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WO2019135405A1 - Detection method for analog derived from trifluridine - Google Patents

Detection method for analog derived from trifluridine Download PDF

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Publication number
WO2019135405A1
WO2019135405A1 PCT/JP2019/000010 JP2019000010W WO2019135405A1 WO 2019135405 A1 WO2019135405 A1 WO 2019135405A1 JP 2019000010 W JP2019000010 W JP 2019000010W WO 2019135405 A1 WO2019135405 A1 WO 2019135405A1
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WO
WIPO (PCT)
Prior art keywords
salt
analog
trifluridine
mobile phase
deoxy
Prior art date
Application number
PCT/JP2019/000010
Other languages
French (fr)
Japanese (ja)
Inventor
武田　大典
章代 小島
Original Assignee
大鵬薬品工業株式会社
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from US16/040,734 external-priority patent/US10816517B2/en
Priority claimed from CN201810821041.5A external-priority patent/CN110007010A/en
Application filed by 大鵬薬品工業株式会社 filed Critical 大鵬薬品工業株式会社
Priority to JP2019563983A priority Critical patent/JP7257969B2/en
Priority to EP19736109.0A priority patent/EP3736568B1/en
Publication of WO2019135405A1 publication Critical patent/WO2019135405A1/en

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/26—Conditioning of the fluid carrier; Flow patterns
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/26—Conditioning of the fluid carrier; Flow patterns
    • G01N30/28—Control of physical parameters of the fluid carrier
    • G01N30/34—Control of physical parameters of the fluid carrier of fluid composition, e.g. gradient
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D239/00—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings
    • C07D239/02—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings not condensed with other rings
    • C07D239/24—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings not condensed with other rings having three or more double bonds between ring members or between ring members and non-ring members
    • C07D239/28—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings not condensed with other rings having three or more double bonds between ring members or between ring members and non-ring members with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, directly attached to ring carbon atoms
    • C07D239/46—Two or more oxygen, sulphur or nitrogen atoms
    • C07D239/52—Two oxygen atoms
    • C07D239/54—Two oxygen atoms as doubly bound oxygen atoms or as unsubstituted hydroxy radicals
    • C07D239/545—Two oxygen atoms as doubly bound oxygen atoms or as unsubstituted hydroxy radicals with other hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, directly attached to ring carbon atoms
    • C07D239/557—Two oxygen atoms as doubly bound oxygen atoms or as unsubstituted hydroxy radicals with other hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, directly attached to ring carbon atoms with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, directly attached to ring carbon atoms, e.g. orotic acid
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H19/00—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
    • C07H19/02—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof sharing nitrogen
    • C07H19/04—Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
    • C07H19/06—Pyrimidine radicals
    • C07H19/073—Pyrimidine radicals with 2-deoxyribosyl as the saccharide radical

Definitions

  • the present invention relates to a method for measuring a related substance of a trifluridine-containing preparation by high performance liquid chromatography.
  • Non-patent Document 1 5-trifluoromethyluracil and 5-carboxyuracil are known as analogues of trifluridine, and there is a report that they are measured by high performance liquid chromatography.
  • Non-patent Document 2 5-trifluoromethyluracil, which is an analogue of trifluridine, can be measured under various reverse phase liquid chromatography conditions.
  • Non-patent Document 3 It has been reported that liquid chromatography is used to measure uptake of trifluridine into tumor cells.
  • Non-patent Document 4 It has been reported that liquid chromatography is used to determine the substrate specificity of trifluridine TK1 (Non-patent Document 4).
  • Non-patent Document 5 It has been reported that liquid chromatography is used in the measurement of trifluridine in enzyme synthesis.
  • Non-patent Document 2 Various high performance liquid chromatography conditions using acetonitrile have been reported for the measurement of 5-trifluoromethyluracil, which is an analogue of trifluridine.
  • Non-patent Document 3 Conditions for high performance liquid chromatography using acetonitrile have been reported to measure uptake of trifluridine into tumor cells.
  • Non-patent Document 5 In order to confirm trifluridine in enzyme synthesis, conditions of high performance liquid chromatography with gradient added with trimethyl ammonium acetate have been reported (Non-patent Document 5).
  • Non-patent Documents 9 and 10 Conditions of high performance liquid chromatography using methanol have been reported for trifluridine as eye drops.
  • Patent Document 7 high performance liquid chromatography is used to confirm the purity of trifluridine, but the conditions are not described.
  • the problem to be solved by the present invention is to provide a novel method capable of detecting trifluridine-derived analogues from a sample containing trifluridine or a salt thereof by high performance liquid chromatography by steps using two gradient conditions. It is.
  • the present inventors are able to detect trifluridine or its salt analogues efficiently by using high performance liquid chromatography under specific conditions, and a detection method suitable for assuring the quality thereof Found out.
  • the present invention provides the following [1] to [31].
  • a method for detecting a related substance derived from trifluridine comprising the step of subjecting a sample containing trifluridine or a salt thereof to high performance liquid chromatography using a mobile phase consisting of an organic phase and an aqueous phase, the method comprising the steps of:
  • the method wherein the step of subjecting to high performance liquid chromatography comprises steps 1 and 2 meeting the following requirements: Step 1: The ratio of the organic phase to the total mobile phase is 1 to 14% by volume. Step 2: After step 1, apply a gradient to increase the ratio of organic phase to total mobile phase.
  • analogue is at least one selected from the group consisting of the following analogues 1 to 6: Related substances 1: 5-carboxyuracil, Analogs 2: 5-Carboxy-2'-deoxy-uridine, Related substances 3: 2'-deoxy-5-methoxycarbonyluridine, Related substances 4: trifluorothymine, Related substances 5: 5-methoxycarbonyluracil, Analogs 6: 5 '-(4-chlorophenylcarboxy) -2'-deoxy-5-trifluoromethyluridine.
  • a method for producing a compounding preparation containing trifluridine or a salt thereof which comprises the step of performing the method according to any one of [1] to [12].
  • a method of quality control of a compounding agent comprising trifluridine or a salt thereof, and tipiracil or a salt thereof which comprises the step of performing the method according to any one of [1] to [12].
  • the present invention is a high-performance liquid chromatography using a mobile phase consisting of an organic phase and an aqueous phase, which is capable of efficiently detecting trifluridine or its salt analogues, and which is suitable for assuring the quality thereof. is there.
  • Example 1 shows a chromatogram in Example 1-1.
  • 7 shows a chromatogram in Example 1-3.
  • the chromatogram in Example 2-1 is shown.
  • Trifluridine (FTD) in the present invention is ⁇ , ⁇ , ⁇ -trifluorothymidine, which is a compound having the following structure.
  • any isomer or a mixture is also a compound of the present invention unless otherwise specified. Is included.
  • a salt means a pharmaceutically acceptable salt and can include a base addition salt or an acid addition salt.
  • the base addition salt examples include alkali metal salts such as sodium salts and potassium salts; alkaline earth metal salts such as calcium salts and magnesium salts; ammonium salts; such as trimethylamine salts, triethylamine salts, dicyclohexylamine salts, ethanolamine And salts thereof, organic amine salts such as diethanolamine salt, triethanolamine salt, procaine salt, N, N'-dibenzylethylenediamine salt and the like.
  • alkali metal salts such as sodium salts and potassium salts
  • alkaline earth metal salts such as calcium salts and magnesium salts
  • ammonium salts such as trimethylamine salts, triethylamine salts, dicyclohexylamine salts, ethanolamine And salts thereof, organic amine salts such as diethanolamine salt, triethanolamine salt, procaine salt, N, N'-dibenzylethylenediamine salt and the like.
  • the acid addition salt examples include inorganic acid salts such as hydrochloride, sulfate, nitrate, phosphate and perchlorate; for example, acetate, formate, maleate, fumarate, tartrate, citric acid Organic acid salts such as salts of ascorbate and trifluoroacetate; and sulfonates such as methanesulfonate, isethionate, benzenesulfonate, p-toluenesulfonate and the like.
  • inorganic acid salts such as hydrochloride, sulfate, nitrate, phosphate and perchlorate
  • Organic acid salts such as salts of ascorbate and trifluoroacetate
  • sulfonates such as methanesulfonate, isethionate, benzenesulfonate, p-toluenesulfonate and the like.
  • trifluridine or a salt thereof to be used in the present invention is preferably a free form of trifluridine which has not formed a salt.
  • the sample in the present invention may contain a trifluridine analogue.
  • the analogues include compounds of analogues 1 to 6.
  • 5-carboxyuracil It may also be referred to as 2,4-dioxo-1,2,3,4-tetrahydropyrimidine-5-carboxylic acid.
  • Analog substance 2 is 5-carboxy-2'-deoxy-uridine. 1-((2R, 4R, 5R) -4-hydroxy-5- (hydroxymethyl) tetrahydrofuran-2-yl) -2,4-dioxo-1,2,3,4-tetrahydropyrimidine-5-carboxylic acid and It is sometimes called.
  • Analog 3 is 2'-deoxy-5-methoxycarbonyluridine. 1-((2R, 4R, 5R) -4-hydroxy-5- (hydroxymethyl) tetrahydrofuran-2-yl) -2,4-dioxo-1,2,3,4-tetrahydropyrimidine-5-carboxylic acid methyl ester It is sometimes called.
  • the related substance 4 is trifluorothymine. Sometimes referred to as 5- (trifluoromethyl) pyrimidine-2,4 (1H, 3H) -dione.
  • the related substance 5 is 5-methoxycarbonyluracil. It may also be referred to as methyl 2,4-dioxo-1,2,3,4-tetrahydropyrimidine-5-carboxylate.
  • Analog 6 is 5 '-(4-chlorophenylcarboxy) -2'-deoxy-5-trifluoromethyluridine.
  • 4-chlorobenzoic acid ((2R, 3R, 5R) -5- (2,4-dioxo-5- (trifluoromethyl) -3,4-dihydropyrimidin-1 (2H) -yl) -3-hydroxytetrahydrofuran Sometimes referred to as -2-yl) methyl.
  • the samples used in the present invention are not only samples prepared from the preparation and drug substance themselves, but also samples prepared in a test for confirming the stability etc., each for confirming the retention time of each related substance, etc. It can also be used as a sample to which a related substance is added, a sample prepared to confirm the preparation process of a drug product or drug substance, and the like.
  • the sample may contain, be substantially free of, or not contain tipiracil or a salt thereof formulated in Ronsurf (registered trademark).
  • Formulations that can be used in the present invention include trifluridine or a salt thereof. And, if necessary, a pharmaceutically acceptable carrier can be blended, and various administration forms can be adopted according to the purpose of prevention or treatment, and as such forms, for example, oral agents, eye drops, injections, Any of suppositories, ointments, patches and the like may be used, and preferably oral preparations are employed. Each of these dosage forms can be prepared by conventional formulation methods known to those skilled in the art.
  • the pharmaceutically acceptable carrier various organic or inorganic carrier substances conventionally used as pharmaceutical ingredients are used, and excipients in solid preparations, binders, disintegrants, lubricants, coloring agents, solvents in liquid preparations, It is formulated as a solubilizer, suspending agent, tonicity agent, buffer, soothing agent and the like.
  • formulation additives such as preservatives, antioxidants, colorants, sweeteners and stabilizers can also be used, if necessary.
  • the eye drop When the eye drop is prepared, it can be produced by using a solubilizing agent, a tonicity agent, a buffer, a preservative and the like with trifluridine or a salt thereof.
  • the daily dose of the drug having the above-mentioned administration form can be appropriately determined depending on the condition, weight, age, sex and the like of the patient.
  • the high performance liquid chromatography column that can be used in the present invention is a silica gel column, a silica gel column whose surface is modified with octadecylsilyl group (ODS column or C18 column), a silica gel column whose surface is modified with octyl group (C8 column), Silica gel column whose surface is modified with cyanopropyl group (CN column), silica gel column whose surface is modified with phenethyl group (Ph column), silica gel column whose surface is modified with aminopropyl group (NH column), dihydroxy surface It is selected from a silica gel column modified with propyl group (Diol column), a column packed with various polymers (polymer column), a column packed with ion exchange resin (ion exchange column), etc. In the present invention, ODS columns are preferred.
  • ODS column various types of silica gel particles different in particle diameter, pore diameter, octadecylsilyl group bonding method, octadecylsilyl group substitution degree and the like can be used.
  • high purity silica gel is used, and an ODS column (end-capped ODS column) in which silanol remaining after octadecylation is treated with a low molecular weight silylating agent is preferable.
  • the average particle diameter of the silica gel is, for example, preferably 2 to 10 ⁇ m, and more preferably 3 to 5 ⁇ m.
  • the average particle size of silica gel can be measured by a laser diffraction method or the like.
  • the average pore diameter of the silica gel is, for example, preferably 6 to 20 nm, and more preferably 8 to 13 nm.
  • the average pore size of silica gel can be measured by a gas adsorption method or the like.
  • bonding method of the octadecyl silyl group in a silica gel for example, a monomeric form, polymeric form etc. are preferable.
  • the degree of substitution of the octadecylsilyl group can be measured by various methods.
  • the amount of carbon in silica gel is, for example, preferably 3% or more, and more preferably 10% or more.
  • the amount of carbon in the silica gel is, for example, preferably 25% or less, more preferably 20% or less.
  • the carbon content of silica gel can be measured by various methods.
  • the organic phase used in the mobile phase of high performance liquid chromatography includes nonpolar solvents such as hexane, cyclohexane, heptane, diethyl ether, tetrahydrofuran, chloroform and methylene chloride; aprotic polar solvents such as acetone, dimethylsulfoxide and acetonitrile; Acetic acid, methanol, ethanol, isopropanol, acetonitrile and the like can be used.
  • One of these solvents may be used alone, or two or more thereof may be used as a mixed solvent.
  • methanol or acetonitrile is preferable, and acetonitrile is more preferable.
  • the organic phase may contain 10% or less of water.
  • the aqueous phase used for the mobile phase of high performance liquid chromatography is not limited to water, but may contain 10% or less of an organic solvent, but contains 10% or less of methanol with respect to the entire aqueous phase.
  • the aqueous phase is preferred, more preferably 5% or less of methanol is contained, 2% or less of methanol is further preferably contained, and 0.1 to 1% of methanol is contained Particularly preferred.
  • acetic acid or a salt thereof citric acid or a salt thereof, tartaric acid or a salt thereof, phosphoric acid or a salt thereof can be added.
  • acetic acid or a salt thereof include acetic acid and sodium acetate.
  • Citric acid or a salt thereof includes citric acid, monosodium citrate, disodium citrate and trisodium citrate.
  • tartaric acid or its salt tartaric acid, sodium tartrate, phosphoric acid or its salt include phosphoric acid, sodium dihydrogen phosphate, disodium hydrogen phosphate, potassium dihydrogen phosphate, dipotassium hydrogen phosphate.
  • a phosphate is preferable, and sodium dihydrogenphosphate is more preferable, in view of the properties of the measurement substance, the shape of the peak obtained in the measurement, and the reproducibility of the measurement.
  • sodium dihydrogenphosphate is more preferable, in view of the properties of the measurement substance, the shape of the peak obtained in the measurement, and the reproducibility of the measurement.
  • the concentration of the buffer that can be used in the present invention can be appropriately adjusted as long as the buffer does not precipitate during measurement of high performance liquid chromatography.
  • it is 1 to 50 mM, more preferably 5 to 40 mM, still more preferably 10 to 30 mM, and particularly preferably 18 to 20 mM.
  • the ratio of the organic phase to the whole mobile phase at the retention time of the analogues 1, 2, 3, 4, 5 and trifluridine is preferably 1 to 14% by volume, more preferably 2 to 10% by volume And 3 to 7% by volume are particularly preferred.
  • the present invention is characterized by including the gradient conditions of step 1 and step 2.
  • Step 1 The ratio of the organic phase to the total mobile phase is 1 to 14% by volume.
  • Step 2 After step 1, apply a gradient to further increase the ratio of organic phase to total mobile phase.
  • the mobile phase may be in a gradient or in an isocratic state, but an isocratic state is preferred.
  • the ratio of the organic phase to the whole mobile phase in step 1 may be 1 to 14% by volume, preferably 2 to 10% by volume, and particularly preferably 3 to 7% by volume.
  • “the ratio of the organic phase to the whole mobile phase in step 1 is X to Y volume%” means that the ratio is in the range of X to Y volume% during step 1.
  • the start of step 1 is within 5 minutes of injecting the sample into high performance liquid chromatography, preferably within 3 minutes, more preferably within 1 minute, and particularly preferably simultaneously with the injection.
  • step 1 is 13 to 30 minutes after injecting the sample into high performance liquid chromatography. Taking into consideration that trifluridine does not overlap with the depressions in the baseline and ghost peaks, the end of step 1 is preferably 15-28 minutes after the injection, and particularly preferably 17-25 minutes.
  • the measurement time in step 1 is not particularly limited as long as it is in the range of the above start and end, but it is preferably 15 to 28 minutes, and more preferably 17 to 25 minutes.
  • the upper limit of the flow rate of the mobile phase in step 1 may be that which is usually used in high performance liquid chromatography, but the flow rate is preferably 2.5 mL / min or less in consideration of separation of the retention time of each related substance. 2.0 mL / min or less is more preferable, 1.5 mL / min or less is more preferable, 1.3 mL / min or less is still more preferable, and 1.1 mL / min or less is particularly preferable. Further, the range of the flow rate of the mobile phase in step 1 is not particularly limited, but is preferably 0.5 to 2.5 mL / min, more preferably 0.5 to 2.0 mL / min, and more preferably 0.7 to 1. 5 mL / min is more preferable, 0.8 to 1.3 mL / min is even more preferable, and 0.9 to 1.1 mL / min is particularly preferable.
  • step 1 one or more trifluridine analogues selected from the group consisting of analogues 1, 2, 3, 4 and 5 can be detected.
  • analogues 1, 2, 3, 4 and 5 can be detected in step 1.
