Method for analyzing diene compounds and method for producing diene compounds

Abstract

To provide a method for analyzing a diene compound using the triazolin dione adduct, enabling a smooth derivatization reaction of a diene compound and as a result, enabling an analysis of a minute amount of diene compound, through emitting a sufficient triazolin dione compound while preventing the diene compound from deteriorating by heat, and a method for producing an ene compound in which the triazolin dione compound is derivatized.SOLUTION: In a method for analyzing a diene compound, a triazolin dione adduct is heated to emit a triazolin dione compound, and the emitted triazolin dione compound and a diene compound are reacted. Specifically, the method for analyzing a diene compound includes: a triazolin dione adduct heating step in which a triazolin dione adduct is heated to generate a triazolin dione compound; an ene compound formation step of reacting the triazolin dione compound and a diene compound to obtain an ene compound; and an ene compound analysis step of analyzing the ene compound to quantify the diene compound.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a method for analyzing a diene compound and a method for producing an ene compound. More specifically, the present invention relates to a method for analyzing an extremely small amount of a diene compound with high accuracy using a stable triazolinedione adduct, and a method for producing an ene compound that is a derivative of a diene compound. [Background technology]

[0002] In recent years, there has been an increasing need to analyze vitamin D and vitamin D metabolites in blood. As a method for analyzing vitamin D and vitamin D metabolites, a method has been proposed in which vitamin D and vitamin D metabolites are derivatized using a Cookson-type derivatization reagent, and then the derivative is analyzed.

[0003] Specifically, under mild conditions, Cookson-type derivatization reagents undergo extremely rapid Diels-Alder reaction with diene compounds such as vitamin D to quantitatively produce ene compounds (see Non-Patent Documents 1 to 4). Taking advantage of this reaction characteristic, vitamin D and other compounds that are difficult to quantify as they are are reacted with Cookson-type derivatization reagents to convert them into ene compounds with high analytical sensitivity, and then the ene compounds are analyzed.

[0004] Examples of the Cookson type derivatization reagent include triazolinedione compounds such as PTAD (4-phenyl-1,2,4-triazoline-3,5-dione) and DAPTAD (4-(4'-dimethylaminophenyl)-1,2,4-triazoline-3,5-dione). [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] S.Ogawa,et al., Rapid Commun.Mass Spectrom, 27(2013) 2453-2460 [Non-Patent Document 2] S.Ogawa,et al., Biomed.Chromatgr., 30(2016) 938-945 [Non-Patent Document 3] S.Ogawa,et al., J.Pharm.Biomed.Anal., 136(2017) 126-133 [Non-Patent Document 4] KDBruycker,et al.,Chem.Rev., 116(2016) 3919-3974 Summary of the Invention [Problem to be solved by the invention]

[0006] However, the triazolinedione compounds used as Cookson-type derivatization reagents are unstable compounds, and higher stability is required for handling as analytical reagents. Therefore, the unstable triazolinedione compounds are converted into stable adducts, which are then converted back to triazolinedione compounds when used, and reacted with diene compounds, allowing them to be used as analytical reagents for diene compounds that are highly stable and easy to handle.

[0007] Usually, triazolinedione adducts generate triazolinedione compounds by applying heat. However, some diene compounds, including vitamin D3, which are the target substances to be analyzed, are sensitive to heat. Therefore, when a triazolinedione adduct is used to perform a derivatization reaction of a diene compound, the measurement sensitivity and accuracy as an analytical reagent may change depending on the conditions, and depending on the method of use, the measurement sensitivity may decrease, and even inaccurate analysis results may be obtained.

[0008] The present invention has been made in view of the above-mentioned background art, and an object of the present invention is to provide a method for analyzing a diene compound using a triazolinedione adduct, which releases a sufficient amount of triazolinedione compound and suppresses deterioration of the diene compound due to heat, thereby smoothly carrying out a derivatization reaction of the diene compound, thereby enabling analysis of trace amounts of the diene compound, and a method for producing an ene compound derived from a triazolinedione compound. [Means for solving the problem]

[0009] The present inventors have conducted extensive research to solve the above problems, and have found that by heating a triazolinedione adduct to release a triazolinedione compound and reacting the released triazolinedione compound with a diene compound, a sufficient amount of the triazolinedione compound can be released and deterioration of the diene compound can be prevented, and a derivative of the diene compound can be measured while maintaining sufficient sensitivity and accuracy required for analyzing a trace amount of the diene compound, thereby completing the present invention.

[0010] That is, a first aspect of the present invention is a method for analyzing a diene compound, comprising: a triazolinedione adduct heating step of heating a triazolinedione adduct represented by the following formula (1) to generate a triazolinedione compound represented by the following formula (2); an ene compound forming step of reacting the triazolinedione compound with a diene compound represented by the following formula (3) to obtain an ene compound represented by the following formula (4); and an ene compound analysis step of quantifying the diene compound by analyzing the ene compound. [ka] (In the formula, R 1 is an organic group, and A is a condensed ring of three or more rings containing at least one aromatic ring. [ka] (In the formula, R 1 is the same as the above formula (1). [ka] (In the formula, R 2 , R 3 , R 4 , R 5 , R 6 , and R 7 is a hydrogen atom, or a group selected from the group consisting of an alkyl group, an aralkyl group, a phenyl group, and a heterocyclic group having 1 to 100 carbon atoms, which may contain an atom selected from the group consisting of an oxygen atom, a nitrogen atom, a sulfur atom, and a phosphorus atom, which may be bonded to each other or independent. [ka] (In the formula, R 1 is the same as in formula (1), and R 2 , R 3 , R 4 , R 5 , R 6 , and R 7 is the same as the above formula (3).

[0011] R in the above formula (1) 1 may be a substituted or unsubstituted phenyl group.

[0012] R in the above formula (1) 1 may be a phenyl group containing an alkylamino group or an alkylaminoalkyl group.

[0013] R in the above formula (1) 1 may be a phenyl group containing a dialkylamino group or a dialkylaminoalkyl group.

[0014] The triazolinedione adduct represented by the formula (1) may be represented by the following formula (5). [ka] (In the formula, X 1 is an organic group, R 8 , R 9 , and R 10are each independently a substituent selected from the group consisting of an alkyl group, an aralkyl group, an alkenyl group, a phenyl group, an acyl group, a halogen group, an amino group which may have a substituent, a nitro group, and a heterocyclic group, each of which may have a substituent and which have 1 to 20 carbon atoms, l and m are each independently an integer of 0 to 4, and n is an integer of 0 to 2.

[0015] X in the formula (5) 1 may be an alkylamino group or an alkylaminoalkyl group.

[0016] X in the formula (5) 1 may be a dialkylamino group or a dialkylaminoalkyl group.

[0017] The triazolinedione adduct represented by the formula (1) may be represented by the following formula (6). [ka] (In the formula, X 1 is an organic group, R 11 , R 12 , R 13 , and R 14 are each independently a substituent selected from the group consisting of an alkyl group, an aralkyl group, an alkenyl group, a phenyl group, an acyl group, a halogen group, an amino group which may have a substituent, a nitro group, and a heterocyclic group, each of which may have a substituent and each of which may have a substituent, each of which has 1 to 20 carbon atoms; p is an integer of 0 to 4; q is an integer of 0 to 2; and r and s are 0 or 1.

