JP2002267650A - High-speed liquid chromatographic apparatus - Google Patents

Abstract

PROBLEM TO BE SOLVED: To efficiently perform accurate melatonin analysis processing in an appropriate high-speed liquid chromatographic apparatus used for analyzing melatonin. SOLUTION: In the high-speed liquid chromatographic apparatus having a sample injection apparatus 5 for introducing a pineal gland body sample into a mobile phase flowing in the apparatus, a first column 16 for separating and processing the introduced pineal body sample, and a detector 18 for detecting melatonin from the separated constituent, a reactor 6 and a first column 15 for performing a derivative processing to the pineal body are provided on the downstream side from the sample injection apparatus 5 in the flow direction of the pineal on body sample and on the upstream side from the first column 16.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a high performance liquid chromatography apparatus, and more particularly to a high performance liquid chromatography apparatus suitable for use in melatonin analysis.

[0002]

2. Description of the Related Art Melatonin is a pineal hormone biosynthesized from tryptophan, and has been known for a long time to have a gonad regulating function. In recent years, it acts on the suprachiasmatic nucleus to regulate the body clock. It is pointed out that there is a possibility. In modern society, diseases involving abnormalities in the internal clock, such as sleep-wake syndrome, are increasing rapidly, and it is desired to elucidate the detailed mechanism of action of melatonin.

However, the generally known melatonin analysis method is insufficient in sensitivity, and there are still many unknown parts. In particular, since the amount of endogenous melatonin in rats and mice commonly used in behavioral and pharmacodynamic studies is extremely small, it has been difficult to quantify melatonin in most tissues. Therefore, the present inventors have attempted to develop a precolumn fluorescent derivatization high-performance liquid chromatography (hereinafter referred to as HPLC) method using hydrogen peroxide under alkaline conditions as a highly sensitive melatonin quantification method. The sensitivity was improved about 10 times or more.

Hereinafter, a method for analyzing melatonin developed by the present inventors (hereinafter referred to as the present analysis method) will be described.
In this analysis method, for example, the pineal gland is excised from a rat, a pineal gland sample is prepared from this, and the prepared pineal gland sample is HP
Analytical processing is performed by introducing into LC. FIG.
1 shows a conventional procedure for preparing a pineal sample.

[0005] To prepare pineal samples, for example, rats (Wistar strain, male, 6 weeks old, SPF) and mice (male of various strains, 8 weeks old, SPF) are used, and ether is used. Under anesthesia, the pineal gland is excised and, as shown in step 30 (step is abbreviated as S in the figure), homogenized with 500 μl of ice-cooled methanol.

Subsequently, 4500 g of the homogenized pineal gland was centrifuged for 5 minutes (step 31), and 50 μl of the supernatant was obtained.
Dispense (200 μl for mice) (step 32).
Next, 5 nM internal standard (MIAA) and methanol (MeOH) are added thereto (step 33), and this is dried (step 34). Subsequently, 40 μl (30 μl for a mouse) of water is added to this (step 35), and the derivatization process from step 36 is performed.

In the derivatization treatment, first, 5 μl of a 2M aqueous solution of Na ₂ CO _{3 and} 5 μl of a 50 mM aqueous solution of H ₂ O ₂
μl is added (steps 36 and 37), and then heat-treated at 100 ° C. for 30 minutes (step 38). Then, to concentrate melatonin, several times (N times)
Extraction of melatonin (steps 39, 4)
0) and dry it (step 41).

Subsequently, a 10% CH ₃ CN aqueous solution 50
After adding μl (step 42), the mixture is introduced into HPLC, and melatonin is analyzed (step 43). Conventionally, all the pineal sample preparation processes shown in steps 30 to 43 have been performed manually (manually).