  • Step 1 when at least one of Related Substances 1, 2, 3, 4 and 5 is detected in Step 1, Related Substances 1, 2, 3,
  • the difference between the maximum value and the minimum value of the ratio of the organic phase to the entire mobile phase at each retention time of each of those detected in step 1 among 4 and 5 is 5% by volume or less of the total mobile phase Preferably, it is 1% by volume or less.
  • step 2 the mobile phase is subjected to a gradient to increase the proportion of the organic phase. If gradients were applied in step 1, step 2 applies gradients to increase the proportion of the organic phase. In the case of isocratic state in step 1, step 2 applies a gradient to increase the proportion of the organic phase.
  • the ratio of the organic phase to the whole mobile phase at the end of Step 2 is 25 to 70% by volume, more preferably 30 to 65% by volume, and particularly preferably 35 to 60% by volume.
  • the end of step 2 means the point at which the application of the gradient of step 2 is stopped and the ratio of the organic phase to the whole mobile phase is started to be reduced.
  • the start of step 2 is not particularly limited as long as it starts within 3 minutes after the end of step 1, but 1 minute or less is preferable, and the end of step 1 and its time are more preferable.
  • Step 2 is not particularly limited as long as it is after the retention time of Related Substance 6, but it is 35 to 65 minutes after injecting the sample into high performance liquid chromatography, and more preferably 40 to 60 minutes. .
  • the measurement time in step 2 is not particularly limited as long as the retention time of the related substance 6 can be measured, but 10 to 50 minutes is preferable, and 15 to 45 minutes is more preferable.
  • the gradient in step 2 is not particularly limited as long as it increases the ratio of the organic phase to the total mobile phase, but increases the ratio of the organic phase to the total mobile phase in one minute by 0.9% or more It is more preferable to increase by 1.0% by volume or more per minute, and it is particularly preferable to increase 2.0% by volume or more by 1 minute.
  • the upper limit of the increase in the ratio of the organic phase to the entire mobile phase is not particularly limited, but for example, 10% by volume or less is preferable, and 5.0% by volume or less is more preferable.
  • the change in the ratio of the organic phase to the whole mobile phase during the measurement time of step 2 (the difference between the maximum value and the minimum value among the ratios of the organic phase to the whole mobile phase in the measurement time) Of the whole mobile phase is preferably 30% by volume or more, and more preferably 50% by volume or more.
  • the upper limit of the change in the ratio of the organic phase to the total mobile phase during the measurement time of step 2 is not particularly limited, but preferably 80% by volume or less of the total mobile phase, for example 70% by volume or less Particularly preferred.
  • the flow rate of the mobile phase in step 2 may be any flow rate commonly used in high performance liquid chromatography, but the flow rate is preferably 2.5 mL / min or less, considering the separation of the retention time of each related substance. 0 mL / min or less is more preferable, and 1.5 mL / min or less is more preferable.
  • the range of the flow rate of the mobile phase in step 2 is not particularly limited, but is preferably 0.5 to 2.5 mL / min, more preferably 0.5 to 2.0 mL / min, and more preferably 1.0 to 1. 5 mL / min is more preferable. From the viewpoint of shortening the overall measurement time, it is also possible to gradually increase the flow rate after the start of step 2, and the flow rate at the end of step 2 is 1.0 to 1.5 of the flow rate at the end of step 1 Double is preferred.
  • step 2 the related substance 6 can be detected.
  • an ion pairing reagent in addition to the above additives, an ion pairing reagent can be added.
  • the ion pairing reagent include sodium pentane sulfonate, sodium hexane sulfonate, sodium heptane sulfonate, sodium octane sulfonate, an alkyl sulfonic acid such as sodium dodecane sulfonate or a salt thereof (preferably sodium alkyl sulfonate), sodium dodecyl sulfate And the like (alkyl sodium sulfate), tetraethylammonium hydroxide, tetrabutylammonium hydroxide, tetrabutylammonium chloride, quaternary ammonium salts such as tetrabutylammonium bromide, trihexylamine, trioctylamine And other tertiary amines can be used.
  • the pH of the mobile phase (typically, the aqueous phase) in the present invention can be appropriately adjusted by the above-mentioned additives and the like, but is preferably 2.0 to 5.0.
  • the detection wavelength that can be used in the method of the present invention can be 208 to 280 nm, preferably 208 to 240 nm, more preferably 208 to 212 nm, in consideration of the properties of each related substance.
  • the temperature of the mobile phase in the column that can be used in the method of the present invention can be set as appropriate.
  • the temperature is preferably kept constant, more preferably 25 to 50 ° C., still more preferably 35 to 45 ° C., particularly preferably 40 to 50 ° C. It is 44 ° C.
  • a preheat mixer etc. can be used in order to keep the said temperature constant.
  • the injection amount, the temperature in the column, and the like can be appropriately changed.
  • analogues 1 to 5 can be synthesized by known methods, or can be obtained as commercial products.
  • the related substance 6 can be synthesized from triflulysine or a commercially available compound according to a known method, or can be synthesized according to the method described later.
  • the high-performance liquid chromatography retention time, mass spectrum, and photodiode array (PDA) of the analogues thus obtained are compared with those of the analogues detected according to the present invention to identify the analogues. can do.
  • these analogues can be quantitatively measured by any method of using an external standard or a method of using an internal standard.
  • analogues can also be detected from trifluridine or salts thereof by the method of the present invention.
  • one or more of the analogues 1 to 6 can be used as a standard product for quality control, preferably three or more of the analogues 1 to 6, and more preferably the analogues It is four or more of 1 to 6, particularly preferably analogues 2, 3, 5 and 6.
  • the trifluridine used in such standard products and the related substances derived therefrom become highly pure. Therefore, each related substance separated under the conditions of the high performance liquid chromatography can be used as a standard. Therefore, the present invention can be reworded as a method for producing the related substance, which is characterized in that it is isolated from a combination drug containing trifluridine or a salt thereof.
  • the related substances include the above-mentioned related substances 1 to 6, and preferably the related substances 2, 3, 5, and 6.
  • the quality control method provided by the present invention is a method for quality control of a compounding agent containing trifluridine or a salt thereof, which comprises the step of carrying out the above-mentioned method of the present invention.
  • the quality control method of the present invention for example, when the content (mass) of the above-mentioned analogue (for example, any one or more of analogues 1 to 6) is not more than a predetermined amount, the combination agent is It can be evaluated that the criteria for
  • the present invention also provides a method for producing a combination drug containing trifluridine or a salt thereof, which comprises the steps of carrying out the above-mentioned method of the present invention.
  • the production method of the present invention performs, for example, the step of producing trifluridine or a salt thereof, the obtained trifluridine or a salt thereof, and the quality control of the compounding agent containing the above method (preferably containing triflulidine or a salt thereof) ) Can be included.
  • the present invention includes the related substances 2, 3, 5 or 6 used for quality control of a pharmaceutical composition containing trifluridine or a salt thereof, and further, an impurity of a pharmaceutical composition containing trifluridine or a salt thereof It contains related substances 2, 3, 5 or 6 for use as a standard in detection.
  • a method of producing analogues 2, 3, 5 or 6 characterized in that it is isolated from a pharmaceutical composition containing trifluridine or a salt thereof is also included in the present invention.
  • the method comprises the conditions of the following Step 1 and Step 2 for detecting at least one trifluridine-derived analogue selected from the group consisting of analogues 1 to 6.
  • Step 1 The ratio of the organic phase to the total mobile phase is 1 to 14% by volume.
  • Step 2 After step 1, apply a gradient to further increase the proportion of the organic phase.
  • the method includes the conditions of step 1 and step 2 below.
  • Step 1 Detect at least one trifluridine-derived analogue selected from the group consisting of analogues 1 to 5, and the ratio of the organic phase to the whole mobile phase is 1 to 14% by volume, in an isocratic state is there.
  • Step 2 After step 1, detect related substance 6 by applying a gradient to further increase the proportion of the organic phase.
  • the method includes the conditions of step 1 and step 2 below.
  • Step 1 The ratio of the organic phase to the total mobile phase is 2 to 10% by volume, is isocratic, and detects Related Substances 1 to 5.
  • Step 2 After step 1, detect related substance 6 by applying a gradient to further increase the ratio of the organic phase to the total mobile phase.
  • Step 1 The ratio of the organic phase to the total mobile phase is 2 to 10% by volume, is isocratic, and detects Related Substances 1 to 5.
  • Step 2 Apply a gradient to further increase the ratio of the organic phase to the total mobile phase after Step 1, and the ratio of the organic phase to the total mobile phase at the end of Step 2 is 25 to 70% by volume, Detect related substance 6
  • the method includes the conditions of the following steps 1 and 2 in which the organic phase in the mobile phase contains acetonitrile, the water phase contains water, and the mobile phase contains phosphate.
  • Step 1 The ratio of the organic phase to the total mobile phase is 2 to 10% by volume, is isocratic, and detects Related Substances 1 to 5.
  • Step 2 Apply a gradient that increases the ratio of the organic phase to the total mobile phase by 0.9% or more per minute after step 1, and the ratio of the organic phase to the total mobile phase at the end of step 2 is 30 It is up to 65% by volume, and the related substance 6 is detected.
  • the method includes the conditions of the following steps 1 and 2 in which the organic phase in the mobile phase contains acetonitrile, the water phase contains water, and the mobile phase contains phosphate.
  • Step 1 The ratio of the organic phase to the total mobile phase is 2 to 10% by volume, is isocratic, and detects Related Substances 1 to 5.
  • Step 2 Apply a gradient that increases the ratio of the organic phase to the total mobile phase by 0.9% or more per minute after step 1, and the ratio of the organic phase to the total mobile phase at the end of step 2 is 30 It is up to 65% by volume, and the related substance 6 is detected.
  • the method includes the conditions of the following steps 1 and 2 in which the organic phase in the mobile phase contains acetonitrile, the water phase contains water, and the mobile phase contains phosphate.
  • Step 1 The ratio of the organic phase to the total mobile phase is 3 to 7% by volume, is isocratic, and detects Related Substances 1 to 5.
  • Step 2 After step 1, apply a gradient that increases the ratio of the organic phase to the total mobile phase by 0.9% by volume or more, and the ratio of the organic phase to the total mobile phase at the end of step 2 is 35 Up to 60% by volume, the related substance 6 is detected.
  • the method includes the conditions of the following steps 1 and 2 in which the organic phase in the mobile phase contains acetonitrile, the water phase contains water, and the mobile phase contains sodium phosphorous dihydrogen.
  • Step 1 The ratio of the organic phase to the whole mobile phase is 3 to 7% by volume, is isocratic, the measurement time is 15 to 28 minutes, and the related substances 1 to 5 are detected.
  • Step 2 After step 1, apply a gradient that increases the ratio of the organic phase to the whole mobile phase by 0.9% by volume or more per minute, and the organic phase to the whole mobile phase at the end of step 2 The ratio is 35 to 60% by volume, the measurement time is 10 to 50 minutes, and the related substance 6 is detected.
  • the method includes the conditions of the following steps 1 and 2 in which the organic phase in the mobile phase contains acetonitrile, the water phase contains water, and the mobile phase contains sodium phosphorous dihydrogen.
  • Step 1 The ratio of the organic phase to the whole mobile phase is 3 to 7% by volume, is isocratic, the measurement time is 15 to 28 minutes, and the related substances 1 to 5 are detected.
  • Step 2 After step 1, apply a gradient that increases the ratio of the organic phase to the total mobile phase by 0.9% by volume or more, and the ratio of the organic phase to the total mobile phase at the end of step 2 is 35 It is ⁇ 60% by volume, the measurement time is 10 to 50 minutes, and the flow rate at the end of step 2 is 1.0 to 1.5 times the flow rate of step 1, and the related substance 6 is detected.
  • Step 1 the organic phase in the mobile phase contains acetonitrile, the water phase contains water, and the mobile phase contains methanol and sodium phosphorous dihydrogen.
  • Step 1 The ratio of the organic phase to the whole mobile phase is 3 to 7% by volume, is isocratic, the measurement time is 15 to 28 minutes, and the related substances 1 to 5 are detected.
  • Step 2 After step 1, apply a gradient that increases the ratio of the organic phase to the total mobile phase by 0.9% by volume or more, and the ratio of the organic phase to the total mobile phase at the end of step 2 is 35 It is ⁇ 60% by volume, the measurement time is 10 to 50 minutes, and the flow rate at the end of step 2 is 1.0 to 1.5 times the flow rate of step 1, and the related substance 6 is detected.
  • the sample to be measured by high performance liquid chromatography was prepared as follows.
  • the solution was dissolved in a solution having the same composition as the mobile phase used in each measurement condition so that the concentration of trifluridine was about 0.8 mg / mL, and a properly diluted solution was used as a sample.
  • “%” indication in each mobile phase means volume%.
  • Example 1-1 Column: Hydrosphere C18, manufactured by YMC (3 ⁇ m) Flow rate: 1.0 mL / min Mobile phase A: 0.05 mol / L aqueous sodium dihydrogenphosphate aqueous solution Mobile phase B: acetonitrile Gradient: The mobile phase A and the mobile phase B were changed as follows to control the concentration gradient. 0 to 18 minutes: A 95% B 5%, 18 to 55 minutes: A 95 to 60% B 5% to 40%, 55 to 55.1 minutes A 60% to 95% B 40% to 5%, 55.1 or later A 95% B 5%.
  • Example 1-2 Gradient Concentration gradient control was performed by changing mobile phase A and mobile phase B as follows. 0 to 18 minutes: A 95% B 5%, 18 to 48 minutes: A 95 to 60% B 5% to 40%, 48 to 48.1 minutes A 60% to 95% B 40% to 5%, 48.1 or later A 95% B 5%.
  • the column, flow rate, mobile phase A and mobile phase B are the same as in Example 1.
  • Example 1-3 Concentration gradient control was performed by changing mobile phase A and mobile phase B as follows. 0 to 20 minutes: A 95% B 5%, 20 to 50 minutes: A 95 to 60% B 5% to 40%, 50 to 50.1 minutes A 60% to 95% B 40% to 5%, 50.1 or later A 95% B 5%.
  • the column, flow rate, mobile phase A and mobile phase B are the same as in Example 1.
  • Example 1-3-1 Gradient The concentration gradient was controlled by changing the mobile phase A and the mobile phase B as follows. 0 to 15 minutes: A 95% B 5%, 15 to 45 minutes: A 95 to 60% B 5% to 40%, 45 to 45.1 minutes A 60% to 95% B 40% to 5%, 45.1 or later A 95% B 5%.
  • the column, flow rate, mobile phase A and mobile phase B are the same as in Example 1.
  • the flow rate, the mobile phase A and the mobile phase B are the same as in Example 1-3.
  • Example 1-3-2 Column: Inertsil ODS-4 GL Science (3 ⁇ m) The flow rate, the mobile phase A and the mobile phase B, and the gradient are the same as in Example 1-3-1.
  • Example 1-3-3 Column: YMC-Pack Pro C18 RS (3 ⁇ m) The flow rate, the mobile phase A and the mobile phase B, and the gradient are the same as in Example 1-3-1.
  • Example 1-3-4 Column: YMC-Pack Pro C18 (3 ⁇ m) The flow rate, the mobile phase A and the mobile phase B, and the gradient are the same as in Example 1-3-1.
  • Example 2-1 Column: Hydrosphere C18, manufactured by YMC (5 ⁇ m) Flow rate: 0 to 21 minutes 1.0 mL / min, 21 to 46 minutes 1.0 ⁇ 1.3 mL / min, 46 to 46.1 minutes 1.3 ⁇ 1.0 mL / min, 46.1 minutes or later 1.0 mL / Min
  • Mobile phase A 3.0 g of sodium dihydrogen phosphate dihydrate was dissolved in 1000 mL of water, adjusted to pH 2.2 by adding phosphoric acid, and then 10 mL of methanol was added.
  • Mobile phase B acetonitrile gradient: Mobile phase A and mobile phase B were changed as follows to control concentration gradient. 0 to 21 minutes: A 96% B 4%, 21 to 46 minutes: A 96 to 45% B 4 to 55%, 46 to 46.1 minutes A 45 to 96% B 55 to 4%, 46.1 or more A 96% B 4%.
  • the retention time of trifluridine was 17.4 minutes. Further, the retention time of Related Substance 1 is 3.1 minutes, the retention time of Related Substance 2 is 5.6 minutes, the retention time of Related Substance 3 is 4.1 minutes, the retention time of Related Substance 4 is 7.9 minutes, The retention time of Related Substance 5 was 8.8 minutes, and the retention time of Related Substance 6 was 42.9 minutes.
  • the detected analogues were compounds showing structures of analogues 1 to 6 because their retention times were identical to those of the analogues purchased or synthesized separately.
  • Example 2-2 Column: Unison UK-C18, manufactured by Intact (5 ⁇ m) Other conditions are the same as in Example 2-1.
  • Example 2-3 Mobile phase A: 3.0 g of sodium dihydrogen phosphate dihydrate was dissolved in 1000 mL of water, adjusted to pH 2.2 with phosphoric acid, and then 9 mL of methanol was added. Other conditions are the same as in Example 2-1.
  • Example 2-4 Flow rate: 0 to 21 minutes 0.95 mL / min, 21 to 46 minutes 0.95 to 1.25 mL / min, 46 to 46.1 minutes 1.25 to 0.95 mL / min, 46.1 minutes to 0.95 mL / Min Other conditions are the same as in Example 2-1.
  • Example 2-5 Concentration gradient control was performed by changing mobile phase A and mobile phase B as follows. 0 to 21 minutes: A 95.8% B 4.2%, 21 to 46 minutes: A 95.8 ⁇ 43% B 4.2 ⁇ 57%, 46 to 46.1 minutes A43 ⁇ 95.8% B57 ⁇ 4.2%, 46.1 or later A 95.8% B 4.2%. Other conditions are the same as in Example 2-1.
  • Example 3 Column: Inertsil ODS-2, manufactured by GL Science (5 ⁇ m) Flow rate: 1.0 mL / min Mobile phase A: 0.05 mol / L sodium dihydrogenphosphate aqueous solution Mobile phase B: acetonitrile Gradient: The mobile phase A and the mobile phase B were changed as follows to control the concentration gradient. 0 to 2 minutes: A 85% B 15%, 2 to 7 minutes: A 85 to 60% B 15 to 40%, 7 minutes or more A 60% B 40%.
