TWI532992B - Method and system for measuring valproic acid and metabolic product thereof - Google Patents

Description

Method and system for measuring valproic acid and its metabolites

The present invention relates to a method, system and kit for measuring valproic acid and its metabolites.

Valproic acid (VPA) is a clinically used small molecule fatty acid drug widely used in the treatment of various types of epilepsy and as a mood-stable drug. Neuromedicine often uses this drug to prevent neurodegenerative diseases and as a protective nerve. System function.

At present, valproic acid can be used as an oral preparation, syrup and injection, and has different blood concentrations in the treatment of various indications. In addition, since valproic acid is mainly used for drug metabolism through the liver, excessive use may cause liver toxicity, and Causes side effects such as fetal malformations and neural tube defects and defects. In recent years, valproic acid has been found in cancer research to inhibit the proliferation of cancer cells as a new use for the treatment of cancer.

In clinical treatment applications, improper doses may cause side effects such as liver failure, pediatric pancreatitis and hyperammonemia. In metabolism, valproic acid is mainly metabolized by liver CYP450 enzyme, producing 50 metabolites, of which the main metabolite 2-propyl-4-pentenoic acid has hepatotoxicity. Secondary metabolism is converted to (E)-2,4-diene VPA ((E)-2,4-diene VPA), causing drug-induced liver injury (DILI). Therefore, whether it is used alone or for combination therapy, it must be clinically necessary. Carefully monitor the blood concentration of the patient's valproic acid.

More than 90% of the valproic acid in the blood binds to the albumin protein in the blood and increases the difficulty of monitoring in the blood. Due to the complex composition of blood samples, including plasma blood cell antibodies and plasma proteins, the past literature uses blood extraction methods, including liquid liquid extraction (LLE) and solid-phase extraction (SPE).

Solid phase extraction is a technique commonly used for filtration and separation. According to the type of sample to be tested, there are different extraction phases. The solid phase extraction tube is used for blood pretreatment. The extraction step includes activation of the column and washing. ), loading (loading) and eluting (elute), obtaining the analyte and then analyzing it by other methods.

On the other hand, valproic acid concentrations can currently be measured by the inclusion of liquid chromatography mass spectrometry and gas chromatography mass spectrometry. In the measurement process, the pretreatment is carried out by solid phase extraction, the internal standard is added and the above mass spectrometer is used to determine the concentration of blood valproic acid; in addition, there is another document (name: Simultaneous determination of mycophenolic acid and valproic acid based on Derivatization by high-performance liquid chromatography with fluorescence detection, Biomedical Chromatography 2006, 20(4): 319-26) Using a liquid chromatography with fluorescence detection to simultaneously measure mycophenolic acid and valproic acid in plasma In the method, the sample needs to be treated with a chlorine-containing organic solvent in the pretreatment, and the structure of the compound cannot be analyzed during the measurement by the analytical instrument; in addition, the time for analyzing the sample using the instrument is rather lengthy.

The above analysis method has the disadvantage that only one sample can be analyzed during the analysis process. The pre-treatment cost is high, and the blood pre-treatment step plus the liquid phase analyzer balance and analysis time is lengthy. Therefore, the market is still looking forward to the development of a rapid and safe method for detecting valproic acid as a basis for clinical use in individual medicine.

In view of the shortcomings of the past methods for analyzing valproic acid and its metabolites, the present invention utilizes a chemical conversion method of valproic acid and utilizes a matrix-assisted laser desorption free method and a mass analyzer for structural analysis and quantification.

The quantitative method of the present invention may be selected from a valproic acid isotope or a valproic acid metabolite isotope such as d ₆ -valproic acid (valproic acid-d ₆ , a valproic acid ruthenium calibration form) as an internal standard.

