JPS6192599A - Method of analyzing prostaglandin and device - Google Patents

Abstract

PURPOSE:To analyze each component of PG (prostaglandin) effectively and efficiently, by combining separation of each PG component by liquid chro matography method using a separation column with a reaction between each PG component and NAD<+> using immobilized 15-OHPGDH. CONSTITUTION:The eluting solution 2 is sent through the syringe 8 to the separa tion column 4 of reversed phase separation type by the pump 6, and a test specimen containing various PG is sent to the column. A reaction solution of NAD<+> (nicotinamide adeine dinucleotide) in the reaction solution reservoir 10 is added through the reaction solution channel 16 to the eluate from the reaction column 4 through the eluate channel 14. Then, the mixed solution is made to flow into the reaction column 18, a PG component and NAD<+> contained in the eluate are reacted by a catalytic action of 15-hydroxyprosta glandin dehydrogenase in the immobilized state in the column 18. NAD<+> formed by the reaction in the column 18 is made to flow out of the column 18, detected and analyzed by the detector 20 set in the downstream side by a common fluores cent measuring means.

Description

DETAILED DESCRIPTION OF THE INVENTION Technical Field The present invention relates to a prostaglandin analysis method and an apparatus thereof, and in particular, to an effective and economical method for analyzing each component of prostaglandins having an OH group at the 15-position. The present invention relates to a method and apparatus for doing so.

Prior Art Prostaglandin
; PG) is a derivative of a monocarboxylic acid having 20 carbon atoms and having two side chains on a 5-membered ring. By the way, there are many types of PC, and each PG has different physiological activities, so it is necessary to separate and quantify each of these components.

In addition, PG has a wide range of physiological effects, such as smooth muscle contraction, blood pressure lowering, anti-lipolysis, and platelet aggregation inhibiting effects, and is useful in clinical medicine.

Although it has attracted attention in fields such as pharmacology, clinical pathology, and biochemistry, conventional methods for analyzing such PCI include
Radioimmunoassay (RIA) method, gas chromatography method, thin layer chromatography method, high performance liquid chromatography method, etc. are mainly used.

Problems However, in the conventional tA method for analyzing PGs, the pretreatment of samples containing PCT is complicated, and analysis is performed for each component of PGs, which requires multiple PCs.
It was not possible to analyze the components simultaneously, and antibodies against all PGs were not available, so there were some PG acid components that could not be analyzed separately. In addition, in the gas chromatography method, the pretreatment of the PC sample is complicated, the derivatization of the PC component is complicated, and there are inherent quantitative problems, and high-sensitivity measurements cannot be performed. There is a problem in that it is difficult to accurately analyze trace amounts of PCIII. Furthermore, the thin layer chromatography method has a fatal problem of lack of quantitative properties.

In addition, there are two methods for analyzing PC using conventional high-performance liquid chromatography: a method in which a PC sample is separated into each PC component in a separation column and then detected directly with a UV detector, and a method in which a PC sample is separated into each PC component in a separation column and then detected directly with a UV detector, and a method in which a PC sample is labeled before introduction into the column is used. There is a pre-label method that uses a fluorescence detector to measure the PG sample, but the former direct method has the problem of low sensitivity, and the latter pre-label method requires complicated pretreatment of the PG sample and its derivatization is complicated. However, due to the lack of stability, there was a problem of lack of quantitative properties, and for example, it was difficult to measure trace amounts of PC in blood.

SUMMARY OF THE INVENTION The present invention was made against the background of the above circumstances, and its purpose is to enable simultaneous analysis of multiple pQIps easily, quickly, and accurately at low cost. An object of the present invention is to provide a method and an apparatus.

In order to achieve the above object, the present invention
After separating a sample containing prostaglandins (PC) having an OH group at the 15th position into each component using a separation column,
The effluent containing each PC component that was sequentially separated was treated with immobilized 15-hydroxyprostaglandin dehydrogenase (15-hydroprostaglandin dehydrogenase) while containing nicotinamide adenine dinucleotide (NAD=).
xy-prostaglandindehydroge
After reacting each PG acid component with the above-mentioned NAD'' in sequence by flowing it through a reaction column having 15-OHP It was designed to detect PG acid components.