  • Example 2 Column: Inertsil ODS-2, manufactured by GL Science (5 ⁇ m) Flow rate: 1.0 mL / min Mobile phase A: water Mobile phase B: acetonitrile gradient: A60% B40% isocratic state.

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Abstract

A method for detecting an analog that is derived from trifluridine, the method including a step for subjecting a sample that includes trifluridine or a salt thereof to high-speed liquid chromatography that uses a mobile phase that comprises an organic phase and an aqueous phase. The step for subjecting the sample to the high-speed liquid chromatography includes a step 1 and a step 2 that satisfy the following conditions. Step 1: The organic phase fraction of the mobile phase is 1–14 volume%. Step 2: After step 1, a gradient is applied to increase the organic phase fraction of the mobile phase.

Description

トリフルリジン由来の類縁物質の検出方法Method for detecting related substances derived from trifluridine
　本発明は、高速液体クロマトグラフィーによるトリフルリジン含有製剤の類縁物質の測定方法に関する。 The present invention relates to a method for measuring a related substance of a trifluridine-containing preparation by high performance liquid chromatography.
　一般的に医薬品の類縁物質の測定は、液体クロマトグラフィーにより行われており、水及び有機溶媒の混液を移動相とした順相又は逆相カラムの系がよく用いられる。古くより、Ｖｉｒｏｐｉｃ（登録商標）の名前でトリフルリジンは感染治療用の点眼剤として用いられている。出願人の製品であるロンサーフ（登録商標）は、トリフルリジン及びチピラシルを含有する製剤である。当該トリフルリジン及びチピラシルを含む医薬品は、抗腫瘍剤として販売されており、その製剤について複数の報告がある（特許文献１、２、３、４）。 In general, measurement of pharmaceutical analogues is carried out by liquid chromatography, and a system of normal phase or reverse phase column using a mixture of water and an organic solvent as a mobile phase is often used. Since ancient times, trifluridine has been used as an eye drop for the treatment of infections under the name of Viropic®. Applicants' product Ronsurf (R) is a formulation containing trifluridine and tipiracil. The medicine containing trifluridine and tipiracil is marketed as an antitumor agent, and there are multiple reports on its preparation ( Patent Documents 1, 2, 3, 4).
　そして、トリフルリジンの類縁物質として、５－トリフルオロメチルウラシル、及び５－カルボキシウラシルが知られており、高速液体クロマトグラフィーでこれらを測定している報告がある（非特許文献１）。 Then, 5-trifluoromethyluracil and 5-carboxyuracil are known as analogues of trifluridine, and there is a report that they are measured by high performance liquid chromatography (Non-patent Document 1).
　トリフルリジンの類縁物質である５－トリフルオロメチルウラシルが、様々な逆相液体クロマトグラフィーの条件で測定できることが報告されている（非特許文献２）。 It is reported that 5-trifluoromethyluracil, which is an analogue of trifluridine, can be measured under various reverse phase liquid chromatography conditions (Non-patent Document 2).
　トリフルリジンの腫瘍細胞への取込みを測定するために、液体クロマトグラフィーを用いることが報告されている（非特許文献３）。 It has been reported that liquid chromatography is used to measure uptake of trifluridine into tumor cells (Non-patent Document 3).
　トリフルリジンのＴＫ１の基質特異性の判断に、液体クロマトグラフィーが用いられていることが報告されている（非特許文献４）。 It has been reported that liquid chromatography is used to determine the substrate specificity of trifluridine TK1 (Non-patent Document 4).
　酵素合成におけるトリフルリジンの測定において、液体クロマトグラフィーが用いられていることが報告されている（非特許文献５）。 It has been reported that liquid chromatography is used in the measurement of trifluridine in enzyme synthesis (Non-patent Document 5).
　また、トリフルリジン及びチピラシルを含む医薬品について、定量的な液体クロマトグラフィーの分析が報告されている（非特許文献６、７、８）。 In addition, quantitative liquid chromatography analysis has been reported for pharmaceuticals containing trifluridine and tipiracil (Non-patent Documents 6, 7 and 8).
　トリフルリジンの類縁物質である５－トリフルオロメチルウラシルの測定のために、アセトニトリルを用いた様々な高速液体クロマトグラフィーの条件が報告されている（非特許文献２）。 Various high performance liquid chromatography conditions using acetonitrile have been reported for the measurement of 5-trifluoromethyluracil, which is an analogue of trifluridine (Non-patent Document 2).
　トリフルリジンの腫瘍細胞への取込みを測定するために、アセトニトリルを用いた高速液体クロマトグラフィーの条件が報告されている（非特許文献３）。 Conditions for high performance liquid chromatography using acetonitrile have been reported to measure uptake of trifluridine into tumor cells (Non-patent Document 3).
　トリフルリジンのＴＫ１の基質特異性の判断のために、アセトニトリルを用いた高速液体クロマトグラフィーの条件が報告されている（非特許文献４）。 Conditions for high-performance liquid chromatography using acetonitrile have been reported for the determination of the substrate specificity of trifluridine TK1 (Non-patent Document 4).
　酵素合成におけるトリフルリジンを確認するために、酢酸トリメチルアンモニウムを添加し、グラジエントをかけた高速液体クロマトグラフィーの条件が報告されている（非特許文献５）。 In order to confirm trifluridine in enzyme synthesis, conditions of high performance liquid chromatography with gradient added with trimethyl ammonium acetate have been reported (Non-patent Document 5).
　ロンサーフに含まれるトリフルリジンとチピラシルの定量的な測定のために、アセトニトリルを用いた高速液体クロマトグラフィーの条件が報告されている（非特許文献６、７、８）。 Conditions for high performance liquid chromatography using acetonitrile have been reported for quantitative determination of trifluridine and tipiracil contained in Ronsurf (Non-patent Documents 6, 7 and 8).
　点眼剤としてのトリフルリジンについて、メタノールを用いた高速液体クロマトグラフィーの条件が報告されている（非特許文献９、１０）。 Conditions of high performance liquid chromatography using methanol have been reported for trifluridine as eye drops (Non-patent Documents 9 and 10).
　トリフルリジンについて、酢酸バッファー、トリフルオロ酢酸、もしくは酢酸を添加した移動相を用いた高速液体クロマトグラフィーの条件が報告されている（非特許文献１１、１２、特許文献６、１３）。 With respect to trifluridine, conditions of high performance liquid chromatography using a mobile phase to which acetic acid buffer, trifluoroacetic acid, or acetic acid is added have been reported (Non-patent documents 11, 12, Patent documents 6, 13).
　また、トリフルリジンの純度を確認するために、高速液体クロマトグラフィーを用いているが、その条件は記載されていない（特許文献７）。 In addition, high performance liquid chromatography is used to confirm the purity of trifluridine, but the conditions are not described (Patent Document 7).
　しかし、これらの報告には、トリフルリジンの類縁物質を２つのグラジェント条件を用いたステップによって高速液体クロマトグラフィーで検出する方法についての示唆はない。 However, these reports do not suggest a method for detecting trifluridine analogues by high performance liquid chromatography by steps using two gradient conditions.
ＷＯ２０１３／１２２１３４WO 2013/122134 ＷＯ２０１３／１２２１３５WO 2013/122135 ＷＯ２００６／０８０３２７WO2006 / 080327 ＷＯ９６／３０３４６WO 96/30346 ＣＮ１０６７４９１９４ＡCN106749194A ＣＮ１０５１９８９４７ＡCN105198947A ＣＮ１０５４６１７７２ＡCN105461772A 日本特許４６０３２７４Japanese Patent 4603274 日本特許４４４１３１３Japanese patent 4441313 日本特許４４３７７８６Japanese patent 4437786
　本発明が解決すべき課題は、トリフルリジン又はその塩を含む試料から、トリフルリジン由来の類縁物質を２つのグラジェント条件を用いたステップにより高速液体クロマトグラフィーで検出し得る新規方法を提供することである。 The problem to be solved by the present invention is to provide a novel method capable of detecting trifluridine-derived analogues from a sample containing trifluridine or a salt thereof by high performance liquid chromatography by steps using two gradient conditions. It is.
　本発明者らは鋭意検討した結果、特定の条件下で高速液体クロマトグラフィーを用いることより、トリフルリジン又はその塩の類縁物質を効率よく検出できると共に、その品質を保証するのに適した検出方法を見出した。 As a result of intensive investigations, the present inventors are able to detect trifluridine or its salt analogues efficiently by using high performance liquid chromatography under specific conditions, and a detection method suitable for assuring the quality thereof Found out.
　したがって、本発明は、次の［１］～［３１］を提供するものである。 Therefore, the present invention provides the following [1] to [31].
　［１］トリフルリジン又はその塩を含む試料を有機相及び水相からなる移動相を用いる高速液体クロマトグラフィーに供する工程を含む、トリフルリジン由来の類縁物質を検出するための方法であって、前記高速液体クロマトグラフィーに供する工程が、以下の要件を満たすステップ１及び２を含む、方法：
ステップ１：移動相全体に対する有機相の割合が１～１４容量％である。
ステップ２：ステップ１の後、移動相全体に対する有機相の割合を増加させるようグラジェントをかける。 [1] A method for detecting a related substance derived from trifluridine, comprising the step of subjecting a sample containing trifluridine or a salt thereof to high performance liquid chromatography using a mobile phase consisting of an organic phase and an aqueous phase, the method comprising the steps of: The method wherein the step of subjecting to high performance liquid chromatography comprises steps 1 and 2 meeting the following requirements:
Step 1: The ratio of the organic phase to the total mobile phase is 1 to 14% by volume.
Step 2: After step 1, apply a gradient to increase the ratio of organic phase to total mobile phase.
　［２］前記類縁物質が下記類縁物質１～６からなる群より選ばれる少なくとも１つである、［１］に記載の方法：
類縁物質１：５－カルボキシウラシル、
類縁物質２：５－カルボキシ－２’－デオキシ－ウリジン、
類縁物質３：２’－デオキシ－５－メトキシカルボニルウリジン、
類縁物質４：トリフルオロチミン、
類縁物質５：５－メトキシカルボニルウラシル、
類縁物質６：５’－（４－クロロフェニルカルボキシ）－２’－デオキシ－５－トリフルオロメチルウリジン。 [2] The method according to [1], wherein the analogue is at least one selected from the group consisting of the following analogues 1 to 6:
Related substances 1: 5-carboxyuracil,
Analogs 2: 5-Carboxy-2'-deoxy-uridine,
Related substances 3: 2'-deoxy-5-methoxycarbonyluridine,
Related substances 4: trifluorothymine,
Related substances 5: 5-methoxycarbonyluracil,
Analogs 6: 5 '-(4-chlorophenylcarboxy) -2'-deoxy-5-trifluoromethyluridine.
　［３］ステップ１で類縁物質１～５を検出し、ステップ２で類縁物質６を検出する、［１］又は［２］に記載の方法。 [3] The method according to [1] or [2], wherein the analogues 1 to 5 are detected in step 1 and the analogue 6 is detected in step 2.
　［４］ステップ１がアイソクラティック状態で行われる、［１］～［３］のいずれか一項に記載の方法。 [4] The method according to any one of [1] to [3], wherein Step 1 is performed in an isocratic state.
　［５］有機相がアセトニトリルである、［１］～［４］のいずれか一項に記載の方法。 [5] The method according to any one of [1] to [4], wherein the organic phase is acetonitrile.
　［６］ステップ１における移動相全体に対する有機相の割合が２～１０容量％である、［１］～［５］のいずれか一項に記載の方法。 [6] The method according to any one of [1] to [5], wherein the ratio of the organic phase to the total mobile phase in Step 1 is 2 to 10% by volume.
　［７］ステップ２の終了時における移動相全体に対する有機相の割合が２５～７０容量％である、［１］～［６］のいずれか一項に記載の方法。 [7] The method according to any one of [1] to [6], wherein the ratio of the organic phase to the total mobile phase at the end of Step 2 is 25 to 70% by volume.
　［８］ステップ２において移動相全体に対する有機相の割合を１分間に０．９容量％以上増加させるグラジェントをかける、［１］～［７］のいずれか一項に記載の方法。 [8] The method according to any one of [1] to [7], wherein a gradient is applied in step 2 to increase the ratio of the organic phase to the total mobile phase by 0.9% by volume or more per minute.
　［９］ステップ２の測定時間が１０～５０分間である、［１］～［８］のいずれか一項に記載の方法。 [9] The method according to any one of [1] to [8], wherein the measurement time of step 2 is 10 to 50 minutes.
　［１０］ステップ２の終了時における流量がステップ１の流量の１．０～１．５倍である、［１］～［９］のいずれか一項に記載の方法。 [10] The method according to any one of [1] to [9], wherein the flow rate at the end of Step 2 is 1.0 to 1.5 times the flow rate of Step 1.
　［１１］水相がさらにリン酸塩を含む、［１］～［１０］のいずれか一項に記載の方法。 [11] The method according to any one of [1] to [10], wherein the aqueous phase further comprises phosphate.
　［１２］水相がさらにメタノールを含む、［１］～［１１］のいずれか一項に記載の方法。 [12] The method according to any one of [1] to [11], wherein the aqueous phase further comprises methanol.
　［１３］トリフルリジン又はその塩を含む配合剤の品質管理に用いられる５－カルボキシ－２’－デオキシ－ウリジン（類縁物質２）。 [13] 5-Carboxy-2'-deoxy-uridine (analog 2) used for quality control of a combination drug containing trifluridine or a salt thereof.
　［１４］トリフルリジン又はその塩を含む配合剤の不純物検出の際に標準品として用いるための５－カルボキシ－２’－デオキシ－ウリジン（類縁物質２）。 [14] 5-Carboxy-2'-deoxy-uridine (analog 2) for use as a standard in the detection of impurities of a combination drug containing trifluridine or a salt thereof.
　［１５］５－カルボキシ－２’－デオキシ－ウリジン（類縁物質２）の、トリフルリジン又はその塩を含む配合剤の不純物検出の際の標準品としての使用。 [15] Use of 5-carboxy-2'-deoxy-uridine (analog 2) as a standard in the detection of impurities of a combination containing trifluridine or a salt thereof.
　［１６］５－カルボキシ－２’－デオキシ－ウリジン（類縁物質２）からなる、トリフルリジン又はその塩を含む配合剤の不純物検出の際の標準品。 [16] A standard preparation for detecting impurities of a compounding preparation containing trifluridine or a salt thereof, which comprises 5-carboxy-2'-deoxy-uridine (analog 2).
　［１７］トリフルリジン又はその塩を含む配合剤の品質管理に用いられる２’－デオキシ－５－メトキシカルボニルウリジン（類縁物質３）。 [17] 2'-Deoxy-5-methoxycarbonyluridine (analog 3) used for quality control of a combination drug containing trifluridine or a salt thereof.
　［１８］トリフルリジン又はその塩を含む配合剤の不純物検出の際に標準品として用いるための２’－デオキシ－５－メトキシカルボニルウリジン（類縁物質３）。 [18] 2'-Deoxy-5-methoxycarbonyluridine (analog 3) for use as a standard in detecting impurities of a compounding agent containing trifluridine or a salt thereof.
　［１９］２’－デオキシ－５－メトキシカルボニルウリジン（類縁物質３）の、トリフルリジン又はその塩を含む配合剤の不純物検出の際の標準品としての使用。 [19] Use of 2'-deoxy-5-methoxycarbonyluridine (analog 3) as a standard in the detection of impurities in a combination containing trifluridine or a salt thereof.
　［２０］２’－デオキシ－５－メトキシカルボニルウリジン（類縁物質３）からなる、トリフルリジン又はその塩を含む配合剤の不純物検出の際の標準品。 [20] A standard preparation for detecting impurities of a compounding preparation containing trifluridine or a salt thereof, which comprises 2'-deoxy-5-methoxycarbonyluridine (analog 3).
　［２１］トリフルリジン又はその塩を含む配合剤の品質管理に用いられる５－メトキシカルボニルウラシル（類縁物質５）。 [21] 5-Methoxycarbonyluracil (analog 5) used for quality control of a combination drug containing trifluridine or a salt thereof.
　［２２］トリフルリジン又はその塩を含む配合剤の不純物検出の際に標準品として用いるための５－メトキシカルボニルウラシル（類縁物質５）。 [22] 5-Methoxycarbonyluracil (analog 5) for use as a standard in the detection of impurities of a compounding agent containing trifluridine or a salt thereof.
　［２３］５－メトキシカルボニルウラシル（類縁物質５）の、トリフルリジン又はその塩を含む配合剤の不純物検出の際の標準品としての使用。 [23] Use of 5-methoxycarbonyluracil (analog 5) as a standard preparation for detecting impurities of a combination agent containing trifluridine or a salt thereof.
　［２４］５－メトキシカルボニルウラシル（類縁物質５）からなる、トリフルリジン又はその塩を含む配合剤の不純物検出の際の標準品。 [24] A standard preparation for impurity detection of a compounding agent containing trifluridine or a salt thereof, which comprises 5-methoxycarbonyluracil (analog 5).
　［２５］５’－（４－クロロフェニルカルボキシ）－２’－デオキシ－５－トリフルオロメチルウリジン（類縁物質６）。 [25] 5 '-(4-Chlorophenylcarboxy) -2'-deoxy-5-trifluoromethyluridine (analog 6).
　［２６］トリフルリジン又はその塩を含む配合剤の品質管理に用いられる５’－（４－クロロフェニルカルボキシ）－２’－デオキシ－５－トリフルオロメチルウリジン（類縁物質６）。 [26] 5 '-(4-Chlorophenylcarboxy) -2'-deoxy-5-trifluoromethyluridine (analog 6) used for quality control of a combination drug containing trifluridine or a salt thereof.
　［２７］トリフルリジン又はその塩を含む配合剤の不純物検出の際に標準品として用いるための５’－（４－クロロフェニルカルボキシ）－２’－デオキシ－５－トリフルオロメチルウリジン（類縁物質６）。 [27] 5 '-(4-Chlorophenylcarboxy) -2'-deoxy-5-trifluoromethyluridine (analogue 6) for use as a standard in the detection of impurities in combinations containing trifluridine or a salt thereof .
