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WO2025157997A1 - Method for determining the purity of a compound by high-performance liquid chromatography - Google Patents

Method for determining the purity of a compound by high-performance liquid chromatography

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Publication number
WO2025157997A1
WO2025157997A1 PCT/EP2025/051804 EP2025051804W WO2025157997A1 WO 2025157997 A1 WO2025157997 A1 WO 2025157997A1 EP 2025051804 W EP2025051804 W EP 2025051804W WO 2025157997 A1 WO2025157997 A1 WO 2025157997A1
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WO
WIPO (PCT)
Prior art keywords
mobile phase
volume
water
acid
sample
Prior art date
Legal status (The legal status 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 status listed.)
Pending
Application number
PCT/EP2025/051804
Other languages
French (fr)
Inventor
Adrian Malcolm CLARKE
Fiona FITZPATRICK
Jose Antonio NAVARRO HUERTA
Filomena PETRUZZIELLO
Luzia SCHENKER
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Novartis AG
Original Assignee
Novartis AG
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
Application filed by Novartis AG filed Critical Novartis AG
Publication of WO2025157997A1 publication Critical patent/WO2025157997A1/en
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

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Classifications

    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
    • B01D15/08—Selective adsorption, e.g. chromatography
    • B01D15/26—Selective adsorption, e.g. chromatography characterised by the separation mechanism
    • B01D15/32—Bonded phase chromatography
    • B01D15/325—Reversed phase
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
    • B01D15/08—Selective adsorption, e.g. chromatography
    • B01D15/10—Selective adsorption, e.g. chromatography characterised by constructional or operational features
    • B01D15/16—Selective adsorption, e.g. chromatography characterised by constructional or operational features relating to the conditioning of the fluid carrier
    • B01D15/166—Fluid composition conditioning, 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
    • 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
    • G01N2030/8809—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample
    • G01N2030/884—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample organic compounds
    • 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
    • G01N2030/8809—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample
    • G01N2030/8872—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample impurities

Definitions

  • This disclosure pertains to the field of analytical chemistry.
  • the present disclosure provides a method for determining the purity of a compound in a sample, as well as the presence and quantity of potential by-products of said compound using high-performance liquid chromatography (HPLC).
  • HPLC high-performance liquid chromatography
  • the compound DOTA-NHS (2,2’,2”-(10- ⁇ 2-[(2,5-dioxopyrrolidin-1 -yl)oxy]-2-oxoethyl ⁇ - 1 ,4,7,10-tetraazacyclododecane-1 ,4,7-triyl)triethanoic acid; CAS Nr: 170908-81 -3) is widely used in drug research and development.
  • the compound DOTA-NHS is composed of two main functional groups: the DOTA (1 ,4,7,10- tetraazacyclododecane-1 ,4,7,10-tetraacetic acid) which may form a stable complex with various metal ions, such as lutecium, gallium and actinium; and the N-hydroxysuccinimide (NHS) ester, which may react with primary amines on biomolecules to form stable amide bonds.
  • DOTA 1,4,7,10- tetraazacyclododecane-1 ,4,7,10-tetraacetic acid
  • various metal ions such as lutecium, gallium and actinium
  • NHS N-hydroxysuccinimide
  • the compound DOTA-NHS ester is widely used in drug research and development, being mainly focused on the development of radiopharmaceuticals for imaging and therapy.
  • An aspect of this disclosure refers to an analytical method for determining, in a sample, the purity of a compound of formula (I): being said compound of formula (I) the compound DOTA-NHS (2,2’,2”-(10- ⁇ 2-[(2,5-dioxopyrrolidin- 1 -yl)oxy]-2-oxoethyl ⁇ -1 ,4,7,10-tetraazacyclododecane-1 ,4,7-triyl)triethanoic acid; CAS Nr: 170908- 81 -3); said method comprising subjecting the sample to a high-performance liquid chromatography (HPLC) process comprising: injecting in an HPLC column a volume (1 ) of water and a volume (2) containing the sample dissolved in a water-free polar solvent not absorbing in the UV region (S1 ) containing an acid (a1 ); eluting with a mobile phase gradient; and determining the purity of the compound of formula (I) in the sample
  • FIG. 1 shows a schematic drawing of the sandwich injection of the sample used in the method of the invention, exemplified with a syringe containing a first layer of a volume (1 ) of 4 pL of water, a second layer of volume (2) containing 0.2 pL of the sample dissolved in water-free acetonitrile ( ⁇ 0.001 % H2O) containing 1 % (vol/vol) trifluoroacetic acid (TFA); and a third layer of volume (1 ) of 4 pL of water.
  • a syringe containing a first layer of a volume (1 ) of 4 pL of water, a second layer of volume (2) containing 0.2 pL of the sample dissolved in water-free acetonitrile ( ⁇ 0.001 % H2O) containing 1 % (vol/vol) trifluoroacetic acid (TFA); and a third layer of volume (1 ) of 4 pL of water.
  • FIG 2 shows a chromatogram of a sample of the compound of formula (I) obtained applying the HPLC method of the invention.
  • the compound of formula (I) i.e., DOTA-NHS ester or 2,2’,2”-(10- ⁇ 2- [(2,5-dioxopyrrolidin-1 -yl)oxy]-2-oxoethyl ⁇ -1 ,4,7,10-tetraazacyclododecane-1 ,4,7-triyl)triethanoic acid; CAS Nr: 170908-81 -3)
  • DOTA-NHS ester or 2,2’,2”-(10- ⁇ 2- [(2,5-dioxopyrrolidin-1 -yl)oxy]-2-oxoethyl ⁇ -1 ,4,7,10-tetraazacyclododecane-1 ,4,7-triyl)triethanoic acid; CAS Nr: 170908-81 -3) is widely used in drug research and development, being said use often focused on the development of radiopharmaceuticals for diagnostic and therapeutic methods. For this reason, it is key to ensure the
  • the development of an analytical methods to determine the purity of the compound of formula (I) encounters several challenges.
  • the compound of formula (I) is an ester which is unstable in water, hydrolyzing quickly.
  • it is a very polar compound, making it difficult to separate/retain the compound of formula (I) when developing a chromatographic analytical method.
  • samples containing the compound of formula (I) may also contain impurities or by-products.
