solution is added to the collected blood and shaken to destroy the blood cell component (hemolysis), and this is used as a sample. First, in the apparatus of FIG. 1, the ion-exchanged water or buffer solution in the water tank 1 is pumped by the sample injector 3
Then, it is sent to the pretreatment column 5 via the connection ends A and B, and then discharged from the system through the connection ends E and F. Next, the sample prepared by the above hemolysis is injected from the sample injector into the system. The injected sample is transferred together with ion-exchanged water or a buffer solution by the pump 2 and reaches the pretreatment column 5. Here, the substance to be measured (which is a non-protein substance) in the sample is adsorbed by the packing material of the column. On the other hand, the protein component in the sample passes through the column 5 without being adsorbed, and is discharged to the outside of the system through the connection ends E and F of the hexagonal valve 4.

The eluent tank 6 contains an eluent capable of melting the substance to be measured adsorbed on the pretreatment column 5. This eluent is sent by the pump 7 to the analytical column 8 via the connection ends D and C. Next, as shown in FIG. 2, when the hexagonal valve 4 is switched to connect the connection end D to E, the eluent in the eluent tank 6 is supplied to the pretreatment column 5 by the pump 7 via the connection ends D and E. It When the eluent passes through the column 5, the adsorbed substance to be measured is eluted, and the analytical column 8 passes through the connecting ends B and C together with the eluent.
Reach The substance to be measured is separated by the analysis column 8 and detected by an appropriate detector (not shown) provided after the analysis column 8. Since the analytical column 8 is packed with a packing material having a higher adsorptivity than the packing material used in the pretreatment column, the substance to be measured adsorbed in the pretreatment column is completely eluted by the eluent, and is highly accurate. Separated and measured.

In the example described above, the advancing direction of the ion-exchanged water by the pump 2 and the advancing direction of the eluent by the pump 7 in the pretreatment column 5 are opposite to each other, but the present invention is not limited to this. A connection method in which the traveling directions are the same may be adopted. After switching to the connection shown in FIG. 2, the substance to be measured is the connecting end C of the hexagonal valve 4.
The six-way valve 4 can be further switched to the connection shown in FIG. By switching in this way, the next sample is analyzed while the analysis column 8 is performing the analysis.
A pretreatment for the next analysis can be carried out through.

In the method of the present invention, a nonionic surfactant having a predetermined HLB value is used as a hemolytic agent. Therefore, it is possible to effectively destroy the blood cell component and release the measured component contained in the blood cell. The disrupted membrane components, other proteins, and lipid components are uniformly dispersed in the sample, and these do not precipitate. for that reason,
It does not require the process of precipitating and removing membrane components and protein components by centrifugation as in the past,
It becomes possible to analyze by using PLC. The blood cell component does not cause the column to be clogged. In the sample treated with the above-mentioned surfactant, the protein component is effectively removed by the pretreatment column using the synthetic polymer packing, so that the component to be measured can be accurately measured after being analyzed by the analytical column. .

(Example) Hereinafter, the present invention will be described with reference to Examples.

Example 1 A high-pressure hexagonal valve 4 was connected with a pretreatment column 5 and an analysis column 8 incorporated in liquid chromatography (manufactured by Shimadzu Corporation, model LC6A) as shown in FIG.
As the pretreatment column 5, a stainless steel column having an inner diameter of 4 mm and a length of 1.5 cm was used. 5 to 20μ particle size as a filler
m acrylic polymer porous particles were used. These acrylic polymer porous particles consist of 40 g of tetraethylene glycol dimethacrylate and 10 ml of tetramethylolmethane triacrylate in 400 ml of a 4% by weight polyvinyl alcohol aqueous solution.
g, 40 g of toluene and 0.5 g of benzoyl peroxide were added, and this was reacted at 80 ° C. for 10 hours while stirring at 400 rpm, and after the reaction, it was obtained by washing with hot water and acetone.

800 n of cyclosporin A (manufactured by Sand Co.) for normal male whole blood
The mixture was added at a rate of g / ml, and 2 ml of the hemolytic reagent was added to 0.5 ml of the mixture and shaken. As a hemolytic reagent, a surfactant (Atlas
Manufactured by Atlas G-2133; HLB value 13.1) in a 2W / V% aqueous solution containing 200n of cyclosporin D (Sand) as an internal standard substance.
The one added at a rate of g / ml was used. The thus obtained 500 μl of hemolyzed blood was used as a sample. The ion exchange water in the water tank 1 is pumped by the pump 2 at a flow rate of 1.0 ml / min.
The sample was injected from the sample injector 3 while flowing through the pretreatment column 5 via B. After 10 minutes, the hexagonal valve 4 was switched and connected as shown in FIG. By the pump 7, the eluent containing 70:30 acetonitrile in water from the eluent tank 6 at a flow rate of 1.5 ml / min via the hexagonal valve connection terminals D, E, the pretreatment column 5, and the hexagonal valve connection terminals B, C. It was passed through an analytical column 8 for liquid chromatography. CyA melted by the flow of the eluent was measured. The analytical column used was a silica-based ODS column (Sekisui Chemical Co., Ltd.).
Made, Medipolar ODS; Column size inner diameter 4.6mm; Length 25
cm), and the detection was performed by absorbance at a wavelength of 210 nm. The obtained chromatogram is shown in FIG. In FIG. 3 (A), a indicates a CyA peak and b indicates a CyD peak.

