JP2768079B2 - High precision analysis method - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a high-precision analytical method using high performance liquid chromatography (hereinafter abbreviated as HPLC) and an analytical apparatus for HPLC 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 for accurately analyzing and quantifying trace components in a solution in a short time. In the analysis by HPLC, it is necessary to set optimal measurement conditions so that the peak of the analyte and the peak of other components can be sufficiently separated in order to enhance the accuracy of the analysis.

[0003] Usually, factors considered for setting the optimum measurement conditions include, for example, the type of the 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 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 other components is considerably higher than the concentration of the analyte in the solution and this peak appears immediately before or immediately after the peak of the analyte, the concentration of the other components is reduced. The peaks of the analyte are affected by the reading or tailing, resulting in a decrease in measurement accuracy, or a phenomenon in which the lower limit of the detectable region is increased and the width of the detection limit is narrowed. For this reason, there is a demand for a new method capable of avoiding such a phenomenon.

[0005]

SUMMARY OF THE INVENTION The present invention has been made in view of the above situation, and clearly separates a peak of an analyte and another peak appearing during the same sample analysis in HPLC analysis. It is an object of the present invention to provide a method for performing the method, a high-precision analysis method provided by the method, and an HPLC analyzer that can be used in the method.

[0006]

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

That is, the inventors of the present invention have made intensive studies in order to achieve the object of the present invention, and have set a flow rate of an eluent at the time of appearance of a peak other than an analyte (hereinafter abbreviated as an unnecessary peak). If the peak of the analyte (hereinafter abbreviated as “main peak”) is made faster than that of the eluent at the time of appearance, unnecessary peak reading and tailing can be prevented from affecting the main peak, and high accuracy can be avoided. The present inventors have found that a simple analysis can be performed, and that the measurement time can be shortened by performing the analysis under such conditions, thereby completing the present invention.

The HPLC apparatus used in the present invention is not particularly limited as long as it is usually used in this field.
The packing of the column to be mounted on the apparatus is also usually used in this field, for example, packing for gel filtration (gel filtration chromatography), packing for ion exchange chromatography, packing for isoelectric focusing, hydrophobic chromatography. It may be appropriately selected from fillers for packing, reversed phase chromatography, hydroxyapatite and the like according to the purpose of analysis.

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

The use of the HPLC apparatus of the present invention makes it possible to automate the analysis of the analyte. When the analyte is a substance having enzyme activity and the peak of the analyte is detected using the enzyme activity,
It is preferable to connect a post-column reaction device to the HPLC device of the present invention. When the present invention is carried out while performing a gradient using two types of eluents, a pump for supplying the eluent is equipped with a check valve, and can maintain a constant flow rate regardless of changes in back pressure. Needless to say, it is preferable to have performance.

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

The analytes that can be analyzed by the method of the present invention are not particularly limited as long as they can be generally analyzed by HPLC. For example, serum, blood,
Representative examples include enzymes, physiologically active substances, cancer-related antigens, substances having sugar chains, etc. contained in biological samples such as biological fluids such as plasma and urine, lymphocytes, blood cells, and various cells. . More specifically, for example, amylase, alkaline phosphatase, acid phosphatase, γ-glutamyltransferase (γ-GTP), lipase, creatine kinase (CK), lactate dehydrogenase (LDH), glutamic oxaloacetate transaminase (GOT), glutamic acid Enzymes such as pyruvate transaminase (GPT), renin, protein kinase, tyrosine kinase, such as steroid hormones, human chorionic gonadotropin (hCG),
Physiologically active substances such as prolactin, thyroid stimulating hormone (TSH) and luteinizing hormone (LH), such as prostate specific antigen (P
SA), α-macroglobulin, carcinoembryonic antigen (CEA), α-
Preferable examples include cancer-related antigens such as fetoprotein.