FIG. 7 is a block diagram showing a conventional HPLC 30 used for melatonin analysis. This HPL
The C30 includes a mobile phase container 31, a pump 33, a sample injection device (sample injection device) 35, a column 10, a detector 37, a waste liquid container 38, and the like. The mobile phase container 31 is a container for storing the mobile phase 32. The pump 33 sucks the mobile phase 32 in the mobile phase container 31 by using the pipe 39 and sends the sucked mobile phase 32 to the pipe 4.
It has the function of discharging at 0 with a predetermined pressure. The pipe 40 from which the mobile phase 32 is discharged from the pump 33 is connected to the sample injection device 35. A pressure gauge 34 is provided at an intermediate position of the pipe 40, and the pump 33 is controlled based on the output of the pressure gauge 34 so that the pressure of the mobile phase 32 in the pipe 40 is constant.

The sample injection device 35 receives (injects) the pineal sample prepared as described above from the injection port 44. The pineal sample injected from the injection port 44 is mixed with the mobile phase 32 and sent to the column 10 via the pipe 41.

The column 10 is filled with a column filler for performing a pineal sample analysis process. The sample separated by the column 10 is injected into the detector 37 via the injection pipe 42 and analyzed for the components, whereby the melatonin can be quantified. As described above, according to the above-described analysis method, it is possible to achieve about 10 times higher sensitivity than the conventional melatonin analysis method.

[0012]

However, in the above-described analysis method, since the pineal sample preparation is all performed manually, the time required for the analysis process becomes longer, and efficient analysis can be performed. There was a problem that it was not possible.

In the heating process in step 38 and the extraction process in step 39, an error is inevitably generated by manual operation, and this error is reflected in the analysis data, thereby lowering the melatonin analysis accuracy. There was a problem that there was a possibility.

The present invention has been made in view of the above points, and an object of the present invention is to provide a high-performance liquid chromatography apparatus capable of efficiently performing high-precision melatonin analysis processing.

[0015]

Means for Solving the Problems In order to solve the above problems, the present invention is characterized by taking the following means.

According to the first aspect of the present invention, there is provided a sample injection device for introducing a biological sample containing melatonin into a mobile phase flowing through the device, a column for separating and processing the introduced biological sample containing melatonin, A high-performance liquid chromatography device having a detector for detecting melatonin from the component, the flow direction of the biological sample containing the melatonin, downstream of the sample injection device, and upstream of the column And a derivatization device for performing a derivatization process on the biological sample containing melatonin.

According to the above invention, the derivatization device is provided on the downstream side of the sample injection device with respect to the flow direction of the biological sample containing melatonin and on the upstream side of the column, thereby derivatizing the biological sample containing melatonin. Processing can be automatically performed in a high performance liquid chromatography apparatus. For this reason, it is possible to suppress a variation in data that occurs when the manual processing is performed as in the related art, and it is possible to perform a highly accurate melatonin analysis. In addition, since the derivatization process is automated, derivatization can be performed in a shorter time than when manually performed,
Analysis efficiency can be improved.

According to a second aspect of the present invention, there is provided the high-performance liquid chromatography apparatus according to the first aspect, wherein the temperature raising / pressure increasing means for increasing the temperature and fluid pressure of the biological sample containing the melatonin passing therethrough, and the melatonin A second column for extracting the melatonin from a biological sample containing: a first state of connecting the sample injection device to the second column; and connecting the second column to the first column. A switching device for performing a process of switching to the second state.

According to the above invention, the derivatization apparatus has the temperature raising / pressure increasing means for increasing the temperature and the fluid pressure of the biological sample containing melatonin. And at a predetermined pressure. Therefore, it is possible to suppress the variation in data caused by the variation in pressure and the variation in temperature caused by the conventional manual derivatization process, and to perform highly accurate melatonin analysis.

In addition, the process of extracting melatonin from a biological sample containing melatonin, which has been conventionally performed manually, can be automatically performed using the second column. Further, by providing the switching device, the biological sample containing melatonin introduced from the sample injection device can be automatically introduced into the second column to perform the extraction process, and the second device can be switched by switching the switching device. Can be automatically introduced into the first column. This makes it possible to reduce the time required for each process and improve the analysis efficiency, as compared with the case where the extraction process is manually performed and the extracted sample is introduced into the high-performance liquid chromatography apparatus.