According to the present invention, first, PGs having an O)[ group at the 15th position of El, E2, etc. are prepared as a sample to be analyzed. If the sample to be analyzed is a biological sample such as blood, the PGs will be extracted through appropriate treatment (e.g., solvent extraction with an organic solvent such as ethyl acetate) and subjected to analysis. , it is not necessary that such removed P(J'JI be conventionally derivatized for analysis.

Next, this 15-0 HPG is separated into each component in a predetermined separation column, and the separation means is liquid chromatography, especially high performance liquid chromatography, which can rapidly separate PC components into each component. One-sided method is adopted. In addition, when separating PC by this liquid chromatography method, the column for separating P G i-Q is a reversed phase distribution type column: internal diameter 1.5 + am ~ 5 mm, length 50 mm ~ 250 am
, for example, JASCO Corporation flJp S-Fin
epak S I L Cg (inner diameter 1.5 fins, length 250 m5) is preferably used. In addition, the eluents that can be passed through the 19 separation column include methanol, acetonitrile, and Net fii solution (phosphate buffer, T
A mixed solvent of ris mild (L liquid, etc.) will be used.

Then, a predetermined amount of the reaction solution containing nicotinamide adenine dinucleotide (NAD) is continuously mixed into the eluate (effluent) containing each PG acid component that is sequentially separated by this liquid chromatography method. The reaction solution containing NAD= is a reaction solution containing disodium ethylenediaminetetraacetate, dithiothreitol, and a buffer solution (phosphate buffer, Tris buffer, etc.).
It is prepared by dissolving D-, and the p 1+ of this reaction solution is preferably 7 to 9, and the concentration is preferably 0.1 to 1 mM. Of course, this NAD" may be mixed in the eluent from the beginning, or it may be supplied to the separation column separately from the eluate and mixed in advance into the eluate containing each PC component flowing out from the separation column. It doesn't make any difference if you pretend like you are.

The thus obtained mixed solution contains 15-hydroxyprostaglandin dehydrogenase (15-OHP G D ) immobilized on a suitable support such as porous glass beads.
H) is caused to flow through a reaction column having H), thereby causing each of the sequentially separated PG acid components NAD" to react sequentially. This reaction is carried out as shown below as a reaction formula: 15- Dehydro-PC+NADH+H”15-0HPG
Due to the catalytic action of DH, the OH group at the 15th position of one chain extending from the 5-membered basic skeleton becomes NAD.
It is dehydrogenated and converted into a dehydro compound, and NADH, which is a fluorescent substance, is produced together with it.This reaction proceeds under neutral or alkaline conditions, and therefore, the above-mentioned mixture The pH of the reaction solution is acidic or the eluent is adjusted so that the solution is neutral or alkaline.
will be controlled.

Further, this 15-0HPGDH is, for example, J, Bi.

chem, 84.1485-94 (1978), and the oxygen immobilization was carried out using known methods, e.g.
．． H, Weetal+.

“Immobilized Hr+zymes,
Antigens, Antibodies and
Peptides'', Marcel Dekke
R, New York, NY (1975) l.

In this way, 15-0 is fixed and retained within the reaction column.
HPCDI+ is added to each PG flowed into the column.
By rapidly reacting with the acid component NAD, a fluorescent substance:
It has the effect of producing NADH. Since this 15-0HPGDH is immobilized on a suitable support, etc., unlike a method in which it is continuously supplied and consumed, it destabilizes the reaction due to changes in the concentration in the supply liquid and the supply rate. Once the elements are removed and fixed, it can be used repeatedly tens to hundreds of times, so the analysis cost is low and it can be advantageously adopted as a general-purpose analysis method. It is.