As used herein, "isotopes of valproic acid isotope or valproic acid metabolite" are isotopic compounds in which valproic acid or its metabolite is synthesized by organic chemistry, and a hydrogen group is replaced by a methyl group (-CH ₃ ) with hydrazine. , d ₆ -valproic acid and valproic acid and its metabolite 2-acet-4-pentenoic acid (4-ene valproic acid) both have a carboxyl group (-COOH), as shown in Figure 1, the present invention utilizes This internal standard, d ₆ -valproic acid, is jointly labeled by a chemical derivatization reaction, a derivatization reagent, a matrix-assisted laser desorption free method, and a mass analyzer. Analyze the peak area of the internal standard according to the mass spectrometer mass analyzer, and bring the value to the calibration curve of valproic acid or its metabolite 2-propyl-4-pentenoic acid compared with the peak area of the sample to be tested. , the content of valproic acid or 2-propyl-4-pentenoic acid in the sample to be tested can be detected.

Therefore, the present invention provides a method for measuring valproic acid and a metabolite thereof, comprising the steps of: (a) providing a sample containing a metabolite of valproic acid or valproic acid; (b) using a solution; Extracting the valproic acid or valproic acid metabolite in the sample to obtain an extract; (c) providing an internal standard and adding the extract, wherein the internal standard is a valproic acid isotope Or a valproic acid metabolite isotope; (d) adding a derivatization reagent to the extract, and heating to carry out a chemical derivatization reaction, a derivatization solution containing a product having a derivatization reagent label, wherein the derivatized reagent-labeled product a valproic acid having a derivatization reagent label, a valproic acid metabolite with a derivatization reagent label or an internal standard with a derivatization reagent label; and (e) a matrix-assisted laser desorption free method and mass analyzer The derivatization solution was assayed by analysis and the amount of valproic acid or valproic acid metabolite was calculated using the internal standard labeled with the derivatization reagent.

In a specific embodiment, the sample is a blood sample or a drug sample. When the sample is a blood sample, the following pre-processing steps may be included between steps (a) and (b): centrifuging the blood sample to separate the bleeding ball portion and the serum portion; and removing the serum portion and An acidic solution is mixed and acidified to obtain a sample to be tested. In another embodiment, the acidic solution can be phosphoric acid, hydrochloric acid or sulfuric acid. When the sample is a drug sample, it may be included between steps (a) and (b): grinding the drug sample to a fine powder.

In a specific embodiment, the solvent used in the present invention to extract the metabolite of valproic acid or valproic acid is n-hexane, toluene, dichloromethane or acetonitrile.

In a specific embodiment, the derivatization reagent used in the present invention is 4-bromomethyl-6,7-dimethoxycoumarin (BrDMC) or 4-bromomethyl-7-methoxycoumarin (BrMMC) at a concentration ranging from about 0.25 to about 5 mM.

In a particular embodiment, the isotopic valproic acid is used in the present invention, d ₆ - propionic acid.

In a specific embodiment, the matrix-assisted laser desorption free method used in the present invention may be a substrate of α-cyano-4-hydroxycinnamic acid, dihydroxybenzoic acid or 3,5-dimethoxy- 4-hydroxycinnamic acid.

In a specific embodiment, the mass analyzers that may be utilized in the present invention include, but are not limited to, a time of flight analyzer (TOF analyzer), a quadrupole analyzer, and ion trap mass analysis. (ion trap analyzer) or Fourier transform-ion cyclotron resonance (FT-ICR).

In another embodiment, the present invention can determine sample concentration using high performance liquid chromatography (HPLC-UV) or liquid chromatography mass spectrometry (LC/MS/MS).

The valproic acid metabolite described herein comprises 2-propyl-4-pentenoic acid (4-ene VPA), 2-propyl-3-hydroxypentenoic acid (3-OH valproic acid), 2-prop 4--4-hydroxypentenoic acid (4-OH valproic acid), 2-propyl-5-hydroxypentenoic acid (5-OH valproic acid), 2-propyl-2-pentenoic acid (2- Ene VPA), 2-propyl-3-pentenoic acid (3-ene VPA), 2-propyl-3-ketopentenoic acid (3-keto VPA) or 2-propyl-4-ketopentyl 4-keto VPA, 2,4-diene VPA.

In a specific embodiment, the extract of the measuring method of the present invention may be an extract obtained by drying an extract or an extract.

