JPH0666778A - High-precision analysis - Google Patents

Abstract

PURPOSE:To improve the accuracy of high performance liquid chromatographic (HPLC) analysis by making the flow velocity of an eluate faster when peaks other than that of an object to be analyzed appear. CONSTITUTION:At the time of using the HPLC analyzing method, the flow velocity of an eluate is made faster as compared with the flow velocity of the eluate used when the peak of an object to be analyzed appears when peaks other than that of the object appear during the analysis of the same sample. Therefore, the influence of the leadings or tailings of unnecessary peaks other than that of the object on main peaks can be eliminated and high-precision analysis can be conducted. In addition, the measuring time can be shortened. When an HPLC device connected with a microcomputer, etc., is used for automatically changing the flow velocity of the eluate in accordance with a preset condition, the analysis can be automated. In addition, in case the object to be analyzed is a substance having enzyme activity and the enzyme activity is utilized for detecting the peak of the object, it is preferable to connect the HPLC device to a post-column reactor.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a highly accurate analysis method by high performance liquid chromatography (hereinafter abbreviated as HPLC) and an HPLC analyzer which can be used in the method.

[0002]

BACKGROUND OF THE INVENTION HPLC is one of the analytical methods used in various fields in recent years as a method capable of accurately analyzing and quantifying a trace component in a solution in a short time. In the analysis by HPLC, in order to improve the accuracy of the analysis, it is necessary to set the optimum measurement conditions such that the peak of the analyte and the peaks of other components can be sufficiently separated.

Usually, factors to be considered for setting the optimum measurement conditions include, for example, the type of filler, the composition of the eluent, the pH of the eluent, and the gradient conditions. However, depending on the sample, it is sometimes difficult to set the measurement conditions that can sufficiently separate the peak of the analyte and the peak of other components even if these factors are variously changed.

For example, when the concentration of the other component is considerably higher than the concentration of the analyte in the solution and this peak appears immediately before or after the peak of the analyte, the concentration of the other component is increased. The peaks of the analyte are affected by the reading or tailing, resulting in a decrease in measurement accuracy, and a phenomenon in which the lower limit of the detectable region is increased and the width of the detection limit is narrowed. Therefore, there is a current demand for the emergence of a new method that can avoid such a phenomenon.

[0005]

SUMMARY OF THE INVENTION The present invention has been made in view of the above situation, and in the analysis by HPLC, the peak of the analyte which appears during the analysis of the same sample and the other peaks are clearly separated. It is an object of the present invention to provide a method for performing the same, a highly accurate analytical method brought about by the method, and an analyzer for HPLC usable in the method.

[0006]

According to the present invention, in the analytical method by HPLC, the flow rate of the eluent at the time of appearance of a peak other than the analyte which appears during the analysis of the same sample, The analysis method characterized by increasing the accuracy of the analysis by making it faster than that of the eluent, and the flow rate of the eluent at the time of appearance of a peak other than the analyte that appears during the same sample analysis, It is an analyzer for HPLC, characterized in that it is provided with a means for automatically changing the eluent of an analyte when a peak appears and in accordance with a preset condition.

[0007] That is, the inventors of the present invention, during the course of intensive research to achieve the above-mentioned object of the present invention, determine the flow rate of the eluent at the time when a peak other than the analyte (hereinafter abbreviated as an unnecessary peak) appears. , If the peak of the analyte (hereinafter abbreviated as the main peak) is made faster than that of the eluent at the time of appearance, it is possible to avoid the influence of leading and tailing of unnecessary peaks on the main peak, and high accuracy. The present invention has been completed based on the finding that various analyzes can be carried out and that the measurement time can be shortened by carrying out the analysis under such conditions.

The HPLC apparatus used in the present invention is not particularly limited as long as it is generally used in this field.
Further, the packing material of the column mounted in the apparatus is also commonly used in this field, for example, packing material for gel filtration (gel filtration chromatography), packing material for ion exchange chromatography, packing material for isoelectric focusing chromatography, hydrophobic chromatography. Depending on the purpose of analysis, it may be appropriately selected and used from among packing materials for packing, reversed-phase chromatography packing, hydroxyapatite and the like.