The NADH produced by this reaction is 340
The absorption spectrum shows a maximum near 460 nm.
The fluorescence spectrum shows a maximum near nm. Therefore, the excitation wavelength was 340 nm.

By performing fluorescence analysis with the fluorescence wavelength set to 460 nm, it is possible to detect the presence of even a trace amount of NADH, in other words, if there is a PG acid component present in the eluate flowing into the reaction column. detection.

It can be analyzed.

In addition, when higher sensitivity detection is required for PG in blood, etc., diaphorase and risazurin (res) can be added to the effluent containing NADH produced by the reaction flowing out from the reaction column.
By mixing the effluent with NAD and causing a reaction, the NADH present in the effluent is converted to NAD, and at the same time, the fluorescent substance resorufin is converted into NAD.
), and by measuring the fluorescence of this resorufin to detect each PC component, even trace amounts of PG can be detected and quantified more effectively. With this system, it is possible to quantify on the order of several tens of pg.

In addition, the solfin produced in this way is 57 O
The absorption spectrum shows a maximum near 580 nm.
The fluorescence spectrum shows a maximum near nm.

Therefore, the excitation wavelength was 570 nm.

By performing fluorescence analysis with the fluorescence wavelength set at 580 nm, solfins can be effectively measured, and thus each PC component can be effectively analyzed and quantified.

Also, although such diaphorase and risazurin are generally present in the same solution and mixed into the effluent flowing out from the reaction column, the compounds are also in separate solutions and mixed into such effluent. Furthermore, diaphorase may be mixed into the 15-
0HPCDI-[ is similarly immobilized on a suitable support and placed in a second reaction column, and the mixture obtained by mixing rizazurin into the effluent is added to the second reaction column. It is also possible to produce a fluorescent substance, resorufin, by allowing the fluorescent substance to flow through the water.

Of course, this risazurin can be introduced into the eluate upstream of the separation column, similar to the above-mentioned NAD, and can also be introduced downstream of the separation column, even before the effluent comes into contact with the diaphorase. For example, they can be introduced into the effluent at any stage.

In addition, if diaphorase is immobilized as described in L, there is no loss of diaphorase, and excellent advantages can be exhibited in terms of analytical operation and economy.

As described above, in the PG analysis method according to the present invention, after separating PGM into each component, a mixture made of PGM in the presence of NAD as a reactant is added to 15-0 as a reaction catalyst.
By passing through a reaction column in which HPGDH is immobilized, fluorescent substances: NAD, H are produced, or in addition to this, diaphorase and risazurin are present to produce a fluorescent substance: resorufin, and then,
This method was designed to detect NADH or solfin, thereby making possible rapid, reliable, and stable analysis of PGs.

Further, suitable apparatuses for carrying out the present invention include:
A separation mechanism using liquid chromatography that sequentially separates a sample containing PGs having (111 groups at the 15th position into each component using a separation column) and (bll
a mixing mechanism that is connected to a channel through which the effluent containing each PG component flowing out from the bone separation mechanism is allowed to flow, and mixes a reaction solution containing NAD into the effluent; 0HPC; has DH, and a mixed liquid of the reaction liquid and the effluent formed by the mixing operation by the mixing mechanism is made to flow, and each PC component in the mixed liquid and the N
A reaction column that sequentially reacts with AD'', and a detection means that is provided downstream of the reaction column and that detects the reaction product contained in the effluent flowing out from the reaction column by fluorescence measurement. , and the above ta), (bl, fc) and fc in fdl)
and fdl, a second mixing mechanism is connected to a channel through which the effluent flowing out from the reaction column flows, and mixes diphophorase and lisazurin into the effluent; There is also a device configured to include a reaction vessel that causes a predetermined reaction to occur while causing a liquid mixture formed by a mixing operation by a mixing mechanism to flow, thereby producing a fluorescent substance: resorufin.