[0018] X in the formula (6) 1 may be an alkylamino group or an alkylaminoalkyl group.

[0019] X in the formula (6) 1 may be a dialkylamino group or a dialkylaminoalkyl group.

[0020] The diene compound may be a vitamin.

[0021] The vitamin may be at least one selected from the group consisting of vitamin D3, 25-hydroxyvitamin D3, 3-epi-25-hydroxyvitamin D3, 24R,25-dihydroxyvitamin D3, vitamin D2, and vitamin A.

[0022] The heating temperature in the triazolinedione adduct heating step may be 50 to 100°C.

[0023] The analysis in the ene compound analysis step may be a method using at least one selected from the group consisting of high performance liquid chromatography, high performance liquid chromatography-mass spectrometry (LC-MS), high performance liquid chromatography-tandem mass spectrometry (LC-MS / MS), and mass spectrometry.

[0024] A second aspect of the present invention includes a triazolinedione adduct heating step of heating a triazolinedione adduct represented by the following formula (1) to generate a triazolinedione compound represented by the following formula (2), and an ene compound-forming step of reacting the triazolinedione compound with a diene compound represented by the following formula (3) to obtain an ene compound represented by the following formula (4). [ka] (In the formula, R 1 is an organic group, and A is a condensed ring of three or more rings containing at least one aromatic ring. [ka] (In the formula, R 1 is the same as the above formula (1). [ka] (In the formula, R 2 , R3 , R 4 , R 5 , R 6 , and R 7 is a hydrogen atom, or a group selected from the group consisting of an alkyl group, an aralkyl group, a phenyl group, and a heterocyclic group having 1 to 100 carbon atoms, which may contain an atom selected from the group consisting of an oxygen atom, a nitrogen atom, a sulfur atom, and a phosphorus atom, which may be bonded to each other or independent. [ka] (In the formula, R 1 is the same as in formula (1), and R 2 , R 3 , R 4 , R 5 , R 6 , and R 7 is the same as the above formula (3). Effect of the Invention

[0025] In the method for analyzing a diene compound of the present invention, the triazolinedione adduct is heated before being mixed with a diene compound to generate the triazolinedione compound, thereby making it possible to generate the triazolinedione compound required for the reaction while suppressing decomposition of the generated triazolinedione compound.

[0026] In addition, since the thermal deterioration of the diene compound to be analyzed can be suppressed, the derivatization reaction of the diene compound can be carried out smoothly, making it possible to analyze trace amounts of the diene compound.

[0027] Therefore, according to the method for analyzing a diene compound of the present invention, the triazolinedione adduct as an analytical reagent can be applied to the quantification of the diene compound with sufficient sensitivity and accuracy. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0028] Hereinafter, an embodiment of the present invention will be described. Note that the present invention is not limited to the embodiment described below.

[0029] <Analysis method for diene compounds> The diene compound analysis method of the present disclosure uses a triazolinedione adduct as an analytical reagent to analyze a trace amount of a diene compound as an analysis target substance, and includes a triazolinedione adduct heating step, an ene compound forming step, and an ene compound analysis step. The diene compound analysis method of the present disclosure may include these steps as essential steps, and may optionally include other steps.

[0030] The most important feature of the diene compound analysis method of the present disclosure is that it includes a triazolinedione adduct heating step, specifically, in the absence of the diene compound to be analyzed, the triazolinedione adduct is heated to generate the triazolinedione compound. The diene compound analysis method of the present disclosure will be described below.

[0031] [Triazolinedione adduct heating process] The triazolinedione adduct heating step is a characteristic step of the diene compound analysis method of the present disclosure, and is a step of heating a triazolinedione adduct represented by the following formula (1) to produce a triazolinedione compound represented by the following formula (2).

[0032] [Triazolinedione adduct] In the method for analyzing a diene compound according to the present disclosure, the triazolinedione adduct used as an analytical reagent is represented by the following formula (1).

[0033] [ka] (In the formula, R 1 is an organic group, and A is a condensed ring of three or more rings containing at least one aromatic ring.

[0034] Preferably, R in the above formula (1) 1is a substituted or unsubstituted group selected from the group consisting of a phenyl group, a naphthyl group, an anthracenyl group, a nitrogen-containing heterocyclic group, and an alkyl group.

[0035] More preferably, R in the above formula (1) 1 is a substituted or unsubstituted phenyl group.

[0036] More preferably, R in the above formula (1) 1 is a phenyl group which may have an oxygen atom or a nitrogen atom and which may have a substituent selected from the group consisting of a di-substituted amino group, a di-substituted aminoalkyl group, a nitro group, an azide group, an alkoxy group, a halogen group, an alkylthio group, a sulfonyl group, a phosphate group, a carboxyl group, an ester group, a nitrile group, an amide group, a ferrocenyl group, and a quinoxalinyl group having a substituent, which may be the same or different.

[0037] More preferably, R in the above formula (1) 1 is a phenyl group containing an alkylamino group or an alkylaminoalkyl group.

[0038] Particularly preferably, R 1 is a phenyl group containing a dialkylamino group or a dialkylaminoalkyl group, and is most preferably a phenyl group containing a group selected from the group consisting of a 4-dimethylaminophenyl group, a 4-diethylaminophenyl group, and a 4-dimethylaminomethylphenyl group.

[0039] An example of the triazolinedione adduct represented by the above formula (1) is that represented by the following formula (5).

[0040] [ka] (In the formula, X 1 is an organic group, R 8 , R 9 , and R10 are each independently a substituent selected from the group consisting of an alkyl group, an aralkyl group, an alkenyl group, a phenyl group, an acyl group, a halogen group, an amino group which may have a substituent, a nitro group, and a heterocyclic group, each of which may have a substituent and which have 1 to 20 carbon atoms, l and m are each independently an integer of 0 to 4, and n is an integer of 0 to 2.

[0041] Preferably, X in the above formula (5) 1 is a group selected from the group consisting of a di-substituted amino group having a substituent selected from the group consisting of an alkyl group, an aralkyl group, and an aryl group, which may contain an oxygen atom or a nitrogen atom, a di-substituted aminoalkyl group, a nitro group, an azide group, an alkoxy group, a halogen group, an alkylthio group, a sulfonyl group, a phosphate group, a carboxyl group, an ester group, a nitrile group, an amide group, a ferrocenyl group, and a quinoxalinyl group having a substituent.

[0042] More preferably, X in the above formula (5) 1 is an alkylamino group or an alkylaminoalkyl group.

[0043] Particularly preferably, X in the above formula (5) 1 is a dialkylamino group or a dialkylaminoalkyl group, most preferably a dimethylamino group or a diethylamino group.