  • the most common by-products present in samples of a compound of formula (I) are the result of the hydrolysis of the N-succinimide ester present in this compound, being said main by-products the compound of formula (II), i.e. free DOTA (2-[4,7,10-tris(carboxymethyl)- 1 ,4,7,10-tetrazacyclododec-1 -yl]acetic acid; CAS number 60239-18-1 ) and the compound of formula (III), i.e. NHS (N-hydroxysuccinimide, CAS number 6066-82-6):
  • Said by-products are also difficult to retain or separate when developing a chromatographic method.
  • solubility of the compound of formula (I) is different from that of the byproducts of formula (II) and (III), making more complex to develop a single analytical method which would determine not only the purity of the compound of formula (I), but which at the same time would allow determining the presence and quantity of the potential by-products of formulae (II) and (III).
  • the compound of formula (I) does not have a good UV absorbance and the UV spectra thereof result in issues linked to the response factor from the impurities or by-products present in the sample.
  • the analytical method of the present invention allows determining the purity of a compound of formula (I), in a sample, overcoming the above referred challenges, while at the same time being also suitable to determine the presence and quantity of the potential by-products of formula (II) and (III).
  • one aspect of the present invention refers to an analytical method for determining, in a sample, the purity of a compound of formula (I): said method comprising subjecting the sample to a high-performance liquid chromatography (HPLC) process comprising: injecting in an HPLC column a volume (1 ) of water and a volume (2) containing the sample dissolved in a water-free polar solvent not absorbing in the UV region (S1 ) containing an acid (a1 ); eluting with a mobile phase gradient; and determining the purity of the compound of formula (I) in the sample obtaining a chromatogram with an UV detector or with a Charged Aerosol Detector (CAD); wherein the HPLC column contains porous silica-based particles comprising aryl groups, preferably the HPLC column contains porous silica-based particles of 1 .5 to 10pm in diameter wherein phenyl groups are chemically bonded to said porous silica particles; and the mobile phase comprises a mobile phase A and
  • HPLC high-performance liquid
  • the method to determine the purity of a compound of formula (I) in a sample further comprises determining the presence and quantity of potential byproducts selected from the group consisting of a compound of formula (II) and a compound of formula (III), or a mixture thereof:
  • the method defined in the present specification comprises includes injecting a volume (1 ) of water and a volume (2) containing the sample dissolved in a water-free polar solvent not absorbing in the UV region (S1 ) containing an acid (a1 ).
  • the volume (1 ) of water and the volume (2) containing the sample are mixed in a vial prior to injecting in an HPLC column, obtaining thus a volume (3) comprising a mixture of a volume (1 ) and a volume (2); and the method comprises injecting in an HPLC column a volume (3) comprising a volume (1 ) of water and a volume (2) containing the sample dissolved in a water- free polar solvent not absorbing in the UV region (S1 ) containing an acid (a1 ).
  • the volume (1 ) of water and the volume (2) containing the sample are included as layers in a syringe, and the method comprises injecting the contents of a syringe comprising, sequentially, a first layer of a volume (1 ) of water and a second layer of a volume (2) containing the sample dissolved in a water-free polar solvent not absorbing in the UV region (S1 ) containing an acid (a1 ).
  • the sample is included in a syringe comprising, sequentially, a first layer which is a volume (1 ) of water and a second layer which is a volume (2) containing the sample dissolved in a water-free polar solvent not absorbing in the UV region (S1 ) containing an acid (a1 ).
  • the volume (1 ) of water and the volume (2) containing the sample are included as layers in a syringe, wherein the syringe comprises, sequentially, a first layer of a volume (1 ) of water, a second layer of a volume (2) containing the sample dissolved in a water- free polar solvent not absorbing in the UV region (S1 ) containing an acid (a1 ), and a third layer of a second volume (1 ) of water.
  • the water free polar solvent not absorbing in the UV region (S1 ) may be an alcohol, for example, methanol, ethanol or isopropanol, or acetonitrile, or mixtures thereof, preferably the water free polar solvent not absorbing in the UV region (S1 ) is aprotic, more preferably is water free acetonitrile.
  • a compound or a media for example, a solvent or an acid
  • a compound or a media is not absorbing in the UV region, when the amount of light absorbed by the compound or media between 200 nm and 400 nm is less than 10%, preferably less than 5%, more preferably less than 1 %, being said % referred to the total amount of light absorbed by said compound or media (being the amounts of light measured, for example, in absorbance units).
  • a polar aprotic solvent refers to a solvent having a dipole moment but not featuring an acidic proton.
  • water-free referred to a solvent is to be understood as referring to a solvent containing a volume of water lower than 5% (vol/vol), for example lower than 1 % (vol/vol), preferably lower than 0.1 % (vol/vol), more preferably lower than 0.01 % (vol/vol), even more preferably lower than 0.001 % (vol/vol) or even lower, being the % (vol/vol) referred to the volume of water in relation to the total volume of the solvent and water contained therein.
  • acid is to be understood for the purposes of the present specification according to the Brbnsted-Lowry definition of acid, i.e. a proton donating species.
  • the inclusion of the acid (a1 ) allows dissolving the potential by-products of formula (II) and (III) present in the sample, so that the analytical method herein disclosed may also determine the potential presence and quantity of said by-products.
  • the acid (a1 ) may be any acid suitable to ensure dissolution of the potential by-products of formula (II) and (III) present in the sample.
  • the acid (a1 ) may be a carboxylic acid R- C(O)OH, wherein R may be H, an alkyl group or a haloalkyl group, for example a fluoralkyl or a perfluoroalkyl group, or may be other suitable acid, such as, an organosulfonic acid, for example, methanesulfonic acid (MSA).
  • acid (a1 ) may be selected from the group consisting of trifluoroacetic acid (TFA), fluoroacetic acid (FA), methanesulfonic acid (MSA) and formic acid.
  • the acid (a1 ) is TFA.
  • the amount of acid (a1 ) may be a quantity adapted to obtain a pH of the volume (1 ) below 5, more preferably a pH between 1 and 3.
  • the acid (a1 ) is in an amount of between 0.1 % to 10% (vol/vol), preferably between 0.5% and 5% (vol/vol), more preferably between 0.7% and 1 .5% (vol/vol), even more preferably 1 % (vol/vol), said % referred to the mL of acid (a1 ) per 100 mL of solvent (S1 ).