According to a third aspect of the present invention, in the high performance liquid chromatography apparatus according to the second aspect, the switching device is a six-way switching valve.

According to the present invention, a high-performance liquid chromatography apparatus capable of performing high-precision melatonin analysis can be realized at low cost by using a six-way switching valve having a simple configuration as the switching apparatus.

[0023]

Next, embodiments of the present invention will be described with reference to the drawings.

FIG. 1 is a configuration diagram showing a high performance liquid chromatography apparatus (HPLC) 1 according to one embodiment of the present invention. This HPLC 1 is basically a first pump 4
The sample injection device 5 and the reactor 6
Pressure switch means), a switching valve 9 (corresponding to a switching device described in claims), a second pump 14, a first column 16, a second column 15, a detector 18, and the like. I have.

The first pump 4 and the second pump 14 are
This function has a function of sucking the first mobile phase 2 or the second mobile phase 12 and discharging the sucked mobile phases 2 and 12 at a predetermined pressure. As the first and second pumps 4 and 14, for example, a plunger type pump,
A syringe type pump, a diaphragm type pump, an air cylinder type pump or the like can be used.
In particular, the first pump 4 is configured to change the flow rate of the first mobile phase 2 to be discharged. In this embodiment,
1.3% methanol (MeOH) was used as the first mobile phase 2, and 100 mM phosphate buffer (pH 7.0) containing 10% CH ₃ CN was used as the second mobile phase 12.

On the other hand, on the upstream side of the first pump 4 with respect to the flow direction of the first mobile phase 2, a first mobile phase deaerator 3
However, a second mobile phase deaerator 13 is disposed upstream of the second pump 14 with respect to the flow direction of the second mobile phase 12. The first and second mobile phase deaerators 3, 1
No. 3 has a function of degassing bubbles which are contained in the mobile phases 2 and 12 and cause noise when analyzing melatonin.

The first pump 4 discharged from the first pump 4
Is sent to the sample injection device 5. The sample injection device 5 is, for example, a universal injector, and a pineal sample (a biological sample containing melatonin) prepared as described later is injected from an injection port 5a. The pineal sample injected from the injection port 5 a is mixed with the first mobile phase 2 in the sample injection device 5 and then sent to the reactor 6.

The reactor 6 comprises a reaction coil 7 and a heater 8
It is composed of The upstream end (the left end in the figure) of the reaction coil 7 is connected to the sample injection device 5. The downstream end (the right end in the figure) is connected to a port P1 of a switching valve 9 described later. In the reactor 6, the pineal sample sent from the sample injector 5 is heated by the heater 8 to a predetermined temperature (for example, 100 ° C.).
Heating is performed until

The switching valve 9 has six ports (P1 to P1).
P6) is a motor-driven six-way switching valve. The switching valve 9 performs a switching process of each of the ports P1 to P6 by a control device 20 described later. The switching valve 9 is provided as an option to the HPLC, and is relatively inexpensive.

In the state shown in FIG. 1, the ports P1 and P2, the port P3 and the port P4, and the port P5 and the port P6 are in communication. In the following description, the state of the switching valve 9 shown in FIG.

As described above, the switching valve 9 has six ports (P1 to P6). The port P1 is connected to the reactor 6, the port P2 is connected to the waste liquid container 10, and the port P3 is connected to the waste liquid container 10. The second column 15 is connected to the lower end in the figure, the port P4 is connected to the second pump 14, the port P5 is connected to the right end of the first column 16 in the figure, and the port P6 is connected to the second column 15 Is connected to the upper end in the figure.