In a specific embodiment, the measuring method of the present invention may further add an activator and/or a catalyst to the extract in the step (d), and the catalyst may increase the efficiency of the chemical derivatization reaction. In a preferred embodiment, the activator is potassium carbonate or potassium bicarbonate in a concentration ranging from about 0.5 to 6 mg; in another preferred embodiment, the catalyst can be 1, 4, 7, 10, 13,16-hexaoxacyclooctadecane in a concentration ranging from about 0.01 to 25 mM.

In another aspect, the invention further provides a method for measuring valproic acid and its metabolites a system comprising: an internal standard which is a valproic acid isotope or a valproic acid metabolite isotope; a derivatization reagent for labeling valproic acid, a valproic acid metabolite or an internal standard in a chemical derivatization reaction a heater for heating the chemical derivatization reaction; a substrate capable of absorbing a laser beam having a wavelength of 330-360 nm, wherein the substrate is α-cyano-4-hydroxycinnamic acid, dihydroxybenzoic acid or 3,5 - Dimethoxy-4-hydroxycinnamic acid; a laser source for emitting a laser beam; and a mass analyzer for analyzing the amount of valproic acid or valproic acid metabolite in the sample.

In a specific embodiment, the derivatization reagent used in the system of the invention is 4-bromomethyl-6,7-dimethoxycoumarin (BrDMC) or 4-bromomethyl-7-methoxy Beansin (BrMMC) at a concentration ranging from about 0.25 to about 5 mM.

In a specific embodiment, the system of the present invention further comprises an activator and/or a catalyst. In a preferred embodiment, the activator used in the system of the present invention is potassium carbonate or potassium bicarbonate in a concentration ranging from about 0.5 to about 6 mg; in another preferred embodiment, the catalyst used in the system of the present invention It is 1,4,7,10,13,16-hexaoxacyclooctadecane in a concentration ranging from about 0.01 to 25 mM.

The invention also provides a kit for measuring valproic acid and its metabolite, comprising: an internal standard comprising a valproic acid isotope or a valproic acid metabolite isotope; a derivatization reagent for A valproic acid, a valproic acid metabolite or an internal standard is labeled in a chemical derivatization reaction; and a matrix that absorbs a laser beam having a wavelength of 330-360 nm.

In a specific embodiment, the derivatization reagent of the kit of the invention is 4-bromomethyl-6,7-dimethoxycoumarin (BrDMC) or 4-bromomethyl-7-methoxycoumarin (BrMMC).

In a specific embodiment, the matrix of the kit of the invention is α-cyano-4-hydroxy laurel Seroic acid, dihydroxybenzoic acid or 3,5-dimethoxy-4-hydroxycinnamic acid.

In a specific embodiment, the kit of the present invention further comprises an activator and/or a catalyst. In a preferred embodiment, the activator used in the kit of the present invention is potassium carbonate or potassium bicarbonate in a concentration ranging from about 0.5 to about 6 mg; in another preferred embodiment, the kit of the present invention is used. The catalyst is 1,4,7,10,13,16-hexaoxacyclooctadecane in a concentration ranging from about 0.01 to 25 mM.

In a specific embodiment, the kit of the present invention further comprises a heater for heating to carry out a chemical derivatization reaction.

In a specific embodiment, the kit of the present invention further comprises a mass analyzer for analyzing the amount of valproic acid or valproic acid metabolite in the assay sample.

In a preferred embodiment, the detection method of the present invention requires only about 50 minutes of detection time, and can be directly analyzed by matrix-assisted laser desorption free time-of-flight mass spectrometry (MALDI-TOF MS) analysis. Blood samples are suitable for a large number of sample tests. On the other hand, in the application of pharmaceutical preparations, it can be used for drug analysis and quality management of pharmaceutical production. In addition, with the measuring kit of the present invention, the sample to be tested is subjected to a chemical derivatization reaction process, and is heated in a general microwave oven to perform analysis.

The method and the kit of the invention can specifically detect valproic acid and its metabolites, and can completely avoid biological interference with respect to other measuring methods, and only need a small amount of samples and reagents to obtain accurate values, regardless of application in medicine Testing or pharmaceutical drug analysis are rapid, efficient and environmentally friendly analytical methods.

The words "including", "having", "including", or "including" are used in this specification and the scope of the claims, and are not intended to be Or method steps.