By connecting a microcomputer or the like for automatically changing the flow rate of the eluent according to preset conditions to such an HPLC apparatus, the H of the present invention can be obtained.
It can be a device for PLC.

By using the apparatus for HPLC of the present invention, it becomes possible to automate the analysis of the analyte. When the analyte is a substance having an enzymatic activity and the peak of the analyte is detected using the enzymatic activity,
It is preferable to connect a post column reaction device to the HPLC device of the present invention. Further, when the present invention is carried out while performing a gradient using two kinds of eluents, the pump for supplying the eluent has a check valve, and can maintain a constant flow rate regardless of changes in back pressure. Needless to say, it is preferable that the material has performance.

The eluent used in the method of the present invention is not particularly limited as long as it is one usually used in this field. For example, phosphate,
Acetates, citrates, Good's buffers, buffers such as tris (hydroxyethyl) aminomethane, salts such as sodium chloride, potassium chloride, ammonium sulfate, such as methanol, ethanol, isopropyl alcohol, acetonitrile, tetrahydrofuran An aqueous solution containing an organic solvent such as trifluoroacetic acid, a surfactant and the like is preferably used.

The substance to be analyzed by the method of the present invention is not particularly limited as long as it can be usually analyzed by HPLC. For example, serum, blood,
Typical examples include enzymes, physiologically active substances, cancer-related antigens, substances having sugar chains, etc., contained in biological body fluids such as plasma and urine, lymphocytes, blood cells, various cells derived from living organisms. . More specifically, for example, amylase, alkaline phosphatase, acid phosphatase, γ-glutamyl transferase (γ-GTP), lipase, creatine kinase (CK), lactate dehydrogenase (LDH), glutamate oxaloacetate transaminase (GOT), glutamate. Enzymes such as pyruvate transaminase (GPT), renin, protein kinase, tyrosine kinase, such as steroid hormones, human chorionic gonadotropin (hCG),
Bioactive substances such as prolactin, thyroid stimulating hormone (TSH), and luteinizing hormone (LH), for example, prostate specific antigen (P
SA), α-macroglobulin, carcinoembryonic antigen (CEA), α-
Preferable examples include cancer-related antigens such as fetoprotein.

To carry out the present invention, for example, the following may be carried out. That is, in the measurement of the analyte by the usual HPLC, the flow rate of the eluent before and after the appearance of the main peak is set higher than that at the appearance of the main peak, by the usual HPLC. It suffices to carry out in the same manner as the measurement. In this case, the method of changing the flow rate may be manual, or may be automatically performed using the HPLC apparatus of the present invention.

Since the upper limit of the flow rate of the eluent is naturally determined by the pressure resistance of the column used, the flow rate when the main peak appears and the flow rate when the unnecessary peak appears are within this range, and The flow rate at the time of appearance and the flow rate at the time of appearance of an unnecessary peak may be appropriately selected from a range in which these peaks can be sufficiently separated, but usually, the flow rate of the eluent at the time of appearance of the peak of the analyte is obtained. In contrast to 1, the eluent at the time of appearance of the other peaks is selected from the range of 1.2 to 10, preferably 1.5 to 5. Further, the change in flow velocity may be stepwise (step) or gradual (gradient).

The method of the present invention is particularly effective when the analyte is a substance having an enzymatic activity and the analyte is detected by the post-column method utilizing the enzymatic activity.

That is, for example, an enzyme-labeled substance (hereinafter, referred to as an analyte) of a specific substance and a substance having an affinity for the specific substance (eg, antibody, lectin, inhibitor for enzyme, polynucleotide complementary to specific nucleic acid, etc.) , Abbreviated as enzyme-labeled antibody, etc.), and is particularly effective when the separation of the complex and free enzyme-labeled antibody and the like is performed by HPLC and the detection is performed by a post-column method. Is.