In the figure, solid lines indicate ports communicating with each other, and broken lines indicate ports not communicating with each other. The specific operation of switching the port by the switching valve 9 will be described later for convenience of explanation.
The first column 16 is a CAPCELL P having an inner diameter of 1.0 mm and a length of 150 mm.
AK C18MG (manufactured by Shiseido Co., Ltd.) is used as a column. The first column 16 has a function of separating melatonin from a pineal sample introduced as described later. Further, the first column 16 is provided in a column oven 17, and the temperature is controlled by the column oven 17. Specifically, the first column 16 is controlled at 40 ° C. by the column oven 17.

The second column 15 has an inner diameter of 1.5 mm and a length of 35 mm
CAPCELL PAK C18MG (manufactured by Shiseido Co., Ltd.) is used as a column. The second column 15 has a function of concentrating melatonin in the pineal sample when the pineal sample is introduced as described later.

As the detector 18, a fluorescence detector is used in this embodiment. This fluorescence detector is configured to detect melatonin by measuring the fluorescence intensity of fluorescence of a specific wavelength generated when the excitation light source is irradiated on melatonin. In this embodiment, this fluorescence detection is performed at the excitation wavelength
The measurement was performed at 245 nm and a fluorescence measurement wavelength of 380 nm.

On the other hand, the control device 20 controls the HPLC 1 in an integrated manner, and is constituted by, for example, a computer. The control device 20 is connected to the above-described first pump 4, sample injection device 5, reactor 6, switching valve 9, second pump 14, column oven 17, and detector 18. Then, a melatonin analysis process is performed according to a melatonin analysis program described later.

Next, the preparation of the pineal sample in this embodiment will be described. FIG. 2 is a flowchart showing the procedure of the pineal sample preparation process in the present embodiment. Here, comparing the pineal sample preparation procedure according to the present embodiment shown in FIG. 2 with the conventional pineal sample preparation procedure described above with reference to FIG. Steps 10 to 17 of the pineal sample preparation procedure shown are the same as steps 30 to 37 of the conventional pineal sample preparation procedure described with reference to FIG.
However, in the preparation processing of this embodiment, the processing of Steps 38 to 42, which has been conventionally performed, is not performed. The present embodiment is characterized in that the processing of steps 38 to 42 is configured to be performed in the HPLC 1.

As described above, steps 30 to 42 shown in FIG. 6 are all manually performed (manually performed). Therefore, conventionally, step 3
The processing from Step 8 to Step 42 was also performed manually. In manual processing, human error and erroneous processing are inevitably generated, and the processing speed is slower than that of mechanical automatic processing.

In the present embodiment, the processing of steps 38 to 42, which has been manually performed in the past, is configured to be performed by the HPLC 1, so that human errors and erroneous processing that may reduce the accuracy of melatonin analysis are reduced. Thus, the melatonin analysis accuracy can be improved. Further, since the time required for the preparation process of the pineal sample can be shortened, the efficiency of the melatonin analysis process can be improved. Note that the processing of Steps 10 to 17 is the same as the processing of Steps 30 to 37 as described above, and the description of each step will be omitted.

Next, the analysis of melatonin by HPLC 1 shown in FIG. 1 will be described. In the description of the melatonin analysis processing, it is clarified in which part of the HPLC 1 the processing of Steps 38 to 42 removed in the pineal sample preparation processing as described above is performed. I do.

As described above, the driving devices 4, 5, 6, 9, 14, 17, and 18 constituting the HPLC 1 are driven and controlled by the control device 20 to perform a melatonin analysis process. FIG. 3 is a flowchart illustrating a melatonin analysis process performed by the control device 20.

When the melatonin analysis process shown in the figure is started, first, the control device 20 executes a startup process (step 20). In this start-up process, the first and second pumps 4 and 14 are started, the heater 8 of the reactor 6 and the column oven 17 are energized, and the switching valve 9 is switched to the valve A state (the state shown in FIG. 1). Is performed. The pineal sample prepared as described above is introduced (injected) into the sample injection device 5. As described above, when the first pump 4 is activated, the first mobile phase 2 is sucked by the first pump 4 and subsequently discharged to the sample injector 5 at, for example, 40 μl / min. At this time, the first mobile phase 2
The deaeration process is performed by passing through the first mobile phase deaerator 3.

