JP6040896B2 - Prelabeled amino acid analysis by liquid chromatography - Google Patents

Description

  The present invention relates to automated prelabeled amino acid analysis by HPLC. More specifically, the present invention relates to a prelabeled amino acid analysis method including an improved sample injection method.

  High resolution is required for amino acid prelabel analysis with orthophthalaldehyde (OPA). For this reason, a column packed with a packing material based on high-performance silica gel is often used for the separation.

  The labeling of amino acids with OPA proceeds efficiently under alkaline conditions with high pH (specifically, around pH 10). For this reason, the sample after the labeling reaction is alkaline. During separation, the sample may be injected into the column while remaining alkaline. Alternatively, since the alkaline sample has a large load on the analytical column, the pH may be adjusted to near neutral with an acidic solution in advance and then injected into the column.

  Non-patent document 1 (Agilend Technologies, "improved Amino Acid Methods using Agilent ZORBAX Eclipse Plus C18 Columns for a Variety of Agilent LC Instrumentation and Separation Goals") The OPA-labeled amino acid sample is introduced in this order, and a buffer of pH 8.2 is used as the mobile phase.

Asylend Technologies, “improved Amino Acid Methods using Agilent ZORBAX Eclipse Plus C18 Columns for a Variety of Agilent LC Instrumentation and Separation Goals”, [online], [Search June 25, 2012], Internet <URL: http: / /www.chem.agilent.com/Library/applications/5990-4547EN.pdf>

  When an alkaline sample solution is directly injected into a column in an amino acid prelabel analysis, the silica gel-based column is hydrolyzed, so that the column life tends to be shortened. On the other hand, when the alkaline sample solution is lowered to near neutrality by mixing it with an acidic solution in advance, and then injected into the column, the OPA-labeled amino acid generated by the labeling reaction becomes unstable and decomposes. It is not possible to ensure sufficient reliability.

  In the amino acid pre-label analysis described in Non-Patent Document 1, there is a problem that the peak of the substance that elutes first tends to collapse.

  Accordingly, an object of the present invention is to provide an OPA amino acid prelabel analysis method that suppresses the degradation of OPA-labeled amino acid sample, suppresses shortening of the column life, and can detect a favorable peak. .

  Prior to the injection of the OPA-labeled sample, the present inventors inject the acidic buffer into the column, and the OPA-labeled sample and the acidic buffer are not brought into contact with each other. The headline and the present invention were completed.

The present invention includes the following inventions.
(1) A liquid chromatograph including introducing an acidic buffer, a gas or a liquid having a pH of 6 to 7, and a sample liquid having a pH of 9 or more containing a labeled amino acid in this order into a column packed with a silica gel packing material. Prelabeled amino acid analysis method.

  In the present invention, when introducing the sample liquid into the column of the liquid chromatograph apparatus, the acidic buffer is introduced prior to the introduction of the alkaline sample liquid, and the alkaline sample liquid and the acidic buffer are not brought into contact with each other. Gas or neutral liquid is interposed in

(2) Pre-labeled amino acid analysis according to (1), wherein the volume (Va) of the acidic buffer is in the range of 0.5 to 1 expressed as a ratio (Va / Vs) to the volume (Vs) of the sample solution. Law.

(3) The prelabeled amino acid according to (1) or (2), wherein the volume (Vg) of the gas is represented by a ratio (Vg / Va) to the volume (Va) of the acidic buffer and is in the range of 1-2. Analytical method.

(4) The volume (Vl) of the liquid having a pH of 6 to 7 is expressed as a ratio (Vl / Va) to the volume (Va) of the acidic buffer, and is in the range of 0.5 to 1, (1) or ( 2) Prelabeled amino acid analysis method.

(5) The prelabeled amino acid analysis method according to any one of (1) to (4), wherein the mobile phase has a pH of 6 to 7.

(6) The prelabeled amino acid analysis method according to any one of (1) to (5), wherein the column has an inner diameter of 2 to 4.6 mm.

(7) The prelabeled amino acid analysis method according to any one of (1) to (6), wherein the labeled amino acid is an amino acid derivative labeled with orthophthalaldehyde (OPA).

(8) The prelabeled amino acid analysis method according to any one of (1) to (7), wherein the acidic buffer is a phosphate buffer.

  According to the present invention, a pre-labeled amino acid sample solution such as OPA-labeled amino acid is injected into the HPLC without coming into contact with the acidic buffer solution, so that degradation of OPA-labeled amino acid is suppressed. Furthermore, by bringing the acidic buffer solution into contact with the silica gel filler before passing through the alkaline OPA-labeled amino acid sample solution, the pH in the vicinity of the filler in contact with the acidic buffer solution is lowered. This improves the durability of the silica gel filler against the alkalinity of the sample liquid that passes thereafter. Moreover, collapse of the peak detected from a sample can also be suppressed because the timing which makes an acidic buffer solution contact a silica gel filler is before passage of the OPA labeled amino acid sample solution which is alkaline.