The present invention may be implemented, for example, as follows. That is, in the measurement of an analyte by ordinary HPLC, except that the flow rate of the eluent before and after the appearance of the main peak is made faster than the flow rate at the time of appearance of the main peak, the HPLC method is used. It is sufficient to carry out the measurement in the same way 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 to be used, the flow rate at the appearance of the main peak and the flow rate at the appearance of the unnecessary peak are within this range, and When the flow velocity at the appearance and the flow velocity at the appearance of unnecessary peaks are changed, these peaks may be appropriately selected from a range capable of sufficiently separating the peaks. In contrast to 1, the eluent at the appearance of the other peaks is selected from the range of 1.2 to 10, preferably 1.5 to 5. The change in the flow velocity may be stepwise (step) or gradually (gradient).

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

That is, for example, an enzyme-labeled product (hereinafter referred to as an antibody, a lectin, an inhibitor for an enzyme, a polynucleotide complementary to a specific nucleic acid, etc.) in which an analyte is a specific substance and a substance having an affinity for the specific substance (hereinafter referred to as a polynucleotide complementary to a specific nucleic acid) , An enzyme-labeled antibody, etc.), which is particularly effective when the complex is separated from a free enzyme-labeled antibody or the like by HPLC and the detection is carried out by a post-column method. It is.

That is, in this case, in the sample to be analyzed by HPLC, a complex of the specific substance to be analyzed and the enzyme-labeled antibody and the like and a free enzyme-labeled antibody and the like coexist, and 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 according to the method of the present invention, unnecessary peaks (peaks of free enzyme-labeled antibodies and the like) are reduced in both peak area and peak height as compared with ordinary HPLC analysis, and the influence on the main peak is reduced. 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 a peak other than the analyte, for example, the degree of diffusion of free enzyme-labeled antibody in the column is reduced. Since the reaction time is shortened and the amount of the enzyme reaction product is reduced, the peak itself is apparently smaller.Because the flow rate of the substrate solution for the enzyme reaction is constant, the concentration of the substrate in the post column is reduced and the enzyme reaction occurs. As the total flow rate increases, the elongation time of the eluent in the detector decreases, and the apparent detector sensitivity decreases. There will be.

In the case where 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 of appearance of an unnecessary peak is reduced (or 0). ), It is possible to increase the detection sensitivity of the main peak. However, this method cannot shorten the measurement time.

In the conventional method, in order to improve the accuracy of the analysis by HPLC, for example, the type of the filler, the composition of the eluent, the pH of the eluent, the gradient conditions, etc. are variously changed by experience to obtain the main peak. Although it was necessary to set the optimum measurement conditions so that unnecessary peaks could be sufficiently separated, as in the present invention, by simply changing the flow rate of the eluent between the time when the main peak appeared and the time when the unnecessary peak appeared, HPLC It was surprising that the analysis could be performed with such high precision and in a short time.

Hereinafter, the present invention will be described more specifically with reference to Examples and Comparative Examples, but the present invention is not limited thereto.

