JPH0643146A - Method for separating and eliminating protein inside living organism specimen for analysis - Google Patents

Abstract

PURPOSE:To obtain a method for simply separating and eliminating protein with a disposable type tool without using a complex device by separating and eliminating protein from a living organism specimen for analysis containing protein and sampling a fraction containing a component to be analyzed efficiently for analyzing a small amount. CONSTITUTION:In the method, a living organism specimen for analysis where protein and a component to be analyzed coexist is loaded on a proper carrier and then the living organism specimen is moved inside the carrier using an elutant. The component to be analyzed with a small retention capacity is exuded before protein with a large retention capacity existing inside the living organism specimen to be analyzed is exuded, thus obtaining an exuded liquid where protein is eliminated and which contains the component to be analyzed.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION The present invention relates to a protein which interferes with quantification of a target component in analysis of a biological sample, for example, high performance liquid chromatography or causes deterioration of a packing material for a column carrier of chromatography. It relates to a method of separating and removing in advance.

[0002]

2. Description of the Related Art As a method for removing proteins in a biological sample, a protein precipitation separation method using an acid or a solvent, an extraction separation method for salt to be analyzed and a salting out separation method are known, but the operation is complicated and It is not suitable as a method for automating the pretreatment of the sample for use. As a method of deproteinizing using a membrane, for example, a polysaccharide type ultrafiltration membrane is used to deproteinize blood. However, this method requires centrifugal separation or membrane permeation under high pressure, and treatment at normal pressure is difficult.

On the other hand, as a method of using a carrier, a method of using a silica gel type carrier such as "Sep-Pak" manufactured by Waters Co. is known. This method is described in W. R
According to Oth's proposal, the whole blood transferred to the pre-column together with the eluent is retained in the carrier, and then the eluent is sent in the direction opposite to the transfer direction so that only the analyte component in the whole blood is transferred. This is a pre-column switch method in which the carrier is eluted with the eluent. T. In the precolumn bendic method proposed by Arvidsson, after the protein is transferred into the precolumn together with the eluent, the analyte and the protein are separated in this column, and the protein passes through the carrier first. This is a method in which an eluate containing only the analyte to be eluted later by switching can be analyzed without interference of proteins. or,
H. In Yoshida, the surface of porous silica is coated with bovine serum albumin, so that only the test substance is adsorbed, and the protein is not adsorbed but is eluted first to perform deproteinization. Kitano uses formaldehyde-hydroxy resin and injects albumin solution with phosphate buffer,
A protein is covalently adsorbed to the resin to adsorb and remove the protein (J. Appl. Biochem., 4,
1982).

The methods using these carriers are all
It requires complicated operations and complicated equipment, and is not suitable as a means for automating deproteinization as a pretreatment of a sample for analysis. Moreover, in all of these methods, the biological sample for analysis is added to the eluent or diluted before the treatment. Therefore, even if the analyte in the biological sample for analysis is once adsorbed on the column, the concentration of the analyte in the eluate becomes relatively low when re-eluting, which also affects the reduction of the recovery rate. However, it was not always suitable for microanalysis.

[0005]

Under such a technical background, in order to obtain a sample solution containing only a component which does not interfere with the analysis of the analyte by separating and removing the protein from the biological sample for analysis, It does not require complicated equipment, does not require complicated operation, does not require separation under high pressure, can be incorporated into a general-purpose analytical sample pretreatment system, and can also automate the pretreatment of analytical samples. It is necessary to develop a carrier that can be filled in disposable type cartridges.

[0006]

Means for Solving the Problems As a result of intensive studies in view of the present situation, the present inventor found that various carriers such as hydroxyapatite and polyamino acid described in JP-A-1-201015 are effective, and based on such findings. Based on this, the present invention has been completed.

That is, according to the present invention, a biological sample for analysis in which a protein and a component to be analyzed coexist is directly loaded on an appropriate carrier, and then the biological sample is moved through the carrier by using an eluent to obtain the biological sample for analysis. A protein of biological shochu for analysis characterized in that an analyte having a small retention volume is eluted before a protein having a large retention volume existing in a sample is eluted to obtain an eluate containing the analyte and deproteinized. It is a method of separating and removing.