DESCRIPTION OF THE PREFERRED EMBODIMENTS An example of this preferred apparatus is shown in FIG. 1 as a system diagram. In FIG. 1, reference numeral 2 denotes an eluent reservoir, which stores an eluent as a mobile phase that sequentially elutes each PG component held in a reversed-phase distribution type separation column 4. And those /8 synergic liquids are /8 synergic pumps 6
Therefore, it is continuously supplied to the separation column 4. Further, a sample injector 8 is provided between the eluent pump 6 and the separation column 4, and the injector 8
The sample containing various PGs injected into the separation column 4 is guided to the separation column 4.

On the other hand, reference numeral 10 denotes a reaction liquid reservoir containing a reaction liquid containing "NAD". Eluate channel 14 through which the liquid flows out
The eluate is mixed into the eluate through a reaction liquid flow path 16 connected to the eluate.

Further, a reaction column 18 is provided on the flow path downstream of the connection point of the eluate flow path 14 with the reaction liquid flow path 16, and each PG separated sequentially by the separation column 4 is provided with a reaction column 18.
A mixed solution of an eluent containing components and a reaction solution is allowed to flow into the reactor.

In this reaction column 18, 15-OTIP CD 11 is held in an immobilized state, and due to the catalytic action of this 15-OHP GDH, the PC component contained in the eluate and NAD' This results in a reaction.

The reaction column 18 containing this 15-0HPGDH adsorbs 15-OHPGDH onto a suitable support.

15-0HPGDH is immobilized by a method such as a covalent bond, and the support is packed into a column, or if a tube is used as a column, the inner surface of the tube itself is immobilized according to the above method. In particular, porous glass beads are used as a support, and after bonding an alkylamine to the surface, 15-(11(P G D A method of chemically immobilizing N on the surface is preferably employed.

The NAD' generated by the reaction within column 18 then flows out of column 18 and is detected and analyzed by a detector 20 provided downstream of column 18 according to conventional fluorometry techniques. That's what happens.

Further, FIG. 2 shows another example of an apparatus suitable for carrying out the present invention. There, a second reaction liquid reservoir 22 and a second reaction liquid pump 24 are further provided in addition to the apparatus of the previous example, and the second reaction liquid in the second reaction liquid reservoir 22 flows out from the reaction column 18. After being added to the effluent, the mixed liquid is made to flow through a reaction coil 26 serving as a reactor, and then guided to a detector 20. It should be noted that parts having the same functions and effects as those of the previous device will be given the same reference numerals and detailed explanations will be omitted.

In short, in the apparatus shown in FIG.
A diaphorase/risazurin solution as a second reaction liquid stored in a second reaction liquid reservoir 22 is mixed into the effluent containing N A D 11 flowing out from the second reaction liquid pump 24. , and reaction coil 26
By flowing through the PC, resorufin is produced, and by measuring the fluorescence of the so-produced sorufin with the detector 20, even trace amounts of each PC component can be analyzed and quantified. -ing

In addition, in the apparatus shown in FIG. 2, a solution containing diaphorase and lisazurin at the same time is used as the second reaction solution, but instead of this, a solution containing lisazurin in the river is used as the second reaction solution. The reaction column 18 is mixed with the effluent flowing out from the reaction column 18.
Similarly, a second reaction column on which diaphorase is immobilized is provided in place of the reaction coil 26, and the second reaction solution containing lisazurin is mixed into the second reaction column. By flowing the liquid, a fluorescent substance: resorufin is generated, and this is sent to the detector 20.
It is also possible to measure by .

In addition, in these devices, in order to effectively generate NADH and resorufin and to make their analysis effective, at least the reaction column 18 portion that generates NADH is heated to a predetermined temperature, generally 10°C to 4
It is necessary to install a constant temperature mechanism to maintain the temperature at about 0°C, and even in the case of the apparatus shown in Fig. 2, at least the reaction column 18 section and the reaction coil 26 section, or at least the reaction column 18 section and the diaphorase It is desirable to provide a constant temperature mechanism for maintaining the immobilized second reaction column portion at a predetermined temperature (approximately 10 to 40'C), either separately or at the same time.