  • the volume (1 ) is between 10 times and 30 times that of the volume (2), more preferably between 15 times and 25 times that of the volume (2), even more preferably the volume (1 ) is 20 times that of the volume (2).
  • a layer refers to an aliquot or volume of a liquid, , being each layer included in a syringe and in contact with another layer which is an aliquot or volume of a different liquid.
  • a layer may be a volume (1 ) comprising water or may be a volume (2) comprising a solvent S1 , the acid a1 , and the sample.
  • An interphase may be formed between two layers. The several layers included in the syringe are drawn sequentially into the syringe from separate vials containing each water or solvent, and optionally comprising the acid a1 and the sample, and which are injected into the HPLC column.
  • more water is injected in the HPLC column by increasing the content of water of volume (1 ).
  • the volume (1 ) of water and the volume (2) containing the sample are included as layers in a syringe and more water is injected in the HPLC column by including two layers of volume (1 ), preferably including a first layer of volume (1 ) of water prior to a second layer of volume (2) comprising the sample, and including a third layer with a second volume (1 ) of water after the second layer of volume (2) comprising the sample, forming in this manner a sandwich injection system where the volume (2) comprising the sample is “sandwiched” between two layers of a volume (1 ) of water.
  • the volume (2) contains the sample dissolved in water-free acetonitrile with trifluoroacetic acid. More preferably the volume (2) contains the sample dissolved in water-free acetonitrile with between 0.5% to 5% (vol/vol) trifluoroacetic acid, more preferably 1 % (vol/vol) trifluoroacetic acid, said % referred to the mL of trifluoroacetic acid per 100 mL of water-free acetonitrile.
  • the method comprises injecting in an HPLC column a volume (1 ) of water and a volume (2) containing the sample dissolved in water-free acetonitrile containing between 0.5% to 5% (vol/vol) trifluoroacetic acid, more preferably 1 % (vol/vol) trifluoroacetic acid, said % referred to the mL of trifluoroacetic acid per 100 mL of water-free acetonitrile.
  • the sample is included in a syringe comprising, sequentially, a first layer of a volume (1 ) of water and a second layer of a volume (2) containing the sample dissolved in water-free acetonitrile containing between 0.5% to 5% (vol/vol) trifluoroacetic acid, more preferably 1 % (vol/vol) trifluoroacetic acid, said % referred to the mL of trifluoroacetic acid per 100 mL of water- free acetonitrile.
  • the sample is included in a syringe comprising, sequentially, a first layer of a volume (1 ) of water, a second layer of a volume (2) containing the sample dissolved in water-free acetonitrile containing between 0.5% to 5% (vol/vol) trifluoroacetic acid, more preferably 1 % (vol/vol) trifluoroacetic acid, said % referred to the mL of trifluoroacetic acid per 100 mL of water- free acetonitrile; and a third layer of a volume (1 ) of water.
  • the volume (2) comprises between 10-25 mg/mL, preferably between 15-17 mg/mL, of the sample dissolved in dissolved in water-free acetonitrile with trifluoroacetic acid, preferably with 1 % (vol/vol) trifluoroacetic acid, said % referred to the mL of trifluoroacetic acid per 100 mL of water-free acetonitrile.
  • the method herein disclosed comprises injecting in an HPLC column a volume (1 ) of water, and a volume (2) containing the sample dissolved in a water-free polar solvent not absorbing in the UV region (S1 ) containing an acid (a1 ); preferably injecting in an HPLC column a volume (1 ) of water and a volume (2) containing the sample, preferably between 10-25 mg/mL, more preferably between 15-17 mg/mL of the sample, dissolved in water-free acetonitrile containing TFA; more preferably containing 1 % (vol/vol) TFA in acetonitrile, said % referred to the mL of TFA per 100 mL acetonitrile.
  • the volume (1 ) of water and the volume (2) containing the sample are included as layers in a syringe
  • the method herein disclosed comprises injecting in an HPLC column the contents of a syringe comprising, sequentially, a first layer of a volume (1 ) of water, and a second layer of a volume (2) containing the sample dissolved in a water-free polar solvent not absorbing in the UV region (S1 ) containing an acid (a1 ), and preferably, also comprising a third layer of a volume (1 ) of water; more preferably injecting in an HPLC column the contents of a syringe comprising sequentially a first layer of a volume (1 ) of water and a second layer of a volume (2) containing the sample, preferably between 10-25 mg/mL, more preferably between 15-17 mg/mL of the sample, dissolved in water-free acetonitrile containing TFA; more preferably containing 1 % (vol/vol)
  • the sample may comprise the compound of formula (I) in the form of a salt or solvate, for example, a HPFe or a TFA salt.
  • the sample comprises the compound of formula (I) forming a salt with HPFe and/or TFA, preferably with between 5% to 25% of HPFe (weight/weight) and between 10% to 40% TFA (weight / weight), more preferably between 10% to 20% of HPFe (weight / weight) and between 25% to 35% TFA (weight /weight), even more preferably with 18% HPFe (weight I weight) and 30% TFA (weight I weight), said % referred respectively to the weight of HPFe and TFA in the total weight of the layer (2).
  • the analytical method disclosed herein comprises using an HPLC column containing porous silica-based particles comprising aryl groups.
  • silicon-based particles comprising aryl groups refers to particles comprising moieties in which a Si (silicon) atom is linked to an aryl group.
  • Said silica-based particles may be also ethylene bridged hybrid silica-based particles (BEH), charged surface hybrid silica-based particles (CSH), high strength silica-based particles (HSS) or solid core silica-based particles.
  • BEH silica-based particles comprising aryl groups refer to silica-based particles comprising aryl groups, where two silicon atoms of a silicon containing moiety are covalently linked by an ethylene bridge
  • CHS silica-based particles refer to surface charged ethylene bridged hybrid silica-based particles
  • HSS silica-based particles feature 100% silica containing particles
  • solid-core silica-based particles include superficially porous particles.
  • the HPLC column contains porous silica-based particles comprising aryl groups, preferably the HPLC column contains porous silica-based particles of 1 .5 to 10pm in diameter wherein phenyl groups are chemically bonded to said porous silica particles. In some embodiments, the HPLC column contains porous silica-based particles of 1 .5 to 10pm in diameter, wherein phenyl groups are chemically bonded to said porous silica particles.