Since the present invention has made it possible to analyze a target component with a small amount of a sample for analysis in accordance with the progress of the analytical technique, it becomes possible to more easily operate.

A feature of the present invention is that a biological sample for analysis is directly loaded onto a carrier without being diluted, and the carrier is soaked in the carrier, and then an eluent is added to the carrier to utilize the difference in retention volume. This means that the analyte can be eluted faster than the protein, and the analyte contained in this can be analyzed using this eluate as a sample.

The present invention will be described in detail below.

Suitable carriers for use in the present invention are those having a smaller retention capacity for the analyte than for the protein. Specific examples thereof include hydroxyapatite and polyamino acids. These carriers are usually used by being filled in a cartridge, and the cartridge is replaced every time a biological sample for analysis is processed once according to the method of the present invention. The filling amount varies depending on the type of carrier, but is usually 0.1 to 1 g.

In this case, the amount of the biological sample for analysis loaded on the carrier cannot be generally determined depending on the contents of the component to be analyzed and the protein, but if it is too small, it may not reach the lower limit of quantification. If the amount is too large compared to the amount, it cannot be temporarily held in the carrier. In a usual analysis of a biological sample, the amount of the biological sample for analysis is 50 to 0.2 g for the carrier amount of 0.2 g.
500 μl is suitable.

The type and amount of the carrier used in the present invention need not completely adsorb the protein in the analytical sample, and the analyte and the protein coexisting in the analytical sample can be chromatographically analyzed. It would be good if they could be separated from each other. This is because the retention capacity of the protein is large, and the fact that the elution time of the protein is delayed compared to the analyte having a small retention capacity is used.

As the eluent used in the present invention, an ordinary eluent used in liquid chromatography can be used. Such eluents include water, saline, aqueous acetonitrile, phosphate buffer and the like.
However, it is necessary to avoid the case where the retention capacity of the protein becomes smaller than the retention capacity of the component to be analyzed in combination with the carrier.

When carrying out a quantitative analysis using the method of the present invention, the type and amount of carrier, the amount of biological sample, the elution rate, the relationship between the amount of eluate and the amount of protein contained in the eluate, etc. Need to be checked in advance. The amount of the eluate to be obtained by the difference between the retention volumes of the analyte and the protein is the same as the amount required for quantitative analysis or qualitative analysis by liquid chromatography, and 100 to 300 μl is usually sufficient. is there.

According to the present invention, since the biological sample for analysis is directly loaded on the carrier, the component to be analyzed temporarily held on the carrier is difficult to diffuse in the single substance, and a high concentration is locally applied to a part of the carrier. Existing. Therefore, when the analyte to be analyzed in this state is eluted with the eluent, the analyte to be analyzed diffuses more than when the biological sample for analysis is diluted with another solution or directly added to the eluent such as chromatography. Therefore, when the analyte component in this state is eluted with the eluent, it can be eluted efficiently, and the analyte component present in the eluate is diluted with the biological sample for analysis. Since the concentration is higher than that in the case of performing the above, the lower limit of quantification can be lowered when quantitatively analyzing the component to be analyzed using this eluate. Moreover, the recovery rate of the component to be analyzed is good, and it becomes possible to perform trace analysis.

There is no particular limitation in subjecting the eluate obtained by the method of the present invention to the deproteinized product and containing the analyte to be subjected to quantitative analysis or qualitative analysis. For example, this eluate can be dispensed into a small test tube and then injected into an analytical instrument by a pipetting operation. Further, this operation can be fully automated by using an automatic pretreatment device, for example, "LC-ROBO COMPO" (manufactured by Ajinomoto Co., Inc.) (see JP-A-1-250071). Further, the carrier is filled in a cartridge, which is set in advance in an automatic pretreatment device, and a biological sample for analysis is injected into the cartridge,
It is also possible to automatically perform a series of operations of injecting an eluent, eluting the component to be analyzed together with the eluent, and when the predetermined amount of the eluate is reached, automatically removing the cartridge from the device. (See Actual Kaihei 1-95208).