The pineal sample is introduced into the first mobile phase 2 discharged from the first pump 4 in the sample injector 5. Then, the introduced pineal sample is supplied to the reactor 6 along the flow of the first mobile phase 2. In FIG. 1, the flow of the pineal sample is indicated by solid arrows.

The pineal sample supplied to the reactor 6 is
Heat treatment is performed by the heater 8 while passing through the reaction coil 7, and the derivatization reaction is completed. This heating process corresponds to the process of step 38 in the conventional pineal sample preparation process described above with reference to FIG.

The reaction coil 7 is completely sealed, and the inside diameter of the reaction coil 7 is smaller than that of the other piping. Therefore, the pressure in the reaction coil 7 increases, and when the pineal sample is heated by the reactor 6, the temperature can be raised to a predetermined temperature (for example, 100 ° C.) in a short time, and the derivatization reaction is completed quickly. can do.
Therefore, the derivatization reaction can be completed to a predetermined temperature in about 6 minutes by using the reactor 6 instead of the heating time of about 30 minutes in the heat treatment (see FIG. 6) in Step 38 conventionally. Thereby, the efficiency of the melatonin analysis processing can be improved.

Although the processing of step 38 in FIG. 6 was performed manually, in the present embodiment, the temperature raising / pressure raising processing for the pineal sample is performed automatically, so that the conventional manual processing in the derivatization reaction is performed. Variations in data due to pressure variations and temperature variations occurring in the processing can be suppressed. The heating temperature of the heater 8 is controlled by the control device 20, and the pineal sample can be stably heated to the predetermined temperature.

As described above, since the switching valve 9 is in the valve A state at the time of the start-up process, the pineal sample flowing out of the reactor 6 flows from the port P1 to the port P2, and then the waste liquid container 10 To be discarded.

On the other hand, the second mobile phase 12 is the second pump 1
4 is activated, and then suction is performed.
50 μl / m toward the second column 15 via P5
Discharged in. At this time, the second mobile phase 12 is deaerated by passing through the second mobile phase deaerator 13.

The second mobile phase 12 that has passed through the second column 15 is introduced into the first column 16 via the ports P6 and P5 because the switching valve 9 is in the valve A state. The column oven 17 is provided so as to cover the first column 16 and heats the first column 16. For this reason, the first column 16 includes the column oven 1
7 to, for example, 40 ° C. The temperature of the column oven 17 is also controlled by the control device 20 so that the temperature of the first column 16 becomes constant. The second mobile phase 12 flowing out of the first column 16
After passing through the detector 18, it is discarded in the waste liquid container 19.

The above-described activation process is performed until the time T1 has elapsed (step 21). In this embodiment, this time T1 is set to 6 minutes. By performing the above-described activation process until the time T1 elapses, the first and second processes are performed.
The operations of the pumps 4 and 12 are stabilized, and the flow rates of the mobile phases 2 and 12 discharged from the pumps 4 and 12 are also stabilized at a predetermined flow rate. In addition, since the temperatures of the heater 8 and the column oven 17 are also stabilized, the pineal sample flowing through the reaction coil 7 and the first
Temperature of the column 16 can be surely raised to a predetermined temperature.

When it is determined in step 21 that the time T1 has elapsed, in other words, the driving devices 4, 5,
If it is determined that 6, 14, 17 are stable, the process proceeds to step 22, where the control device 20 changes the flow rate of the first pump 4. Specifically, the flow rate of the first mobile phase 2 is set to 40
Change from μl / min to 200 μl / min.

In the following step 23, the control device 20 drives the switching valve 9 to be in the state shown in FIG. 4 (hereinafter, the state of the switching valve 9 shown in FIG. 4 is referred to as the valve B state). When the switching valve 9 is switched to the valve B state, the port P1, the port P6, and the port P2 are switched.
And the port P3, and the port P4 and the port P5.