The invention is further illustrated by the following detailed description of the embodiments of the invention.

Figure 1 is a flow chart of the chemical derivatization reaction of the present invention.

2 is a process of the chemical derivatization reaction step for measuring valproic acid and its metabolites according to the present invention.

Figure 3 is a valproic acid blood test curve.

Figure 4 is a 2-propyl-4-pentenoic acid blood test curve.

Figure 2 is a process of the steps of measuring valproic acid and its metabolites in accordance with the present invention. In addition to analyzing the blood of patients taking valproic acid, the present invention can also be applied to analyze the purity and content of valproic acid drugs.

Example 1: Blood sample

Sample acquisition and pre-processing (step 101)

Obtain the blood sample of the test subject and perform pretreatment: separate the blood sample from the hemorrhage ball part and the serum part, and take 20 μL of serum as the sample to be tested. Since the serum is a complex biochemical sample, the acidic solution needs to be further (Phosphoric acid, hydrochloric acid or sulfuric acid) is added to the blood sample to be tested to adjust the pH to acidify it to remove valproic acid or 2-propyl-4-pentenoic acid from the serum fraction. 1 μL of 85% phosphoric acid is uniformly mixed with the sample to be tested, the purpose of which is to acidify free valproic acid in the blood to reduced valproic acid, and the free state of 2-propyl-4-pentenoic acid is acidified. It is then reduced to 2-propyl-4-pentenoic acid.

Sample extraction (step 102)

After solvent extraction, a fat-soluble solvent such as n-hexane, toluene or dichloromethane can be selected. In this example, 100 μL of toluene is used as the extraction solvent. After shaking the solvent and sample for 30 seconds to 2 minutes, centrifuge in a high-speed refrigerated centrifuge for 1-30 minutes (10000-13000 rpm, 4 ° C), take 80 μL of the extract to another microcentrifuge tube, and vacuum the removed organic layer. After the air concentrator is dried, the chemical derivatization reaction step is continued.

Internal standard and chemical derivatization reactions (steps 103, 104)

Add 5 μL of valproic acid isotope or valproic acid metabolite isotope (eg 80-100 μM d ₆ -valproic acid) to the extract containing valproic acid and 2-propyl-4-pentenoic acid as internal standard ( In step 103), an activator, a catalyst, and a derivatization reagent are added for chemical derivatization (step 104) to increase detection sensitivity.

The activator used in this example was 2 mg potassium carbonate, the catalyst was 5 μL 1,4,7,10,13,16-hexaoxacyclooctadecane (18-crown-6, 10 mM) and the derivatization reagent was 5 μL 4 -bromomethyl-6,7-dimethoxy coumarin (BRDMC).

Next, the chemical derivatization reaction is carried out by dry heating (30 ° C - 80 ° C, 10-60 minutes) or microwave heating (150 - 190 W, 2 - 10 minutes) to obtain a labeled derivative solution, wherein the solution comprises valproic acid derivative having derivatization reagent labeled with derivatization reagents labeled 2-propyl-4-pentenoic acid and / or with derivatization reagents labeled d ₆ - propionic acid.

Sample analysis determination (step 105)

Take 0.5 μL of the derivatization solution for matrix-assisted laser desorption free method, load the derivatization solution onto the sample tray, cover 0.5 μL of the matrix solution (5 mg/mL), and mix the derivatized solution and the matrix solution evenly. After volatilization, the product with the derivatization reagent label in the matrix and the derivatization solution forms a crystalline solid.

The substrate solution used in the present invention can be selected from α-cyano-4-hydroxycinnamic acid (CHCA) and dihydroxybenzoic acid (2,5-) which absorb a laser beam having a wavelength of 330-360 nm. Dihydroxybenzoic acid, DHB) or 3,5-dimethoxy-4-hydroxycinnamic acid (sinapic acid, SA). In this example, the substrate solution used 5-10 mg/mL α-cyano-4-hydroxycinnamic acid (CHCA).

The crystalline solid is then irradiated with a laser beam (wavelength 330-360 nm) to excite the crystalline solid to form a gas phase ion that can be accelerated into the mass analyzer via an electric field to further detect the The charge-to-mass ratio (m/z) of the gas phase ions to obtain the molecular weight of the analyte.