[0018]

EXAMPLES The present invention will be further described below with reference to examples.

Example 1 (Separation and removal of proteins in human plasma by hydroxyapatite) 100 μl of human plasma (containing 2.006 mg of γ-globulin and 6.694 mg of albumin) 0.4 g of hydroxyapatite (manufactured by Koken Co., Ltd.) The cartridge (filled portion 9 mmφ × 15 mmL) filled with was injected and loaded.

The various eluents shown in Table 1 were passed through the respective cartridges, and the eluate was collected for each fraction (each 300 μl of the fraction), and γ-globulin (γ-GLB), transferrin and albumin contained therein ( ALB) was quantified using a high performance liquid chromatograph (“Gaskuro Kogyo Co., Ltd.“ 576 type high performance liquid chromatograph ”and the same company“ UV detector 502 type ”). The results are also shown in Table 1. From this result, it is possible to determine the optimal amount of eluent that can be deproteinized depending on the type of eluent to be analyzed.

[0021]

[Table 1]

Example 2 (Removal of Protein in Human Plasma by Hydroxyapatite) The same procedure as in Example 1 was repeated except that 300 μl of human plasma was used.
The relationship between the type of eluent and the deproteinization effect was examined. The results are shown in Table 2.

[0023]

[Table 2]

Example 3 (Analysis of caffeine in human plasma) A cartridge (filled portion 9 mm) in which 50 μl of human plasma was filled with 0.4 g of hydroxyapatite (manufactured by Koken Co., Ltd.)
Injection load was applied to the upper part of (φ × 15 mmL). 3 ml of a 10% aqueous acetonitrile solution was passed through the upper part of the cartridge to obtain 400 μl of a deproteinized eluate initial flow.

Using 20 μl of this initial flow, caffeine was analyzed by a high performance liquid chromatograph (“Hitachi 655A-12 type high performance liquid chromatograph” and “Hitachi L-4000 type UV detector”). Figure 1
Shown in. In the figure, 1 indicates the peak of caffeine.

Example 4 (Analysis of theophylline in human plasma) 7- was used as an internal standard substance for quantifying theophylline.
A 2.5% aqueous solution of (2-hydroxyethyl) theophylline was prepared. 100 μl of this solution was analyzed by the high performance liquid chromatography of Example 3 to determine the retention volume in advance.

Blood was taken from an asthma patient after taking theophylline, and 50 μl of this plasma and 2.5 of internal standard substance were collected.
% Aqueous solution (100 μl) was mixed and loaded onto the upper part of a cartridge (filling portion 9 mmφ × 15 mmL) filled with 0.4 g of hydroxyapatite (manufactured by Koken Co., Ltd.). 3 ml of a 10% acetonitrile aqueous solution was passed through the top of this cartridge. Deproteinized initial liquid 400
μl was obtained. 20 μl of this initial flow was used for the same high performance liquid chromatography as in Example 3. The obtained chromatogram is shown in FIG. In the figure, 1 shows the peak of theophylline and 2 shows the peak of the internal standard substance.

When theophylline was quantified by a calibration curve method based on the height of its peak, the amount of theophylline in the human plasma was 25 μg / ml.

Example 5 (Analysis of ascorbic acid in human plasma) A cartridge (20 mm of human plasma) filled with 0.4 g of hydroxyapatite (manufactured by Koken Co., Ltd.) (filled portion 9 mm)
(φ × 15 mmL). Next, 0.25%
Cysteine-containing 10 mM phosphate buffer (pH 6.
8) 3 ml was passed through to obtain 800 μl of the initial flow solution. 20 μl of this solution was applied to high performance liquid chromatography (“Hitachi 65
5A-12 type high performance liquid chromatograph "and" electroscience detector Σ875 type "(manufactured by Irika Co., Ltd.) were used for analysis.

The obtained chromatogram is shown in FIG. In the figure, 1 indicates the peak of ascorbic acid. The analytical value obtained by the standard addition method of ascorbic acid was 7.79 μg /
It's ml.