When using such an analyzer, separation of PG, reaction of each separated PC component with NAD'', furthermore, a reaction to produce purfin, and NAD I-1 or NAD I-1 generated by these reactions. Detection of lysorufin can be carried out continuously using expensive 15-0HPGDH.
There is no need to continuously supply (consume)
By simply using -0HPGDH and diaphorase, it has become possible to analyze PG extremely economically, rapidly, and stably.

Incidentally, in order to confirm the effect of the method of the present invention, the results of analysis performed on standard samples containing each PG using the apparatus shown in FIG. 1 are shown in FIG.

The measurement conditions are as follows.

Separation column (4): Co reverse phase column (packed with spherical silica gel of around 10 μm to which octyl groups are chemically bonded) (2.1 dragons, 250 fins long) /8 Synergy: 40 mM phosphorus Acid potassium buffer, pH 6.0
0/ace1-nitrile (75/25, volume ratio)/8 Kagata liquid flow rate: 0.1 ml/min reaction column (
18): Inner diameter 2.1 mm, length 50 mm (15-0HP
Enzyme column with immobilized GDH) Reaction solution: 0.1 M potassium phosphate buffer (pH 7,
Reaction liquid flow rate: 0.1mM/min Detector (20): Fluorescence detector (F, P-210, JASCO Corporation) Excitation wavelength: 340 nm Fluorescence wavelength: 470 nm Measurement temperature: 20"C (both separation column and reaction column) Eluent pump (6): BfP-I C JASCO Corporation ■
] Reaction liquid pump 42): BIP-1 (JASCO Corporation (41)
] Note that 15-OHP G D l-[ was immobilized by the following method.

■ 150mg of aminopropyl CPG (200-40
0 Menshi, Electronucleo, manufactured by Nix Corporation), 2.
Add 1.5 mM of 5% glutaraldehyde.

■ Degas at room temperature for 30 minutes.

■ Further react for 1 hour at room temperature under normal pressure.

■ Wash with distilled water.

■ 15-OHPGDH* (70 units, 0.1M IJ acid buffer (pT) containing 1mM disodium ethylenediaminetetraacetate and 1mM dithiothreitol)
7, 30) dissolved in 1 m/ml].

■ Degas for 30 minutes under water cooling.

■ Further react for 30 minutes at normal pressure under water cooling.

■ Wash with distilled water.

*H, 0hno, Y, Morikawa, and
P. Hirata.

J.Biochem, 84.1485-1494 (
197B).

Furthermore, the analysis was conducted as follows.

That is, 160 ng and 80 ng of PGE1 and PGE2, respectively.
3 .mu.l of an ethanol solution containing .ng was injected at once from the sample injector 8, passed through the separation column 4 and the reaction column 18 set to the measurement conditions described above, and was subjected to fluorescence analysis using the detector 20. Measurements were carried out in the same manner using samples containing various concentrations of PC, and the calibration curve shown in FIG. 4 was obtained.

From this calibration curve, the measurement range of each PG in the measurement method of the present invention is PGE1: 2ng to 100ng1100n
: 1 ng to I00 ng. From this result, it can be said that the analysis method of the present invention is a very sensitive measurement method.