  • the silica-based particles may be superficially porous or fully porous particles and contain one or two phenyls, such as biphenyl, attached to one silicon atom.
  • the HPLC column contains porous silica-based particles of 1 .5 to 10pm in diameter, wherein biphenyl groups are chemically bonded to said porous silica particles.
  • the silica-based particles comprise groups -O- Si(CH3)2(biphenyl):
  • the HPLC column porous silica-based particles have a particle size of between 1 pm and 5 pm, for example between 2 pm and 4 pm, preferably 3 pm; a pore size of between 75 A to 120 A, preferably of between 85 A and 1 10 A, for example 90 A or 100 A; a carbon load of between 5% to 25%, preferably of about 15%; and a surface area of between 80 m 2 /g to 400 m 2 /g, for example between 100 m 2 /g to 350 m 2 /g, or for example between 280 m 2 /g to 320 m 2 /g.
  • the HPLC column contains fully porous particles (FPP) comprising biphenyl groups and have a particle size of between 2 pm and 5 pm, preferably 3 pm; a pore size of between 75 A to 150 A, preferably between 85 A and 1 10 A, for example 90 A or 100 A; a carbon load of between 5% to 25%, preferably of about 15%; and a surface area of between 200 m2/g to 400 m2/g, preferably of between 250 m2/g to 350 m2/g, more preferably of between 280 m2/g to 320 m2/g, even more preferably of 300 m2/g.
  • FPP fully porous particles
  • the column is used at a temperature between 10 s C to 30 s C, preferably between 12 S C and at 25 S C, more preferably at 15 S C.
  • HPLC columns for use in the method disclosed herein include the Ultra Biphenyl column from Restek, Halo Biphenyl column from Advanced Materials Technologies, Allure Biphenyl column from Restek, Ultra II Biphenyl column from Restek, Viva Biphenyl column from Restek, Pinnacle II Biphenyl column from Restek, Shim-pack Velox Biphenyl column from Shimadzu, Accucore Biphenyl column from Thermo/Hypersyl, Cosmosil piNap column from Nacalai Tesque, Ascentis Express Biphenyl column from Supelco, Ultra II Aromax column from Restek, Pronto SIL 120 Phenyl column from Bischoff, Pinnacle DB Biphenyl column from Restek, Ultra Aromax column from Restek, ACE 5 Phenyl column from ACT, ACE Phenyl-300 column from ACT, Inertsil Ph-3 column from GL Sciences, Genesis Phenyl column from Grace/Jones, Ultimate XB
  • the HPLC column is a Restek Ultra Biphenyl 3 pm, 100 x 2.1 mm HPLC column of 100 mm length and 2.1 mm of internal diameter, comprising fully porous silica particles of a particle size of 3.0 pm having a pore size of 100 A and a surface area of 300 m2/g.
  • a skilled person will know how to modify column dimensions, particle size, flow rate and isocratic elution time, referring to the current version of the European Pharmacopoeia (Chapter 2.2.46).
  • the analytical methods disclosed herein comprise eluting with a mobile phase gradient, wherein the mobile phase comprises a mobile phase A and a mobile phase B; and wherein the mobile phase A comprises water, and the mobile phase B comprises a polar solvent not absorbing in the UV region (S2).
  • the mobile phase further comprises a mobile phase C comprising water and an acid (a2).
  • the method disclosed herein comprises eluting with a mobile phase gradient, wherein the mobile phase comprises a mobile phase A and a mobile phase B; and wherein the mobile phase A is water comprising an acid (a2), and the mobile phase B is a polar solvent not absorbing in the UV region (S2) comprising the acid (a2).
  • the mobile phase further comprises a mobile phase C
  • the method disclosed herein comprises eluting with a mobile phase gradient, wherein the mobile phase comprises a mobile phase A, a mobile phase B and a mobile phase C; and wherein the mobile phase A is water, the mobile phase B comprises a polar solvent not absorbing in the UV region (S2); and the mobile phase C is water comprising the acid (a2).
  • the polar solvent not absorbing in the UV region (S2) may be an alcohol, for example, methanol, ethanol or isopropanol, or acetonitrile, or mixtures thereof, preferably the polar solvent not absorbing in the UV region (S2) is aprotic, more preferably the polar solvent not absorbing in the UV region (S2) is acetonitrile.
  • both the solvents (S1 ) and (S2) are both acetonitrile.
  • the acid (a2) may be any acid suitable to ensure dissolution of the potential by-products of formula (II) and (III) present in the sample.
  • the acid a2 may be a carboxylic acid R- C(O)OH, wherein R may be H, an alkyl group or a haloalkyl group, or other acids such as, organosulfonic acids, for example, methanesulfonic acid (MSA).
  • the acid (a2) may be selected from fluoroacetic acid, methanesulfonic acid, or mixtures thereof.
  • the acid (a2) is an acid not absorbing in the UV region; more preferably the acid (a2) is methanesulfonic acid,
  • the total amount of acid a2 (including the amount of acid a2 included in different the mobile phases) is a quantity adapted to obtain a pH below 5, more preferably a pH between 2 and 4.
  • the amount of acid (a2) in the mobile phase A and the amount of acid (a2) in the mobile phase B is the same and said amount is a quantity adapted to obtain a pH of the mobile phase A below 5, more preferably a pH between 2 and 4. Adjusting the pH to a value lower than 5 allows retaining (and detecting) the presence and quantity of the compounds of formula (II) and of formula (III).
  • the mobile phase A is an aqueous solution comprising methanesulfonic acid
  • the mobile phase B is acetonitrile comprising methanesulfonic acid, wherein the amount of methanesulfonic acid in the mobile phase A and the amount of methanesulfonic acid in the mobile phase B is the same and is a quantity adapted to obtain a pH of the mobile phase A below 5, more preferably a pH between 1 and 3.
  • the mobile phase A and the mobile phase B contain between 0.01 % and 0.1 % (vol/vol) methanesulfonic acid, more preferably 0.05% methanesulfonic acid (vol/vol), said % referred to the mL of MSA in 100 mL of water for the mobile phase A or in 100 mL of acetonitrile for the mobile phase B.