[0044] Above R 8 , R 9 , and R 10In the above, examples of the substituent that may be possessed by the substituent selected from the group consisting of an alkyl group, an aralkyl group, an alkenyl group, a phenyl group, an acyl group, a halogen group, an amino group that may be substituted, a nitro group, and a heterocyclic group include alkyl groups such as a methyl group, an ethyl group, a propyl group, a butyl group, an isobutyl group, a t-butyl group, a chloromethyl group, a 2-chloroethyl group, a 3-chloropropyl group, a 2-chloropropyl group, a 1-chloropropyl group, a bromomethyl group, a 2-bromoethyl group, a 3-bromopropyl group, a 2-bromopropyl group, and a 1-bromopropyl group; a benzyl group, a phenethyl group, a 3-phenylpropyl group, a 4-methylbenzyl group, a 4-methylphenethyl group, and the like. alkenyl groups such as vinyl group, allyl group, propenyl group, cyclopropenyl group, 2-chlorovinyl group, 3-chloroallyl group, cinnamyl group, styryl group, etc.; phenyl groups such as phenyl group, tolyl group, xylyl group, trityl group, etc.; acyl groups such as formyl group, acetyl group, propionyl group, benzoyl group, acryloyl group, methacryloyl group, etc.; halogen groups such as chloro group, bromo group, fluoro group, iodo group, etc.; amino groups such as amino group, methylamino group, dimethylamino group, ethylamino group, diethylamino group, etc.; nitro group; heterocyclic groups such as quinolyl group, pyridyl group, pyrrolidyl group, pyrrolyl group, furyl group, thienyl group, lutidyl group, etc.;

[0045] Examples of the triazolinedione adduct represented by the above formula (5) include those represented by the following formulae (5-1) and (5-2).

[0046] [ka]

[0047] [ka]

[0048] Moreover, an example of the triazolinedione adduct represented by the above formula (1) is that represented by the following formula (6).

[0049] [ka] (In the formula, X 1 is an organic group, R 11 , R 12 , R 13 , and R 14 are each independently a substituent selected from the group consisting of an alkyl group, an aralkyl group, an alkenyl group, a phenyl group, an acyl group, a halogen group, an amino group which may have a substituent, a nitro group, and a heterocyclic group, each of which may have a substituent and each of which may have a substituent, each of which has 1 to 20 carbon atoms; p is an integer of 0 to 4; q is an integer of 0 to 2; and r and s are 0 or 1.

[0050] Preferably, X in the above formula (6) 1 is a group selected from the group consisting of a di-substituted amino group having a substituent selected from the group consisting of an alkyl group, an aralkyl group, and an aryl group, which may contain an oxygen atom or a nitrogen atom, a di-substituted aminoalkyl group, a nitro group, an azide group, an alkoxy group, a halogen group, an alkylthio group, a sulfonyl group, a phosphate group, a carboxyl group, an ester group, a nitrile group, an amide group, a ferrocenyl group, and a quinoxalinyl group having a substituent.

[0051] More preferably, X in the above formula (6) 1 is an alkylamino group or an alkylaminoalkyl group.

[0052] Particularly preferably, X in the above formula (6) 1 is a dialkylamino group or a dialkylaminoalkyl group, most preferably a dimethylamino group or a diethylamino group.

[0053] Above R 11 , R 12 , R 13 , and R 14In the above, examples of the substituent that may be possessed by the substituent selected from the group consisting of an alkyl group, an aralkyl group, an alkenyl group, a phenyl group, an acyl group, a halogen group, an amino group that may be substituted, a nitro group, and a heterocyclic group include alkyl groups such as a methyl group, an ethyl group, a propyl group, a butyl group, an isobutyl group, a t-butyl group, a chloromethyl group, a 2-chloroethyl group, a 3-chloropropyl group, a 2-chloropropyl group, a 1-chloropropyl group, a bromomethyl group, a 2-bromoethyl group, a 3-bromopropyl group, a 2-bromopropyl group, and a 1-bromopropyl group; a benzyl group, a phenethyl group, a 3-phenylpropyl group, a 4-methylbenzyl group, a 4-methylphenethyl group, and the like. alkenyl groups such as vinyl group, allyl group, propenyl group, cyclopropenyl group, 2-chlorovinyl group, 3-chloroallyl group, cinnamyl group, styryl group, etc.; phenyl groups such as phenyl group, tolyl group, xylyl group, trityl group, etc.; acyl groups such as formyl group, acetyl group, propionyl group, benzoyl group, acryloyl group, methacryloyl group, etc.; halogen groups such as chloro group, bromo group, fluoro group, iodo group, etc.; amino groups such as amino group, methylamino group, dimethylamino group, ethylamino group, diethylamino group, etc.; nitro group; heterocyclic groups such as quinolyl group, pyridyl group, pyrrolidyl group, pyrrolyl group, furyl group, thienyl group, lutidyl group, etc.;

[0054] [Method for producing triazolinedione adduct] In the diene compound analysis method of the present invention, the triazolinedione adduct represented by the above formula (1) used as an analytical reagent can be produced by a known method without any particular limitation. For example, a triazolinedione adduct obtained by reacting a triazolinedione compound represented by the following formula (7) with a fused ring compound containing at least two aromatic rings in the presence of an oxidizing agent can be used. In producing the triazolinedione adduct, additives such as a drying agent can be added as long as they do not interfere with the reaction.

[0055] [ka] (In the formula, R1 is the same as the above formula (1).

[0056] (Triazolidinedione compounds) The compound of the above formula (7) which is the raw material of the triazolidinedione adduct is a triazolidinedione compound containing a urazole group, that is, a triazolidinedione compound having a 1,2,4-triazolidine-3,5-dione group.

[0057] Preferably, R in the above formula (7) 1 is a substituted or unsubstituted group selected from the group consisting of a phenyl group, a naphthyl group, an anthracenyl group, a nitrogen-containing heterocyclic group, and an alkyl group.

[0058] More preferably, R in the above formula (7) 1 is a substituted or unsubstituted phenyl group.

[0059] More preferably, R in the above formula (7) 1 is a phenyl group which may have an oxygen atom or a nitrogen atom and which may have a substituent selected from the group consisting of a di-substituted amino group, a di-substituted aminoalkyl group, a nitro group, an azide group, an alkoxy group, a halogen group, an alkylthio group, a sulfonyl group, a phosphate group, a carboxyl group, an ester group, a nitrile group, an amide group, a ferrocenyl group, and a quinoxalinyl group having a substituent, which may be the same or different.

[0060] More preferably, R in the above formula (7) 1 is a phenyl group containing an alkylamino group or an alkylaminoalkyl group.

[0061] Particularly preferably, R in the above formula (7) 1is a phenyl group containing a dialkylamino group or a dialkylaminoalkyl group, and is most preferably a phenyl group containing a group selected from the group consisting of a 4-diethylaminophenyl group, a 4-dimethylaminophenyl group, and a 4-dimethylaminomethylphenyl group.

[0062] Therefore, the most preferred compounds of formula (7) above are 4-(4'-diethylaminophenyl)-1,2,4-triazolidine-3,5-dione, 4-(4'-dimethylaminophenyl)-1,2,4-triazolidine-3,5-dione (DMU), or 4-(4'-dimethylaminomethylphenyl)-1,2,4-triazolidine-3,5-dione.