Effects of the Invention As described in detail above, the present invention separates each PG acid component by one method of liquid chromatography using a separation column,
By cleverly combining each PC component with the reaction of NAD= using immobilized 15-0 HPGDI, NAD II can be effectively produced depending on the presence of the PG acid component, and depending on the amount of NADH. Alternatively, in the presence of such generated NADH, it is attempted to convert it into "NAD" and at the same time generate a fluorescent substance: resolufin, and analyze each -PG acid component based on the amount of generation. This simplifies the pretreatment (al); since the enzyme's high substrate specificity is used for reaction detection, the pretreatment is simpler than that of conventional high-performance liquid chromatography, RIA, and gas chromatography methods. (bl specificity is high (high sensitivity); methods other than the conventional RIA method are detection methods that utilize Lachelation of all carboxylic acids or methods that detect without Lachelation, and multi-component systems. However, in the present invention, 15-0HPG
This is an analysis that uses the reaction only for PG with DH,
Because it performs fluorescence measurement of NAD tl or sorufin, it has ten times or more sensitivity than conventional methods. (C) Low analysis cost; 15-OH
By immobilizing expensive enzymes such as PGDH and diaphorase, it is possible to measure four or more PG components in one analysis, and continuous use is possible up to the analysis of about 200 samples. It has excellent features such as , etc., and has great significance therein.

[Brief explanation of drawings]

FIGS. 1 and 2 are system diagrams showing one embodiment of the PG analyzer according to the present invention, FIG. 3 is a chromatogram obtained according to the method of the present invention, and FIG. 4 is a diagram showing a calibration method. Show the line. 2: /8 syneresis reservoir 4: Separation column 6' /5i
PiIl liquid pump 8: Sample injector lO: Reaction liquid reservoir
12; Reaction liquid pump 14: Eluent flow path 16: Reaction liquid flow path 18 Two reaction columns 20: Detector 22: Second reaction liquid reservoir 24: Second reaction liquid pump 26: Reaction coil

Claims (14)