  That is, according to the present invention, there is provided a prelabeled amino acid analysis method capable of suppressing shortening of the column life and detecting a favorable peak while suppressing degradation of a prelabeled amino acid sample such as OPA-labeled amino acid. Is done.

It is the flow-path block diagram which showed schematically the structure of an example of the autosampler which can implement this invention with the liquid chromatograph. It is a result of progress observation of sample deterioration at the time of adding phosphoric acid aqueous solution to OPA labeling amino acid sample solution before injection to a column in comparative example 1. 2 is a chromatogram showing the results of a continuous test in Example 1. FIG. 6 is a chromatogram showing the results of a continuous test in Comparative Example 2.

  The sample solution used for analysis in the present invention is an alkaline liquid containing labeled amino acids. Specifically, the specific pH of the sample solution is pH 9.0 or higher at which the labeled amino acid can stably exist. The labeled amino acid is preferably an amino acid derivative that dissolves only under such alkaline conditions. For example, it may be an amino acid derivative obtained by labeling with a typical precolumn derivatization reagent for amino acid analysis. The pre-column derivatization reagent can be appropriately selected by those skilled in the art depending on the detection means after column chromatography. Examples of pre-column derivatization reagents include o-phthalaldehyde (OPA), phenyl isothiocyanate (PITC), fluoresamine, dansyl chloride, 2,4-dinitrofluorobenzene (DNFB), and Nα (2,4-dinitro-5 -Fluorophenyl) -L-alaninamide (FDAA) and the like.

  Thus, in the present invention, amino acids are labeled before introduction into the column. In amino acid labeling, a functional group having high hydrophobicity is usually added. For this reason, the separation by the reverse phase method can be performed. Specifically, a column filled with a silica gel filler can be used.

  The column has an inner diameter of preferably 2.0 to 4.6 mm, and more preferably 3.0 mm. If the column has an inner diameter in such a range, good resolution can be obtained. Although the length of a column is not specifically limited, For example, it is 50-150 mm.

  In the present invention, the acidic buffer is introduced before the sample solution is introduced into the column. The pH of the acidic buffer is, for example, 2 to 4, preferably 2.1 to 2.5. If the pH is within this range, the pH in the vicinity of the column filler in contact with the acidic buffer solution can be sufficiently lowered, and deterioration of the column filler relative to the alkaline sample liquid can be effectively prevented. An example of the acidic buffer is a phosphate buffer.

  Further, in order not to bring the sample solution and the acidic buffer into contact with each other, a gas or neutral liquid is introduced between the introduction of the acidic buffer and the introduction of the sample solution, and a spacer is interposed between the two liquids. That is, an acidic buffer, a gas or neutral liquid, and a sample liquid are introduced into the column in this order.

  The gas may be an inert gas or other pure gas that does not have a chemical effect on the analytical system, but air is usually sufficient. The neutral liquid has a pH of 6 to 7, for example. The neutral liquid is preferably a buffer. Specifically, the liquid used for the mobile phase in the analysis system of the present invention can be used.

  The liquid used in the mobile phase of the present invention is preferably a neutral buffer having a pH of 6 or more, preferably 6-7. An example of a neutral buffer is a phosphate buffer.

  The amount of the sample solution introduced is, for example, 1 to 5 μL, preferably 1 to 2 μL from the viewpoint of resolution. The amount of sample solution introduced can be appropriately selected by those skilled in the art depending on the inner diameter of the column. More specifically, the amount of sample solution introduced when using a column having an inner diameter of 2 to 3 mm can be 1 to 2 μL. The amount of sample solution introduced when using a column having an inner diameter exceeding the above range may be, for example, 5 μL.

  The introduction amount (Va) of the acidic buffer is expressed by a ratio (Va / Vs) to the volume (Vs) of the sample solution, for example, Va / Vs = 0.5 to 1, preferably Va / Vs = 1. is there. By introducing the acidic buffer within this range, the pH in the vicinity of the filler in contact with the acidic buffer is lowered, and the durability of the silica gel filler with respect to the alkalinity of the sample liquid passing thereafter is improved.

  Since the gas or the neutral liquid of pH 6 to 7 is a spacer between the two liquids for the purpose of preventing contact between the sample liquid and the acidic buffer, a minimum amount is sufficient. The introduction amount (Vl) of the neutral liquid is expressed by a ratio (Vl / Va) to the volume (Va) of the acidic buffer, for example, a range of Vl / Va− = 0.5 to 1, preferably Vl / Va = 1 can be used. The amount of gas introduced (Vg) is preferably larger than that in the case of using a neutral liquid in consideration of being transported in the flow path under a pressurized condition. For example, the ratio (Va) of the acidic buffer to the volume (Va) Vg / Va), for example, in the range of Vg / Va = 1 to 2, preferably Vg / Va = 2 at normal pressure.