[0063] (Fused ring compounds containing at least two aromatic rings) In producing a triazolinedione adduct, examples of the fused ring compound containing at least two aromatic rings to be reacted with the triazolidinedione compound represented by the above formula (7) include compounds having skeletons represented by the following formulas (7-1) to (7-12). Some hydrogen atoms of the fused ring compounds having these skeletons may be substituted with a substituent selected from the group consisting of an alkyl group, an aralkyl group, an alkenyl group, a phenyl group, an acyl group, a halogen group, an amino group which may have a substituent, a nitro group, and a heterocyclic group, all of which have 1 to 20 carbon atoms and which may have a substituent. Examples of the substituent that may be possessed by the substituent selected from the group consisting of an alkyl group, an aralkyl group, an alkenyl group, a phenyl group, an acyl group, a halogen group, an amino group which may be substituted, a nitro group, and a heterocyclic group include alkyl groups such as a methyl group, an ethyl group, a propyl group, a butyl group, an isobutyl group, a t-butyl group, a chloromethyl group, a 2-chloroethyl group, a 3-chloropropyl group, a 2-chloropropyl group, a 1-chloropropyl group, a bromomethyl group, a 2-bromoethyl group, a 3-bromopropyl group, a 2-bromopropyl group, and a 1-bromopropyl group; a benzyl group, a phenethyl group, a 3-phenylpropyl group, a 4-methylbenzyl group, and a 4-methylphenethyl group. aralkyl groups such as vinyl, allyl, propenyl, cyclopropenyl, 2-chlorovinyl, 3-chloroallyl, cinnamyl, styryl, and other alkenyl groups; phenyl groups such as phenyl, tolyl, xylyl, and trityl; acyl groups such as formyl, acetyl, propionyl, benzoyl, acryloyl, and methacryloyl; halogen groups such as chloro, bromo, fluoro, and iodine; amino groups such as amino, methylamino, dimethylamino, ethylamino, and diethylamino; nitro groups; heterocyclic groups such as quinolyl, pyridyl, pyrrolidyl, pyrrolyl, furyl, thienyl, and lutidyl; and the like. In addition, at least one selected from a cyclic alkyl group having 1 to 20 carbon atoms, a phenyl group, and a heterocyclic group may be condensed with the condensed ring compound having such a skeleton.

[0064] The fused ring compound containing at least two aromatic rings to be reacted with the triazolidinedione compound represented by the above formula (7) is preferably one having a skeleton represented by the following formula (7-5) because of the ease of the retro-Diels-Alder reaction occurring in the heating step of the triazolidinedione adduct.

[0065] [ka]

[0066] (Oxidizing agent) In producing the triazolidinedione adduct, the oxidizing agent present when reacting the triazolidinedione compound represented by the above formula (7) with the above fused ring compound containing at least two aromatic rings is not particularly limited as long as it can generate the triazolidinedione adduct represented by the above formula (1), but is preferably a hypervalent iodine compound. If the hypervalent iodine compound is used, it is possible to smoothly convert the triazolidinedione compound to triazolidinedione, and the subsequent reaction with the fused ring compound containing at least two aromatic rings can proceed smoothly.

[0067] (Additives) In producing the triazolidinedione adduct, an additive such as a desiccant may be added as long as it does not interfere with the reaction between the triazolidinedione compound represented by the above formula (7) and the fused ring compound containing at least two aromatic rings. Examples of the additive include magnesium sulfate, sodium sulfate, potassium carbonate, calcium chloride, silica gel, and molecular sieves.

[0068] [Reaction conditions] (Reaction Solvent) In the production of the triazolidinedione adduct, the reaction solvent used when reacting the triazolidinedione compound represented by the above formula (7) with a fused ring compound containing at least two aromatic rings in the presence of an oxidizing agent is preferably at least one selected from the group consisting of esters, halogen-containing hydrocarbons, aromatic hydrocarbons, ketones, amides, alkylnitriles, dialkyl ethers, and ureas. The diene compound itself to be reacted can also be used as the solvent.

[0069] The amount of the solvent used in the reaction is preferably 5 to 1000 parts by volume per part by mass of the triazolidinedione compound.

[0070] (Reaction temperature) The temperature at which the triazolidinedione compound represented by the above formula (7) is reacted with the fused ring compound containing at least two aromatic rings in the presence of an oxidizing agent is preferably in the range of -10 to 60°C, more preferably in the range of 0 to 40°C.

[0071] (Reaction time) The time for reacting the triazolidinedione compound represented by the above formula (7) with the fused ring compound containing at least two aromatic rings in the presence of an oxidizing agent is preferably 10 minutes to 48 hours, and more preferably in the range of 1 to 10 hours.

[0072] (Amount charged) The amount of the fused ring compound containing at least two aromatic rings used is preferably within the range of 1.0 to 10,000 equivalents per equivalent of the triazolidinedione compound.

[0073] The amount of the oxidizing agent used is preferably 1 to 10 molar equivalents, more preferably 1 to 5 molar equivalents, relative to the triazolidinedione compound. If the amount is within the range of 1 to 5 molar equivalents, the oxidizing agent and its decomposition products remaining after the reaction can be easily removed.

[0074] When an additive is used, the amount used can be without particular limitation as long as the additive does not interfere with the reaction. For example, when the additive is magnesium sulfate, the amount is preferably in the range of 1 to 50 molar equivalents.

[0075] (Solidification and purification) The resulting triazolinedione adduct of the above formula (1) can also be obtained as a solid by crystallization.

[0076] After the crystallization, further purification can be carried out to obtain a high purity product. Examples of the purification method include recrystallization and column purification. The obtained solid triazolinedione adduct can be isolated by solid-liquid separation by solvent distillation, filtration, centrifugation, or the like, and drying by natural drying, air drying, vacuum drying, or the like.

[0077] [Triazolinedione compounds] In the triazolinedione adduct heating step of the present disclosure, the triazolinedione adduct represented by the above formula (1) is heated to produce a triazolinedione compound represented by the following formula (2).

[0078] [ka] (In the formula, R 1 is the same as the above formula (1).

[0079] More specifically, the triazolinedione adduct heating step heats the triazolinedione adduct represented by the above formula (1) to cause a back reaction (retro-Diels-Alder reaction) between the triazolinedione compound represented by the above formula (2) and the fused ring compound containing at least two aromatic rings. That is, the triazolinedione adduct heating step of the present disclosure produces the triazolinedione compound from the triazolinedione adduct by the back reaction.

[0080] Therefore, the triazolinedione compound represented by the above formula (2) produced in the triazolinedione adduct heating step is generated from the triazolinedione adduct represented by the above formula (1). For this reason, examples of the triazolinedione compound represented by the above formula (2) include 4-phenyl-1,2,4-triazoline-3,5-dione (PTAD), 4-methyl-1,2,4-triazoline-3,5-dione (MTAD), 4-[2-(6,7-dimethoxy-4-methyl-3-oxo-3,4-dihydroquinoxalinyl)ethyl]-1,2,4-triazoline-3,5-dione (DMEQTAD), 4-(4-nitrophenyl)-1,2,4-triazoline-3,5-dione (NPTAD), ... -ferrocenylmethyl-1,2,4-triazoline-3,5-dione (FMTAD), 4-(6-quinolyl)-1,2,4-triazoline-3,5-dione (QTAD), 4-(4'-diethylaminophenyl)-1,2,4-triazoline-3,5-dione (DEAPTAD), 4-(4'-dimethylaminophenyl)-1,2,4-triazoline-3,5-dione (DAPTAD), and 4-(4'-dimethylaminomethylphenyl)-1,2,4-triazoline-3,5-dione. These triazolinediones can be used as Cookson-type derivatizing agents.