【Example】

Embodiment 1 (Eluent 1) 4.2 g of sodium phosphate and 80.0 g of disodium phosphate
Was dissolved in purified water to make a total volume of 5 l, and this was used as eluent 1. (Eluent 2) Eluent 2 was prepared by dissolving ammonium sulfate in eluent 1 so as to have a concentration of 1.70 M and adjusting the pH to 7.5. (Reaction Buffer) 0.2 g of bovine serum albumin (manufactured by Sigma) was dissolved in eluent 1 to make a total volume of 100 ml, which was used as a reaction buffer. (Antibody solution) An anti-human α-fetoprotein (mouse) monoclonal antibody (manufactured by Wako Pure Chemical Industries, Ltd.) is treated in a conventional manner to give F
ab 'and peroxidase (POD)
(Manufactured by TOYOBO), and a POD-labeled anti-human α-fetoprotein-Fab ′ obtained by dissolving it in a reaction buffer at a concentration of 100 nM was used as an antibody solution. (Substrate solution) 3- (p-hydroxyphenyl) -propionic acid 16.6 g and 35%
630 mg of hydrogen peroxide solution (220 mg as hydrogen peroxide) was dissolved in eluent 1 and the total amount was made 1 l to be used as a substrate solution. (Sample) A sample was prepared by dissolving human α-fetoprotein (manufactured by Sigma) in a reaction buffer at a concentration of 150 ng / ml. (Conditions for Using HPLC) An outline of the system is shown in FIG.・ Column: φ4.6 × 30mm. 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 in flow rate is shown by a dotted line in FIG. 2 (1). -Substrate liquid 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) After 50 µl of the antibody solution and 50 µl of the sample were mixed and reacted at room temperature, 10 µl of the reaction solution was analyzed by HPLC under the above conditions. (Results) The obtained elution pattern is shown in FIG. 1 (1). In the figure, 1
Indicates the peak of free POD-labeled anti-human α-fetoprotein-Fab ′, and 2 indicates the peak of the complex of human α-fetoprotein and POD-labeled anti-human α-fetoprotein-Fab ′.

Comparative Example 1 The flow rate of the eluent was 1 ml / min,
Except that the gradient pattern was shown by a solid line in FIG. 2 (2), the same operation was performed using the same samples and reagents as in Example 1, and HPL was performed under the same conditions as in Example 1.
Analysis by C was performed. Fig. 1 shows the elution pattern obtained.
It is shown in (2). In the figures, 1 indicates the peak of free POD-labeled anti-human α-fetoprotein-Fab ′, and 2 indicates human α-fetoprotein and POD-labeled anti-human α-fetoprotein-Fab ′.
The peaks of the complex with are shown respectively. As is evident from the results of FIG. 1, it can be understood that the method of the present invention can analyze an object to be analyzed with higher accuracy and in a shorter time than in the conventional method.

Embodiment 2 FIG. Measurement of renin activity The abbreviations used below have the following formal names. His: histidine residue, Pro: proline residue, Ph
e: phenylalanine residue, Leu: leucine residue, V
al: valine residue, Ile: isoleucine residue, Al
a: Alanine residue. (Eluent 1) 50 mM acetic acid-ammonium acetate buffer (pH 6.0) containing 10% acetonitrile was used as eluent 1. (Eluent 2) 50 mM acetic acid-ammonium acetate buffer (pH 6.0) containing 60% acetonitrile was used as eluent 2. (Substrate solution) 500 μM pyridylglycyl-His-Pro-Ph
e-His-Leu-Val-Ile-His-βAl
a-OH, 0.1 mM 3- (N) containing 3 mM o-phenanthroline, 1 mM phenylmethylsulfonyl fluoride
-Morpholino) propanesulfonic acid (MOPS) buffer (pH 7.0) was used as a substrate solution. (Sample) Fresh human serum was used as a sample. (Use Conditions of HPLC) FIG. 6 shows an outline of the system. Column: φ4.6 × 10 mm (40 ° C.). -Filler: Wakosil 5C18 (manufactured by Wako Pure Chemical Industries, Ltd.)-Eluent and flow rate: The gradient pattern is shown by a solid line, the change in flow rate is shown by a dotted line, and FIG . . Detection: excitation wavelength; 306 nm, fluorescence wavelength: 358 nm
The fluorescence was measured with. (Measurement procedure) After mixing 100 µl of the substrate solution and 10 µl of the sample at room temperature, the mixture was reacted at 37 ° C for 1 hour. 60 of the obtained reaction solution
μl was analyzed by HPLC under the above conditions. (Result) The obtained solution pattern is shown in FIG . In the figure,
1 is the peak of o-phenanthroline, 2 is the enzymatic product of renin (pyridylglycyl-His-Pro-P
he-His-Leu-OH), and 3 is the renin substrate (pyridylglycyl-His-Pro-Phe-).
His-Leu-Val-Ile-His-βAla-
OH) are shown.