Example 6 (Analysis of diazepam in human plasma) Diazepam is a minor tranquilizer (anxiolytic drug)
It is a drug that is applied to anxiety, nervousness, depression in neurosis, muscle spasms associated with brain and kidney diseases, and is listed in the Japanese Pharmacopoeia and the American Pharmacopoeia. In treatment,
It is extremely important to establish a rapid and simple analytical method for the blood concentration of diazepam.

The analysis of diazepam is carried out in the pharmacopoeia by the potentiometric titration method (perchloric acid). The measurement of diazepam in blood is mainly performed by high performance liquid chromatography. As a pretreatment method, Bond Elut. Sep. Pak C ₁₈ or column switch method, solvent extraction, centrifugation, etc. are used,
It is not always easy to say. Since diazepam has a relatively low blood concentration (0.1 to 1.0 μg / ml), development of an excellent analytical method is desired.

According to the present invention, plasma can be deproteinized simply by attaching it to a deproteinizing cartridge "PCPure cartridge" (manufactured by Moritex Co., Ltd.) filled with hydroxyapatite, and the eluate can be subjected to operations such as solvent extraction and centrifugation. The sample for analysis of diazepam can be prepared without adding. This preparation method is simpler and more rapid than conventional methods, and is suitable for routine analysis and automated analysis.

In this example, as will be described below, mainly diazepam as a standard product and 4, as an internal standard substance, were used.
4'-Difluorobenzophenone was added to plasma, the elution behavior of diazepam and internal standard substances by various eluents was investigated, and the conventional method and this method were compared.

(A) Sample preparation method and diazepam analysis method (outline) 10 μl of diazepam standard solution (0.1 μl) was added to 100 μl of plasma.
g) and 10 .mu.l (1 .mu.g) of 4,4'-difluorobenzophenone standard solution as an internal standard substance, shaken well and left to stand at room temperature for 30 minutes.
Inject into the cartridge. Next, 2 to 3 ml of a 50% acetonitrile aqueous solution as an eluent was passed through to prepare an initial eluent 6
Use 00 μl as a sample for analysis. 20 μl of this eluate is injected into a high performance liquid chromatograph, and diazepam is analyzed from the ratio of peak heights of diazepam and internal standard substance.

As the diazepam standard solution, 10 mg of diazepam was accurately weighed and adjusted to 100 ml with a 10% aqueous solution of acetonitrile.
Make up to 10 ml with aqueous acetonitrile (10 μg
/ Ml) and the 4,4-difluorobenzophenone standard solution was 4,4'-difluorobenzophenone 10
Accurately weigh mg to 10 with 10% aqueous acetonitrile.
The volume is adjusted to 0 ml (100 μg / ml).

(B) Determination of eluent In consideration of the results in (i) and (ii) below and the conditions of high performance liquid chromatography, a 50% acetonitrile aqueous solution was selected as an eluent in (a).

(I) Recovery rate of standard diazepam and internal standard by various eluents Standard diazepam (0.1 μg) and internal standard 4,4′-difluorobenzophenone (1 μg) were put in a “PCPure cartridge”. After injection, each eluent 2
After passing 3 ml, the behavior regarding the recovery rate of each fraction (300 μl) was examined. The results are shown in Table 3.

[0039]

[Table 3]

From the results shown in Table 3, diazepam and 4,4'-difluorobenzophenone were almost always obtained in 600 μl of the initial flow solution containing the first and second fractions, whichever eluent was used. It was found to elute. Diazepam also eluted in the third fraction when saline or 10 mM phosphate buffer was used as the eluent. In acetonitrile, when 50% and 60% aqueous solutions were used, it was possible to recover the whole amount of siazepam and the internal standard substance in the first fraction.

(Ii) Confirmation of deproteinization in plasma When diazepam in plasma is analyzed using "PCPure cartridge", it is necessary that the eluate contains no diazepam and no protein. Is. In this example, the conditions for deproteinization with an eluent were investigated in the same manner as in Examples 1 and 2 above, but with the range expanded.