[0081] Preferably, R in the above formula (2) 1 is a substituted or unsubstituted group selected from the group consisting of a phenyl group, a naphthyl group, an anthracenyl group, a nitrogen-containing heterocyclic group, and an alkyl group.

[0082] More preferably, R in the above formula (2) 1 is a substituted or unsubstituted phenyl group.

[0083] More preferably, R in the above formula (2) 1is a phenyl group which may have an oxygen atom or a nitrogen atom and which may have a substituent selected from the group consisting of a di-substituted amino group, a di-substituted aminoalkyl group, a nitro group, an azide group, an alkoxy group, a halogen group, an alkylthio group, a sulfonyl group, a phosphate group, a carboxyl group, an ester group, a nitrile group, an amide group, a ferrocenyl group, and a quinoxalinyl group having a substituent, which may be the same or different.

[0084] More preferably, R in the above formula (2) 1 is a phenyl group containing an alkylamino group or an alkylaminoalkyl group.

[0085] Particularly preferably, R 1 is a phenyl group containing a dialkylamino group or a dialkylaminoalkyl group, and is most preferably a phenyl group containing a group selected from the group consisting of a 4-diethylaminophenyl group, a 4-dimethylaminophenyl group, and a 4-dimethylaminomethylphenyl group.

[0086] Therefore, the triazolinedione compound represented by the above formula (2) is particularly preferably 4-phenyl-1,2,4-triazoline-3,5-dione, 4-(4'-diethylaminophenyl)-1,2,4-triazoline-3,5-dione, 4-(4'-dimethylaminophenyl)-1,2,4-triazoline-3,5-dione (DAPTAD), or 4-(4'-dimethylaminomethylphenyl)-1,2,4-triazoline-3,5-dione.

[0087] [Heating conditions] (Reaction Solvent) In the triazolinedione adduct heating step, the solvent used for the retro-Diels-Alder reaction can be any commonly used solvent without any particular limitation. Aprotic solvents that make the resulting triazolinedione compound more stable are preferred, specifically ethyl acetate, acetonitrile, and dioxane. Among these, ethyl acetate is more preferred due to its good handling properties.

[0088] (Reaction temperature) In the triazolinedione adduct heating step, the temperature for the retro-Diels-Alder reaction is preferably in the range of 50 to 100°C. From the viewpoint of the reaction rate of the retro-Diels-Alder reaction, the temperature is more preferably in the range of 70 to 100°C. Further, from the viewpoints of the sufficient rate of the retro-Diels-Alder reaction, the stability of the generated triazolinedione compound, and the boiling point of a general solvent, the temperature is still more preferably in the range of 70 to 80°C. For example, when the solvent is ethyl acetate, the suitable temperature is 77 to 80°C.

[0089] (Reaction time) In the triazolinedione adduct heating step, the reaction time for the retro-Diels-Alder reaction is preferably 10 minutes to 5 hours. If the reaction time is too long, the triazolinedione compound will decompose. On the other hand, if the reaction time is too short, a sufficient amount of triazolinedione compound will not be generated to react with the diene compound in the next ene compound formation step. More preferably, the reaction time is 5 to 30 minutes.

[0090] [Ene compound formation process] The ene compound formation step is a step in which the triazolinedione compound produced in the triazolinedione adduct heating step is reacted with a diene compound represented by the following formula (3) to obtain an ene compound represented by the following formula (4).

[0091] [ka] (In the formula, R 2 , R 3 , R 4 , R 5 , R 6 , and R 7 is a hydrogen atom, or a group selected from the group consisting of an alkyl group, an aralkyl group, a phenyl group, and a heterocyclic group having 1 to 100 carbon atoms, which may contain an atom selected from the group consisting of an oxygen atom, a nitrogen atom, a sulfur atom, and a phosphorus atom, which may be bonded to each other or independent.

[0092] [ka] (In the formula, R 1 is the same as in formula (1), and R 2 , R 3 , R 4 , R 5 , R 6 , and R 7 is the same as the above formula (3).

[0093] [Diene Compounds] The diene compound represented by the above formula (3) which is reacted with the produced triazolinedione compound is the substance to be analyzed in the diene compound analysis method of the present disclosure.

[0094] Because of their high usefulness, the diene compound represented by the above formula (3) is preferably a compound selected from the group consisting of high molecular or low molecular weight natural products, physiologically active compounds including pharmaceuticals, functional materials, and intermediates thereof.

[0095] Therefore, the diene compound represented by the above formula (3) to be analyzed is R 2 , R 3 , R 4 , R 5 , R 6 , and R 7 is preferably an anthracene derivative, a quinoline derivative, a vitamin, an amino acid, a steroid, or the like, having the above-mentioned substituent.

[0096] Among them, since the triazolinedione adduct represented by the above formula (1) acts as a Cookson-type derivatization reagent, the diene compound represented by the above formula (3) to be analyzed is preferably a vitamin, specifically at least one selected from the group consisting of vitamin D2, vitamin D3, vitamin A, 25-hydroxyvitamin D3, 3-epi-25-hydroxyvitamin D3, and 24R,25-dihydroxyvitamin D3.

[0097] [Ene compounds] The ene compound represented by the above formula (4) obtained by reacting the triazolinedione compound with the diene compound represented by the above formula (3) is the substance to be analyzed in the ene compound analysis step described below.

[0098] The ene compound represented by the above formula (4) is derived from the diene compound represented by the above formula (3). Therefore, R of the ene compound represented by the above formula (4) 2 , R 3 , R 4 , R 5 , R 6 , and R 7 has the same structure as the diene compound represented by the above formula (3). That is, R of the ene compound represented by the above formula (4) 2 , R 3 , R 4 , R 5 , R 6 , and R 7 is preferably an anthracene derivative, a quinoline derivative, a vitamin, an amino acid, a steroid, or the like having the above-mentioned substituent.

[0099] [Reaction conditions] (Reaction Solvent) The solvent used in reacting the triazolinedione compound represented by the formula (2) with the diene compound represented by the formula (3) is not particularly limited as long as it is a commonly used solvent, but from the viewpoint of the solution stability of the triazolinedione compound, it is preferably an aprotic solvent and a hydrocarbon solvent, for example, ethyl acetate, acetonitrile, dioxane, toluene, hexane, etc.

[0100] In the method for analyzing a diene compound of the present disclosure, an ene compound-forming step of reacting with a diene compound is carried out subsequent to a triazolinedione adduct heating step for generating a triazolinedione compound. Therefore, it is preferable to use the same solvent as that used in the ene compound-forming step as the solvent used in the triazolinedione adduct heating step.

[0101] The amount of the solvent used in the reaction is preferably 5 to 1000 parts by volume per part by mass of the triazolinedione compound.

[0102] (Reaction temperature) The temperature at which the triazolinedione compound represented by the formula (2) is reacted with the diene compound represented by the formula (3) is preferably in the range of −20 to 200° C., and from the viewpoint of the stability of the diene compound and the triazolinedione compound, is more preferably in the range of 0 to 50° C.

[0103] (Reaction time) The time for reacting the triazolinedione compound represented by the formula (2) with the diene compound represented by the formula (3) is preferably 1 minute to 12 hours, and more preferably 15 minutes to 1 hour.