[Claims] (1) After separating a sample containing prostaglandins having an OH group at the 15-position into each component using a separation column, the effluent containing each prostaglandin component separated sequentially is added to the nicotinamide adenine diluted sample. After sequentially reacting each prostaglandin component with the nicotinamide adenine dinucleotide by flowing it through a reaction column containing immobilized 15-hydroxyprostaglandin dehydrogenase in a state containing nucleotides. A method for analyzing prostaglandins, which comprises detecting each prostaglandin component by measuring the fluorescence of a substance produced by such a reaction. (2) the nicotinamide adenine dinucleotide,
It is mixed into the effluent from the separation column as a reaction solution containing the immobilized 1
The analytical method according to claim 1, wherein the analytical method is caused to flow through a reaction column containing 5-hydroxyprostaglandin dehydrogenase. (3) After separating a sample containing prostaglandins having an OH group at the 15th position into each component using a separation column, the effluent containing each prostaglandin component separated sequentially is added to nicotinamide adenine dichloromethane. After sequentially reacting each prostaglandin component with the nicotinamide adenine dinucleotide by flowing it through a reaction column containing immobilized 15-hydroxyprostaglandin dehydrogenase in a state containing nucleotides. The method is further characterized in that diaphorase is mixed with this and reacted in the presence of risazurin to generate a fluorescent substance: resorufin, and the fluorescence is measured to detect each prostaglandin component. Method for analyzing prostaglandins. (4) the nicotinamide adenine dinucleotide,
It is mixed into the effluent from the separation column as a reaction solution containing the immobilized 1
The analytical method according to claim 3, wherein the analyte is passed through a reaction column containing 5-hydroxyprostaglandin dehydrogenase. (5) The rizazurin is added to the effluent containing the reaction product of each of the prostaglandin components and the nicotinamide adenine dinucleotide, together with or separately from the diaphorase. Analysis method described in Section 4. (6) After separating a sample containing prostaglandins having an OH group at the 15th position into each component using a separation column, the effluent containing each prostaglandin component separated sequentially is added to nicotinamide adenine dichloromethane. Each prostaglandin component and the nicotinamide adenine dinucleotide are sequentially reacted by flowing the mixture into a first reaction column containing immobilized 15-hydroxyprostaglandin dehydrogenase in a state containing nucleotides. After that, this is further passed through a second reaction column having immobilized diaphorase in the presence of risazurin to cause a reaction.
A prostaglandin analysis method characterized by producing a fluorescent substance: resorufin and measuring its fluorescence to detect each prostaglandin component. (7) The nicotinamide adenine dinucleotide is
It is mixed into the effluent from the separation column as a reaction solution containing the immobilized 1
7. The analytical method according to claim 6, wherein the analyte is passed through a reaction column containing 5-hydroxyprostaglandin dehydrogenase. (8) The rizazurin is added together with the diaphorase or separately to the effluent containing the reaction product of each of the prostaglandin components and the nicotinamide adenine dinucleotide. Analysis method described in Section 7. (9) A separation mechanism using a liquid chromatography method that sequentially separates a sample containing prostaglandins having an OH group at the 15th position into each component using a separation column, and each prostaglandin component flowing out from the separation mechanism. a mixing mechanism connected to a flow path through which the effluent is allowed to mix a reaction solution containing nicotinamide adenine dinucleotide into the effluent; and an immobilized 15-hydroxyprostaglandin dehydrogenase; a reaction column through which a mixture of a reaction solution and an effluent formed by a mixing operation is caused to sequentially react with each prostaglandin component in the mixture with the nicotinamide adenine dinucleotide; 1. A prostaglandin analysis device, comprising: a detection means provided on the downstream side of a column for detecting reaction products contained in the effluent flowing out from the reaction column by fluorescence measurement. (10) The apparatus according to claim 9, further comprising a constant temperature mechanism that maintains at least the reaction column portion at a predetermined temperature. (11) A separation mechanism using a liquid chromatography method that sequentially separates a sample containing prostaglandins having an OH group at the 15th position into each component using a separation column, and each prostaglandin component flowing out from the separation mechanism. a first mixing mechanism connected to a flow path through which the effluent is caused to mix a reaction solution containing nicotinamide adenine dinucleotide into the effluent; and immobilized 15-hydroxyprostaglandin dehydrogenase; A mixed solution of the reaction solution and the effluent formed by the mixing operation by the first mixing mechanism is circulated, and each prostaglandin component in the mixed solution is caused to react with the nicotinamide adenine dinucleotide in sequence. a reaction column; a second mixing mechanism that is connected to a flow path through which the effluent flowing out from the reaction column flows and mixes diaphorase and risazurin into the effluent; and a second mixing mechanism that is formed by a mixing operation by the second mixing mechanism. A reactor that causes a predetermined reaction to flow through the mixed solution to produce a fluorescent substance: resorufin; 1. A prostaglandin analysis device comprising: detection means for detecting each prostaglandin component by measuring fluorescence. (12) The apparatus according to claim 11, further comprising a constant temperature mechanism for maintaining at least the reaction column portion and the reactor portion at a predetermined temperature separately or simultaneously. (13) A separation mechanism using a liquid chromatography method that sequentially separates a sample containing prostaglandins having an OH group at the 15th position into each component using a separation column, and each prostaglandin component flowing out from the separation mechanism. a first mixing mechanism connected to a flow path through which the effluent is caused to mix a reaction solution containing nicotinamide adenine dinucleotide into the effluent; and immobilized 15-hydroxyprostaglandin dehydrogenase; A mixed solution of the reaction solution and the effluent formed by the mixing operation by the first mixing mechanism is circulated, and each prostaglandin component in the mixed solution is caused to react with the nicotinamide adenine dinucleotide in sequence. A first reaction column, a second mixing mechanism connected to a flow path through which an effluent flowing out from the first reaction column is allowed to flow, and which mixes risazurin into the effluent, and has an immobilized diaphorase. a second reaction column that generates a fluorescent substance: resorufin by causing a predetermined reaction to flow through the mixed liquid formed by the mixing operation by the second mixing mechanism; and the second reaction column. and a detection means for detecting each prostaglandin component by measuring fluorescence of resorufin contained in the effluent flowing out from the second reaction column. Prostaglandin analyzer. (14) The apparatus according to claim 13, further comprising a constant temperature mechanism that maintains at least the first reaction column portion and the second reaction column portion at a predetermined temperature separately or simultaneously.