Comparative 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. 5 (2) , the same operation was performed using the same samples and reagents as in Example 2. Analysis by HPLC was performed under the same conditions as in Example 2. The obtained elution pattern is shown in FIG .
In the figure, 1 is the peak of o-phenanthroline, 2 is the enzymatic action product of renin (pyridylglycyl-His-
Pro-Phe-His-Leu-OH)
3 is a substrate of renin (pyridylglycyl-His-Pro
-Phe-His-Leu-Val-Ile-His-
(Ala-OH) peaks are shown. As is clear from the results of FIG. 4 , it can be understood that the method of the present invention can analyze an object to be analyzed with higher accuracy and in a shorter time than in the conventional method.

[0025]

As described above, the present invention relates to the conventional HP
In an analytical method using LC, for example, the type of the filler, the composition of the eluent, the pH of the eluent, the gradient conditions, etc. are variously changed by experience, so that the main peak and the unnecessary peak can be sufficiently separated. Analysis that could not be performed unless the measurement conditions were set can be performed with high precision and in a short time simply by 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 which has a remarkable effect, and is a great invention which contributes to the industry.

[Brief description of the drawings]

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

FIG. 2A shows a gradient pattern in Example 1 and a change in flow velocity, and FIG. 2B shows a gradient pattern in Comparative Example 1, respectively.

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

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

FIG. 5A shows a gradient pattern and a change in flow velocity in Example 2, and FIG. 5B shows a gradient pattern in Comparative Example 2, respectively.

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 indicates the following peak, respectively. 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, a solid line represents a gradient pattern,
Dotted lines represent changes in flow velocity. Each number in each figure of FIG. 4 indicates the following peak, respectively. 1: o-phenanthroline. 2: Enzymatic product of renin (pyridylglycyl-Hi
s-Pro-Phe-His-Leu-oH). 3: Substrate of renin (pyridylglycyl-His-Pro
-Phe-His-Leu-Val-Ile-His-
βAla-OH). In each figure of FIG. 5 , a solid line represents a gradient pattern,
Dotted lines represent changes in flow velocity.

──────────────────────────────────────────────────続 き Continuation of the front page (56) References JP-A-57-194351 (JP, A) JP-A-59-38651 (JP, A) JP-B-43-27079 (JP, B1) Jiko-sho 44- 1040 (JP, Y1) (58) Field surveyed (Int. Cl. ⁶ , DB name) G01N 30/32

Claims (7)

(57) [Claims] In an analysis method using high performance liquid chromatography, the flow rate of an eluent at the time of appearance of a peak other than the analyte appearing during the same sample analysis is determined by the eluent at the time of appearance of the peak of the analyte. The method according to claim 1, wherein the accuracy of the analysis is improved by making the analysis faster. 2. The method according to claim 1, wherein said sample has a concentration relative to an analyte concentration in said sample.
High concentration of other components
In the case of analysis by chromatography, this peak
Appears before and after the elephant peak, analyzed by other components
Claims where the peak of the object is affected
Item 7. The analysis method according to Item 1. 3. The method according to claim 1, wherein the analysis by high performance liquid chromatography is performed by a post-column method. 4. is a substance analyte possess enzymatic activity, assay according to any one of claims 1 to 3. 5. The method according to claim 1 , wherein the analyte is a substance having an enzyme activity, and the detection of the analyte is performed using the enzyme activity.
The analysis method according to claim 4 . 6. The method according to claim 1, wherein the flow rate of the eluent at the time of appearance of a peak other than the analyte appearing during the same sample analysis and that of the eluate at the time of appearance of the analyte peak are determined according to preset conditions. An analyzer for high-performance liquid chromatography, characterized by comprising means for automatically changing the temperature and pressure. 7. A post-column reactor is connected,
The high-performance liquid chromatography analyzer according to claim 6 .