[0104] (Amount charged) In the reaction of the triazolinedione compound represented by the formula (2) with the diene compound represented by the formula (3), the amount of the triazolinedione compound represented by the formula (2) used is not particularly limited as long as it is an excess amount relative to the diene compound represented by the formula (3). For example, when the diene compound represented by the formula (3) is a vitamin, the amount is preferably in the range of 1 to 1,000,000 equivalents relative to 1 equivalent of the diene compound, more preferably in the range of 10 to 1,000,000 equivalents, and most preferably in the range of 1,000 to 1,000,000 equivalents.

[0105] [Ene compound analysis process] The ene compound analysis step is a step of quantifying the diene compound reacted in the ene compound formation step by analyzing the ene compound obtained in the ene compound formation step.

[0106] The ene compound represented by the above formula (4) formed in the ene compound formation step has strong ultraviolet-visible absorption and is easily ionized due to its structure, which improves the sensitivity in various analytical methods.

[0107] [Analysis method] Although the method for analyzing the ene compound represented by the above formula (4) is not particularly limited, from the viewpoints of high sensitivity, accuracy, and ease of handling, a method using mass spectrometry or high performance liquid chromatography is preferable. A combination of these two methods, high performance liquid chromatography-mass spectrometry (LC-MS) and high performance liquid chromatography-tandem mass spectrometry (LC-MS / MS), can also be used.

[0108] (High Performance Liquid Chromatography) The detector for high performance liquid chromatography is not particularly limited as long as it can measure the analyte, and examples thereof include an ultraviolet-visible spectroscopic detector, a photodiode array detector, a fluorescence detector, an RI detector, and an evaporative light scattering detector.

[0109] The column for high performance liquid chromatography is not particularly limited as long as it is a column capable of measuring the analyte, but due to its high versatility, a column using chemically bonded porous spherical silica gel whose surface is modified with octadecylsilyl groups is preferred.

[0110] The analytical conditions for the high performance liquid chromatography are not particularly limited as long as the analyte can be measured under the conditions.

[0111] As the eluent, a commonly used solvent can be used. For example, methanol, acetonitrile, hexane, chloroform, ethyl acetate, tetrahydrofuran, distilled water, formic acid aqueous solution, carbonic acid aqueous solution, and phosphoric acid aqueous solution can be mentioned. One or more types of solvents can be used in combination, and it is preferable to use distilled water or an aqueous solution in combination with an organic solvent because it is easy to adjust the measurement time of the analyte. Among them, the combinations of distilled water / acetonitrile, distilled water / methanol, formic acid aqueous solution / acetonitrile, and formic acid aqueous solution / methanol are preferable because they can be easily combined with mass spectrometry.

[0112] (Mass Spectrometry) The mass spectrometry is not particularly limited as long as it is commercially available.

[0113] Examples of ion sources that can be used in mass spectrometry include electron ionization (EI), chemical ionization (CI), field desorption (FD), fast atom bombardment (FAB), matrix-assisted laser desorption ionization (MALDI), electrospray ionization (ESI), atmospheric pressure chemical ionization (APCI), direct analysis in real time (DART), ion attachment, inductively coupled plasma (ICP), etc. Among these, electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI) are preferred because they can be easily combined with high performance liquid chromatography.

[0114] Examples of mass separation units for mass spectrometry include magnetic deflection type, quadrupole type, ion trap type, time-of-flight type, Fourier transform ion cyclotron resonance type, and tandem type.

[0115] <Method of producing ene compound> The method for producing an ene compound according to the present disclosure uses a triazolinedione adduct as an analytical reagent to convert a trace amount of a diene compound, which is an analysis target substance, into an ene compound that is easily analyzable, and includes a triazolinedione adduct heating step and an ene compound forming step. The method for producing an ene compound according to the present disclosure may include these steps as essential steps, and may optionally include other steps.

[0116] The greatest feature of the method for producing an ene compound according to the present disclosure is that it includes a step of heating a triazolinedione adduct, specifically, in which the triazolinedione adduct is heated in the absence of a diene compound, which is the substance to be analyzed, to generate a triazolinedione compound.

[0117] The triazolinedione adduct heating step and the ene compound forming step in the production method of the ene compound of the present disclosure are similar to the triazolinedione adduct heating step and the ene compound forming step in the above-mentioned method for analyzing a diene compound of the present invention. EXAMPLES

[0118] Next, examples of the present invention will be described, but the present invention is not limited to these examples.

[0119] <Production Example 1> [Synthesis of 9-phenylanthracene adduct (DAP-PA)] A mixture of 4-(4'-dimethylaminophenyl)-1,2,4-triazolidine-3,5-dione (0.429 g, 1.95 mmol), iodosylbenzene (0.429 g, 1.95 mmol), 9-phenylanthracene (0.496 g, 1.95 mmol), magnesium sulfate (0.429 g, 3.56 mmol), and ethyl acetate (21.5 ml) was stirred at room temperature for 24 hours.

[0120] The mixture was filtered, and 300 ml of hexane was added to the filtrate. The mixture was stirred under ice cooling for 2 hours. The precipitate was collected by filtration and dried under reduced pressure to obtain 5,10-dihydro-2-(4-dimethylaminophenyl)-5-phenyl-5,10[1',2']-benzeno-1H-[1,2,4]triazolo[1,2-b]phthalazine-1,3(2H)-dione (DAP-PA) (0.54 g, 58.6%).

[0121] [Reaction formula] The reaction scheme carried out in Production Example 1 is shown below.

[0122] [ka]

[0123] [Physical property evaluation] The obtained DAP-PA was subjected to various analyses, and the results are shown below. Mp: >200℃ IR(KBr): 1774,1714cm -1 1 H-NMR(CDCl3): δ 6.25-8.10(m,17H), 2.80(s,6H)

[0124] <Production Example 2> [Synthesis of 9,10-diphenylanthracene adduct (DAP-DPA)] A mixture of 4-(4'-dimethylaminophenyl)-1,2,4-triazolidine-3,5-dione (0.50 g, 2.27 mmol), iodosylbenzene (0.50 g, 2.27 mmol), 9,10-diphenylanthracene (0.75 g, 2.27 mmol), magnesium sulfate (0.50 g, 4.15 mmol), and toluene (25 ml) was stirred at room temperature for 24 hours.

[0125] The mixture was filtered, and 300 ml of hexane was added to the filtrate. The mixture was stirred under ice-cooling for 2 hours. The precipitate was collected by filtration and dried under reduced pressure to obtain 12-dihydro-2-(4-dimethylaminophenyl)-6,11-diphenyl-5,12-etheno-1H-benzo[g][1,2,4]triazolo[1,2-b]phthalazine-1,3(2H)-dione (DAP-DPA) (43 mg, 3.4%).

[0126] [Reaction formula] The reaction scheme carried out in Production Example 2 is shown below.