　［２８］５’－（４－クロロフェニルカルボキシ）－２’－デオキシ－５－トリフルオロメチルウリジン（類縁物質６）の、トリフルリジン又はその塩を含む配合剤の不純物検出の際の標準品としての使用。 [28] 5 '-(4-Chlorophenylcarboxy) -2'-deoxy-5-trifluoromethyluridine (analog 6) as a standard for the detection of impurities in combinations containing trifluridine or a salt thereof use.
　［２９］５’－（４－クロロフェニルカルボキシ）－２’－デオキシ－５－トリフルオロメチルウリジン（類縁物質６）からなる、トリフルリジン又はその塩を含む配合剤の不純物検出の際の標準品。 [29] A standard preparation for detecting an impurity of a compounding agent containing trifluridine or a salt thereof, which comprises 5 '-(4-chlorophenylcarboxy) -2'-deoxy-5-trifluoromethyluridine (analog 6).
　［３０］［１］～［１２］のいずれか１項に記載の方法を実施する工程を含む、トリフルリジン又はその塩を含む配合剤の製造方法。 [30] A method for producing a compounding preparation containing trifluridine or a salt thereof, which comprises the step of performing the method according to any one of [1] to [12].
　［３１］［１］～［１２］のいずれか１項に記載の方法を実施する工程を含む、トリフルリジン又はその塩、及びチピラシル又はその塩を含む配合剤の品質管理方法。 [31] A method of quality control of a compounding agent comprising trifluridine or a salt thereof, and tipiracil or a salt thereof, which comprises the step of performing the method according to any one of [1] to [12].
　本発明は、有機相及び水相からなる移動相を用いる高速液体クロマトグラフィーにより、トリフルリジン又はその塩の類縁物質を効率よく検出可能であり、かつ、その品質を保証するのに適したものである。 The present invention is a high-performance liquid chromatography using a mobile phase consisting of an organic phase and an aqueous phase, which is capable of efficiently detecting trifluridine or its salt analogues, and which is suitable for assuring the quality thereof. is there.
実施例１－１におけるクロマトグラムを示す。1 shows a chromatogram in Example 1-1. 実施例１－３におけるクロマトグラムを示す。7 shows a chromatogram in Example 1-3. 実施例２－１におけるクロマトグラムを示す。The chromatogram in Example 2-1 is shown.
　本発明におけるトリフルリジン（ＦＴＤ）とは、α，α，α－トリフルオロチミジンであり、下記の構造を持つ化合物である。 Trifluridine (FTD) in the present invention is α, α, α-trifluorothymidine, which is a compound having the following structure.
Figure JPOXMLDOC01-appb-C000001
Figure JPOXMLDOC01-appb-C000001
　本発明におけるトリフルリジンには、光学異性体、立体異性体、回転異性体、互変異性体等の異性体を有する場合には、特に明記しない限り、いずれの異性体も混合物も本発明化合物に包含される。 In the present invention, in the case of having an isomer such as an optical isomer, a stereoisomer, a rotational isomer, a tautomer and the like in trifluridine in the present invention, any isomer or a mixture is also a compound of the present invention unless otherwise specified. Is included.
　本発明において、特にそうでないことを明記しない限り、塩とは、薬学的に許容される塩を意味し、塩基付加塩又は酸付加塩を挙げることができる。 In the present invention, unless otherwise specified, a salt means a pharmaceutically acceptable salt and can include a base addition salt or an acid addition salt.
　該塩基付加塩としては、例えばナトリウム塩、カリウム塩等のアルカリ金属塩；例えばカルシウム塩、マグネシウム塩等のアルカリ土類金属塩；例えばアンモニウム塩；例えばトリメチルアミン塩、トリエチルアミン塩、ジシクロヘキシルアミン塩、エタノールアミン塩、ジエタノールアミン塩、トリエタノールアミン塩、プロカイン塩、Ｎ，Ｎ’－ジベンジルエチレンジアミン塩等の有機アミン塩等が挙げられる。 Examples of the base addition salt include alkali metal salts such as sodium salts and potassium salts; alkaline earth metal salts such as calcium salts and magnesium salts; ammonium salts; such as trimethylamine salts, triethylamine salts, dicyclohexylamine salts, ethanolamine And salts thereof, organic amine salts such as diethanolamine salt, triethanolamine salt, procaine salt, N, N'-dibenzylethylenediamine salt and the like.
　該酸付加塩としては、例えば塩酸塩、硫酸塩、硝酸塩、リン酸塩、過塩素酸塩等の無機酸塩；例えば酢酸塩、ギ酸塩、マレイン酸塩、フマル酸塩、酒石酸塩、クエン酸塩、アスコルビン酸塩、トリフルオロ酢酸塩等の有機酸塩；例えばメタンスルホン酸塩、イセチオン酸塩、ベンゼンスルホン酸塩、ｐ－トルエンスルホン酸塩等のスルホン酸塩等が挙げられる。 Examples of the acid addition salt include inorganic acid salts such as hydrochloride, sulfate, nitrate, phosphate and perchlorate; for example, acetate, formate, maleate, fumarate, tartrate, citric acid Organic acid salts such as salts of ascorbate and trifluoroacetate; and sulfonates such as methanesulfonate, isethionate, benzenesulfonate, p-toluenesulfonate and the like.
　本発明に用いられるトリフルリジン又はその塩として、好ましくは塩を形成していないトリフルリジンのフリー体である。 As trifluridine or a salt thereof to be used in the present invention, it is preferably a free form of trifluridine which has not formed a salt.
　本発明の方法において、トリフルリジン又はその塩と溶媒を調整して試料とし、これを高速液体クロマトグラフィーへ注入することにより、トリフルリジンの類縁物質を検出することができる。 In the method of the present invention, it is possible to detect trifluridine analogues by adjusting trifluridine or a salt thereof and a solvent to prepare a sample and injecting it into high performance liquid chromatography.
　本発明における試料は、トリフルリジンの類縁物質を含みうる。類縁物質としては類縁物質１～６の化合物が挙げられる。 The sample in the present invention may contain a trifluridine analogue. Examples of the analogues include compounds of analogues 1 to 6.
Figure JPOXMLDOC01-appb-T000002
Figure JPOXMLDOC01-appb-T000002
　類縁物質１は、５－カルボキシウラシルである。２，４－ジオキソ－１，２，３，４－テトラヒドロピリミジン－５－カルボン酸と称されることもある。 Related substance 1 is 5-carboxyuracil. It may also be referred to as 2,4-dioxo-1,2,3,4-tetrahydropyrimidine-5-carboxylic acid.
　類縁物質２は、５－カルボキシ－２’－デオキシ－ウリジンである。１－（（２Ｒ，４Ｒ，５Ｒ）－４－ヒドロキシ－５－（ヒドロキシメチル）テトラヒドロフラン－２－イル）－２，４－ジオキソ－１，２，３，４－テトラヒドロピリミジン－５－カルボン酸と称されることもある。 Analog substance 2 is 5-carboxy-2'-deoxy-uridine. 1-((2R, 4R, 5R) -4-hydroxy-5- (hydroxymethyl) tetrahydrofuran-2-yl) -2,4-dioxo-1,2,3,4-tetrahydropyrimidine-5-carboxylic acid and It is sometimes called.
　類縁物質３は、２’－デオキシ－５－メトキシカルボニルウリジンである。１－（（２Ｒ，４Ｒ，５Ｒ）－４－ヒドロキシ－５－（ヒドロキシメチル）テトラヒドロフラン－２－イル）－２，４－ジオキソ－１，２，３，４－テトラヒドロピリミジン－５－カルボン酸メチルと称されることもある。 Analog 3 is 2'-deoxy-5-methoxycarbonyluridine. 1-((2R, 4R, 5R) -4-hydroxy-5- (hydroxymethyl) tetrahydrofuran-2-yl) -2,4-dioxo-1,2,3,4-tetrahydropyrimidine-5-carboxylic acid methyl ester It is sometimes called.
　類縁物質４は、トリフルオロチミンである。５－（トリフルオロメチル）ピリミジン－２，４（１Ｈ，３Ｈ）－ジオンと称されることもある。 The related substance 4 is trifluorothymine. Sometimes referred to as 5- (trifluoromethyl) pyrimidine-2,4 (1H, 3H) -dione.
　類縁物質５は、５－メトキシカルボニルウラシルである。２，４－ジオキソ－１，２，３，４－テトラヒドロピリミジン－５－カルボン酸メチルと称されることもある。 The related substance 5 is 5-methoxycarbonyluracil. It may also be referred to as methyl 2,4-dioxo-1,2,3,4-tetrahydropyrimidine-5-carboxylate.
　類縁物質６は、５’－（４－クロロフェニルカルボキシ）－２’－デオキシ－５－トリフルオロメチルウリジンである。４－クロロ安息香酸（（２Ｒ，３Ｒ，５Ｒ）－５－（２，４－ジオキソ－５－（トリフルオロメチル）－３，４－ジヒドロピリミジン－１（２Ｈ）－イル）－３－ヒドロキシテトラヒドロフラン－２－イル）メチルと称することもある。 Analog 6 is 5 '-(4-chlorophenylcarboxy) -2'-deoxy-5-trifluoromethyluridine. 4-chlorobenzoic acid ((2R, 3R, 5R) -5- (2,4-dioxo-5- (trifluoromethyl) -3,4-dihydropyrimidin-1 (2H) -yl) -3-hydroxytetrahydrofuran Sometimes referred to as -2-yl) methyl.
　本発明に用いられる試料としては、製剤や原薬そのものから調製した試料だけでなく、安定性などを確認するための試験において調製された試料、各類縁物質の保持時間等を確認するために各類縁物質を添加した試料、製剤や原薬の製造工程を確認するために調製された試料などにも用いることができる。試料は、ロンサーフ（登録商標）に配合されるチピラシル又はその塩を含むものであっても、これを実質的に含まないものであっても、含まないものであってもよい。 The samples used in the present invention are not only samples prepared from the preparation and drug substance themselves, but also samples prepared in a test for confirming the stability etc., each for confirming the retention time of each related substance, etc. It can also be used as a sample to which a related substance is added, a sample prepared to confirm the preparation process of a drug product or drug substance, and the like. The sample may contain, be substantially free of, or not contain tipiracil or a salt thereof formulated in Ronsurf (registered trademark).
　本発明で用いることができる製剤には、トリフルリジン又はその塩を含む。そして、必要に応じて薬学的に許容される担体を配合し、予防又は治療目的に応じて各種の投与形態を採用可能であり、該形態としては、例えば、経口剤、点眼剤、注射剤、坐剤、軟膏剤、貼付剤等のいずれでもよく、好ましくは、経口剤が採用される。これらの投与形態は、各々当業者に公知慣用の製剤方法により製造できる。 Formulations that can be used in the present invention include trifluridine or a salt thereof. And, if necessary, a pharmaceutically acceptable carrier can be blended, and various administration forms can be adopted according to the purpose of prevention or treatment, and as such forms, for example, oral agents, eye drops, injections, Any of suppositories, ointments, patches and the like may be used, and preferably oral preparations are employed. Each of these dosage forms can be prepared by conventional formulation methods known to those skilled in the art.
　薬学的に許容される担体としては、製剤素材として慣用の各種有機或いは無機担体物質が用いられ、固形製剤における賦形剤、結合剤、崩壊剤、滑沢剤、着色剤、液状製剤における溶剤、溶解補助剤、懸濁化剤、等張化剤、緩衝剤、無痛化剤等として配合される。また、必要に応じて防腐剤、抗酸化剤、着色剤、甘味剤、安定化剤等の製剤添加物を用いることもできる。 As the pharmaceutically acceptable carrier, various organic or inorganic carrier substances conventionally used as pharmaceutical ingredients are used, and excipients in solid preparations, binders, disintegrants, lubricants, coloring agents, solvents in liquid preparations, It is formulated as a solubilizer, suspending agent, tonicity agent, buffer, soothing agent and the like. In addition, formulation additives such as preservatives, antioxidants, colorants, sweeteners and stabilizers can also be used, if necessary.
　経口用固形製剤を調製する場合は、トリフルリジン又はその塩に賦形剤、必要に応じて、結合剤、崩壊剤、滑沢剤、着色剤、矯味・矯臭剤等を加えた後、常法により錠剤、被覆錠剤、顆粒剤、散剤、カプセル剤等を製造することができる。 When preparing a solid preparation for oral use, after adding trifluridine or a salt thereof with an excipient, if necessary, a binder, a disintegrant, a lubricant, a coloring agent, a flavoring agent, a flavoring agent, etc. Thus, tablets, coated tablets, granules, powders, capsules and the like can be produced.
　点眼剤を調整する場合は、トリフルリジン又はその塩に溶解補助剤、等張化剤、緩衝剤、防腐剤などを使用することにより製造することができる。 When the eye drop is prepared, it can be produced by using a solubilizing agent, a tonicity agent, a buffer, a preservative and the like with trifluridine or a salt thereof.
　また、上記投与形態を有する薬剤の１日あたりの投与量は、患者の症状、体重、年齢、性別等によって適宜決定することができる。 The daily dose of the drug having the above-mentioned administration form can be appropriately determined depending on the condition, weight, age, sex and the like of the patient.
　本発明において用いられる高速液体クロマトグラフィーは、通常市販されているものを用いることができる。 As the high performance liquid chromatography used in the present invention, those commercially available can be used.
　クロマトグラフィーで用いられるカラムには、有機相を移動相にして脂溶性のある化合物を分離する順相カラムと、水相を移動相にして化合物を分離する逆相カラムが知られているが、高速液体クロマトグラフィーでは、逆相カラムが使用されることが多く、本発明においても逆相クロマトグラフィーが好ましい。 As columns used in chromatography, there are known a normal phase column that separates a lipid-soluble compound by using an organic phase as a mobile phase and a reverse phase column that separates a compound by using an aqueous phase as a mobile phase. In high performance liquid chromatography, reverse phase columns are often used, and in the present invention, reverse phase chromatography is preferred.
　本発明において使用できる高速液体クロマトグラフィーのカラムは、シリカゲルカラム、表面をオクタデシルシリル基で修飾されたシリカゲルカラム（ＯＤＳカラム又はＣ１８カラム）、表面をオクチル基で修飾されたシリカゲルカラム（Ｃ８カラム）、表面をシアノプロピル基で修飾されたシリカゲルカラム（ＣＮカラム）、表面をフェネチル基で修飾されたシリカゲルカラム（Ｐｈカラム）、表面をアミノプロピル基で修飾されたシリカゲルカラム（ＮＨカラム）、表面をジヒドロキシプロピル基で修飾されたシリカゲルカラム（Ｄｉｏｌカラム）、各種ポリマーで充填されたカラム（ポリマーカラム）、イオン交換樹脂で充填されたカラム（イオン交換カラム）、などから選択されるが、本発明においてはＯＤＳカラムが好ましい。 The high performance liquid chromatography column that can be used in the present invention is a silica gel column, a silica gel column whose surface is modified with octadecylsilyl group (ODS column or C18 column), a silica gel column whose surface is modified with octyl group (C8 column), Silica gel column whose surface is modified with cyanopropyl group (CN column), silica gel column whose surface is modified with phenethyl group (Ph column), silica gel column whose surface is modified with aminopropyl group (NH column), dihydroxy surface It is selected from a silica gel column modified with propyl group (Diol column), a column packed with various polymers (polymer column), a column packed with ion exchange resin (ion exchange column), etc. In the present invention, ODS columns are preferred.
　ＯＤＳカラムとしては、シリカゲルの粒径、細孔径、オクタデシルシリル基の結合方法、オクタデシルシリル基の置換度等が異なる種々のものを使用することができる。本発明においては、高純度シリカゲルが使用され、オクタデシル化後に残存したシラノールを低分子のシリル化剤で処理したＯＤＳカラム（エンドキャッピングされたＯＤＳカラム）が好ましい。 As the ODS column, various types of silica gel particles different in particle diameter, pore diameter, octadecylsilyl group bonding method, octadecylsilyl group substitution degree and the like can be used. In the present invention, high purity silica gel is used, and an ODS column (end-capped ODS column) in which silanol remaining after octadecylation is treated with a low molecular weight silylating agent is preferable.
　ＯＤＳカラムとしては、シリカゲルの粒径、細孔径、オクタデシルシリル基の結合方法、オクタデシルシリル基の置換度等が異なる種々のものを使用することができる。本発明においては、シリカゲルの平均粒径は、例えば、２～１０μｍが好ましく、３～５μｍがより好ましい。シリカゲルの平均粒径は、レーザー回折法等により測定することができる。シリカゲルの平均細孔径は、例えば、６～２０ｎｍが好ましく、８～１３ｎｍがより好ましい。シリカゲルの平均細孔径は、ガス吸着法等により測定することができる。シリカゲルにおけるオクタデシルシリル基の結合様式としては、例えば、モノメリック様式、ポリメリック様式等が好ましい。オクタデシルシリル基の置換度は、種々の方法により測定することができる。シリカゲルの炭素量は、例えば、３％以上が好ましく、１０％以上がより好ましい。シリカゲルの炭素量は、例えば、２５％以下が好ましく、２０％以下がより好ましい。シリカゲルの炭素量は、種々の方法により測定することができる。 As the ODS column, various types of silica gel particles different in particle diameter, pore diameter, octadecylsilyl group bonding method, octadecylsilyl group substitution degree and the like can be used. In the present invention, the average particle diameter of the silica gel is, for example, preferably 2 to 10 μm, and more preferably 3 to 5 μm. The average particle size of silica gel can be measured by a laser diffraction method or the like. The average pore diameter of the silica gel is, for example, preferably 6 to 20 nm, and more preferably 8 to 13 nm. The average pore size of silica gel can be measured by a gas adsorption method or the like. As a coupling | bonding method of the octadecyl silyl group in a silica gel, for example, a monomeric form, polymeric form etc. are preferable. The degree of substitution of the octadecylsilyl group can be measured by various methods. The amount of carbon in silica gel is, for example, preferably 3% or more, and more preferably 10% or more. The amount of carbon in the silica gel is, for example, preferably 25% or less, more preferably 20% or less. The carbon content of silica gel can be measured by various methods.