That is, in this case, in the sample to be analyzed by HPLC, the complex of the specific substance to be analyzed and the enzyme-labeled antibody and the free enzyme-labeled antibody coexist, and usually, Since the concentration of the free enzyme-labeled antibody or the like is often higher than the concentration of the complex, the main peak (peak of the complex) is often affected by the free enzyme-labeled antibody or the like. When the sample is analyzed by the method of the present invention, unnecessary peaks (peaks of free enzyme-labeled antibody, etc.) are smaller in both peak area and peak height than during normal HPLC analysis, and influence on the main peak. Is almost gone. The reason for this is not clear, but by increasing the flow rate of the eluent at the time of appearance of peaks other than the analyte, for example, the degree of diffusion of free enzyme-labeled antibody in the column is reduced, and post-column Since the reaction time is shortened, the amount of enzyme reaction products is reduced, so the peak itself is apparently reduced.Since the flow rate of the substrate solution for enzyme reaction is constant, the substrate concentration in the post column decreases and enzyme reaction occurs. It becomes difficult, and the total flow velocity increases, so that the retention time of the eluent in the detector decreases, and the apparent sensitivity of the detector decreases. Ah

When the analyte is detected by utilizing the enzyme activity possessed by the analyte as in the above case, the flow rate of the substrate solution at the time when the unwanted peak appears is decreased (or 0). It is also possible to increase the detection sensitivity of the main peak by However, this method cannot reduce the measurement time.

In the conventional method, in order to improve the accuracy of the analysis by HPLC, for example, the kind of the packing material, the composition of the eluent, the pH of the eluent, the gradient condition, etc. can be variously changed by experience to determine the main peak. It was necessary to set the optimum measurement conditions so that the unnecessary peaks can be sufficiently separated, but by HPLC by simply changing the flow rate of the eluent between the appearance of the main peak and the appearance of the unnecessary peak as in the present invention. It was surprising that the analysis could be performed with such high accuracy and in a short time.

The present invention will be described in more detail below with reference to Examples and Comparative Examples, but the present invention is not limited thereto.

【Example】

Example 1. (Eluent 1) 4.2 g of sodium phosphate and 80.0 g of disodium phosphate were dissolved in purified water to make the total amount 5 l, which was designated as eluent 1. (Eluent 2) Ammonium sulfate was dissolved in eluent 1 so that the concentration was 1.70 M, and pH was adjusted to 7.5 to give eluent 2. (Reaction buffer) Bovine serum albumin (Sigma) 0.2g
Was dissolved in eluent 1 to make the total amount 100 ml, which was used as a reaction buffer. (Antibody solution) Anti-human α-fetoprotein (mouse) monoclonal antibody (manufactured by Wako Pure Chemical Industries, Ltd.) was processed into Fab 'by a conventional method, and peroxidase (POD) (manufactured by TOYOBO) ) Was obtained by dissolving POD-labeled anti-human α-fetoprotein-Fab ′ in a reaction buffer at 100 nM to give an antibody solution. (Substrate solution) 3- (p-hydroxyphenyl) -propionic acid 16.
6g and 630mg of 35% hydrogen peroxide (220m as hydrogen peroxide)
g) and were dissolved in eluent 1 to make the total amount 1 l, which was used as a substrate solution. (Sample) Human α-fetoprotein (Sigma) 150
What was dissolved in the reaction buffer so that the concentration was ng / ml was used as a sample. (Use condition of HPLC) The outline of the system is shown in FIG. -Column: φ 4.6 x 30 mm. -Filler: MCAgel ethyl (manufactured by Wako Pure Chemical Industries, Ltd.)-Eluent and flow rate: The gradient pattern is shown by a solid line, and the change in flow rate is shown by a dotted line in Fig. 2 (1). -Substrate solution flow rate: 0.1 ml / min. -Detection: Fluorescence was measured at an excitation wavelength of 320 nm and a fluorescence wavelength of 404 nm. (Measurement procedure) 50 μl of the antibody solution and 50 μl of the sample were mixed and reacted at room temperature, and 10 μl of the reaction solution was mixed with HPL under the above conditions.
Analyzed by C. (Results) The obtained elution pattern is shown in Fig. 1 (1). still,
In the figure, 1 is a free POD-labeled anti-human α-fetoprotein-
Fab 'peak is 2 for human α-fetoprotein and POD
The peaks of the complex with the labeled anti-human α-fetoprotein-Fab ′ are shown, respectively.