[0127] [ka]

[0128] [Physical property evaluation] The obtained DAP-DPA was subjected to various analyses, and the results are shown below. Mp: >200℃ IR(KBr): 1764,1705cm -1 1 H-NMR(DMSO-d6): δ 7.30-8.00(m,16H), 6.70-7.30(m,4H), 5.50-5.80(m,2H), 2.90(s,6H)

[0129] <Production Example 3> [Synthesis of 9-methylanthracene adduct (DAP-MA)] A mixture of 4-(4'-dimethylaminophenyl)-1,2,4-triazolidine-3,5-dione (99.1 mg, 0.45 mmol), iodosylbenzene (99.0 mg, 0.45 mmol), 9-methylanthracene (86.5 mg, 0.45 mmol), magnesium sulfate (99.0 mg, 0.82 mmol), and ethyl acetate (5.0 ml) was stirred at room temperature for 24 hours.

[0130] The mixture was filtered, and 100 ml of hexane was added to the filtrate. The mixture was stirred under ice cooling for 2 hours. The precipitate was collected by filtration and dried under reduced pressure to obtain 5,10-dihydro-2-(4-dimethylaminophenyl)-5-methyl-5,10[1',2']-benzeno-1H-[1,2,4]triazolo[1,2-b]phthalazine-1,3(2H)-dione (DAP-MA) (95 mg, 51.4%).

[0131] [Reaction formula] The reaction scheme carried out in Production Example 3 is shown below.

[0132] [ka]

[0133] [Physical property evaluation] The obtained DAP-MA was subjected to various analyses, and the results are shown below. Mp: >218℃ IR(KBr): 1765,1708cm -1 1 H-NMR(CDCl3): δ 7.10-7.75(m,8H), 6.50-7.20(m,4H), 6.25(s,1H), 3.00(s,3H), 2.70(s,3H)

[0134] Example 1: Use of 9-phenylanthracene adduct (DAP-PA) [Triazolinedione adduct heating process] 5.00 mg (0.0106 mmol) of the 9-phenylanthracene adduct (DAP-PA) obtained in Production Example 1 was placed in 10 ml of ethyl acetate and heated at 80° C. for 30 minutes to prepare an ethyl acetate solution. The color of the ethyl acetate solution changed from colorless to light reddish purple.

[0135] [Ene compound formation process] To 10 ml of the ethyl acetate solution obtained in the triazolinedione adduct heating step, 0.400 mg (0.00104 mmol) of vitamin D3 was added and reacted at 25° C. for 1 hour.

[0136] [Ene compound analysis process] Next, the ethyl acetate solution that had undergone the ene compound formation step was analyzed by high performance liquid chromatography (HPLC) under the following analytical conditions.

[0137] [High performance liquid chromatography analysis conditions] Column: Inertsil ODS-3 (5 μm, 4.6 × 250 mm) (GL Science) Detector: UV-visible spectrometer (Waters 2489) Detection wavelength: 210nm Eluent: Mobile phase A: Acetonitrile Mobile phase B: distilled water Mobile phase A / mobile phase B=50 / 50 (0~10min) Mobile phase A / mobile phase B=50 / 50→95 / 5 (10~20min) Mobile phase A / Mobile phase B=95 / 5 (20~50min) Mobile phase A / mobile phase B=95 / 5→50 / 50(50~55min) Mobile phase A / mobile phase B=50 / 50(55~60min) Flow rate: 1.0ml / min Sample temperature: 25℃ Column temperature: 35℃ Injection volume: 10μL Dilution solvent: Acetonitrile Holding time:DAPTAD-VD3 21.4min

[0138] Analysis revealed that the sample contained 0.615 mg of vitamin D3 adduct (DAPTAD-VD3), an ene compound of the following formula: (5S,7S)-2-[4-(dimethylamino)phenyl]-5-[(E)-[(1R,3aS,7aR)-1-[(1R)-1,5-dimethylhexyl]octahydro-7a-methyl-4H-inden-4-ylidene]methyl]-5,6,7,8,9,10-hexahydro-7-hydroxy-1H-[1,2,4]triazolo[1,2-b]phthalazine-1,3(2H)-dione. The recovery rate calculated as vitamin D3 was 98.1%.

[0139] [ka]

[0140] The operations and analytical results carried out in the examples are shown in Tables 1 and 2.

[0141] [Table 1] [Table 2]

[0142] Example 2: Use of 9,10-diphenylanthracene adduct (DAP-DPA) [Triazolinedione adduct heating process] 10.0 mg (0.0182 mmol) of the 9,10-diphenylanthracene adduct (DAP-DPA) obtained in Production Example 2 was placed in 10 ml of ethyl acetate and heated at 70° C. for 15 minutes to prepare an ethyl acetate solution. The color of the ethyl acetate solution changed to a light reddish purple.

[0143] [Ene compound formation process] To 10 ml of the ethyl acetate solution obtained in the triazolinedione adduct heating step, 0.700 mg (0.00182 mmol) of vitamin D3 was added and reacted at 30° C. for 1 hour.

[0144] [Ene compound analysis process] Next, the ethyl acetate solution that had been subjected to the ene compound formation step was analyzed by high performance liquid chromatography (HPLC) under the same analytical conditions as in Example 1.

[0145] Analysis revealed that the sample contained 10.4 mg of vitamin D3 adduct (DAPTAD-VD3), an ene compound, with a recovery rate of 95.2% calculated as vitamin D3.

[0146] Example 3: Use of 9-methylanthracene adduct (DAP-MA) [Triazolinedione adduct heating process] 41.0 mg (0.100 mmol) of the 9-methylanthracene adduct (DAP-MA) obtained in Production Example 3 was placed in 10 ml of toluene and heated at 120° C. for 2 hours to prepare a toluene solution. The color of the solution changed to a light reddish purple.

[0147] [Ene compound formation process] To 10 ml of the toluene solution obtained in the triazolinedione adduct heating step, 3.84 mg (0.0100 mmol) of vitamin D3 was added and reacted at 20° C. for 30 minutes.

[0148] [Ene compound analysis process] Next, the toluene solution that had been subjected to the ene compound formation step was analyzed by high performance liquid chromatography (HPLC) under the same analytical conditions as in Example 1.

[0149] Analysis revealed that the sample contained 5.24 mg of vitamin D3 adduct (DAPTAD-VD3), an ene compound, and the recovery rate calculated as vitamin D3 was 87.1%.

[0150] Example 4: Use of 9,10-diphenylanthracene adduct (DAP-DPA) [Triazolinedione adduct heating process] 10.0 mg (0.0182 mmol) of the 9,10-diphenylanthracene adduct (DAP-DPA) obtained in Production Example 2 was placed in 10 ml of ethyl acetate and heated at 45° C. for 1 hour to prepare an ethyl acetate solution. The color of the solution changed to a light reddish purple.

[0151] [Ene compound formation process] To 10 ml of the ethyl acetate solution obtained in the triazolinedione adduct heating step, 0.4 mg (0.00104 mmol) of vitamin D3 was added and reacted at 15° C. for 1 hour.

[0152] [Ene compound analysis process] The ethyl acetate solution that had been subjected to the ene compound formation step was analyzed by high performance liquid chromatography (HPLC) under the same analytical conditions as in Example 1.

[0153] Analysis revealed that the sample contained 0.440 mg of vitamin D3 adduct (DAPTAD-VD3), an ene compound, and the recovery rate calculated as vitamin D3 was 70.3%.