　高速液体クロマトグラフィーの移動相において用いられる有機相としては、ヘキサン、シクロヘキサン、ヘプタン、ジエチルエーテル、テトラヒドロフラン、クロロホルム、塩化メチレンなどの非極性溶媒、アセトン、ジメチルスルホキシド、アセトニトリルなどの非プロトン性極性溶媒、酢酸、メタノール、エタノール、イソプロパノール、アセトニトリルなどを用いることができる。これらの溶媒は、１種単独で、又は２種以上を混合溶媒として用いることができる。本発明における有機相としては、メタノール、又はアセトニトリルが好ましく、アセトニトリルがより好ましい。また、有機相には１０％以下の水が含まれていてもよい。 The organic phase used in the mobile phase of high performance liquid chromatography includes nonpolar solvents such as hexane, cyclohexane, heptane, diethyl ether, tetrahydrofuran, chloroform and methylene chloride; aprotic polar solvents such as acetone, dimethylsulfoxide and acetonitrile; Acetic acid, methanol, ethanol, isopropanol, acetonitrile and the like can be used. One of these solvents may be used alone, or two or more thereof may be used as a mixed solvent. As the organic phase in the present invention, methanol or acetonitrile is preferable, and acetonitrile is more preferable. In addition, the organic phase may contain 10% or less of water.
　高速液体クロマトグラフィーの移動相に用いられる水相は水のみだけでなく、１０％以下の有機溶媒が含まれていてもよいが、水相全体に対して１０％以下のメタノールが含まれている水相が好ましく、５％以下のメタノールが含まれていることがより好ましく、２％以下のメタノールが含まれていることがさらに好ましく、０．１～１％のメタノールが含まれていることが特に好ましい。 The aqueous phase used for the mobile phase of high performance liquid chromatography is not limited to water, but may contain 10% or less of an organic solvent, but contains 10% or less of methanol with respect to the entire aqueous phase. The aqueous phase is preferred, more preferably 5% or less of methanol is contained, 2% or less of methanol is further preferably contained, and 0.1 to 1% of methanol is contained Particularly preferred.
　高速液体クロマトグラフィーの移動相には、再現性を確保する観点から様々な緩衝剤を添加することができる。例えば、酢酸又はその塩、クエン酸又はその塩、酒石酸又はその塩、リン酸又はその塩を添加することができる。酢酸又はその塩としては、酢酸、酢酸ナトリウムが挙げられる。クエン酸又はその塩としては、クエン酸、クエン酸一ナトリウム、クエン酸二ナトリウム、クエン酸三ナトリウムが挙げられる。酒石酸又はその塩には、酒石酸、酒石酸ナトリウム、リン酸又はその塩としては、リン酸、リン酸二水素ナトリウム、リン酸水素二ナトリウム、リン酸二水素カリウム、リン酸水素二カリウムが挙げられる。本発明における水相の添加剤としては、測定物質の持つ性質、測定の際に得られるピークの形状、及び測定の再現性のから、リン酸塩が好ましく、リン酸二水素ナトリウムがより好ましい。これらの添加剤は、１種単独で、又は２種以上を組み合わせて用いることができる。 Various buffers can be added to the mobile phase of high performance liquid chromatography from the viewpoint of ensuring reproducibility. For example, acetic acid or a salt thereof, citric acid or a salt thereof, tartaric acid or a salt thereof, phosphoric acid or a salt thereof can be added. Examples of acetic acid or a salt thereof include acetic acid and sodium acetate. Citric acid or a salt thereof includes citric acid, monosodium citrate, disodium citrate and trisodium citrate. As tartaric acid or its salt, tartaric acid, sodium tartrate, phosphoric acid or its salt include phosphoric acid, sodium dihydrogen phosphate, disodium hydrogen phosphate, potassium dihydrogen phosphate, dipotassium hydrogen phosphate. As the additive of the aqueous phase in the present invention, a phosphate is preferable, and sodium dihydrogenphosphate is more preferable, in view of the properties of the measurement substance, the shape of the peak obtained in the measurement, and the reproducibility of the measurement. These additives can be used singly or in combination of two or more.
　本発明に用いることができる緩衝剤の濃度は、高速液体クロマトグラフィーの測定中に緩衝剤が析出しない濃度であれば適宜調整することができる。好ましくは、１～５０ｍＭであり、より好ましくは５～４０ｍＭであり、さらに好ましくは１０～３０ｍＭであり、特に好ましく１８～２０ｍＭである。 The concentration of the buffer that can be used in the present invention can be appropriately adjusted as long as the buffer does not precipitate during measurement of high performance liquid chromatography. Preferably, it is 1 to 50 mM, more preferably 5 to 40 mM, still more preferably 10 to 30 mM, and particularly preferably 18 to 20 mM.
　高速液体クロマトグラフィーの移動相では、有機相と水相の混液が用いられる。本発明においては、類縁物質１、２、３、４、５、及びトリフルリジンの保持時間における移動相全体に対する有機相の割合が、１～１４容量％が好ましく、２～１０容量％がより好ましく、３～７容量％が特に好ましい。 In the high performance liquid chromatography mobile phase, a mixture of an organic phase and an aqueous phase is used. In the present invention, the ratio of the organic phase to the whole mobile phase at the retention time of the analogues 1, 2, 3, 4, 5 and trifluridine is preferably 1 to 14% by volume, more preferably 2 to 10% by volume And 3 to 7% by volume are particularly preferred.
　高速液体クロマトグラフィーの移動相では、有機相と水相の混液が用いられるが、それらの割合は測定中に変化させることが多く、グラジェントをかけると呼ばれる。通常、目的とされる化合物の保持時間及び目的とする化合物と類縁物質との分離を考慮して、グラジェントをかけることが多い。 In the mobile phase of high performance liquid chromatography, a mixture of an organic phase and an aqueous phase is used, but their ratio is often changed during measurement and is called gradient application. In general, gradients are often applied in consideration of the retention time of the target compound and the separation of the target compound and the related substance.
　本発明においては、ステップ１とステップ２のグラジェント条件を含むことを特徴とする。
ステップ１：移動相全体に対する有機相の割合が、１～１４容量％である。
ステップ２：ステップ１の後、移動相全体に対する有機相の割合をさらに増加させるようグラジェントをかける。 The present invention is characterized by including the gradient conditions of step 1 and step 2.
Step 1: The ratio of the organic phase to the total mobile phase is 1 to 14% by volume.
Step 2: After step 1, apply a gradient to further increase the ratio of organic phase to total mobile phase.
　ステップ１においては、移動相にグラジェントをかけてもアイソクラティック状態でもよいが、アイソクラティック状態が好ましい。また、ステップ１における移動相全体に対する有機相の割合は１～１４容量％が挙げられるが、２～１０容量％がより好ましく、３～７容量％が特に好ましい。本発明において、「ステップ１における移動相全体に対する有機相の割合がＸ～Ｙ容量％である」とは、ステップ１の間、上記割合がＸ～Ｙ容量％の範囲にあることを意味する。 In Step 1, the mobile phase may be in a gradient or in an isocratic state, but an isocratic state is preferred. The ratio of the organic phase to the whole mobile phase in step 1 may be 1 to 14% by volume, preferably 2 to 10% by volume, and particularly preferably 3 to 7% by volume. In the present invention, “the ratio of the organic phase to the whole mobile phase in step 1 is X to Y volume%” means that the ratio is in the range of X to Y volume% during step 1.
　ステップ１の開始は、試料を高速液体クロマトグラフィーに注入してから５分以内であり、３分以内が好ましく、１分以内がより好ましく、注入と同時が特に好ましい。 The start of step 1 is within 5 minutes of injecting the sample into high performance liquid chromatography, preferably within 3 minutes, more preferably within 1 minute, and particularly preferably simultaneously with the injection.
　ステップ１の終了は、試料を高速液体クロマトグラフィーに注入してから１３～３０分後である。トリフルリジンが、ベースラインの凹み及びゴーストピークと重複しないように考慮し、ステップ１の終了は、当該注入から１５～２８分後が好ましく、１７～２５分後が特に好ましい。 The end of step 1 is 13 to 30 minutes after injecting the sample into high performance liquid chromatography. Taking into consideration that trifluridine does not overlap with the depressions in the baseline and ghost peaks, the end of step 1 is preferably 15-28 minutes after the injection, and particularly preferably 17-25 minutes.
　ステップ１における測定時間は、上記の開始と終了の範囲にあれば特に制限はないが、１５～２８分間が好ましく、１７～２５分間がより好ましい。 The measurement time in step 1 is not particularly limited as long as it is in the range of the above start and end, but it is preferably 15 to 28 minutes, and more preferably 17 to 25 minutes.
　ステップ１における移動相の流量の上限は、高速液体クロマトグラフィーで通常用いられるものであればよいが、各類縁物質の保持時間の分離を考慮し、流量は、２．５ｍＬ／ｍｉｎ以下が好ましく、２．０ｍＬ／ｍｉｎ以下がより好ましく、１．５ｍＬ／ｍｉｎ以下がさら好ましく、１．３ｍＬ／ｍｉｎ以下がなおさらに好ましく、１．１ｍＬ／ｍｉｎ以下が特に好ましい。また、ステップ１における移動相の流量の範囲は特に限定されないが、例えば、０．５～２．５ｍＬ／ｍｉｎが好ましく、０．５～２．０ｍＬ／ｍｉｎがより好ましく、０．７～１．５ｍＬ／ｍｉｎがさらに好ましく、０．８～１．３ｍＬ／ｍｉｎがなおさらに好ましく、０．９～１．１ｍＬ／ｍｉｎが特に好ましい。 The upper limit of the flow rate of the mobile phase in step 1 may be that which is usually used in high performance liquid chromatography, but the flow rate is preferably 2.5 mL / min or less in consideration of separation of the retention time of each related substance. 2.0 mL / min or less is more preferable, 1.5 mL / min or less is more preferable, 1.3 mL / min or less is still more preferable, and 1.1 mL / min or less is particularly preferable. Further, the range of the flow rate of the mobile phase in step 1 is not particularly limited, but is preferably 0.5 to 2.5 mL / min, more preferably 0.5 to 2.0 mL / min, and more preferably 0.7 to 1. 5 mL / min is more preferable, 0.8 to 1.3 mL / min is even more preferable, and 0.9 to 1.1 mL / min is particularly preferable.
　このようにして、ステップ１では類縁物質１、２、３、４、及び５からなる群より選ばれる１つ以上のトリフルリジンの類縁物質を検出することができる。好ましくは、ステップ１では類縁物質１、２、３、４、及び５を検出することができる。 Thus, in step 1, one or more trifluridine analogues selected from the group consisting of analogues 1, 2, 3, 4 and 5 can be detected. Preferably, analogues 1, 2, 3, 4 and 5 can be detected in step 1.
　また、ステップ１をよりアイソクラティック状態な状態で行う観点から、ステップ１において類縁物質１、２、３、４及び５のうち少なくとも１種が検出された場合、類縁物質１、２、３、４及び５のうちステップ１において検出されたもののそれぞれの保持時間における移動相全体に対する有機相の割合のうち、最大値と最小値との差が、移動相全体の５容量％以下であることが好ましく、１容量％以下であることが特に好ましい。 Also, from the viewpoint of performing Step 1 in a more isocratic state, when at least one of Related Substances 1, 2, 3, 4 and 5 is detected in Step 1, Related Substances 1, 2, 3, The difference between the maximum value and the minimum value of the ratio of the organic phase to the entire mobile phase at each retention time of each of those detected in step 1 among 4 and 5 is 5% by volume or less of the total mobile phase Preferably, it is 1% by volume or less.
　ステップ２においては、移動相にグラジェントをかけて有機相の割合を増加させる。ステップ１でグラジェントをかけていた場合、ステップ２では、より有機相の割合を増加させるようグラジェントをかける。ステップ１でアイソクラティック状態の場合、ステップ２では有機相の割合を増加させるようグラジェントをかける。 In step 2, the mobile phase is subjected to a gradient to increase the proportion of the organic phase. If gradients were applied in step 1, step 2 applies gradients to increase the proportion of the organic phase. In the case of isocratic state in step 1, step 2 applies a gradient to increase the proportion of the organic phase.
　ステップ２の終了時における移動相全体に対する有機相の割合は、２５～７０容量％であり、３０～６５容量％がより好ましく、３５～６０容量％が特に好ましい。本発明において、ステップ２の終了時とは、ステップ２のグラジェントをかけるのを止めて移動相全体に対する有機相の割合を下げ始めた時点を意味する。 The ratio of the organic phase to the whole mobile phase at the end of Step 2 is 25 to 70% by volume, more preferably 30 to 65% by volume, and particularly preferably 35 to 60% by volume. In the present invention, the end of step 2 means the point at which the application of the gradient of step 2 is stopped and the ratio of the organic phase to the whole mobile phase is started to be reduced.
　ステップ２の開始は、ステップ１の終了後３分以内に開始すれば特に制限はないが、１分以内が好ましく、ステップ１の終了と当時がより好ましい。 The start of step 2 is not particularly limited as long as it starts within 3 minutes after the end of step 1, but 1 minute or less is preferable, and the end of step 1 and its time are more preferable.
　ステップ２の終了は、類縁物質６の保持時間より後であれば特に制限はないが、試料を高速液体クロマトグラフィーに注入してから３５～６５分後であり、４０～６０分後がより好ましい。 The end of Step 2 is not particularly limited as long as it is after the retention time of Related Substance 6, but it is 35 to 65 minutes after injecting the sample into high performance liquid chromatography, and more preferably 40 to 60 minutes. .
　ステップ２における測定時間は、類縁物質６の保持時間が測定できれば特に制限はないが、１０～５０分間が好ましく、１５～４５分間がより好ましい。 The measurement time in step 2 is not particularly limited as long as the retention time of the related substance 6 can be measured, but 10 to 50 minutes is preferable, and 15 to 45 minutes is more preferable.
　ステップ２におけるグラジェントは、移動相全体に対する有機相の割合を増加させるものであれば特に制限はないが、１分間に移動相全体に対する有機相の割合を０．９容量％以上増加させることが好ましく、１分間に１．０容量％以上増加させることがより好ましく、１分間に２．０容量％以上増加させることが特に好ましい。移動相全体に対する有機相の割合の増加の上限は特に限定されないが、例えば、１分間に１０容量％以下等が好ましく、１分間に５．０容量％以下等がより好ましい。本明細書において、時間当たり（例えば、１分間に）移動相全体に対する有機相の割合をＸ容量％増加するとは、単位時間当たり、移動相全体に対する有機相の割合を、移動相全体１００容量％に対し、Ｘ容量％増加することを意味する。また、本発明においては、ステップ２の測定時間中における、移動相全体に対する有機相の割合の変化（測定時間中における移動相全体に対する有機相の割合のうち、最大値と最小値との差）を移動相全体の３０容量％以上にすることが好ましく、５０容量％以上にすることが特に好ましい。ステップ２の測定時間中における、移動相全体に対する有機相の割合の変化の上限は特に限定されないが、例えば、移動相全体の８０容量％以下にすることが好ましく、７０容量％以下にすることが特に好ましい。 The gradient in step 2 is not particularly limited as long as it increases the ratio of the organic phase to the total mobile phase, but increases the ratio of the organic phase to the total mobile phase in one minute by 0.9% or more It is more preferable to increase by 1.0% by volume or more per minute, and it is particularly preferable to increase 2.0% by volume or more by 1 minute. The upper limit of the increase in the ratio of the organic phase to the entire mobile phase is not particularly limited, but for example, 10% by volume or less is preferable, and 5.0% by volume or less is more preferable. In this specification, increasing the ratio of the organic phase to the total mobile phase by X volume% per hour (for example, 1 minute) means the ratio of the organic phase to the total mobile phase per unit time, 100 volume% of the total mobile phase In contrast, it means that X capacity% is increased. In the present invention, the change in the ratio of the organic phase to the whole mobile phase during the measurement time of step 2 (the difference between the maximum value and the minimum value among the ratios of the organic phase to the whole mobile phase in the measurement time) Of the whole mobile phase is preferably 30% by volume or more, and more preferably 50% by volume or more. The upper limit of the change in the ratio of the organic phase to the total mobile phase during the measurement time of step 2 is not particularly limited, but preferably 80% by volume or less of the total mobile phase, for example 70% by volume or less Particularly preferred.
　ステップ２における移動相の流量は、高速液体クロマトグラフィーで通常用いられるものであればよいが、各類縁物質の保持時間の分離を考慮し、流量は、２．５ｍＬ／ｍｉｎ以下が好ましく、２．０ｍＬ／ｍｉｎ以下がより好ましく、１．５ｍＬ／ｍｉｎ以下がさら好ましい。また、ステップ２における移動相の流量の範囲は特に限定されないが、例えば、０．５～２．５ｍＬ／ｍｉｎが好ましく、０．５～２．０ｍＬ／ｍｉｎがより好ましく、１．０～１．５ｍＬ／ｍｉｎがさらに好ましい。全体の測定時間を短縮させる観点から、ステップ２の開始後から徐々に流量を上げることも可能であり、ステップ２の終了時における流量がステップ１の終了時の流量の１．０～１．５倍が好ましい。 The flow rate of the mobile phase in step 2 may be any flow rate commonly used in high performance liquid chromatography, but the flow rate is preferably 2.5 mL / min or less, considering the separation of the retention time of each related substance. 0 mL / min or less is more preferable, and 1.5 mL / min or less is more preferable. In addition, the range of the flow rate of the mobile phase in step 2 is not particularly limited, but is preferably 0.5 to 2.5 mL / min, more preferably 0.5 to 2.0 mL / min, and more preferably 1.0 to 1. 5 mL / min is more preferable. From the viewpoint of shortening the overall measurement time, it is also possible to gradually increase the flow rate after the start of step 2, and the flow rate at the end of step 2 is 1.0 to 1.5 of the flow rate at the end of step 1 Double is preferred.