Comparative Example 1. The flow rate of the eluent is 1 ml / min,
Further, except that the gradient pattern is shown by the solid line in FIG. 2 (2), the same sample and reagent as in Example 1 are used, the same operation is performed, and HPL is performed under the same conditions as in Example 1.
Analysis by C was performed. Figure 1 shows the elution pattern obtained.
Shown in (2). In the figure, 1 is the peak of free POD-labeled anti-human α-fetoprotein-Fab 'and 2 is the peak of human α-fetoprotein and POD-labeled anti-human α-fetoprotein-Fab'.
The respective peaks of the complex with are shown. As is clear from the results shown in FIG. 1, the method of the present invention can perform the analysis of the analyte with higher accuracy and in a shorter time than the conventional method.

Example 2. Measurement of renin activity The official names of the abbreviations used below are as follows. His: Histidine residue, Pro: Proline residue, Phe: Phenylalanine residue, Leu: Leucine residue, Val: Valine residue, Ile: Isoleucine residue, Ala: Alanine residue. (Eluent 1) 50 mM acetic acid containing 10% acetonitrile-
The eluent 1 was ammonium acetate buffer (pH 6.0). (Eluent 2) 50 mM acetic acid containing 60% acetonitrile-
The eluent 2 was ammonium acetate buffer (pH 6.0). (Substrate solution) 500 μM pyridylglycyl-His-Pro-Phe-His
-Leu-Val-Ile-His-βAla-OH, 0.1 mM containing 3 mM o-phenanthroline, 1 mM phenylmethylsulfonyl fluoride
3- (N-morpholino) propanesulfonic acid (MOPS) buffer (p
H7.0) was used as the substrate solution. (Sample) Fresh human serum was used as a sample. (Use condition of HPLC) The outline of the system is shown in FIG.・ Column: φ4.6 × 10mm (40 ℃ heat retention). -Filler: Wakosil 5C18 (manufactured by Wako Pure Chemical Industries, Ltd.)-Eluent and flow rate: The gradient pattern is shown by a solid line, and the change of flow rate is shown by a dotted line in Fig. 4 (1). -Detection: Fluorescence was measured at an excitation wavelength of 306 nm and a fluorescence wavelength of 358 nm. (Measurement procedure) 100 μl of the substrate solution and 10 μl of the sample were mixed at room temperature and then reacted at 37 ° C. for 1 hour. Of the obtained reaction solution
60 μl was analyzed by HPLC under the above conditions. (Result) The obtained elution pattern is shown in FIG. 5 (1). still,
In the figure, 1 is the peak of o-phenanthroline, and 2 is the enzymatic action product of renin (pyridylglycyl-His-Pro-Phe-His).
-Leu-OH) peak, 3 is a renin substrate (pyridylglycyl-His-Pro-Phe-His-Leu-Val-Ile-His-βAla-OH)
The respective peaks are shown.

Comparative Example 2. Using the same sample and reagents as in Example 2, except that the flow rate of the eluent was 1.5 ml / min and the gradient pattern was as shown by the solid line in FIG. HP under the same conditions as in Example 2
Analysis by LC was performed. The elution pattern obtained is shown in FIG.
Shown in (2). In the figure, 1 is the peak of o-phenanthroline, and 2 is the enzymatic action product of renin (pyridylglycyl).
-His-Pro-Phe-His-Leu-OH), 3 is a renin substrate (pyridylglycyl-His-Pro-Phe-His-Leu-Val-Ile)
-His-βAla-OH) peaks are shown. As is clear from the results shown in FIG. 5, the method of the present invention can perform the analysis of the analyte with higher accuracy and in a shorter time than the conventional method.