Thus, the pineal sample injected by the sample injection device 5 is heated in the reactor 6 along the flow of the first mobile phase 2 and then passed through the ports P1 and P6.
Is introduced into the column 15. This second column 15
When the pineal sample is introduced together with the first mobile phase 2, it has a function of removing interfering substances (substances that become noise when detecting melatonin) contained in the pineal sample and concentrating melatonin. . The function of the second column 15 corresponds to the processing of steps 39 and 40 in the conventional pineal sample preparation processing described above with reference to FIG.

In this state of the valve B, the second mobile phase 12 sucked by the second pump 14 is supplied to the port P
The liquid is introduced into the first column 16 via P4 and P5, and then passes through the detector 18 and is discarded in the waste liquid container 19.

The above-described state of the valve B is executed until the time T2 elapses (step 24). This time T2
Is set to a time sufficient for the first column 16 to concentrate the melatonin in the pineal sample. In this embodiment, the time T2 is set to 2 minutes.

If it is determined in step 24 that the time T2 has elapsed, in other words, if it is determined that the concentration of melatonin in the pineal sample in the first column 16 has been ensured, the control device 20 proceeds to step S24. In step 25, the switching valve 9 is switched again to the valve A state, and the following step 26
In the above, the flow rate of the first pump 4 is increased from 200 μl / min to 4
Change the flow rate to return to 0 μl / min.

FIG. 5 shows a state in which the switching valve 9 is switched back to the valve A state. When the switching valve 9 is switched to the valve A state, a state is established in which the ports P1 and P2, the port P3 and the port P4, and the port P5 and the port P6 are in communication. Thereby, melatonin in the pineal sample concentrated in the second column 15 is:
In accordance with the flow of the second mobile phase 12, it is introduced into the first column 16 via the ports P6 and P5. That is, step 25
Corresponds to the processing of steps 42 and 43 in FIG.

The first column 16 has a function of separating melatonin from the pineal sample to be introduced.
During the separation of melatonin, the first column 16 is heated to a predetermined temperature by the column oven 17, and the melatonin introduced into the first column 16 is subjected to an extraction (concentration) treatment in the second column 15. Has been
Melatonin can be smoothly and reliably separated.

Further, in this embodiment, the pineal sample is introduced into the second column 15 having a large inner diameter, and then the pineal sample is introduced into the first column 16 having a small inner diameter. Even if the flow rate of the fruit sample is increased, melatonin can be reliably separated. In addition, since the separation process is performed in the first column 16 having a small inner diameter, melatonin can be separated in a short time.

The pineal sample that has been subjected to the separation process in the first column 16 is subsequently sent to the detector 18 where the process for detecting melatonin is performed (step 27). As described above, the detector 18 uses a fluorescence detector, and is configured to detect melatonin by measuring the fluorescence intensity of fluorescence having a specific wavelength generated when the excitation light source is irradiated to melatonin. .

In this embodiment, as described above, melatonin is reacted with hydrogen peroxide under alkaline conditions. As a result, a new phosphor is generated in melatonin, and its excitation maximum wavelength is 245 nm and fluorescence maximum wavelength is 380 nm. In addition to having excellent stability, this phosphor has a very high detection limit of 500 amol. For this reason, by using the HPLC 1 according to the present example, it is possible to perform highly accurate melatonin analysis.

The HPLC 1 comprises a reactor 6 for performing a part of the derivatization process on the downstream side of the sample injection device 5 and an upstream side of the first column 16 with respect to the flow direction of the pineal sample, and the second and third reactors. Column 15 (derivatizing device), part of the derivatization process for the pineal gland
Automatically implemented in C1. For this reason, it is possible to suppress the variation in data that occurs when the manual processing is performed as in the related art, and it is possible to perform highly accurate melatonin analysis. In addition, derivation can be performed in a shorter time than in the case of performing manually, and the analysis efficiency can be improved.

[0062]

According to the present invention as described above, the following various effects can be realized.