　このようにして、ステップ２では類縁物質６を検出することができる。 Thus, in step 2, the related substance 6 can be detected.
　本発明においては、上記の添加剤の他に、イオン対試薬を添加することができる。イオン対試薬には、ペンタンスルホン酸ナトリウム、ヘキサンスルホン酸ナトリウム、ヘプタンスルホン酸ナトリウム、オクタンスルホン酸ナトリウム、ドデカンスルホン酸ナトリウムなどのアルキルスルホン酸又はその塩（好ましくはアルキルスルホン酸ナトリウム）、ドデシル硫酸ナトリウムなどのアルキル硫酸又はその塩（好ましくはアルキル硫酸ナトリウム）、水酸化テトラエチルアンモニウム、水酸化テトラブチルアンモニウム、塩化テトラブチルアンモニウム、臭化テトラブチルアンモニウムなどの４級アンモニウム塩、トリヘキシルアミン、トリオクチルアミンなどの３級アミンを用いることができる。 In the present invention, in addition to the above additives, an ion pairing reagent can be added. Examples of the ion pairing reagent include sodium pentane sulfonate, sodium hexane sulfonate, sodium heptane sulfonate, sodium octane sulfonate, an alkyl sulfonic acid such as sodium dodecane sulfonate or a salt thereof (preferably sodium alkyl sulfonate), sodium dodecyl sulfate And the like (alkyl sodium sulfate), tetraethylammonium hydroxide, tetrabutylammonium hydroxide, tetrabutylammonium chloride, quaternary ammonium salts such as tetrabutylammonium bromide, trihexylamine, trioctylamine And other tertiary amines can be used.
　本発明における移動相（典型的には水相）のｐＨは、上記の添加剤などにより適宜調整することができるが、好ましくは２．０～５．０である。 The pH of the mobile phase (typically, the aqueous phase) in the present invention can be appropriately adjusted by the above-mentioned additives and the like, but is preferably 2.0 to 5.0.
　本発明における方法で用いることができる検出波長は、各類縁物質の性質を考慮して２０８～２８０ｎｍとすることができ、好ましくは２０８～２４０ｎｍであり、より好ましくは２０８～２１２ｎｍである。 The detection wavelength that can be used in the method of the present invention can be 208 to 280 nm, preferably 208 to 240 nm, more preferably 208 to 212 nm, in consideration of the properties of each related substance.
　本発明における方法で用いることができるカラム内の移動相の温度は適宜設定することができる。外部環境の影響、再現性などを考慮し、好ましくは当該温度を一定に保つことであり、より好ましくは２５～５０℃であり、さらに好ましくは３５～４５℃であり、特に好ましくは、４０～４４℃である。なお、当該温度を一定に保つにあたり、カラム全体の温度を制御するだけでなく、プレヒートミキサーなどを用いることができる。 The temperature of the mobile phase in the column that can be used in the method of the present invention can be set as appropriate. In consideration of the influence of the external environment, reproducibility, etc., the temperature is preferably kept constant, more preferably 25 to 50 ° C., still more preferably 35 to 45 ° C., particularly preferably 40 to 50 ° C. It is 44 ° C. In addition, in order to keep the said temperature constant, not only the temperature of the whole column can be controlled but a preheat mixer etc. can be used.
　本発明における高速液体クロマトグラフィーでは、適宜、注入量、カラム内温度、などを変更することができる。 In the high performance liquid chromatography in the present invention, the injection amount, the temperature in the column, and the like can be appropriately changed.
　これらの類縁物質のうち、類縁物質１～５は、公知の方法により合成できるか、又は市販品により入手することができる。また、類縁物質６はトリフルリジンや市販の化合物から公知の方法により合成することができるか、後述の方法により合成することができる。このようにして得られた類縁物質の高速液体クロマトグラフィーの保持時間、マススペクトル、フォトダイオードアレイ（ＰＤＡ）と、本発明により検出された類縁物質のそれらとを比較することで、類縁物質を特定することができる。 Among these analogues, analogues 1 to 5 can be synthesized by known methods, or can be obtained as commercial products. The related substance 6 can be synthesized from triflulysine or a commercially available compound according to a known method, or can be synthesized according to the method described later. The high-performance liquid chromatography retention time, mass spectrum, and photodiode array (PDA) of the analogues thus obtained are compared with those of the analogues detected according to the present invention to identify the analogues. can do.
　さらに、これらの類縁物質は、外部標準を用いる方法、内部標準を用いる方法のいずれの方法によっても定量的に測定することができる。 Furthermore, these analogues can be quantitatively measured by any method of using an external standard or a method of using an internal standard.
　これらの類縁物質が不純物として医薬品や製剤に含まれうる場合、これらの類縁物質は医薬品規制調和国際会議におけるガイドライン（ＩＣＨ－Ｑ３）に従って制御されるものである。本発明の方法は、当該ガイドラインの基準を満たすかどうか確認できるため、非常に有用である。 When these related substances can be contained as impurities in pharmaceuticals and preparations, these related substances are controlled in accordance with the guidelines (ICH-Q3) at the International Conference on Regulation of Pharmaceutical Regulations. The method of the present invention is very useful because it can be confirmed whether the criteria of the guidelines are met.
　また、これらの類縁物質は、本発明の方法によりトリフルリジン又はその塩から検出することができる。さらに、本発明は、類縁物質１～６のうち１つ又は２以上を標準品として品質管理に用いることができ、好ましくは類縁物質１～６のうち３つ以上であり、より好ましくは類縁物質１～６のうち４つ以上であり、特に好ましくは類縁物質２、３、５及び６である。 These analogues can also be detected from trifluridine or salts thereof by the method of the present invention. Furthermore, in the present invention, one or more of the analogues 1 to 6 can be used as a standard product for quality control, preferably three or more of the analogues 1 to 6, and more preferably the analogues It is four or more of 1 to 6, particularly preferably analogues 2, 3, 5 and 6.
　このような標準品に用いられるトリフルリジン及びこれら由来の類縁物質は、高純度のものになる。従って、当該高速液体クロマトグラフィーの条件により分離された各類縁物質は、標準品として用いることができる。よって、本発明は、トリフルリジン又はその塩を含む配合剤から単離することを特徴とする当該類縁物質の製造方法と言い換えることもできる。当該類縁物質は上記の類縁物質１～６が挙げられ、好ましくは類縁物質２、３、５、６である。 The trifluridine used in such standard products and the related substances derived therefrom become highly pure. Therefore, each related substance separated under the conditions of the high performance liquid chromatography can be used as a standard. Therefore, the present invention can be reworded as a method for producing the related substance, which is characterized in that it is isolated from a combination drug containing trifluridine or a salt thereof. The related substances include the above-mentioned related substances 1 to 6, and preferably the related substances 2, 3, 5, and 6.
　本発明が提供する品質管理方法は、トリフルリジン又はその塩を含む配合剤の品質管理方法であって、上記の本発明の方法を実施する工程を含む。本発明の品質管理方法は、例えば、上記類縁物質（例えば、類縁物質１～６のいずれか１つ以上）の含有量（質量）が、所定の量以下である場合に、配合剤は品質管理に係る基準を満たすと評価することができる。 The quality control method provided by the present invention is a method for quality control of a compounding agent containing trifluridine or a salt thereof, which comprises the step of carrying out the above-mentioned method of the present invention. In the quality control method of the present invention, for example, when the content (mass) of the above-mentioned analogue (for example, any one or more of analogues 1 to 6) is not more than a predetermined amount, the combination agent is It can be evaluated that the criteria for
　本発明は、上記の本発明の方法を実施する工程を含む、トリフルリジン又はその塩を含む配合剤の製造方法をも提供する。本発明の製造方法は、例えば、トリフルリジン又はその塩を製造する工程、得られたトリフルリジン又はその塩、上記方法を実施する（好ましくはトリフルリジン又はその塩を含む配合剤の品質管理を行う）工程を含むことができる。 The present invention also provides a method for producing a combination drug containing trifluridine or a salt thereof, which comprises the steps of carrying out the above-mentioned method of the present invention. The production method of the present invention performs, for example, the step of producing trifluridine or a salt thereof, the obtained trifluridine or a salt thereof, and the quality control of the compounding agent containing the above method (preferably containing triflulidine or a salt thereof) ) Can be included.
　本発明において、トリフルリジンに類縁物質２、３、５又は６が含まれていることは知られておらず、特に類縁物質６は新規の物質である。そのため、本発明には、トリフルリジン又はその塩を含む医薬組成物の品質管理に用いられる類縁物質２、３、５又は６を含み、さらには、トリフルリジン又はその塩を含む医薬組成物の不純物検出の際に標準品として用いるための類縁物質２、３、５又は６を含む。併せて、トリフルリジン又はその塩を含む医薬組成物から単離することを特徴とする類縁物質２、３、５又は６の製造方法も本発明に含まれる。 In the present invention, it is not known that trifluridine contains analogues 2, 3, 5 or 6, and in particular, analogue 6 is a novel substance. Therefore, the present invention includes the related substances 2, 3, 5 or 6 used for quality control of a pharmaceutical composition containing trifluridine or a salt thereof, and further, an impurity of a pharmaceutical composition containing trifluridine or a salt thereof It contains related substances 2, 3, 5 or 6 for use as a standard in detection. In addition, a method of producing analogues 2, 3, 5 or 6 characterized in that it is isolated from a pharmaceutical composition containing trifluridine or a salt thereof is also included in the present invention.
　本発明におけるトリフルリジン由来の類縁物質の検出において、高速液体クロマトグラフィーによる方法が用いられる。 In the detection of trifluridine-derived analogues in the present invention, a high performance liquid chromatography method is used.
　好ましくは、類縁物質１～６からなる群より選ばれる少なくとも１つのトリフルリジン由来の類縁物質を検出する、以下のステップ１とステップ２の条件を含む方法である。
ステップ１：移動相全体に対する有機相の割合が、１～１４容量％である。
ステップ２：ステップ１の後、有機相の割合をさらに増加させるようグラジェントをかける。 Preferably, the method comprises the conditions of the following Step 1 and Step 2 for detecting at least one trifluridine-derived analogue selected from the group consisting of analogues 1 to 6.
Step 1: The ratio of the organic phase to the total mobile phase is 1 to 14% by volume.
Step 2: After step 1, apply a gradient to further increase the proportion of the organic phase.
　より好ましくは、以下のステップ１とステップ２の条件を含む方法である。
ステップ１：類縁物質１～５からなる群より選ばれる少なくとも１つのトリフルリジン由来の類縁物質を検出し、移動相全体に対する有機相の割合が、１～１４容量％であり、アイソクラティック状態である。
ステップ２：ステップ１の後、有機相の割合をさらに増加させるようグラジェントをかけて、類縁物質６を検出する。 More preferably, the method includes the conditions of step 1 and step 2 below.
Step 1: Detect at least one trifluridine-derived analogue selected from the group consisting of analogues 1 to 5, and the ratio of the organic phase to the whole mobile phase is 1 to 14% by volume, in an isocratic state is there.
Step 2: After step 1, detect related substance 6 by applying a gradient to further increase the proportion of the organic phase.
　より好ましくは、以下のステップ１とステップ２の条件を含む方法である。
ステップ１：移動相全体に対する有機相の割合が、２～１０容量％であり、アイソクラティック状態であり、類縁物質１～５を検出する。
ステップ２：ステップ１の後、移動相全体に対する有機相の割合をさらに増加させるようグラジェントをかけて、類縁物質６を検出する。 More preferably, the method includes the conditions of step 1 and step 2 below.
Step 1: The ratio of the organic phase to the total mobile phase is 2 to 10% by volume, is isocratic, and detects Related Substances 1 to 5.
Step 2: After step 1, detect related substance 6 by applying a gradient to further increase the ratio of the organic phase to the total mobile phase.
　より好ましくは、移動相における有機相にアセトニトリルを含む、以下のステップ１とステップ２の条件を含む方法である。
ステップ１：移動相全体に対する有機相の割合が、２～１０容量％であり、アイソクラティック状態であり、類縁物質１～５を検出する。
ステップ２：ステップ１の後、移動相全体に対する有機相の割合をさらに増加させるようグラジェントをかけ、ステップ２の終了時における移動相全体に対する有機相の割合は、２５～７０容量％であり、類縁物質６を検出する。 More preferably, it is a method including the conditions of the following Step 1 and Step 2 including acetonitrile in the organic phase in the mobile phase.
Step 1: The ratio of the organic phase to the total mobile phase is 2 to 10% by volume, is isocratic, and detects Related Substances 1 to 5.
Step 2: Apply a gradient to further increase the ratio of the organic phase to the total mobile phase after Step 1, and the ratio of the organic phase to the total mobile phase at the end of Step 2 is 25 to 70% by volume, Detect related substance 6
　より好ましくは、移動相における有機相にアセトニトリルを含み、水相に水を含み、移動相にリン酸塩を含む、以下のステップ１とステップ２の条件を含む方法である。
ステップ１：移動相全体に対する有機相の割合が、２～１０容量％であり、アイソクラティック状態であり、類縁物質１～５を検出する。
ステップ２：ステップ１の後、移動相全体に対する有機相の割合を１分間に０．９容量％以上増加させるグラジェントをかけ、ステップ２の終了時における移動相全体に対する有機相の割合は、３０～６５容量％であり、類縁物質６を検出する。 More preferably, the method includes the conditions of the following steps 1 and 2 in which the organic phase in the mobile phase contains acetonitrile, the water phase contains water, and the mobile phase contains phosphate.
Step 1: The ratio of the organic phase to the total mobile phase is 2 to 10% by volume, is isocratic, and detects Related Substances 1 to 5.
Step 2: Apply a gradient that increases the ratio of the organic phase to the total mobile phase by 0.9% or more per minute after step 1, and the ratio of the organic phase to the total mobile phase at the end of step 2 is 30 It is up to 65% by volume, and the related substance 6 is detected.
　より好ましくは、移動相における有機相にアセトニトリルを含み、水相に水を含み、移動相にリン酸塩を含む、以下のステップ１とステップ２の条件を含む方法である。
ステップ１：移動相全体に対する有機相の割合が、の２～１０容量％であり、アイソクラティック状態であり、類縁物質１～５を検出する。
ステップ２：ステップ１の後、移動相全体に対する有機相の割合を１分間に０．９容量％以上増加させるグラジェントをかけ、ステップ２の終了時における移動相全体に対する有機相の割合は、３０～６５容量％であり、類縁物質６を検出する。 More preferably, the method includes the conditions of the following steps 1 and 2 in which the organic phase in the mobile phase contains acetonitrile, the water phase contains water, and the mobile phase contains phosphate.
Step 1: The ratio of the organic phase to the total mobile phase is 2 to 10% by volume, is isocratic, and detects Related Substances 1 to 5.
Step 2: Apply a gradient that increases the ratio of the organic phase to the total mobile phase by 0.9% or more per minute after step 1, and the ratio of the organic phase to the total mobile phase at the end of step 2 is 30 It is up to 65% by volume, and the related substance 6 is detected.
　より好ましくは、移動相における有機相にアセトニトリルを含み、水相に水を含み、移動相にリン酸塩を含む、以下のステップ１とステップ２の条件を含む方法である。
ステップ１：移動相全体に対する有機相の割合が、３～７容量％であり、アイソクラティック状態であり、類縁物質１～５を検出する。
ステップ２：ステップ１の後、移動相全体に対する有機相の割合を１分間に０．９容量％以上増加させるグラジェントをかけ、ステップ２の終了時における移動相全体に対する有機相の割合は、３５～６０容量％であり、類縁物質６を検出する。 More preferably, the method includes the conditions of the following steps 1 and 2 in which the organic phase in the mobile phase contains acetonitrile, the water phase contains water, and the mobile phase contains phosphate.
Step 1: The ratio of the organic phase to the total mobile phase is 3 to 7% by volume, is isocratic, and detects Related Substances 1 to 5.
Step 2: After step 1, apply a gradient that increases the ratio of the organic phase to the total mobile phase by 0.9% by volume or more, and the ratio of the organic phase to the total mobile phase at the end of step 2 is 35 Up to 60% by volume, the related substance 6 is detected.
　より好ましくは、移動相における有機相にアセトニトリルを含み、水相に水を含み、移動相にリン二水素ナトリウムを含む、以下のステップ１とステップ２の条件を含む方法である。
ステップ１：移動相全体に対する有機相の割合が、３～７容量％であり、アイソクラティック状態であり、測定時間が１５～２８分間であり、類縁物質１～５を検出する。
ステップ２：ステップ１の後、移動相全体に対する有機相の割合を１分間に有機相の割合を０．９容量％以上増加させるグラジェントをかけ、ステップ２の終了時における移動相全体に対する有機相の割合は、３５～６０容量％であり、測定時間が１０～５０分間であり、類縁物質６を検出する。 More preferably, the method includes the conditions of the following steps 1 and 2 in which the organic phase in the mobile phase contains acetonitrile, the water phase contains water, and the mobile phase contains sodium phosphorous dihydrogen.
Step 1: The ratio of the organic phase to the whole mobile phase is 3 to 7% by volume, is isocratic, the measurement time is 15 to 28 minutes, and the related substances 1 to 5 are detected.
Step 2: After step 1, apply a gradient that increases the ratio of the organic phase to the whole mobile phase by 0.9% by volume or more per minute, and the organic phase to the whole mobile phase at the end of step 2 The ratio is 35 to 60% by volume, the measurement time is 10 to 50 minutes, and the related substance 6 is detected.
　より好ましくは、移動相における有機相にアセトニトリルを含み、水相に水を含み、移動相にリン二水素ナトリウムを含む、以下のステップ１とステップ２の条件を含む方法である。
ステップ１：移動相全体に対する有機相の割合が、３～７容量％であり、アイソクラティック状態であり、測定時間が１５～２８分間であり、類縁物質１～５を検出する。
ステップ２：ステップ１の後、移動相全体に対する有機相の割合を１分間に０．９容量％以上増加させるグラジェントをかけ、ステップ２の終了時における移動相全体に対する有機相の割合は、３５～６０容量％であり、測定時間が１０～５０分間であり、ステップ２の終了時における流量がステップ１の流量の１．０～１．５倍であり、類縁物質６を検出する。 More preferably, the method includes the conditions of the following steps 1 and 2 in which the organic phase in the mobile phase contains acetonitrile, the water phase contains water, and the mobile phase contains sodium phosphorous dihydrogen.