  • % referred to the mL of MSA in 100 mL of water for the mobile phase A or in 100 mL of acetonitrile for the mobile phase B.
  • the mobile phase A further comprises EDTA, preferably EDTA 1 -10 pM, more preferably EDTA 5 pM.
  • the analytical method to determine the purity, in a sample, of a compound of formula (I) disclosed herein comprises subjecting the sample to a high liquid performance chromatography (HPLC) process using a sandwich injection system, the HPLC process comprising: injecting in an HPLC column a volume (1 ) of water and a volume (2) containing the sample dissolved in a water-free polar solvent not absorbing in the UV region (S1 ) containing an acid (a1 ); eluting with a mobile phase gradient, wherein the mobile phase comprises a mobile phase A and a mobile phase B; and determining the purity of the compound of formula (I) in the sample obtaining a chromatogram with an UV detector or with a Charged Aerosol Detector (CAD); wherein the compound of formula (I), the volume (1 ), volume (2), solvent (S1 ), acid (a1 ), the HPLC column, the mobile phase gradient and the mobile phase A and mobile phase B, are as described and defined in the present specification.
  • HPLC high liquid performance chromatography
  • the determining the purity of the compound of formula (I) in the sample obtaining a chromatogram with an UV detector.
  • the volume (1 ) of water and the volume (2) containing the sample are included as layers in a syringe, wherein the syringe comprises, sequentially, a first layer of a volume (1 ) of water, a second layer of a volume (2) containing the sample dissolved in a water-free polar solvent not absorbing in the UV region (S1 ) containing an acid (a1 ), and preferably a third layer of a second volume (1 ) of water.
  • the step of eluting is carried out immediately after the step of injecting the contents of the syringe containing the sample.
  • step is carried out “immediately after” another step when there is no other steps carried out between said steps and the time between said steps is the usual time for a skilled person to carry out said steps sequentially.
  • the eluting comprises a gradient of from 98% of a mobile phase A:2% of a mobile phase B, to 2% of a mobile phase A:98% of a mobile phase B, and returning to 98% of a mobile phase A:2% of a mobile phase B, being each % referred to the volume of a mobile phase in relation to the total volume of mobile phase A and mobile phase B. If the composition of the mobile phases used to make a gradient are adjusted (e.g. ratio of aqueous to organic solvent), the gradient must be adapted to ensure that the mobile phase composition at the head of the column remains equivalent according to the gradient described above herein.
  • the eluting comprises conducting an elution sequence of 0 minutes, 98% mobile phase A and 2% mobile phase B; 1 .5 minutes, 98% mobile phase A and 2% mobile phase B; 15 minutes, 2% mobile phase A and 98% mobile phase B; 17 minutes, 2% mobile phase A and 98% mobile phase B; 17.1 minutes, 98% mobile phase A and 2% mobile phase B; and 20 minutes 98% mobile phase A and 2% mobile phase B, being each % referred to the volume of a mobile phase in relation to the total volume of mobile phase A and mobile phase B.
  • the method further comprises conducting an isocratic elution phase after injecting and prior to eluting with a mobile phase gradient.
  • the isocratic elution phase is carried out using 98% mobile phase A and 2% mobile phase B, being each % referred to the volume of a mobile phase in relation to the total volume of mobile phase A and mobile phase B.
  • the HPLC column is a Ultra Biphenyl 3.0 pm, 100 x 2.1 mm HPLC column of 100 mm length and 2.1 mm of internal diameter, having a dwell volume of 0.44 mL and comprising fully porous silica particles of a particle size of 3.0 pm having a pore size of 100 A and a surface area of 300 m2/g, the isocratic elution phase is carried out for 15 minutes; and, the eluting is carried out at a flow rate lower than 1 mL/min, preferably a flow rate of between 0.2 mL/min to 0.7 mL/min, more preferably a flow rate of 0.5 mL/min.
  • the skilled person will know how to modify column dimensions, particle size, flow rate and isocratic elution time, referring to the current version of the European Pharmacopoeia (Chapter 2.2.46).
  • the method does not comprise an initial isocratic step and the step of eluting with the gradient elution sequence is started before the step of injecting the sample, i.e. using a so-called injection delay or pre-injection volume.
  • This compensation ensures consistent gradient profiles independent of the instrument type and is therefore not considered to be a deviation from the control procedure.
  • the sample comprises the compound of formula (I) in a theoretical concentration.
  • determining the purity of the compound of formula (I) in the sample refers to the ratio between the real concentration of the compound of formula (I) determined with the method to determine the purity of the compound of formula (I) herein disclosed and the theoretical concentration of the sample, said ratio expressed as a %, being 100% the theoretical concentration of the sample.
  • the step of determining the purity of the compound of formula (I) in a sample containing a theoretical concentration of the compound of formula (I) is carried out obtaining a chromatogram with an UV detector; and comprises: determining if the sample contains the compound of formula (I) if the difference between the retention time (RTss) of the main peak in the chromatogram of the sample and the retention time (RTcs) of the main peak in a reference sample containing the same theoretical concentration of the compound of formula (I) in the sample is below a certain threshold value; calculating the real concentration of the compound of formula (I) in the sample by calculating the ratio between the area of the main peak of the sample and the area of the main peak of a reference sample containing the same theoretical concentration of the compound of formula (I); and determining the % purity of the compound of formula (I) in the sample by obtaining the ratio between the real concentration of the compound of formula (I) and the theoretical concentration of the sample, said ratio expressed as a %, being 100% the theoretical concentration
  • the step of determining the potential presence and quantity of the byproducts of formula (II) and (III) comprises determining the concentration of the compounds of formula (II) and (III) present in the sample by: o calculating the concentration of the compound of formula (II) in the sample by calculating the ratio between the area of the peak belonging to the compound of formula (II) in the chromatogram of the sample and the area of the peak of a reference sample containing a known concentration of the compound of formula (II); and o calculating the concentration of the compound of formula (III) in the sample by calculating the ratio between the area of the peak belonging to the compound of formula (III) in the chromatogram of the sample and the area of the peak of a reference sample containing a known concentration of the compound of formula (III).
  • the steps of determining the purity of the compound of formula (I), determining the presence and quantity of the potential by-products of formula (II) and (III), and determining the concentration of formula (I), are carried out with a diode array detector at a wavelength of 201 nm.