According to the first aspect of the present invention, it is possible to suppress a variation in data generated when the conventional manual processing is performed, and to perform a highly accurate melatonin analysis. Further, since the derivatization process is automated, derivatization can be performed in a shorter time than in the case where the derivatization process is performed manually, and the analysis efficiency can be improved.

According to the second aspect of the present invention, since the derivatization process can be performed on the pineal body sample at the predetermined temperature and the predetermined pressure, the pressure fluctuation and the data fluctuation due to the temperature fluctuation can be suppressed. It is possible,
Highly accurate melatonin analysis can be performed.

In addition, since the process of extracting melatonin from the pineal sample and the process of introducing the sample extracted in the second column into the first column can be automatically performed, the extraction process and the manual process can be performed manually. Compared with the case where the extracted sample is introduced into a high-performance liquid chromatography apparatus, the time for each processing can be reduced, and the analysis efficiency can be improved.

According to the third aspect of the present invention, a high-performance liquid chromatography apparatus capable of performing high-precision melatonin analysis can be realized at low cost by using a six-way switching valve having a simple configuration as the switching apparatus. can do.

[Brief description of the drawings]

FIG. 1 is a configuration diagram showing a startup processing state of a high-performance liquid chromatography apparatus according to one embodiment of the present invention.

FIG. 2 is a flowchart showing a preparation process of a pineal sample to be introduced into a high-performance liquid chromatography apparatus according to one embodiment of the present invention.

FIG. 3 is a flowchart showing the procedure of a melatonin analysis process performed by the high performance liquid chromatography apparatus according to one embodiment of the present invention.

FIG. 4 is a configuration diagram showing a high-performance liquid chromatography apparatus according to one embodiment of the present invention, showing a state where a pineal sample is supplied to a first column.

FIG. 5 is a configuration diagram illustrating a high-performance liquid chromatography apparatus according to one embodiment of the present invention, illustrating a state in which a melatonin analysis process is being performed.

FIG. 6 is a flowchart showing a conventional example of a pineal sample preparation process.

FIG. 7 is a configuration diagram of a conventional high-performance liquid chromatography apparatus.

[Explanation of symbols]

 DESCRIPTION OF SYMBOLS 1 HPLC (high performance liquid chromatography apparatus) 2 1st mobile phase 3 1st mobile phase deaerator 4 1st pump 5 Sample injection apparatus 6 Reactor 7 Reaction coil 9 Switching valve 10 Waste liquid container 12 2nd mobile phase 13 Second Mobile Phase Deaerator 14 Second Pump 15 Second Column 16 First Column 17 Column Oven 18 Detector 19 Waste Liquid Container 20 Controller

 ──────────────────────────────────────────────────続 き Continuing on the front page (72) Kiyoshi Zaitsu, 3-1-1 Made, Higashi-ku, Fukuoka City, Fukuoka Prefecture On-Campus (72) Kenji Hamase 3-1-1, Made, Higashi-ku, Fukuoka City, Fukuoka Kyushu Univ. Campus

Claims (3)

[Claims] 1. A sample injection device for introducing a biological sample containing melatonin into a mobile phase flowing through the device, a first column for separating the introduced biological sample containing melatonin, and a separated component. A high-performance liquid chromatography apparatus having a detector for detecting more melatonin, the flow direction of the biological sample containing melatonin, downstream of the sample injection device, and upstream of the first column A high-performance liquid chromatography apparatus, further comprising a derivatization device for performing a derivatization process on the biological sample containing melatonin. 2. The high-performance liquid chromatography device according to claim 1, wherein the derivatization device includes a temperature-raising / pressure-raising means for raising the temperature and fluid pressure of the passing biological sample containing melatonin, and the melatonin. A second column for extracting the melatonin from a biological sample, a first state in which the sample injection device is connected to the second column, and a second state in which the second column is connected to the first column. A high-performance liquid chromatography apparatus, comprising: a switching device for performing a switching process with the state described above. 3. The high-performance liquid chromatography apparatus according to claim 2, wherein the switching device is a six-way switching valve.