Step 1: The ratio of the organic phase to the whole mobile phase is 3 to 7% by volume, is isocratic, the measurement time is 15 to 28 minutes, and the related substances 1 to 5 are detected.
Step 2: After step 1, apply a gradient that increases the ratio of the organic phase to the total mobile phase by 0.9% by volume or more, and the ratio of the organic phase to the total mobile phase at the end of step 2 is 35 It is ̃60% by volume, the measurement time is 10 to 50 minutes, and the flow rate at the end of step 2 is 1.0 to 1.5 times the flow rate of step 1, and the related substance 6 is detected.
　より好ましくは、移動相における有機相にアセトニトリルを含み、水相に水を含み、移動相にメタノール及びリン二水素ナトリウムを含む、以下のステップ１とステップ２の条件を含む方法である。
ステップ１：移動相全体に対する有機相の割合が、３～７容量％であり、アイソクラティック状態であり、測定時間が１５～２８分間であり、類縁物質１～５を検出する。
ステップ２：ステップ１の後、移動相全体に対する有機相の割合を１分間に０．９容量％以上増加させるグラジェントをかけ、ステップ２の終了時における移動相全体に対する有機相の割合は、３５～６０容量％であり、測定時間が１０～５０分間であり、ステップ２の終了時における流量がステップ１の流量の１．０～１．５倍であり、類縁物質６を検出する。 More preferably, it is a method including the conditions of the following steps 1 and 2 in which the organic phase in the mobile phase contains acetonitrile, the water phase contains water, and the mobile phase contains methanol and sodium phosphorous dihydrogen.
Step 1: The ratio of the organic phase to the whole mobile phase is 3 to 7% by volume, is isocratic, the measurement time is 15 to 28 minutes, and the related substances 1 to 5 are detected.
Step 2: After step 1, apply a gradient that increases the ratio of the organic phase to the total mobile phase by 0.9% by volume or more, and the ratio of the organic phase to the total mobile phase at the end of step 2 is 35 It is ̃60% by volume, the measurement time is 10 to 50 minutes, and the flow rate at the end of step 2 is 1.0 to 1.5 times the flow rate of step 1, and the related substance 6 is detected.
　以下の試験条件により高速液体クロマトグラフィーの測定を行った。
・検出器：紫外吸光光度計（波長は２１０ｎｍ）
・カラム：内径４．６ｍｍ，長さ１５ｃｍのステンレス管に３μｍ又は５μｍの液体クロマトグラフィー用オクタデシルシリル化シリカゲルカラム
・カラム温度：４０℃
・流量：各実施例に記載
・移動相：各実施例に記載
・グラジェント：各実施例に記載 The measurement of high performance liquid chromatography was performed under the following test conditions.
・ Detector: Ultraviolet absorptiometer (wavelength: 210 nm)
Column: Octadecylsilylated silica gel column for liquid chromatography of 3 μm or 5 μm in a stainless steel tube having an inner diameter of 4.6 mm and a length of 15 cm. Column temperature: 40 ° C.
Flow rate: described in each example Mobile phase: described in each example Gradient: described in each example
　高速液体クロマトグラフィーで測定する試料は以下の通り調製した。 The sample to be measured by high performance liquid chromatography was prepared as follows.
　トリフルリジンの濃度が約０．８ｍｇ／ｍＬとなるよう各測定条件で用いられる移動相と同じ組成の溶液に溶解させ、適宜希釈した溶液を試料とした。 The solution was dissolved in a solution having the same composition as the mobile phase used in each measurement condition so that the concentration of trifluridine was about 0.8 mg / mL, and a properly diluted solution was used as a sample.
　本実施例において、各移動相における「％」表示は、容量％を意味する In the present embodiment, “%” indication in each mobile phase means volume%.
　実施例１－１
　カラム：Ｈｙｄｒｏｓｐｈｅｒｅ　Ｃ１８、ＹＭＣ社製（３μｍ）
　流量：１．０ｍＬ／ｍｉｎ
　移動相Ａ：０．０５ｍｏｌ／Ｌのリン酸二水素ナトリウム水溶液
　移動相Ｂ：アセトニトリル
　グラジェント：移動相Ａと移動相Ｂを次の様に変えて濃度勾配制御した。０～１８分：Ａ　９５％　Ｂ　５％、１８～５５分：Ａ　９５→６０％　Ｂ　５％→４０％、５５～５５．１分　Ａ６０％→９５％　Ｂ４０％→５％、５５．１以降　Ａ　９５％　Ｂ　５％。 Example 1-1
Column: Hydrosphere C18, manufactured by YMC (3 μm)
Flow rate: 1.0 mL / min
Mobile phase A: 0.05 mol / L aqueous sodium dihydrogenphosphate aqueous solution Mobile phase B: acetonitrile Gradient: The mobile phase A and the mobile phase B were changed as follows to control the concentration gradient. 0 to 18 minutes: A 95% B 5%, 18 to 55 minutes: A 95 to 60% B 5% to 40%, 55 to 55.1 minutes A 60% to 95% B 40% to 5%, 55.1 or later A 95% B 5%.
　測定結果を図１に示す。これより、トリフルリジンの保持時間が１７．２分に確認された。本測定方法において、トリフルリジンが、ベースラインの凹み及びゴーストピークと重複しないことを確認した。 The measurement results are shown in FIG. From this, the retention time of trifluridine was confirmed at 17.2 minutes. In this measurement method, it was confirmed that trifluridine did not overlap with the depressions in the baseline and ghost peaks.
　実施例１－２
　グラジェント：移動相Ａと移動相Ｂを次の様に変えて濃度勾配制御した。０～１８分：Ａ　９５％　Ｂ　５％、１８～４８分：Ａ　９５→６０％　Ｂ　５％→４０％、４８～４８．１分　Ａ６０％→９５％　Ｂ４０％→５％、４８．１以降　Ａ　９５％　Ｂ　５％。 Example 1-2
Gradient: Concentration gradient control was performed by changing mobile phase A and mobile phase B as follows. 0 to 18 minutes: A 95% B 5%, 18 to 48 minutes: A 95 to 60% B 5% to 40%, 48 to 48.1 minutes A 60% to 95% B 40% to 5%, 48.1 or later A 95% B 5%.
　カラム、流量、移動相Ａ及び移動相Ｂは実施例１と同じ条件である。 The column, flow rate, mobile phase A and mobile phase B are the same as in Example 1.
　本測定により、トリフルリジンが、ゴーストピークと重複しないことを確認した。 By this measurement, it was confirmed that trifluridine did not overlap with the ghost peak.
　実施例１－３
　グラジェント：移動相Ａと移動相Ｂを次の様に変えて濃度勾配制御した。０～２０分：Ａ　９５％　Ｂ　５％、２０～５０分：Ａ　９５→６０％　Ｂ　５％→４０％、５０～５０．１分　Ａ６０％→９５％　Ｂ４０％→５％、５０．１以降　Ａ　９５％　Ｂ　５％。カラム、流量、移動相Ａ及び移動相Ｂは実施例１と同じ条件である。 Example 1-3
Gradient: Concentration gradient control was performed by changing mobile phase A and mobile phase B as follows. 0 to 20 minutes: A 95% B 5%, 20 to 50 minutes: A 95 to 60% B 5% to 40%, 50 to 50.1 minutes A 60% to 95% B 40% to 5%, 50.1 or later A 95% B 5%. The column, flow rate, mobile phase A and mobile phase B are the same as in Example 1.
　測定結果を図２に示す。これより、トリフルリジンの保持時間が１７．１分に確認された。本測定方法において、トリフルリジンが、ベースラインの凹み及びゴーストピークと重複しないことを確認した。 The measurement results are shown in FIG. From this, the retention time of trifluridine was confirmed at 17.1 minutes. In this measurement method, it was confirmed that trifluridine did not overlap with the depressions in the baseline and ghost peaks.
　実施例１－３－１　グラジェント：移動相Ａと移動相Ｂを次の様に変えて濃度勾配制御した。０～１５分：Ａ　９５％　Ｂ　５％、１５～４５分：Ａ　９５→６０％　Ｂ　５％→４０％、４５～４５．１分　Ａ６０％→９５％　Ｂ４０％→５％、４５．１以降　Ａ　９５％　Ｂ　５％。カラム、流量、移動相Ａ及び移動相Ｂは実施例１と同じ条件である。
　カラム：Ｉｎｅｒｔｓｉｌ　ＯＤＳ－３　ＧＬサイエンス社製（３μｍ）
　流量、移動相Ａ及び移動相Ｂは実施例１－３と同じ条件である。 Example 1-3-1 Gradient: The concentration gradient was controlled by changing the mobile phase A and the mobile phase B as follows. 0 to 15 minutes: A 95% B 5%, 15 to 45 minutes: A 95 to 60% B 5% to 40%, 45 to 45.1 minutes A 60% to 95% B 40% to 5%, 45.1 or later A 95% B 5%. The column, flow rate, mobile phase A and mobile phase B are the same as in Example 1.
Column: Inertsil ODS-3 GL Science (3 μm)
The flow rate, the mobile phase A and the mobile phase B are the same as in Example 1-3.
　本測定により、トリフルリジンが、ゴーストピークと重複しないことを確認した。 By this measurement, it was confirmed that trifluridine did not overlap with the ghost peak.
　実施例１－３－２
　カラム：Ｉｎｅｒｔｓｉｌ　ＯＤＳ－４　ＧＬサイエンス社製（３μｍ）
　流量、移動相Ａ及び移動相Ｂ、グラジェントは実施例１－３－１と同じ条件である。 Example 1-3-2
Column: Inertsil ODS-4 GL Science (3 μm)
The flow rate, the mobile phase A and the mobile phase B, and the gradient are the same as in Example 1-3-1.
　本測定により、トリフルリジンが、ゴーストピークと重複しないことを確認した。 By this measurement, it was confirmed that trifluridine did not overlap with the ghost peak.
　実施例１－３－３
　カラム：ＹＭＣ－Ｐａｃｋ　Ｐｒｏ　Ｃ１８　ＲＳ（３μｍ）
流量、移動相Ａ及び移動相Ｂ、グラジェントは実施例１－３－１と同じ条件である。 Example 1-3-3
Column: YMC-Pack Pro C18 RS (3 μm)
The flow rate, the mobile phase A and the mobile phase B, and the gradient are the same as in Example 1-3-1.
　本測定により、トリフルリジンが、ゴーストピークと重複しないことを確認した。 By this measurement, it was confirmed that trifluridine did not overlap with the ghost peak.
　実施例１－３－４
　カラム：ＹＭＣ－Ｐａｃｋ　Ｐｒｏ　Ｃ１８（３μｍ）
流量、移動相Ａ及び移動相Ｂ、グラジェントは実施例１－３－１と同じ条件である。 Example 1-3-4
Column: YMC-Pack Pro C18 (3 μm)
The flow rate, the mobile phase A and the mobile phase B, and the gradient are the same as in Example 1-3-1.
　本測定により、トリフルリジンが、ゴーストピークと重複しないことを確認した。 By this measurement, it was confirmed that trifluridine did not overlap with the ghost peak.
　実施例２－１
　カラム：Ｈｙｄｒｏｓｐｈｅｒｅ　Ｃ１８、ＹＭＣ社製（５μｍ）
　流量：０～２１分　１．０ｍＬ／ｍｉｎ、２１～４６分　１．０→１．３ｍＬ／ｍｉｎ、４６～４６．１分　１．３→１．０ｍＬ／ｍｉｎ、４６．１分以降　１．０ｍＬ／ｍｉｎ
　移動相Ａ：リン酸二水素ナトリウム二水和物３．０ｇを水１０００ｍＬに溶かし、リン酸を加えてｐＨ２．２に調整した後にメタノール１０ｍＬを加えた。
　移動相Ｂ：アセトニトリル
　グラジェント：移動相Ａと移動相Ｂを次の様に変えて濃度勾配制御した。０～２１分：Ａ　９６％　Ｂ　４％、２１～４６分：Ａ　９６→４５％　Ｂ　４→５５％、４６～４６．１分　Ａ４５→９６％　Ｂ５５→４％、４６．１以降　Ａ　９６％　Ｂ　４％。 Example 2-1
Column: Hydrosphere C18, manufactured by YMC (5 μm)
Flow rate: 0 to 21 minutes 1.0 mL / min, 21 to 46 minutes 1.0 → 1.3 mL / min, 46 to 46.1 minutes 1.3 → 1.0 mL / min, 46.1 minutes or later 1.0 mL / Min
Mobile phase A: 3.0 g of sodium dihydrogen phosphate dihydrate was dissolved in 1000 mL of water, adjusted to pH 2.2 by adding phosphoric acid, and then 10 mL of methanol was added.
Mobile phase B: acetonitrile gradient: Mobile phase A and mobile phase B were changed as follows to control concentration gradient. 0 to 21 minutes: A 96% B 4%, 21 to 46 minutes: A 96 to 45% B 4 to 55%, 46 to 46.1 minutes A 45 to 96% B 55 to 4%, 46.1 or more A 96% B 4%.
　測定結果を図３に示す。これにより、トリフルリジンの保持時間が１７．４分であることがわかった。また、類縁物質１の保持時間が３．１分、類縁物質２の保持時間が５．６分、類縁物質３の保持時間が４．１分、類縁物質４の保持時間が７．９分、類縁物質５の保持時間が８．８分、類縁物質６の保持時間が４２．９分であった。 The measurement results are shown in FIG. From this, it was found that the retention time of trifluridine was 17.4 minutes. Further, the retention time of Related Substance 1 is 3.1 minutes, the retention time of Related Substance 2 is 5.6 minutes, the retention time of Related Substance 3 is 4.1 minutes, the retention time of Related Substance 4 is 7.9 minutes, The retention time of Related Substance 5 was 8.8 minutes, and the retention time of Related Substance 6 was 42.9 minutes.
　検出された各類縁物質は、別途購入又は合成した各類縁物質と保持時間が一致したことから、類縁物質１～６の構造を示した化合物であることを確認した。 It was confirmed that the detected analogues were compounds showing structures of analogues 1 to 6 because their retention times were identical to those of the analogues purchased or synthesized separately.
　結論としては、本測定方法において、トリフルリジンが、ベースラインの凹み及びゴーストピークと重複しないことを確認した。併せて、トリフルリジンの各類縁物質及びトリフルリジンが分離できることがわかった。さらに、類縁物質６のピークが、後述の合成した類縁物質６と保持時間が一致したため、本測定における保持時間４２．９分のピークは類縁物質６と判断した。 In conclusion, in this measurement method, it was confirmed that trifluridine did not overlap with the depressions in the baseline and ghost peaks. In addition, it was found that trifluridine analogues and trifluridine could be separated. Furthermore, since the peak of relative substance 6 matched the retention time with the later-described synthesized relative substance 6, the peak for retention time 42.9 minutes in this measurement was determined to be relative substance 6.
　実施例２－２
　カラム：Ｕｎｉｓｏｎ　ＵＫ－Ｃ１８、インタクト社製（５μｍ）
その他の条件は実施例２－１と同一である。 Example 2-2
Column: Unison UK-C18, manufactured by Intact (5 μm)
Other conditions are the same as in Example 2-1.
　本測定による結論は、実施例２－１と同様であった。 The conclusions of this measurement were the same as in Example 2-1.
　実施例２－３
　移動相Ａ：リン酸二水素ナトリウム二水和物３．０ｇを水１０００ｍＬに溶かし、リン酸を加えてｐＨ２．２に調整した後にメタノール９ｍＬを加えた。
その他の条件は実施例２－１と同一である。 Example 2-3
Mobile phase A: 3.0 g of sodium dihydrogen phosphate dihydrate was dissolved in 1000 mL of water, adjusted to pH 2.2 with phosphoric acid, and then 9 mL of methanol was added.
Other conditions are the same as in Example 2-1.
　本測定による結論は、実施例２－１と同様であった。 The conclusions of this measurement were the same as in Example 2-1.
　実施例２－４
　流量：０～２１分　０．９５ｍＬ／ｍｉｎ、２１～４６分　０．９５→１．２５ｍＬ／ｍｉｎ、４６～４６．１分　１．２５→０．９５ｍＬ／ｍｉｎ、４６．１分以降　０．９５ｍＬ／ｍｉｎ
その他の条件は実施例２－１と同一である。 Example 2-4
Flow rate: 0 to 21 minutes 0.95 mL / min, 21 to 46 minutes 0.95 to 1.25 mL / min, 46 to 46.1 minutes 1.25 to 0.95 mL / min, 46.1 minutes to 0.95 mL / Min
Other conditions are the same as in Example 2-1.
　本測定による結論は、実施例２－１と同様であった。 The conclusions of this measurement were the same as in Example 2-1.
　実施例２－５
　グラジェント：移動相Ａと移動相Ｂを次の様に変えて濃度勾配制御した。０～２１分：Ａ　９５．８％　Ｂ　４．２％、２１～４６分：Ａ　９５．８→４３％　Ｂ　４．２→５７％、４６～４６．１分　Ａ４３→９５．８％　Ｂ５７→４．２％、４６．１以降　Ａ　９５．８％　Ｂ　４．２％。
その他の条件は実施例２－１と同一である。 Example 2-5
Gradient: Concentration gradient control was performed by changing mobile phase A and mobile phase B as follows. 0 to 21 minutes: A 95.8% B 4.2%, 21 to 46 minutes: A 95.8 → 43% B 4.2 → 57%, 46 to 46.1 minutes A43 → 95.8% B57 → 4.2%, 46.1 or later A 95.8% B 4.2%.
Other conditions are the same as in Example 2-1.
　本測定による結論は、実施例２－１と同様であった。 The conclusions of this measurement were the same as in Example 2-1.