[0025]

As described above, according to the present invention, the conventional HP
In an analytical method using LC, for example, the type of packing material, the composition of the eluent, the pH of the eluent, the gradient conditions, etc. can be variously changed by experience to optimize the main peak and the unnecessary peak. The point that the analysis that could not be carried out without setting the measurement conditions described above can be carried out with high accuracy and in a short time by simply changing the flow rate of the eluent between the appearance of the main peak and the appearance of the unnecessary peak. It is an invention that has a remarkable effect on the invention, and is a great invention that contributes to the industry.

[Brief description of drawings]

FIG. 1 (1) shows the results of high performance liquid chromatography (HPLC) obtained in Example 1, as shown in FIG.
Shows the results of HPLC obtained in Comparative Example 1, respectively.

FIG. 2 (1) shows a gradient pattern and a change in flow velocity in Example 1, and FIG. 2 (2) shows a gradient pattern in Comparative Example 1.

FIG. 3 is a schematic diagram of a system of an HPLC apparatus used in Example 1 and Comparative Example 1.

FIG. 4 (1) shows the HPL obtained in Example 2.
The result of C is shown in FIG. 4 (2) as H obtained in Comparative Example 2.
The results of PLC are shown respectively.

5 (1) shows a gradient pattern and a change in flow velocity in Example 2, and FIG. 5 (2) shows a gradient pattern in Comparative Example 2.

FIG. 6 shows an outline of a system of an HPLC apparatus used in Example 2 and Comparative Example 2.

[Explanation of symbols]

Each number in each figure of FIG. 1 shows the following peaks, respectively. 1: Free POD-labeled anti-human α-fetoprotein-Fab ′. 2: A complex of human α-fetoprotein and POD-labeled anti-human α-fetoprotein-Fab ′. In each figure of FIG. 2, the solid line represents the gradient pattern,
Dotted lines represent changes in flow velocity. Each number in each figure of FIG. 4 shows the following peaks, respectively. 1: o-phenanthroline. 2: Enzymatic product of renin (pyridylglycyl-His-P
ro-Phe-His-Leu-OH). 3: substrate of renin (pyridylglycyl-His-Pro-Phe-His-Leu-Val-Ile-His-βAla-O
H). In each figure of FIG. 4, the solid line represents the gradient pattern,
Dotted lines represent changes in flow velocity.

─────────────────────────────────────────────────── ───

[Procedure amendment]

[Submission date] August 7, 1992

[Procedure Amendment 1]

[Document name to be amended] Statement

[Correction target item name] 0021

[Correction method] Change

[Correction content]

Example 1. (Eluent 1) 4.2 g of sodium phosphate and 80.0 g of disodium phosphate were dissolved in purified water to make the total amount 5 l, which was designated as eluent 1. (Eluent 2) Ammonium sulfate was dissolved in eluent 1 so that the concentration was 1.70 M, and pH was adjusted to 7.5 to give eluent 2. (Reaction buffer) Bovine serum albumin (Sigma) 0.2g
Was dissolved in eluent 1 to make the total amount 100 ml, which was used as a reaction buffer. (Antibody solution) Anti-human α-fetoprotein (mouse) monoclonal antibody (manufactured by Wako Pure Chemical Industries, Ltd.) was processed into Fab 'by a conventional method, and peroxidase (POD) (manufactured by TOYOBO) ) Was obtained by dissolving POD-labeled anti-human α-fetoprotein-Fab ′ in a reaction buffer at 100 nM to give an antibody solution. (Substrate solution) 3- (p-hydroxyphenyl) -propionic acid 16.
6g and 630mg of 35% hydrogen peroxide (220m as hydrogen peroxide)
g) and were dissolved in eluent 1 to make the total amount 1 l, which was used as a substrate solution. (Sample) Human α-fetoprotein (Sigma) 150
What was dissolved in the reaction buffer so that the concentration was ng / ml was used as a sample. (Usage Conditions of HPLC) An outline of the system is shown in FIG. -Column: φ 4.6 x 30 mm. -Filler: MCIgel ethyl (manufactured by Mitsubishi Kasei Co., Ltd.)-Eluent and flow rate: The gradient pattern is shown by a solid line, and the change of flow rate is shown by a dotted line in Fig. 2 (1). -Substrate solution flow rate: 0.1 ml / min. -Detection: Fluorescence was measured at an excitation wavelength of 320 nm and a fluorescence wavelength of 404 nm. (Measurement procedure) 50 μl of the antibody solution and 50 μl of the sample were mixed and reacted at room temperature, and 10 μl of the reaction solution was mixed with HPL under the above conditions.
Analyzed by C. (Results) The obtained elution pattern is shown in Fig. 1 (1). still,
In the figure, 1 is a free POD-labeled anti-human α-fetoprotein-
Fab 'peak is 2 for human α-fetoprotein and POD
The peaks of the complex with the labeled anti-human α-fetoprotein-Fab ′ are shown, respectively. ─────────────────────────────────────────────────── ───