  In the analysis flow path of the liquid chromatograph capable of implementing the present invention, the acidic buffer, the gas or neutral liquid, and the sample liquid can be automatically introduced into the column by the autosampler. The autosampler inserts needles in order into a container containing a sample liquid, a container containing a neutral liquid, and a container containing an acidic buffer, and a predetermined amount of the sample liquid and neutral liquid (or gas) are measured by a metering pump. ) And acidic buffer liquids are inhaled in this order and are introduced by injecting the inhaled liquids (or liquids and gases) into the column of the liquid chromatograph.

FIG. 1 shows an example of an autosampler together with a liquid chromatograph.
This autosampler is configured by switching the low-pressure valve 11 and the high-pressure valve 12 and driving the needle 13 in conjunction with the driving of the metering pump 16 (that is, sample liquid 15a, gas or neutral liquid 15b, and acidic liquid). Buffer 15c) A flow path for executing a suction process, a sample liquid introduction process to the analysis column 26, a cleaning process for the needle 13, a cleaning liquid discharge from the cleaning port 14 and a cleaning liquid replenishment process is configured.

  The metering pump 16 is connected to the low pressure valve 11 by a flow path. The low pressure valve 11 includes a central common port G connected to the suction port 16a of the metering pump 16 by a flow path, a port A connected to the port b of the high pressure valve 12 by a flow path, and a discharge port 16b of the metering pump 16. And port B connected by a flow path, ports C, D and E connected to the containers 18a, 18b and 18c containing the cleaning liquid by the flow path via the deaeration unit 27, and ports connected to the cleaning port 14 by the flow path, respectively. F is provided. The low pressure valve 11 can connect the common port G to any one of the ports A to F, and connect any of the ports A to F at different timings. Can do.

  The suction port 16 a of the metering pump 16 is connected to the common port G of the low-pressure valve 11 via a manual prime valve 19 through a flow path. The discharge port 16b of the metering pump 16 is connected to the port B of the low pressure valve 11 by a flow path.

  The high pressure valve 12 is connected to the needle 13 by a flow path, a port a connected to the port A of the low pressure valve 11 by a flow path, a port c to which the drain flow path 20 is connected, and an injection port 21 by a flow path. Port d and ports e and f to which a flow path constituting an analysis flow path of the liquid chromatograph is connected are provided. The high pressure valve 12 switches the connection between adjacent ports. Specifically, the mode is switched between a state in which ports a-b, cd, and ef are connected, and a state in which ports bc, dh, and fa are connected. Is. In FIG. 1, the ports bc and de are connected. In FIG. 1, each flow path CHP of the high-pressure mobile phase is highlighted with a bold line.

  The needle 13 moves between the position of the container 15a containing the sample liquid, the position of the container 15b containing the neutral liquid, the position of the container 15c containing the acidic buffer, the position of the washing port 14, and the position of the injection port 21. can do. A sample loop 17 for retaining the liquid sucked from the tip of the needle 13 is provided on the flow path connecting the port a of the high-pressure valve 12 and the needle 13.

  The drain flow path 20 is a flow path for discharging the liquid in the flow path of the autosampler to the outside, and is opened and closed by a drain valve 22.

  For example, when the sample liquid is inhaled, the low pressure valve 11 is connected to the common port G and the port A to which the suction port 16a of the metering pump 16 is connected while the needle 13 is inserted into the container 15a containing the sample liquid. And the high pressure valve 12 is connected between the ports a and b. When the metering pump 16 is driven to suck in this state, the sample liquid is sucked from the tip of the needle 13 and the sample liquid is placed in the sample loop 17 provided on the flow path connecting the port a of the high-pressure valve 12 and the needle 13. Stays.

  When replenishing the cleaning liquid to the cleaning port 14, the common port G to which the suction port 16a of the metering pump 16 is connected and any one of the ports C, D and E connected to the cleaning liquid storage containers 18a, 18b and 18c, respectively. When the metering pump 16 is driven with the port B and the port F connected to the discharge port 16b of the metering pump 16 being connected, the cleaning liquid is discharged to the cleaning port 14.

  The port f of the high-pressure valve 12 receives the mobile phase degassed from the containers 23a and 23b containing the mobile phase A and mobile phase B through the degassing unit 28, respectively, by the liquid feeding units 24a and 24b. A flow path for pumping, mixing with the mixer 25 and feeding the solution is connected. A flow path including an analysis column 26 is connected to the port e of the high pressure valve 12.