[0154] Example 5: Use of 9-phenylanthracene adduct (DAP-PA) [Triazolinedione adduct heating process] 10 mg (0.0212 mmol) of the 9-phenylanthracene adduct (DAP-PA) obtained in Production Example 1 was placed in 100 ml of ethyl acetate and heated at 60° C. for 1 hour to prepare an ethyl acetate solution. The color of the solution changed to a light reddish purple.

[0155] [Ene compound formation process] A vitamin D3 solution was prepared by dissolving 1.00 mg (0.00260 mmol) of vitamin D3 in 100 ml of ethyl acetate. Next, 1 ml (0.0000260 mmol) of the vitamin D3 solution was added to 100 ml of the ethyl acetate solution obtained in the triazolinedione adduct heating step, and the mixture was allowed to react at 25°C for 1 hour.

[0156] [Ene compound analysis process] The ethyl acetate solution after the ene compound formation step was analyzed by high performance liquid chromatography-mass spectrometry (LC-MS) under the following analytical conditions.

[0157] [LC-MS analysis conditions] (Device) LC: ACQUITY UPLC (Waters) MS: Xevo QTof MS (Waters) (LC conditions) The high performance liquid chromatography conditions were the same as those described in Example 1. (MS conditions) Source temperature: 120℃ Cone gas: 50L / h Desolvation temperature: 500℃ Desolvation gas: 1000L / h (positive) Capillary voltage: 3.0kV Sample cone voltage: 20V

[0158] Analysis revealed that the sample contained 0.0143 mg of vitamin D3 adduct (DAPTAD-VD3), an ene compound, and the recovery rate calculated as vitamin D3 was 91.3%.

[0159] Example 6: Use of 9-phenylanthracene adduct (DAP-PA) [Triazolinedione adduct heating process] 5.00 mg (0.106 mmol) of the 9-phenylanthracene adduct (DAP-PA) obtained in Production Example 1 was placed in 10 ml of toluene and heated at 100° C. for 30 minutes to prepare a toluene solution. The color of the solution changed to a light reddish purple.

[0160] [Ene compound formation process] To 10 ml of the toluene solution obtained in the triazolinedione adduct heating step, 0.400 mg (0.00104 mmol) of vitamin D3 was added and reacted at 30° C. for 1 hour.

[0161] [Ene compound analysis process] The toluene solution that had been subjected to the ene compound formation step was analyzed by high performance liquid chromatography (HPLC) under the same analytical conditions as in Example 1.

[0162] Analysis revealed that the sample contained 0.610 mg of vitamin D3 adduct (DAPTAD-VD3), an ene compound, and the recovery rate calculated as vitamin D3 was 93.2%.

[0163] Example 7: Use of 9-phenylanthracene adduct (DAP-PA) [Triazolinedione adduct heating process] 5.00 mg (0.106 mmol) of the 9-phenylanthracene adduct (DAP-PA) obtained in Production Example 1 was placed in 10 ml of toluene and heated at 110° C. for 30 minutes to prepare a toluene solution. The color of the solution changed to a light reddish purple.

[0164] [Ene compound formation process] To 10 ml of the toluene solution obtained in the triazolinedione adduct heating step, 0.400 mg (0.00104 mmol) of vitamin D3 was added and reacted at 10° C. for 1 hour.

[0165] [Ene compound analysis process] The toluene solution that had been subjected to the ene compound formation step was analyzed by high performance liquid chromatography (HPLC) under the same analytical conditions as in Example 1.

[0166] Analysis revealed that the sample contained 0.565 mg of vitamin D3 adduct (DAPTAD-VD3), an ene compound, with a recovery rate of 90.2% calculated as vitamin D3.

[0167] Example 8: Use of 9-phenylanthracene adduct (DAP-PA) [Triazolinedione adduct heating process] 5.00 mg (0.0106 mmol) of the 9-phenylanthracene adduct (DAP-PA) obtained in Production Example 1 was placed in 10 ml of ethyl acetate and heated at 75° C. for 30 minutes to prepare an ethyl acetate solution. The color of the solution changed to a light reddish purple.

[0168] [Ene compound formation process] A vitamin D3 solution was prepared by dissolving 0.500 mg (0.00130 mmol) of vitamin D3 in 100 ml of ethyl acetate. Then, 1 ml of this vitamin D3 solution was weighed out and diluted to 10 ml with ethyl acetate to prepare a diluted vitamin D3 solution.

[0169] To 10 ml of the ethyl acetate solution obtained in the triazolinedione adduct heating step, 1 ml (0.00000130 mmol) of the diluted vitamin D3 solution obtained above was added, and the mixture was allowed to react at 30° C. for 1 hour.

[0170] [Ene compound analysis process] The ethyl acetate solution that had been subjected to the ene compound formation step was analyzed by a high performance liquid chromatography-mass spectrometer (LC-MS) under the same analytical conditions as in Example 5.

[0171] Analysis revealed that the sample contained 0.00754 mg of vitamin D3 adduct (DAPTAD-VD3), an ene compound, and the recovery rate calculated as vitamin D3 was 96.4%.

Claims

1. a triazolinedione adduct heating step of heating the triazolinedione adduct represented by the following formula (5) or the following formula (6) to generate a triazolinedione compound represented by the following formula (2); an ene compound formation step of subjecting the triazolinedione compound and a diene compound to a Diels-Alder reaction to obtain an ene compound; and an ene compound analysis step of quantifying the diene compound by analyzing the ene compound, The method for analyzing a diene compound, wherein the diene compound is at least one selected from the group consisting of vitamin D3, 25-hydroxyvitamin D3, 3-epi-25-hydroxyvitamin D3, 24R,25-dihydroxyvitamin D3, and vitamin D2. 【Chemistry 5】 (In the formula, X 1 is an alkylamino group, a dialkylamino group, an alkylaminoalkyl group or a dialkylaminoalkyl group; R 8 , R 9 and R 10 are each independently a substituent selected from the group consisting of an alkyl group having 1 to 20 carbon atoms, an aralkyl group, an alkenyl group, a phenyl group, an acyl group, a halogen group, an amino group which may have a substituent, a nitro group and a heterocyclic group, which may have a substituent; l and m are each independently an integer of 0 to 4, and n is an integer of 0 to 2.) 【Chemistry 6】 (In the formula, X 1 is an alkylamino group, a dialkylamino group, an alkylaminoalkyl group or a dialkylaminoalkyl group; R 11 , R 12 , R 13 and R 14 are each independently a substituent selected from the group consisting of an alkyl group having 1 to 20 carbon atoms, an aralkyl group, an alkenyl group, a phenyl group, an acyl group, a halogen group, an amino group which may have a substituent, a nitro group and a heterocyclic group; p is an integer of 0 to 4; q is an integer of 0 to 2; and r and s are each 0 or 1.) 【Chemistry 2】 (In the formula, R １ is a phenyl group substituted with X 1 .

2. The method for analyzing a diene compound according to claim 1, wherein the heating temperature in the triazolinedione adduct heating step is 50 to 100° C.

3. The method for analyzing a diene compound according to claim 1 or 2, wherein the analysis in the ene compound analysis step is a method using at least one method selected from the group consisting of high performance liquid chromatography, high performance liquid chromatography-mass spectrometry (LC-MS), high performance liquid chromatography-tandem mass spectrometry (LC-MS / MS), and mass spectrometry.

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