Separately, when the measurement was carried out by the same method without adding cyclosporin A, the chromatogram shown in FIG. 3 (B) was obtained. Comparing FIGS. 3 (A) and (B),
It can be seen that by the method of the present invention, cyclosporin A and D are separated and measured as individual peaks. From the separately prepared calibration curve, the blood concentration of CyA was calculated to be 760 ng / ml, and the recovery rate was 95%, which was excellent.

Example 2 Tween-20 (manufactured by Atlas; H, which is a surfactant containing polyethylene glycol sorbitan monopalmitate as a main component)
LB value 15.6) at a ratio of 2 W / V% water-ethanol mixture (9
0:10 v / v) to obtain a hemolytic reagent. When operated in the same manner as in Example 1 except that this was used, the peaks of CyA and D were confirmed as in Example 1. The recovery rate was 93% in all cases.

Example 3-1 Altas G-3705 (manufactured by Atlas; HLB value 10.
2) was added to 100 ml of a 10% aqueous ethanol solution, and the mixture was stirred with a stirrer for 5 minutes at room temperature, and its solubility was visually observed. Next, this solution was injected from the injector of the apparatus of Example 1, and the column pressure and whether or not clogging occurred were observed. Separately, 0.5 ml of anticoagulated whole blood was added to 2 ml of this surfactant, and the mixture was shaken and visually and microscopically observed to confirm whether hemolysis was completely performed. The results are shown in the table below. The results of Examples 3-2 to 3-3 and Comparative Examples 1-1 and 1-2 are also shown in the table below.

Example 3-2 As a surfactant, Altas G-2133 (manufactured by Atlas; HLB value 13.
Same as Example 2-1 except that 1) was used.

Example 3-3 The same as Example 2-1 except that Brij 35 (manufactured by Atlas; HLB value 16.9) was used as the surfactant.

Comparative Example 1-1 The same as Example 3-1 except that Brij 30 (manufactured by Atlas; HLB value 9.5) was used.

Comparative Example 1-2 The same as Example 2-1 except that Altas G-2159 (manufactured by Atlas; HLB value 18.8) was used.

Example 4 After collecting whole blood of a healthy person, 1 drop of an anticoagulant (manufactured by Nippon Shoji Co., Ltd .; Anglot) was immediately added to 1 ml of the blood. Cyclosporin A was added to this at a ratio of 800 ng / ml to prepare CyA-containing whole blood. This was heated at 37 ° C for 3 hours and immediately separated into blood cells (sediment) and plasma (supernatant) by centrifugation. The volume ratio of blood cells to plasma was 55:45.

The obtained plasma was measured by the method described in Medical History Vol. 129, No. 9, p. 623, and the concentration of cyclosporin A in plasma was 640 ng / ml. Next, 0.25 ml of the above blood cells
On the other hand, when 2.25 ml of the hemolytic reagent similar to that used in Example 1 was added and cyclosporin A was measured by the same method and apparatus as in Example 1, the concentration was 840 ng / ml. From these results, it was confirmed that cyclosporin A was initially dissolved in plasma but gradually dissolved in blood cells by incubation at 37 ° C. for 3 hours.

〔The invention's effect〕

According to the method of the present invention, the components contained in plasma and blood cells can be easily measured with high accuracy. The method of the present invention is particularly suitable for the measurement of blood components by automated high performance liquid chromatography, and can be used in a wide range of applications such as monitoring the blood concentration of CyA, which is difficult to predict the blood concentration easily from the dose. It is possible.

[Brief description of drawings]

1 and 2 are connection diagrams showing an example of a measuring apparatus used for carrying out the method of the present invention, and FIGS. 3 (A) and 3 (B).
Is a chromatogram obtained when cyclosporin A and D in blood was measured by the method of the present invention. 1 …… water tank, 2,7 …… pump, 3 …… sample injector, 4
…… Hexagon valve, 5 …… Pretreatment column, 6 …… Eluent tank, 8…
… Analysis column.

Claims (2)

[Claims] 1. A method for measuring a substance to be measured in a sample containing blood or blood cell components by high performance liquid chromatography, which comprises a solution containing a nonionic surfactant having an HLB value of 10 to 17, and the sample. To destroy the blood cell components in the sample, guide the mixed sample solution to a pretreatment column to adsorb the substance to be measured, and pass without adsorbing the protein component in the sample solution. And a step of causing an eluent to flow through the pretreatment column to desorb the substance to be measured and introducing the substance to a separation column, and measuring the same. 2. The measuring method according to claim 1, wherein the substance to be measured is a cyclosporin derivative.