  • DOTA-NHS compound of formula (I), 2,2’,2”-(10- ⁇ 2-[(2,5-dioxopyrrolidin-1 - yl)oxy]-2-oxoethyl ⁇ -1 ,4,7,10-tetraazacyclododecane-1 ,4,7-triyl)triethanoic acid; CAS Nr: 170908-81 - 3) from ChemMatech was used to exemplify the method disclosed in the present specification.
  • Acetonitrile HPLC grade e.g. Merck 1 .00030.
  • MSA Methane sulfonic acid
  • Trifluoro acetic acid (TFA) HPLC grade e.g. Sigma-Aldrich 91707
  • Acetonitrile anhydrous HPLC grade e.g. Sigma-Aldrich 271004
  • Apparatus e.g. Agilent 1290 HPLC with high pressure mixing and compatible with sandwich injection.
  • Response time width (response time ⁇ 25% of the peak width at half height).
  • Sample solution Weigh about 30 - 34 mg of the sample accurately to 0.1 mg into a 2 mL volumetric flask, dissolve in and dilute to volume with solvent.
  • Results are shown in the table 1 below: [0086] Table 1 (Channel: A / Reporting limit (RL): 0.100%)

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Abstract

An analytical method for determining, in a sample, the purity of a compound of formula (I): is disclosed, being said compound of formula (I) the compound DOTA-NHS (2,2',2"-(10-{2-[(2,5- dioxopyrrolidin-1 -yl)oxy]-2-oxoethyl}-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triethanoic acid.

Description

Description
Title: METHOD FOR DETERMINING THE PURITY OF A COMPOUND BY HIGH- PERFORMANCE LIQUID CHROMATOGRAPHY
Technical Field
[0001] This disclosure pertains to the field of analytical chemistry. In particular the present disclosure provides a method for determining the purity of a compound in a sample, as well as the presence and quantity of potential by-products of said compound using high-performance liquid chromatography (HPLC).
Backg ound Art
[0002] The compound DOTA-NHS (2,2’,2”-(10-{2-[(2,5-dioxopyrrolidin-1 -yl)oxy]-2-oxoethyl}- 1 ,4,7,10-tetraazacyclododecane-1 ,4,7-triyl)triethanoic acid; CAS Nr: 170908-81 -3) is widely used in drug research and development.
[0003] The compound DOTA-NHS is composed of two main functional groups: the DOTA (1 ,4,7,10- tetraazacyclododecane-1 ,4,7,10-tetraacetic acid) which may form a stable complex with various metal ions, such as lutecium, gallium and actinium; and the N-hydroxysuccinimide (NHS) ester, which may react with primary amines on biomolecules to form stable amide bonds.
[0004] Due to those properties, the compound DOTA-NHS ester is widely used in drug research and development, being mainly focused on the development of radiopharmaceuticals for imaging and therapy.
[0005] There is thus a need to provide reliable and accurate analytical methods to ensure the purity of the compound DOTA-NHS used in the development of subsequent drugs and, in particular, in the development of radiopharmaceuticals.
Brief description of the invention
[0006] An aspect of this disclosure refers to an analytical method for determining, in a sample, the purity of a compound of formula (I): being said compound of formula (I) the compound DOTA-NHS (2,2’,2”-(10-{2-[(2,5-dioxopyrrolidin- 1 -yl)oxy]-2-oxoethyl}-1 ,4,7,10-tetraazacyclododecane-1 ,4,7-triyl)triethanoic acid; CAS Nr: 170908- 81 -3); said method comprising subjecting the sample to a high-performance liquid chromatography (HPLC) process comprising: injecting in an HPLC column a volume (1 ) of water and a volume (2) containing the sample dissolved in a water-free polar solvent not absorbing in the UV region (S1 ) containing an acid (a1 ); eluting with a mobile phase gradient; and determining the purity of the compound of formula (I) in the sample obtaining a chromatogram with an UV detector or with a Charged Aerosol Detector (CAD); wherein the HPLC column contains porous silica-based particles comprising aryl groups, preferably the HPLC column contains porous silica-based particles of 1 .5 to 10pm in diameter wherein phenyl groups are chemically bonded to said porous silica particles; and the mobile phase comprises a mobile phase A and a mobile phase B; and wherein the mobile phase A comprises water, and the mobile phase B comprises a polar solvent not absorbing in the UV region (S2).
Brief Description of Drawings
[0007] Other features, details and advantages will be shown in the following detailed description and on the figures, on which:
Fig. 1
[0008] [Fig. 1 ] shows a schematic drawing of the sandwich injection of the sample used in the method of the invention, exemplified with a syringe containing a first layer of a volume (1 ) of 4 pL of water, a second layer of volume (2) containing 0.2 pL of the sample dissolved in water-free acetonitrile (<0.001 % H2O) containing 1 % (vol/vol) trifluoroacetic acid (TFA); and a third layer of volume (1 ) of 4 pL of water.
Fig. 2
[0009] [Fig 2] shows a chromatogram of a sample of the compound of formula (I) obtained applying the HPLC method of the invention.
Detailed description
[0010] As previously indicated, the compound of formula (I) (i.e., DOTA-NHS ester or 2,2’,2”-(10-{2- [(2,5-dioxopyrrolidin-1 -yl)oxy]-2-oxoethyl}-1 ,4,7,10-tetraazacyclododecane-1 ,4,7-triyl)triethanoic acid; CAS Nr: 170908-81 -3) is widely used in drug research and development, being said use often focused on the development of radiopharmaceuticals for diagnostic and therapeutic methods. For this reason, it is key to ensure the purity of the compound of formula (I) for use in the development of drugs or diagnostic agents.
[0011] However, the development of an analytical methods to determine the purity of the compound of formula (I) encounters several challenges. In particular, the compound of formula (I) is an ester which is unstable in water, hydrolyzing quickly. On the other hand, it is a very polar compound, making it difficult to separate/retain the compound of formula (I) when developing a chromatographic analytical method.