[Procedure amendment]

[Submission date] August 6, 1993

[Procedure Amendment 2]

[Document name to be amended] Statement

[Name of item to be corrected] Brief description of the drawing

[Correction method] Change

[Correction content]

[Brief description of drawings]

FIG. 1 (1) shows the results of high performance liquid chromatography (HPLC) obtained in Example 1.
(2) shows the results of the HPLC obtained in Comparative Example 1, respectively.

2 (1) shows a gradient pattern and a change in flow velocity in Example 1, and FIG. 2 (2) shows a gradient pattern in Comparative Example 1.

FIG. 3 shows an outline of a system of an HPLC apparatus used in Example 1 and Comparative Example 1.

FIG. 4 (1) shows the HP obtained in Example 2.
The LC results and FIG. 4 (2) show the HPLC results obtained in Comparative Example 2, respectively.

5 (1) shows a gradient pattern and a change in flow velocity in Example 2, and FIG. 5 (2) shows a gradient pattern in Comparative Example 2.

FIG. 6 shows an outline of a system of an HPLC apparatus used in Example 2 and Comparative Example 2.

[Explanation of Codes] Each number in each figure of FIG. 1 indicates the following peaks. 1: Free POD-labeled anti-human α-fetoprotein-F
ab '. 2: Human α-fetoprotein and POD-labeled anti-human α-
Complex with fetoprotein-Fab '. In each figure of FIG. 2, the solid line represents the gradient pattern,
Dotted lines represent changes in flow velocity. Each number in each figure of FIG. 4 shows the following peaks, respectively. 1: o-phenanthroline. 2: Enzyme action product of renin (pyridylglycyl-Hi
s-Pro-Phe-His-Leu-OH). 3: substrate for renin (pyridylglycyl-His-Pro
-Phe-His-Leu-Val-Ile-His-
βAla-OH). In each figure of FIG. 4, the solid line represents the gradient pattern,
Dotted lines represent changes in flow velocity.

[Procedure 3]

[Document name to be corrected] Drawing

[Correction target item name] All drawings

[Correction method] Change

[Correction content]

[Figure 1]

[Fig. 2]

[Figure 3]

[Figure 5]

[Figure 4]

[Figure 6]

Claims (6)

[Claims] 1. A high-performance liquid chromatography analysis method, wherein the flow rate of the eluent at the time of appearance of a peak other than the analyte that appears during the analysis of the same sample is the eluent at the time of the peak of the analyte. The analytical method is characterized by increasing the accuracy of the analysis by making it faster than that. 2. The analysis method according to claim 1, wherein the analysis by high performance liquid chromatography is performed by a post-column method. 3. The analysis method according to claim 1, wherein the analyte is a substance having an enzymatic activity. 4. The analyte is a substance having an enzymatic activity, and the analyte is detected by utilizing the enzymatic activity.
The analysis method according to claim 3. 5. The flow rate of the eluent at the time of appearance of a peak other than the analyte which appears during the analysis of the same sample, and that of the eluent at the time of appearance of the peak of the analyte are determined according to preset conditions. An analyzer for high-performance liquid chromatography, characterized in that it comprises means for automatically changing the temperature. 6. A post-column reactor is connected,
The high performance liquid chromatography analyzer according to claim 5.