The results were as follows.

(A) Saline solution (0.9%, pH 4.5
-8.0): 50-300 μl of plasma was injected into the cartridge, then eluted with physiological saline, and the amount of protein in each fraction (300 μl) of the eluate was examined. No protein was observed in any of the ~ 5 fractions. Furthermore, when the plasma volume was increased and 300 μl was injected into the cartridge, no protein was observed in the first fraction,
Although transferrin was slightly detected in the ~ 5 fractions, the deproteinization rate was as high as about 99%.

(B) In case of 10 mM phosphate buffer: After injecting plasma into 50 or 100 μl cartridge, pH
The buffer solution of 6.8 or 8.6 was passed through, and the protein in each fraction of the eluate was confirmed in the same manner as above. Although there were some fractions in which albumin, globumin and transferrin were detected, the deproteinization rate was as high as about 99%. There was almost no difference due to the pH of the eluent.

(C) Aqueous acetonitrile solution: 100 μl of plasma was injected into each cartridge, and then aqueous acetonitrile solutions of various concentrations were passed as eluents, and the amount of protein in each fraction of the eluate was examined. 5%
When using an aqueous solution, some albumin, globumin and transferrin were detected, but
The deproteinization rate was a high value of about 99%. On the other hand, 10
At a concentration of -100%, the deproteinization rate was 100%. Next, a plasma amount of 300 μl was examined with an eluent having the same concentration as the mobile phase of diazepam at 50%. As a result, it was slightly different from the case of the above-mentioned physiological saline, and the protein was not contained in any of the first to fifth fractions. I was not able to admit.

(D) In the case of aqueous alcohol solution: plasma 10
After injecting 0 μl into each cartridge, water was passed through as an eluent with 10% methanol and ethanol, and the amount of protein in each fraction of the obtained eluate was examined. No protein was detected in the first fraction in water and in the first and second fractions in methanol. In addition, albumin and transferrin were slightly detected, and the deproteinization rate was a high value of about 99%. In ethanol, no protein was observed in any of the first to fifth fractions.

(E) In the case of cysteine-containing 10 mM phosphate buffer (pH 6.8): In the chromatogram, the presence or absence of albumin could not be clearly observed due to the interference of cysteine. When the plasma volume is 100 μl, albumin is considered to be eluted since globulin was detected. In the case of 20 μl, albumin was not eluted since globulin was not detected.

(F) 10 mM phosphate buffer (pH 6.
8) + physiological saline (1: 1): 100 μl of plasma was injected into the cartridge, and when this mixture was used as an eluent, 10 mM phosphate buffer (pH 6.8) was used more than when it was used alone. The deproteinization rate was good. Although some albumin was detected in the third fraction, the deproteinization rate was about 9
It was 9%.

Incidentally, the operating conditions of high performance liquid chromatography used for confirming deproteinization were as shown in Table 4 below.

[0050]

[Table 4]

(C) Analysis of diazepam in plasma and comparison with conventional method A calibration curve was obtained from the ratio of the peak heights of diazepam and the internal standard substance. The calibration curve showed linearity in the range of 0 to 6.6 ng (see FIG. 4).

When analyzing diazepam in plasma using the "PCPure cartridge", it is important to make a comparison with conventional deproteinization methods using acetonitrile, methanol and the like. Further, in the present Example, since diazepam and an internal standard substance were added to plasma for the examination, it was incubated at 37 ° C. for 30 minutes to examine the binding property between plasma protein and diazepam. We also conducted a comparative examination.

That is, 0.1 μg of the standard product diazepam and 1 μg of the internal standard substance were added to 100 μl of plasma, and this was left at room temperature and 37 ° C. for 30 minutes to prepare a sample.

Diazepam was analyzed from the ratio of the peak heights of diazepam and the internal standard, and as a result of comparison, there was almost no difference between the "PCP cartridge" and the conventional method as shown in Table 5 below. I knew that.

[0055]

[Table 5]

When 50 μl of plasma was subjected to deproteinization by the “PC Pure cartridge” (eluate 40
The recovery rate of 0 μl) was about 84%.