[0012] In addition, samples containing the compound of formula (I) may also contain impurities or by-products. In general, the most common by-products present in samples of a compound of formula (I) are the result of the hydrolysis of the N-succinimide ester present in this compound, being said main by-products the compound of formula (II), i.e. free DOTA (2-[4,7,10-tris(carboxymethyl)- 1 ,4,7,10-tetrazacyclododec-1 -yl]acetic acid; CAS number 60239-18-1 ) and the compound of formula (III), i.e. NHS (N-hydroxysuccinimide, CAS number 6066-82-6):
[0013] Said by-products are also difficult to retain or separate when developing a chromatographic method. In addition, the solubility of the compound of formula (I) is different from that of the byproducts of formula (II) and (III), making more complex to develop a single analytical method which would determine not only the purity of the compound of formula (I), but which at the same time would allow determining the presence and quantity of the potential by-products of formulae (II) and (III).
[0014] In addition, the compound of formula (I) does not have a good UV absorbance and the UV spectra thereof result in issues linked to the response factor from the impurities or by-products present in the sample.
[0015] The analytical method of the present invention allows determining the purity of a compound of formula (I), in a sample, overcoming the above referred challenges, while at the same time being also suitable to determine the presence and quantity of the potential by-products of formula (II) and (III).
[0016] In that regard, one aspect of the present invention refers to an analytical method for determining, in a sample, the purity of a compound of formula (I): said method comprising subjecting the sample to a high-performance liquid chromatography (HPLC) process comprising: injecting in an HPLC column a volume (1 ) of water and a volume (2) containing the sample dissolved in a water-free polar solvent not absorbing in the UV region (S1 ) containing an acid (a1 ); eluting with a mobile phase gradient; and determining the purity of the compound of formula (I) in the sample obtaining a chromatogram with an UV detector or with a Charged Aerosol Detector (CAD); wherein the HPLC column contains porous silica-based particles comprising aryl groups, preferably the HPLC column contains porous silica-based particles of 1 .5 to 10pm in diameter wherein phenyl groups are chemically bonded to said porous silica particles; and the mobile phase comprises a mobile phase A and a mobile phase B; and wherein the mobile phase A comprises water, and the mobile phase B comprises a polar solvent not absorbing in the UV region (S2).
[0017] In some embodiments, the method to determine the purity of a compound of formula (I) in a sample, herein disclosed, further comprises determining the presence and quantity of potential byproducts selected from the group consisting of a compound of formula (II) and a compound of formula (III), or a mixture thereof: [0018] The method defined in the present specification comprises includes injecting a volume (1 ) of water and a volume (2) containing the sample dissolved in a water-free polar solvent not absorbing in the UV region (S1 ) containing an acid (a1 ).
[0019] In some embodiments, the volume (1 ) of water and the volume (2) containing the sample are mixed in a vial prior to injecting in an HPLC column, obtaining thus a volume (3) comprising a mixture of a volume (1 ) and a volume (2); and the method comprises injecting in an HPLC column a volume (3) comprising a volume (1 ) of water and a volume (2) containing the sample dissolved in a water- free polar solvent not absorbing in the UV region (S1 ) containing an acid (a1 ).
[0020] In some embodiments, the volume (1 ) of water and the volume (2) containing the sample are included as layers in a syringe, and the method comprises injecting the contents of a syringe comprising, sequentially, a first layer of a volume (1 ) of water and a second layer of a volume (2) containing the sample dissolved in a water-free polar solvent not absorbing in the UV region (S1 ) containing an acid (a1 ).
[0021] Accordingly, in one embodiment, the sample is included in a syringe comprising, sequentially, a first layer which is a volume (1 ) of water and a second layer which is a volume (2) containing the sample dissolved in a water-free polar solvent not absorbing in the UV region (S1 ) containing an acid (a1 ).
[0022] In some preferred embodiments, the volume (1 ) of water and the volume (2) containing the sample are included as layers in a syringe, wherein the syringe comprises, sequentially, a first layer of a volume (1 ) of water, a second layer of a volume (2) containing the sample dissolved in a water- free polar solvent not absorbing in the UV region (S1 ) containing an acid (a1 ), and a third layer of a second volume (1 ) of water.
[0023] In some embodiments, the water free polar solvent not absorbing in the UV region (S1 ) may be an alcohol, for example, methanol, ethanol or isopropanol, or acetonitrile, or mixtures thereof, preferably the water free polar solvent not absorbing in the UV region (S1 ) is aprotic, more preferably is water free acetonitrile.
[0024] For the purposes of the present specification, it is to be understood that a compound or a media (for example, a solvent or an acid) is not absorbing in the UV region, when the amount of light absorbed by the compound or media between 200 nm and 400 nm is less than 10%, preferably less than 5%, more preferably less than 1 %, being said % referred to the total amount of light absorbed by said compound or media (being the amounts of light measured, for example, in absorbance units).
[0025] In addition, a polar aprotic solvent refers to a solvent having a dipole moment but not featuring an acidic proton.
[0026] The term “water-free” referred to a solvent is to be understood as referring to a solvent containing a volume of water lower than 5% (vol/vol), for example lower than 1 % (vol/vol), preferably lower than 0.1 % (vol/vol), more preferably lower than 0.01 % (vol/vol), even more preferably lower than 0.001 % (vol/vol) or even lower, being the % (vol/vol) referred to the volume of water in relation to the total volume of the solvent and water contained therein.
[0027] The term acid is to be understood for the purposes of the present specification according to the Brbnsted-Lowry definition of acid, i.e. a proton donating species.
[0028] Dissolving the sample in a water-free polar solvent (S1 ), as defined herein, results in that the stability of the compound of formula (I) is ensured while avoiding stability issues (hydrolysis of the NHS ester of the compound of formula (I).
[0029] On the other hand, the inclusion of the acid (a1 ) allows dissolving the potential by-products of formula (II) and (III) present in the sample, so that the analytical method herein disclosed may also determine the potential presence and quantity of said by-products.
[0030] The acid (a1 ) may be any acid suitable to ensure dissolution of the potential by-products of formula (II) and (III) present in the sample. For example, the acid (a1 ) may be a carboxylic acid R- C(O)OH, wherein R may be H, an alkyl group or a haloalkyl group, for example a fluoralkyl or a perfluoroalkyl group, or may be other suitable acid, such as, an organosulfonic acid, for example, methanesulfonic acid (MSA). In some embodiments acid (a1 ) may be selected from the group consisting of trifluoroacetic acid (TFA), fluoroacetic acid (FA), methanesulfonic acid (MSA) and formic acid. Preferably the acid (a1 ) is TFA. The amount of acid (a1 ) may be a quantity adapted to obtain a pH of the volume (1 ) below 5, more preferably a pH between 1 and 3.