In this example, the mass analyzer may select a time of flight analyzer (TOF analyzer), a quadrupole analyzer, an ion trap analyzer, or a Fourier. Fourier transform-ion cyclotron resonance (FT-ICR).

Finally, according to the mass spectrometer mass analyzer, the peak area of the d ₆ -valproic acid labeled with the derivatization reagent is analyzed, and the valproic acid labeled with the derivatization reagent and the 2-propyl-4-pentane with the derivatization reagent are labeled. The ratio of the peak area of the sample of 4-ene valproic acid to the calibration curve of valproic acid or 2-propyl-4-pentenoic acid can be calculated in the sample to be tested. The content of valproic acid or 2-propyl-4-pentenoic acid.

On the other hand, this example can also detect the concentration of a sample to be tested using a high performance liquid chromatography (HPLC-UV) or a liquid chromatography mass spectrometer (LC/MS/MS).

Example 2: Drug sample

Sample acquisition and pre-processing (step 101)

The drug in a lozenge form is physically pulverized to a fine powder, such as a mortar tool.

Sample extraction (step 102)

Take the fine powder of the medicine, add solvent to dissolve and extract, such as toluene and acetonitrile. In this example, acetonitrile (drug to solvent ratio: 1 mg/μL) is used, and ultrasonic vibration is used for 5-15 minutes to extract valproic acid, followed by 10000. Centrifugation at 13,000 rpm for 3-10 minutes followed by a chemical derivatization reaction step.

Internal standard and chemical derivatization reactions (steps 103, 104)

10 μL of the extract was taken out to add a valproic acid isotope or a valproic acid metabolite isotope as an internal standard (step 103), and an activator, a catalyst, and a derivatization reagent were added for chemical derivatization (step 104).

The activator used in this example was 2 mg potassium carbonate, the catalyst was 10 μL 1,4,7,10,13,16-hexaoxacyclooctadecane (18-crown-6, 0-25 mM) and the derivatization reagent was 5 μL of 4-bromomethyl-6,7-dimethoxy coumarin (BRDMC).

The chemical derivatization reaction is carried out by dry heating (30 ° C - 80 ° C, 10-60 minutes) or microwave heating (150-190 W, 2-10 minutes) to obtain a labeled derivative solution, wherein the derivative derivatization reagent solution containing labeled with valproate and d ₆ / or with derivatization reagents labeled - propionic acid.

Sample analysis determination (step 105)

Take 0.5 μL of the derivatized solution for matrix-assisted laser desorption free method, place the derivatized solution on the sample tray, cover 0.5 μL of the matrix solution (10 mg/mL), and uniformly mix the derivatized solution with the matrix solution. The product with the derivatization reagent label in the matrix and the derivatization solution forms a crystalline solid.

The substrate solution used in the present invention can be selected from α-cyano-4-hydroxycinnamic acid (CHCA) and dihydroxybenzoic acid (2,5-) which absorb a laser beam having a wavelength of 330-360 nm. Dihydroxybenzoic acid, DHB) or 3,5-dimethoxy-4-hydroxycinnamic acid (sinapic acid, SA). In this example, the substrate solution used 5-10 mg/mL α-cyano-4-hydroxycinnamic acid (CHCA).

The crystalline solid is then irradiated with a laser beam (wavelength 330-360 nm) to excite the crystalline solid to form a gas phase ion that can be accelerated into the mass analyzer via an electric field to further detect the The charge-to-mass ratio (m/z) of the gas phase ions to obtain the molecular weight of the analyte.

In this example, the mass analyzer may select a time of flight analyzer (TOF analyzer), a quadrupole analyzer, an ion trap analyzer, or a Fourier transform. Fourier transform-ion cyclotron resonance (FT-ICR).

Finally, according to the mass spectrometer mass analyzer, the peak area of the d ₆ -valproic acid labeled with the derivatization reagent is analyzed, and the value is brought into the valproic acid compared with the peak area of the sample of the valproic acid labeled with the derivatization reagent. The calibration curve can calculate the content of valproic acid in the sample to be tested.