For reference, FIGS. 5 and 6 show examples of chromatograms of diazepam in plasma thus analyzed. That is, FIG. 5 shows a chromatogram of diazepam in plasma (blank), and FIG. 6 shows a chromatogram of diazepam in plasma (actual solution). In each figure, 1 and 2 are diazepam and 4, respectively.
4'-difluorobenzophenone (internal standard substance) is shown.

Incidentally, the operating conditions of high performance liquid chromatography used for the analysis of diazepam are as follows:
It was as shown in the table.

[0059]

[Table 6]

(D) Overall Evaluation From the above results, it was found that diazepam in plasma can be quantified by using the method of the present invention with almost no difference from the conventional method. The recovery rate was about 93%. Also,
Relative Standard Deviation, RS
D.) was 3.2 (n = 5), which is slightly worse than 1.2 (the recovery rate is about 100%) of the drug with high blood concentration, theophylline, but is good as a drug with low blood concentration. Conceivable. For theophylline, see Example 4 above.

Example 7 (Analysis of amino acids in human plasma) In research and development of so-called amino acid infusion, it is important to measure amino acids in plasma pharmacologically,
Establishing a rapid and simple method for analyzing amino acids in human plasma is extremely important.

According to the present invention, as in the previous example, plasma can be deproteinized simply by attaching it to the "PCPure cartridge", and the eluate can be treated with amino acids without being subjected to operations such as solvent extraction and centrifugation. An analytical sample can be prepared. It was found that this method of preparation allows deproteinization to be simpler and faster than conventional methods, and that tryptophan, which has a strong binding property to proteins, can be quantitatively analyzed. Suitable for routine and automated analysis.

In this example, as described below, first, in order to analyze amino acids in plasma more accurately using a “PCPure cartridge”, examination of internal standard substances and eluents, and eluent standards The recovery rates of product amino acids and internal standard substances were examined. Next, amino acids in plasma were analyzed, and further compared with the analysis results when the deproteinization was performed by a conventionally used method such as sulfosalicylic acid.

(A) Sample Preparation Method and Amino Acid Analysis Method 150 μl of plasma and 10 μl of internal standard substance solution were added to “PCPu
After injecting into the "re cartridge", 2-3 ml of physiological saline is passed through. Dissolve 1,000 μl of the initial eluate with 1N hydrochloric acid-20
Add μl to adjust the pH to 2 and use it as a sample for amino acid analysis. The amino acid analysis of this sample is performed using a conventional amino acid analyzer (ninhydrin method). The operating conditions are as shown in Table 7 below.

[0065]

[Table 7]

As the internal standard substance solution, 45 mg of S-carboxymethyl-L-cysteine (SCM), 30 mg of D-phenylglycine (PG) and 45 mg of S-aminoethyl-L-cysteine (SAE) were accurately prepared. Weigh them, combine them, and add water to adjust the volume to 50 ml.

(B) Examination of internal standard substances In order to accurately analyze amino acids in plasma by deproteinizing using "PCPure cartridge", substances such as various amino acids of more than 10 kinds are suitable as internal standard substances. I inspected if there was. Since the internal standard substance does not exist in plasma, it needs to be separated from the amino acid on the chromatogram.

Of the substances tested, the three amino acids SCM, PG and SAE were found to be suitable for use as internal standards in terms of retention time.

(C) Examination of Eluent In order to examine the eluent, the standard amino acid solution and the internal standard substance solution were each put in a “PC Pure cartridge” at 150 μm.
After injecting 1 and 10 μl, (a) physiological saline, (b)
10 mM phosphate buffer (pH 6.8), (c) physiological saline + 10 mM phosphate buffer (1: 1, pH 6.8),
The examination was carried out using five kinds of eluents: (d) 10% ethanol aqueous solution and (e) 10% acetonitrile aqueous solution. In the standard amino acid solution, first, L-asparagine (Asn), L-glutamine (Gln), and L-tryptophan (Trp) were accurately weighed at 40 mg each, and these were combined and quantified to 100 ml by adding water. (4
0 mg / 100 ml), then accurately measure 2 ml each of Beckman standard reagents (STD, AN +, B +) and 2 ml of the above solution, and add 0.2 N to the combined product.
4 ml of hydrochloric acid is added, and finally, 2 ml of this product and 2 ml of a 10-fold diluted solution of the above-mentioned internal standard substance solution are accurately weighed and 0.2 N hydrochloric acid is added to adjust the volume to 20 ml.