　実施例３
　カラム：Ｉｎｅｒｔｓｉｌ　ＯＤＳ－２、ＧＬサイエンス社製（５μｍ）
　流量：１．０ｍＬ／ｍｉｎ
　移動相Ａ：０．０５ｍｏｌ／Ｌ　リン酸二水素ナトリウム水溶液
　移動相Ｂ：アセトニトリル
　グラジェント：移動相Ａと移動相Ｂを次の様に変えて濃度勾配制御した。０～２分：Ａ　８５％　Ｂ　１５％、２～７分：Ａ　８５→６０％　Ｂ　１５→４０％、７分以降　Ａ６０％　Ｂ４０％。 Example 3
Column: Inertsil ODS-2, manufactured by GL Science (5 μm)
Flow rate: 1.0 mL / min
Mobile phase A: 0.05 mol / L sodium dihydrogenphosphate aqueous solution Mobile phase B: acetonitrile Gradient: The mobile phase A and the mobile phase B were changed as follows to control the concentration gradient. 0 to 2 minutes: A 85% B 15%, 2 to 7 minutes: A 85 to 60% B 15 to 40%, 7 minutes or more A 60% B 40%.
　測定結果より、トリフルリジンの保持時間が３．６分であることがわかった。また、類縁物質１～５の保持時間が１．０～２．５分に集中し、それらのピーク面積を算出できなかった。 From the measurement results, it was found that the retention time of trifluridine is 3.6 minutes. In addition, the retention times of Related Substances 1 to 5 were concentrated to 1.0 to 2.5 minutes, and their peak areas could not be calculated.
　結論としては、本測定方法では、トリフルリジンの類縁物質１～５の検出はかならずしも正確ではないものの、類縁物質６の検出ができたことがわかった。 As a conclusion, it was found that, with this measurement method, although the detection of analogues 1 to 5 of trifluridine was not necessarily accurate, the analogue 6 could be detected.
　実施例２
　カラム：Ｉｎｅｒｔｓｉｌ　ＯＤＳ－２、ＧＬサイエンス社製（５μｍ）
　流量：１．０ｍＬ／ｍｉｎ
　移動相Ａ：水
　移動相Ｂ：アセトニトリル
　グラジェント：　Ａ６０％　Ｂ４０％のアイソクラティック状態。 Example 2
Column: Inertsil ODS-2, manufactured by GL Science (5 μm)
Flow rate: 1.0 mL / min
Mobile phase A: water Mobile phase B: acetonitrile gradient: A60% B40% isocratic state.
　測定結果より、トリフルリジンの保持時間が１．８分、類縁物質６の保持時間が７．６分であることがわかった。類縁物質１～５の保持時間が１．６～２．５分に集中し、それらのピーク面積を算出できなかったものの、上記のように類縁物質６を検出することはできた。 From the measurement results, it was found that the retention time of trifluridine was 1.8 minutes and that of related substance 6 was 7.6 minutes. Although the retention times of related substances 1 to 5 were concentrated to 1.6 to 2.5 minutes, and their peak areas could not be calculated, it was possible to detect related substance 6 as described above.
　参考例　５’－（４－クロロフェニルカルボキシ）－２’－デオキシ－５－トリフルオロメチルウリジン（類縁物質６）の合成 Reference Example Synthesis of 5 '-(4-chlorophenylcarboxy) -2'-deoxy-5-trifluoromethyluridine (analog 6)
Figure JPOXMLDOC01-appb-C000003
Figure JPOXMLDOC01-appb-C000003
　市販のトリフルリジン（２．００ｇ）をピリジン（８ｍＬ）に溶解させ、氷浴下ｐ－クロロベンゾイルクロリド（１．１８ｇ）をゆっくり加え、室温にて１時間撹拌した。その後、溶媒を留去し、残渣をシリカゲルカラムクロマトグラフィー（クロロホルム／メタノール＝１００／０～９１／９）にて精製し、５’－（４－クロロフェニルカルボキシ）－２’－デオキシ－５－トリフルオロメチルウリジン（１．４９ｇ、類縁物質６）及び３’－（４－クロロフェニルカルボキシ）－２’－デオキシ－５－トリフルオロメチルウリジン（２３８ｍｇ、参考化合物１）を得た。 Commercially available trifluridine (2.00 g) was dissolved in pyridine (8 mL), p-chlorobenzoyl chloride (1.18 g) was slowly added in an ice bath, and the mixture was stirred at room temperature for 1 hour. Thereafter, the solvent is distilled off, and the residue is purified by silica gel column chromatography (chloroform / methanol = 100/0 to 91/9) to obtain 5 '-(4-chlorophenylcarboxy) -2'-deoxy-5-tri Fluoromethyl uridine (1.49 g, analog 6) and 3 '-(4-chlorophenylcarboxy) -2'-deoxy-5-trifluoromethyluridine (238 mg, reference compound 1) were obtained.
　類縁物質６：１Ｈ－ＮＭＲ（ＤＭＳＯ－ｄ６、２７０ＭＨｚ）　δ（ｐｐｍ）１１．９１（１Ｈ、ｓ）、８．０３（１Ｈ、ｓ）、７．９５（２Ｈ、ｄ、Ｊ＝６．７５Ｈｚ）、７．６０（２Ｈ、ｄ、Ｊ＝６．７５Ｈｚ）、６．０７（１Ｈ、ｔ、Ｊ＝６．５５Ｈｚ）、５．４８（１Ｈ、ｄ、Ｊ＝４．２９Ｈｚ）、４．５６－４．３８（３Ｈ、ｍ）、４．１８－４．１６（１Ｈ、ｍ）
参考化合物１：１Ｈ－ＮＭＲ（ＤＭＳＯ－ｄ６、２７０ＭＨｚ）　δ（ｐｐｍ）１１．９２（１Ｈ、ｓ）、８．７２（１Ｈ、ｓ）、８．０３（２Ｈ、ｄ、Ｊ＝８．０７Ｈｚ）、７．６３（２Ｈ、ｄ、Ｊ＝８．０７Ｈｚ）、６．２７（１Ｈ、ｔ、Ｊ＝６．７５Ｈｚ）、５．４８－５．４２（２Ｈ、ｍ）、４．２８（１Ｈ、ｓ）、３．７２（２Ｈ、ｓ） Analogs 6: 1 H-NMR (DMSO-d6, 270 MHz) δ (ppm) 11.91 (1 H, s), 8.03 (1 H, s), 7.95 (2 H, d, J = 6.75 Hz) , 7.60 (2 H, d, J = 6. 75 Hz), 6.07 (1 H, t, J = 6. 55 Hz), 5. 48 (1 H, d, J = 4. 29 Hz), 4.56- 4.38 (3H, m), 4.18-4.16 (1H, m)
Reference compound 1: 1 H-NMR (DMSO-d6, 270 MHz) δ (ppm) 11.92 (1 H, s), 8.72 (1 H, s), 8.03 (2 H, d, J = 8.07 Hz) , 7.63 (2H, d, J = 8.07 Hz), 6.27 (1 H, t, J = 6.75 Hz), 5.48-5.42 (2 H, m), 4.28 (1 H, 1 H, m) s), 3.72 (2H, s)

Claims (29)

  1. トリフルリジン又はその塩を含む試料を有機相及び水相からなる移動相を用いる高速液体クロマトグラフィーに供する工程を含む、トリフルリジン由来の類縁物質を検出するための方法であって、前記高速液体クロマトグラフィーに供する工程が、以下の要件を満たすステップ１及び２を含む、方法：
    ステップ１：移動相全体に対する有機相の割合が１～１４容量％である。
    ステップ２：ステップ１の後、移動相全体に対する有機相の割合を増加させるようグラジェントをかける。     A method for detecting a related substance derived from trifluridine, comprising the step of subjecting a sample containing trifluridine or a salt thereof to high-performance liquid chromatography using a mobile phase consisting of an organic phase and an aqueous phase, wherein the high-performance liquid chromatography The method wherein the steps of applying to graphy comprise steps 1 and 2 meeting the following requirements:
    Step 1: The ratio of the organic phase to the total mobile phase is 1 to 14% by volume.
    Step 2: After step 1, apply a gradient to increase the ratio of organic phase to total mobile phase.
  2. 前記類縁物質が下記類縁物質１～６からなる群より選ばれる少なくとも１つである、請求項１に記載の方法：
    類縁物質１：５－カルボキシウラシル、
    類縁物質２：５－カルボキシ－２’－デオキシ－ウリジン、
    類縁物質３：２’－デオキシ－５－メトキシカルボニルウリジン、
    類縁物質４：トリフルオロチミン、
    類縁物質５：５－メトキシカルボニルウラシル、
    類縁物質６：５’－（４－クロロフェニルカルボキシ）－２’－デオキシ－５－トリフルオロメチルウリジン。     The method according to claim 1, wherein the related substance is at least one selected from the group consisting of the following related substances 1 to 6:
    Related substances 1: 5-carboxyuracil,
    Analogs 2: 5-Carboxy-2'-deoxy-uridine,
    Related substances 3: 2'-deoxy-5-methoxycarbonyluridine,
    Related substances 4: trifluorothymine,
    Related substances 5: 5-methoxycarbonyluracil,
    Analogs 6: 5 '-(4-chlorophenylcarboxy) -2'-deoxy-5-trifluoromethyluridine.
  3. ステップ１で類縁物質１～５を検出し、ステップ２で類縁物質６を検出する、請求項１又は２に記載の方法。 The method according to claim 1 or 2, wherein analogues 1 to 5 are detected in step 1, and analogue 6 is detected in step 2.
  4. ステップ１がアイソクラティック状態で行われる、請求項１～３のいずれか一項に記載の方法。 The method according to any one of the preceding claims, wherein step 1 is carried out isocratically.
  5. 有機相がアセトニトリルである、請求項１～４のいずれか一項に記載の方法。 The method according to any one of the preceding claims, wherein the organic phase is acetonitrile.
  6. ステップ１における移動相全体に対する有機相の割合が２～１０容量％である、請求項１～５のいずれか一項に記載の方法。 The method according to any one of claims 1 to 5, wherein the ratio of the organic phase to the total mobile phase in step 1 is 2 to 10% by volume.
  7. ステップ２の終了時における移動相全体に対する有機相の割合が２５～７０容量％である、請求項１～６のいずれか一項に記載の方法。 The method according to any one of the preceding claims, wherein the ratio of organic phase to total mobile phase at the end of step 2 is 25 to 70% by volume.
  8. ステップ２において移動相全体に対する有機相の割合を１分間に０．９容量％以上増加させるグラジェントをかける、請求項１～７のいずれか一項に記載の方法。 The method according to any one of claims 1 to 7, wherein in step 2, a gradient is applied to increase the ratio of the organic phase to the total mobile phase by 0.9% by volume or more per minute.
  9. ステップ２の測定時間が１０～５０分間である、請求項１～８のいずれか一項に記載の方法。 The method according to any one of claims 1 to 8, wherein the measurement time of step 2 is 10 to 50 minutes.
  10. ステップ２の終了時における流量がステップ１の流量の１．０～１．５倍である、請求項１～９のいずれか一項に記載の方法。 The method according to any one of the preceding claims, wherein the flow rate at the end of step 2 is 1.0 to 1.5 times the flow rate of step 1.
  11. 水相がさらにリン酸塩を含む、請求項１～１０のいずれか一項に記載の方法。 The method according to any one of the preceding claims, wherein the aqueous phase further comprises phosphate.
  12. 水相がさらにメタノールを含む、請求項１～１１のいずれか一項に記載の方法。 The method according to any one of the preceding claims, wherein the aqueous phase further comprises methanol.
  13. トリフルリジン又はその塩を含む配合剤の品質管理に用いられる５－カルボキシ－２’－デオキシ－ウリジン（類縁物質２）。 5-carboxy-2'-deoxy-uridine (analog 2) used for quality control of a combination drug containing trifluridine or a salt thereof.
  14. トリフルリジン又はその塩を含む配合剤の不純物検出の際に標準品として用いるための５－カルボキシ－２’－デオキシ－ウリジン（類縁物質２）。 5-carboxy-2'-deoxy-uridine (analog 2) for use as a standard in the detection of impurities of a compounding agent containing trifluridine or a salt thereof.
  15. ５－カルボキシ－２’－デオキシ－ウリジン（類縁物質２）の、トリフルリジン又はその塩を含む配合剤の不純物検出の際の標準品としての使用。 Use of 5-carboxy-2'-deoxy-uridine (analog 2) as a standard in the detection of impurities of a combination containing trifluridine or a salt thereof.
  16. ５－カルボキシ－２’－デオキシ－ウリジン（類縁物質２）からなる、トリフルリジン又はその塩を含む配合剤の不純物検出の際の標準品。 A standard preparation for detecting impurities of a compounding agent containing trifluridine or a salt thereof, which comprises 5-carboxy-2'-deoxy-uridine (analog 2).
  17. トリフルリジン又はその塩を含む配合剤の品質管理に用いられる２’－デオキシ－５－メトキシカルボニルウリジン（類縁物質３）。 2'-Deoxy-5-methoxycarbonyluridine (analog 3) used for quality control of a combination drug containing trifluridine or a salt thereof.
  18. トリフルリジン又はその塩を含む配合剤の不純物検出の際に標準品として用いるための２’－デオキシ－５－メトキシカルボニルウリジン（類縁物質３）。 2'-Deoxy-5-methoxycarbonyluridine (analog 3) for use as a standard in the detection of impurities of a compounding agent containing trifluridine or a salt thereof.
  19. ２’－デオキシ－５－メトキシカルボニルウリジン（類縁物質３）の、トリフルリジン又はその塩を含む配合剤の不純物検出の際の標準品としての使用。 Use of 2'-deoxy-5-methoxycarbonyluridine (analog 3) as a standard in the detection of impurities in combination containing trifluridine or a salt thereof.
  20. ２’－デオキシ－５－メトキシカルボニルウリジン（類縁物質３）からなる、トリフルリジン又はその塩を含む配合剤の不純物検出の際の標準品。 A standard preparation for detecting impurities of a compounding agent containing trifluridine or a salt thereof, which comprises 2'-deoxy-5-methoxycarbonyluridine (analog 3).
  21. トリフルリジン又はその塩を含む配合剤の品質管理に用いられる５－メトキシカルボニルウラシル（類縁物質５）。 5-methoxycarbonyluracil (analog 5) which is used for quality control of a combination drug containing trifluridine or a salt thereof.
  22. トリフルリジン又はその塩を含む配合剤の不純物検出の際に標準品として用いるための５－メトキシカルボニルウラシル（類縁物質５）。 5-Methoxycarbonyluracil (analog 5) to be used as a standard in the detection of impurities in formulations containing trifluridine or a salt thereof.
  23. ５－メトキシカルボニルウラシル（類縁物質５）の、トリフルリジン又はその塩を含む配合剤の不純物検出の際の標準品としての使用。 Use of 5-methoxycarbonyluracil (analog 5) as a standard preparation for detecting impurities of a compound containing trifluridine or a salt thereof.
  24. ５－メトキシカルボニルウラシル（類縁物質５）からなる、トリフルリジン又はその塩を含む配合剤の不純物検出の際の標準品。 A standard preparation for detecting impurities of a compounding agent containing trifluridine or a salt thereof, which comprises 5-methoxycarbonyluracil (analog 5).
  25. ５’－（４－クロロフェニルカルボキシ）－２’－デオキシ－５－トリフルオロメチルウリジン（類縁物質６）。 5 '-(4-Chlorophenylcarboxy) -2'-deoxy-5-trifluoromethyluridine (analog 6).
  26. トリフルリジン又はその塩を含む配合剤の品質管理に用いられる５’－（４－クロロフェニルカルボキシ）－２’－デオキシ－５－トリフルオロメチルウリジン（類縁物質６）。 5 '-(4-Chlorophenylcarboxy) -2'-deoxy-5-trifluoromethyluridine (analog 6) used for quality control of a combination drug containing trifluridine or a salt thereof.
  27. トリフルリジン又はその塩を含む配合剤の不純物検出の際に標準品として用いるための５’－（４－クロロフェニルカルボキシ）－２’－デオキシ－５－トリフルオロメチルウリジン（類縁物質６）。 5 '-(4-Chlorophenylcarboxy) -2'-deoxy-5-trifluoromethyluridine (analog 6) for use as a standard in the detection of impurities in combination agents containing trifluridine or a salt thereof.
  28. ５’－（４－クロロフェニルカルボキシ）－２’－デオキシ－５－トリフルオロメチルウリジン（類縁物質６）の、トリフルリジン又はその塩を含む配合剤の不純物検出の際の標準品としての使用。 Use of 5 '-(4-chlorophenylcarboxy) -2'-deoxy-5-trifluoromethyluridine (analog 6) as a standard in the detection of impurities in combination containing trifluridine or a salt thereof.
  29. ５’－（４－クロロフェニルカルボキシ）－２’－デオキシ－５－トリフルオロメチルウリジン（類縁物質６）からなる、トリフルリジン又はその塩を含む配合剤の不純物検出の際の標準品。 A standard preparation for detecting impurities of a compounding agent containing trifluridine or a salt thereof, which comprises 5 '-(4-chlorophenylcarboxy) -2'-deoxy-5-trifluoromethyluridine (analog 6).
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US10866219B2 (en) 2017-12-22 2020-12-15 Taiho Pharmaceutical Co., Ltd. Method for detecting trifluridine- and/or tipiracil-related substance
US10809237B2 (en) 2018-01-05 2020-10-20 Taiho Pharmaceutical Co., Ltd. Method for detecting trifluridine-related substance by high-performance liquid chromatography
US10816517B2 (en) 2018-01-05 2020-10-27 Taiho Pharmaceutical Co., Ltd. Method for detecting trifluridine-related substance by high-performance liquid chromatography
CN117871740A (en) * 2024-03-11 2024-04-12 炉霍雪域俄色有限责任公司 Russian color fruit raw pulp liquid chromatography quality detection method
CN117871740B (en) * 2024-03-11 2024-05-10 炉霍雪域俄色有限责任公司 Russian color fruit raw pulp liquid chromatography quality detection method

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