On the other hand, this example can also detect the concentration of a sample to be tested using a high performance liquid chromatography (HPLC-UV) or a liquid chromatography mass spectrometer (LC/MS/MS).

Table 1 is a list of reaction reagents in the chemical derivatization step and the analysis step for the different samples in Example 1 and Example 2.

Example 3: Analysis of valproic acid or its metabolite directly into blood

Through the same day and different day analysis results, the valproic acid in the blood and its metabolite 2-isopropyl-4-pentenoic acid (4-ene valproic acid) are labeled by the derivatization reaction through the measurement method of the present invention. The linear relationship between the six concentrations of valproic acid and 2-propyl-4-pentenoic acid was determined as shown in Figures 3 and 4.

The valproic acid blood quantitation range is 10-1000 μM, the toxic metabolite 2-propyl-4-pentenoic acid has a blood quantification range of 5-500 μM, the coefficient of determination (R ² ) is greater than 0.999, and the relative standard deviation (relative standard) Deviation, RSD) is less than 5.73%. The above results demonstrate that the method of the present invention has high stability and reproducibility, and can measure the content of valproic acid and metabolites in the blood of patients after using valproic acid drugs, and can monitor the concentration of drug treatment as an individual medical treatment. in accordance with.

Example 4

The average concentration of valproic acid and its metabolites was measured by the method of the present invention 4 hours after oral administration of the 500 mg valproic acid preparation of the subject of the present example, and the results are shown in Table 2. As can be seen from the results of Table 2, the smaller the value of the relative standard deviation, the more accurate the detection step using the present invention and the higher the data reliability.

Example 5

In addition, the measurement method of the present invention is applied to drug analysis, the clinical drug is used as a sample, the tablet assay uniformity test is performed, and the standard product calibration curve is established for quantification and determination coefficient ( The coefficient of determination, R ² ) is greater than 0.998, and from the results of Table 3, the relative standard deviation (RSD) in the present example is less than 2.77%, and the drug purity is within the range indicated by the pharmaceutical company, so the present invention The analysis results caused by the testing steps are in line with production management standards.

The "recovery rate" as used in the present example means that the actual weight of the valproic acid-containing drug by the method of the present invention/the pharmaceutical manufacturing trademark shows the weight of the valproic acid-containing drug × 100%, and the higher the recovery rate and the closer to 100%, the representative The more precise the method of the invention.

The specific embodiments described in the present invention are intended to illustrate and describe the invention. Many possible modifications and variations are possible in the above teachings.

The above-mentioned facts and examples are chosen merely to explain the principles of the invention and its practical use, and to enable those skilled in the art or the art to use or practice the invention, A person skilled in the art or a person skilled in the art may thus invent other applications, as long as they do not depart from the gist of the present invention. The scope of the invention is therefore intended to be defined in the scope of the claims

Claims (22)