In addition to the rapid elution of amino acids, it is important that each eluate contains no protein in the eluate. The main results are described below. (1) Asn, Gln, Pro, Cit, G
ABA, Met, PG, Phe, Trp, etc. were easily eluted. On the other hand, Asp, Ser, (Cys) _2, etc. were difficult to elute. (2) When 10% methanol and 10% acetonitrile were used, SAE was difficult to elute among the internal standard substances. (3) Using physiological saline and 10 mM phosphate buffer (pH 6.8),
Elution of the three internal standards and amino acids was better than 10% methanol and 10% acetonitrile. The first three fractions of the eluate (each fraction 300
.mu.l), that is, most of the amino acids could be recovered in the first to third fractions of the initial flow solution (total initial flow solution 900 .mu.l).
(4) Saline + 10 mM phosphate buffer (pH 6.
8) Better results were obtained when each was used alone than (1: 1). (5) When physiological saline and 10% acetonitrile were used, no protein was eluted up to the fifth fraction, that is, up to 1500 μl of the initial flow. Meanwhile, 10 mM phosphate buffer (pH 6.8) and 10%
In methanol, protein was eluted from the third fraction.

In consideration of the above elution results of amino acids and proteins, physiological saline was used as the eluent in this example. When physiological saline was used as the eluent, the recovery rate of the internal standard substance when the first-flow three fractions of the “PCPure cartridge” were combined (total 900 μl), the results of three trials were 95.8 for SMC, respectively. , 9
7.2 and 120%, 97.2, 9 for PG
5.3 and 96.4%, and 9 for SAE
The results were good at 7.3, 97.3 and 103%. In addition, when physiological saline was used, even if 200 μl of plasma was injected into the “PCPure cartridge”, no protein was eluted until the 5th fraction of the first flow of the eluate.

(D) Preparation of sample and other analytical conditions From the result of the examination using the standard amino acid in the previous item (c), the initial eluate 9 from the "PC Pure cartridge" was obtained.
It was found that when 90 μl was used as the sample for analysis, a recovery rate of amino acids of about 90% was obtained, but in order to improve the elution, 1,000 μl of the initial eluate was used as the sample for analysis. The plasma injection volume into the “PCPure cartridge” was considered to be 50, 100, 150 and 200 μl, but it was set to 150 μl considering the amino acid concentration in the eluate and deproteinization. For other items, see (a) above.

Incidentally, in the "PCPure cartridge", the filler is hydroxyapatite (Ca ₁₀ (PO ₄ )).
_{Since it is 6} (OH) ₂ ), it may dissolve in the eluate at the time of elution, which may affect the amino acid analyzer. Therefore, when the amount of calcium in the sample solution of the method of the present invention and the method of the present invention, which is a conventional deproteinization method, was investigated to make sure that the calcium contained in the sample solution of the method of the present invention was It was concluded that it was almost the same as the blank, lower than the sulfosalicylic acid method, and did not adversely affect the analyzer. In addition, regarding the sample preparation, in the case of the “PCPure cartridge” method, 150 μl of plasma and internal standard solution 1
After injecting 0 μl into the cartridge, physiological saline was passed through, and 1,000 μl of the initial flow solution was prepared by adding 20 μl of 1N hydrochloric acid. In the case of the sulfosalicylic acid method, 150 μl of plasma was prepared.
And 1 ml of 3.75% sulfosalicylic acid was added to 10 μl of the internal standard solution, and this was then centrifuged (10,0%).
00 rpm), the upper layer liquid was used as a sample, and calcium was quantified by an atomic absorption method for each sample.