  The above figure is an example, and various modifications are possible.

  EXAMPLES Hereinafter, the present invention will be specifically described with reference to examples. However, the present invention is not limited to the following examples.

[Preparation of OPA-labeled amino acid sample solution]
An amino acid mixed standard solution containing glycine, aspartic acid, glutamic acid, serine, lysine, arginine, proline and the like is reacted with o-phthalaldehyde (OPA) in the presence of basic, 2-mercaptoethanol by a conventional method, and OPA A labeled amino acid sample solution was obtained. This OPA-labeled amino acid sample solution was used in the following Examples and Comparative Examples.

[Comparative Example 1]
In this comparative example, the progress of sample deterioration was observed when an aqueous phosphoric acid solution was added to the OPA-labeled amino acid sample solution before injection into the column.

  To the OPA-labeled amino acid sample solution, 20 μL of a 100 mmol / L phosphoric acid aqueous solution was added to prepare a sample mixture solution with a total amount of 105.5 μL. In this sample mixture, significant amino acid degradation was confirmed.

  Separately, 10 μL of a 100 mmol / L phosphoric acid aqueous solution was added to the OPA-labeled amino acid sample solution to prepare a sample mixture solution having a total amount of 90 μL. This sample mixed solution was continuously injected 6 times into HPLC (manufactured by Shimadzu Corporation, ultra-high performance liquid chromatograph Nexera). The time per analysis was 8.5 minutes. FIG. 2 is a graph showing the relationship between the number of injections and the change in the area value of the glycine peak. As shown in FIG. 2, the glycine peak area value decreased by 3 to 4% for each analysis.

[Example 1]
The autosampler and liquid chromatograph of FIG. 1 (manufactured by Shimadzu Corporation, ultra high performance liquid chromatograph Nexera) were used. First, 1.0 μL of the OPA-labeled amino acid sample solution is aspirated with the needle 13, then 0.5 μL of the mobile phase solution [20 mmol / L phosphate buffer (pH 6.5)] is aspirated, and finally the acid phosphate buffer. 1.0 μL of the liquid (pH 2.1) was sucked. Then, these liquids were introduced into the analytical column by injecting acidic phosphate buffer (pH 2.1), phosphate buffer (pH 6.5), and OPA-labeled amino acid sample solution in this order. The OPA-labeled amino acid sample solution and the acidic phosphate buffer are not mixed.

  A chromatogram obtained by continuously performing the above test is shown in FIG. Horizontal axis: retention time (min), vertical axis: fluorescence intensity (μV). Table 1 shows the relationship between the number of injections and the number of theoretical histidine plates.

  As shown in FIG. 3 and Table 1, column deterioration was suppressed even at the 500th injection, and good peaks corresponding to each amino acid were obtained.

[Comparative Example 2]
The same operation as in Example 1 was performed except that the acidic phosphate buffer solution was not aspirated and injected into the analytical column. The chromatogram obtained by the continuous test is shown in FIG. Table 2 shows the relationship between the number of injections and the number of theoretical histidine plates.

  As shown in FIG. 4 and Table 2, when the number of injections exceeded 40, the deterioration of the column became severe and a good peak corresponding to each amino acid could not be obtained.

Claims (8)

  Pre-labeling by liquid chromatography including introducing an acidic buffer, a gas or a liquid having a pH of 6 to 7, and a sample liquid having a pH of 9 or more containing a labeled amino acid in this order into a column packed with a silica gel packing material. Amino acid analysis.   The prelabeled amino acid analysis method according to claim 1, wherein the volume (Va) of the acidic buffer is expressed as a ratio (Va / Vs) to the volume (Vs) of the sample solution and is in the range of 0.5 to 1.   The prelabeled amino acid analysis method according to claim 1 or 2, wherein the volume (Vg) of the gas is represented by a ratio (Vg / Va) to the volume (Va) of the acidic buffer and is in the range of 1-2.   The prelabel according to claim 1 or 2, wherein the volume (Vl) of the liquid having a pH of 6 to 7 is expressed as a ratio (Vl / Va) to the volume (Va) of the acidic buffer and is in the range of 0.5 to 1. Amino acid analysis.   The prelabeled amino acid analysis method according to any one of claims 1 to 4, wherein the mobile phase has a pH of 6 to 7.   The prelabeled amino acid analysis method according to any one of claims 1 to 5, wherein an inner diameter of the column is 2 to 4.6 mm.   The amino acid analysis method according to claim 1, wherein the labeled amino acid is an amino acid derivative labeled with orthophthalaldehyde (OPA).   The prelabeled amino acid analysis method according to claim 1, wherein the acidic buffer is a phosphate buffer.

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