A method for measuring valproic acid and its metabolites, which is measured by the following steps: (a) providing a sample containing a valproic acid or a valproic acid metabolite; (b) performing a pretreatment of the sample; c) separating the valproic acid or valproic acid metabolite in the sample by a solvent extraction to obtain an extract; (d) providing an internal standard and adding the extract, wherein the internal standard is a valproic acid isotope or a valproic acid metabolite isotope; (e) adding a derivatization reagent to the extract and heating to carry out a chemical derivatization reaction to obtain a derivatized solution containing a product having a derivatization reagent label, wherein the The derivative reagent-labeled product comprises valproic acid labeled with a derivatizing reagent, a valproic acid metabolite with a derivatizing reagent label or an internal standard with a derivatization reagent label; and (f) matrix-assisted laser desorption The free method and mass analyzer are used to analyze the derivatization solution and calculate the content of the valproic acid or valproic acid metabolite using the internal standard labeled with the derivatization reagent. The method of measuring according to claim 1, wherein the sample is a blood sample or a drug sample. The method of measuring according to claim 2, wherein the pretreatment of the blood sample is: centrifuging the blood sample to separate the hemosphere portion and the serum portion; and removing the serum portion and being acidic The solution is mixed and acidified to obtain a sample to be tested. The measuring method according to claim 3, wherein the acidic solution is phosphoric acid or hydrochloric acid. Or sulfuric acid. The method of measuring according to claim 2, wherein the pretreatment of the drug sample is: grinding the drug sample into a fine powder. The method of measuring according to claim 1, wherein the solvent is n-hexane, toluene, dichloromethane or acetonitrile. The method of measuring according to claim 1, wherein the derivatization reagent is 4-bromomethyl-6,7-dimethoxycoumarin (BrDMC) or 4-bromomethyl-7- Oxycoumarin (BrMMC). The method of measuring according to claim 7, wherein the concentration of the derivatization reagent ranges from 0.25 to 5 mM. The method of measuring according to claim 1, wherein the valproic acid isotope is d ₆ -valproic acid. The measuring method according to claim 1, wherein the mass analyzer is a flying time quality analyzer, a quadrupole mass analyzer, an ion trap mass analyzer, and a Fourier transform mass analyzer. The method of measuring according to claim 1, wherein the valproic acid metabolite comprises 2-propyl-4-pentenoic acid (4-ene VPA), 2-propyl-3-hydroxypentenoic acid (3-OH valproic acid), 2-propyl-4-hydroxypentenoic acid (4-OH valproic acid), 2-propyl-5-hydroxypentenoic acid (5-OH valproic acid), 2 -propyl-2-pentenoic acid (2-ene VPA), 2-propyl-3-pentenoic acid (3-ene VPA), 2-propyl-3-ketopentenoic acid (3-keto VPA) Or 2-propyl-4-ketopentanoic acid (4-keto VPA), 2,4-dienyl valeric acid (2,4-diene VPA). The measuring method according to claim 1, wherein the activator may be further added in the step (d) And/or a catalyst is added to the extract. The method of measuring according to claim 12, wherein the activator is potassium carbonate or potassium hydrogencarbonate; and the catalyst is 1,4,7,10,13,16-hexaoxacyclooctadecane. The method of measuring according to claim 13, wherein the activator has a concentration ranging from 0.5 to 6 mg; and the catalyst has a concentration ranging from 0.01 to 25 mM. A system for measuring valproic acid and its metabolites comprising: an internal standard which is a valproic acid isotope or a valproic acid metabolite isotope; a derivatization reagent for labeling in a chemical derivatization reaction a valproic acid, a valproic acid metabolite or an internal standard; a heater for heating for a chemical derivatization reaction; a substrate capable of absorbing a laser beam having a wavelength of 330-360 nm, wherein the substrate is α-cyano-4 - hydroxy cinnamic acid, dihydroxybenzoic acid or 3,5-dimethoxy-4-hydroxycinnamic acid; a laser source for emitting a laser beam; and a mass analyzer for analyzing the sample C The content of valeric acid or valproic acid metabolites. The system of claim 15 wherein the derivatization reagent is 4-bromomethyl-6,7-dimethoxycoumarin (BrDMC) or 4-bromomethyl-7-methoxy. Coumarin (BrMMC). The system of claim 15 further comprising an activator and/or a catalyst. The system of claim 17, wherein the activator is potassium carbonate or potassium hydrogencarbonate; and the catalyst is 1,4,7,10,13,16-hexaoxacyclooctadecane. A kit for measuring valproic acid and its metabolites comprising: an internal standard comprising a valproic acid isotope or a valproic acid metabolite isotope; a derivatization reagent for use in a chemical derivatization reaction Mark valproic acid, valproic acid metabolite Or an internal standard, the derivatization reagent is 4-bromomethyl-6,7-dimethoxycoumarin (BrDMC) or 4-bromomethyl-7-methoxycoumarin (BrMMC); A substrate which absorbs a laser beam having a wavelength of 330-360 nm and which is α-cyano-4-hydroxycinnamic acid, dihydroxybenzoic acid or 3,5-dimethoxy-4-hydroxycinnamic acid. The kit of claim 19, further comprising an activator and/or a catalyst. The kit of claim 19, further comprising a heater for heating for chemical derivatization. The kit of claim 19, further comprising a mass analyzer for analyzing the amount of valproic acid or valproic acid metabolite in the assay sample.