When the amino acids in plasma were measured by the above-mentioned analysis method, there was no significant difference in the measured values due to the difference in the internal standard substance. In general, choose an internal standard that is close to the one to be analyzed, but among the three, SCM and PG
Is the standard deviation value (R.
S. D. ) Showed a good value. The reason for this is not clear, but it is considered that it can be improved by increasing the SAE concentration a little and increasing the peak height. Asp. S. D. It is not good to use any of these, but it is thought to be because the content is low. R.S. with SCM and PG. S. D. There was almost no difference in PG, but PG showed a slightly better value.
PG eluted almost at the center of the chromatogram, and R.
S. D. Therefore, it is considered to be suitable as an internal standard substance for simultaneous analysis of all amino acids. Therefore, in this example, PG was used to compare the recovery rate by the standard addition method and the conventionally used deproteinization method (see (f) below).

The recoveries by the standard addition method were as follows. That is, incubate to examine the binding property between amino acid and protein (37 ° C for 30 minutes)
The recovery rate (%) by the standard addition method was measured for those that did and did not. The average recovery rate by the method of the present invention was 93.5% without incubation and 94.3% with incubation, showing no difference between the two. In particular, Trp is an amino acid that strongly binds to proteins, but since the recovery rate by the standard addition method (with incubation) according to the method of the present invention is 97.9%,
The method of the present invention is sufficiently applicable to amino acids having protein binding. Therefore, it is expected to be applied to pharmaceuticals that strongly bind to proteins.

(E) Results of Measuring Amino Acids in Plasma The results of measuring amino acids in plasma three times using three kinds of internal standard substances are shown in Table 8 below.

[0077]

[Table 8]

(F) Comparison between conventional method of deproteinization and method of the present invention Deproteinization method using "PCPure cartridge" of the present invention and conventional deproteinization method (method of deproteinizing using sulfosalicylic acid or ethanol) The results of amino acid analysis in plasma were compared and examined.

Although the conventional deproteinization method does not use an internal standard substance, in this example, a sample was prepared by adding an internal standard substance for comparison with the method of the present invention. . The conventional deproteinization method involves adding a sample to plasma, centrifuging and filtering to prepare a sample. On the other hand, in the method of the present invention, “PCPu
The sample can be prepared simply by injecting plasma into the "re cartridge" and then passing a physiological saline solution, and the operation is simple and quick.

From the results obtained by both deproteinization methods, there is almost no difference in the amino acid analysis results between the two. S. D.
Was also found to be extremely good, except for Asp.
It was also found that the use of PG as the internal standard substance did not result in any difference in the amino acid analysis results even if the pretreatment method was different. It can be said that PG is suitable as an internal standard substance for the amino acid analyzer method (ninhydrin method).

[0081]

EFFECTS OF THE INVENTION By utilizing the present invention, it is possible to efficiently analyze the components to be analyzed contained in the biological sample, and the quantitative analysis thereof can be easily performed. Further, when the biological sample for analysis is processed according to the embodiment of the present invention in which the carrier is filled in the cartridge and used, the pretreatment operation can be automated without complicating the apparatus.

[Brief description of drawings]

FIG. 1 is a liquid chromatogram of caffeine in human plasma obtained in Example 3.

FIG. 2 is a liquid chromatogram of theophylline in human plasma obtained in Example 4.

FIG. 3 is a liquid chromatogram of ascorbic acid in human plasma obtained in Example 5.

FIG. 4 is a calibration curve for diazepam in Example 6 (c).

FIG. 5 is an example of a chromatogram of diazepam (blank) in Example 6 (c).

FIG. 6 is an example of a chromatogram of diazepam (actual liquid) in Example 6 (c).

Claims (1)

[Claims] 1. A biological sample for analysis in which a protein and a component to be analyzed coexist is loaded on an appropriate carrier as it is, and then the biological sample is moved in the carrier by using an eluent to exist in the biological sample for analysis. Separation and removal of protein in biological sample for analysis characterized in that the analyte having a small retention volume is eluted before the protein having a large retention volume is eluted to obtain an eluate containing deproteinized and analyte. Method.