[0031] In some embodiments the acid (a1 ) is in an amount of between 0.1 % to 10% (vol/vol), preferably between 0.5% and 5% (vol/vol), more preferably between 0.7% and 1 .5% (vol/vol), even more preferably 1 % (vol/vol), said % referred to the mL of acid (a1 ) per 100 mL of solvent (S1 ).
[0032] Preferably, the volume (1 ) is between 10 times and 30 times that of the volume (2), more preferably between 15 times and 25 times that of the volume (2), even more preferably the volume (1 ) is 20 times that of the volume (2).
[0033] For the purposes of the present specification the term layer refers to an aliquot or volume of a liquid, , being each layer included in a syringe and in contact with another layer which is an aliquot or volume of a different liquid. In particular, a layer may be a volume (1 ) comprising water or may be a volume (2) comprising a solvent S1 , the acid a1 , and the sample. An interphase may be formed between two layers. The several layers included in the syringe are drawn sequentially into the syringe from separate vials containing each water or solvent, and optionally comprising the acid a1 and the sample, and which are injected into the HPLC column.
[0034] Adding more water in the volume ratios disclosed herein allows retaining the compound of formula (II) more appropriately, while not resulting in stability issues for the compound of formula (I).
[0035] In some embodiments, more water is injected in the HPLC column by increasing the content of water of volume (1 ).
[0036] In other embodiments, the volume (1 ) of water and the volume (2) containing the sample are included as layers in a syringe and more water is injected in the HPLC column by including two layers of volume (1 ), preferably including a first layer of volume (1 ) of water prior to a second layer of volume (2) comprising the sample, and including a third layer with a second volume (1 ) of water after the second layer of volume (2) comprising the sample, forming in this manner a sandwich injection system where the volume (2) comprising the sample is “sandwiched” between two layers of a volume (1 ) of water.
[0037] In a preferred embodiment the volume (2) contains the sample dissolved in water-free acetonitrile with trifluoroacetic acid. More preferably the volume (2) contains the sample dissolved in water-free acetonitrile with between 0.5% to 5% (vol/vol) trifluoroacetic acid, more preferably 1 % (vol/vol) trifluoroacetic acid, said % referred to the mL of trifluoroacetic acid per 100 mL of water-free acetonitrile.
[0038] Accordingly, in one embodiment the method comprises injecting in an HPLC column a volume (1 ) of water and a volume (2) containing the sample dissolved in water-free acetonitrile containing between 0.5% to 5% (vol/vol) trifluoroacetic acid, more preferably 1 % (vol/vol) trifluoroacetic acid, said % referred to the mL of trifluoroacetic acid per 100 mL of water-free acetonitrile.
[0039] In a particular embodiment, the sample is included in a syringe comprising, sequentially, a first layer of a volume (1 ) of water and a second layer of a volume (2) containing the sample dissolved in water-free acetonitrile containing between 0.5% to 5% (vol/vol) trifluoroacetic acid, more preferably 1 % (vol/vol) trifluoroacetic acid, said % referred to the mL of trifluoroacetic acid per 100 mL of water- free acetonitrile.
[0040] In another particular embodiment, the sample is included in a syringe comprising, sequentially, a first layer of a volume (1 ) of water, a second layer of a volume (2) containing the sample dissolved in water-free acetonitrile containing between 0.5% to 5% (vol/vol) trifluoroacetic acid, more preferably 1 % (vol/vol) trifluoroacetic acid, said % referred to the mL of trifluoroacetic acid per 100 mL of water- free acetonitrile; and a third layer of a volume (1 ) of water.
[0041] In some preferred embodiments the volume (2) comprises between 10-25 mg/mL, preferably between 15-17 mg/mL, of the sample dissolved in dissolved in water-free acetonitrile with trifluoroacetic acid, preferably with 1 % (vol/vol) trifluoroacetic acid, said % referred to the mL of trifluoroacetic acid per 100 mL of water-free acetonitrile.
[0042] In an embodiment, the method herein disclosed comprises injecting in an HPLC column a volume (1 ) of water, and a volume (2) containing the sample dissolved in a water-free polar solvent not absorbing in the UV region (S1 ) containing an acid (a1 ); preferably injecting in an HPLC column a volume (1 ) of water and a volume (2) containing the sample, preferably between 10-25 mg/mL, more preferably between 15-17 mg/mL of the sample, dissolved in water-free acetonitrile containing TFA; more preferably containing 1 % (vol/vol) TFA in acetonitrile, said % referred to the mL of TFA per 100 mL acetonitrile.
[0043] In a particularly preferred embodiment the volume (1 ) of water and the volume (2) containing the sample are included as layers in a syringe, and the method herein disclosed comprises injecting in an HPLC column the contents of a syringe comprising, sequentially, a first layer of a volume (1 ) of water, and a second layer of a volume (2) containing the sample dissolved in a water-free polar solvent not absorbing in the UV region (S1 ) containing an acid (a1 ), and preferably, also comprising a third layer of a volume (1 ) of water; more preferably injecting in an HPLC column the contents of a syringe comprising sequentially a first layer of a volume (1 ) of water and a second layer of a volume (2) containing the sample, preferably between 10-25 mg/mL, more preferably between 15-17 mg/mL of the sample, dissolved in water-free acetonitrile containing TFA; more preferably containing 1 % (vol/vol) TFA in acetonitrile, said % referred to the mL of TFA per 100 mL acetonitrile, even more preferably also comprising a third layer of a volume (1 ) of water.
[0044] The sample may comprise the compound of formula (I) in the form of a salt or solvate, for example, a HPFe or a TFA salt. In some embodiments the sample comprises the compound of formula (I) forming a salt with HPFe and/or TFA, preferably with between 5% to 25% of HPFe (weight/weight) and between 10% to 40% TFA (weight / weight), more preferably between 10% to 20% of HPFe (weight / weight) and between 25% to 35% TFA (weight /weight), even more preferably with 18% HPFe (weight I weight) and 30% TFA (weight I weight), said % referred respectively to the weight of HPFe and TFA in the total weight of the layer (2).
Stationary phase
[0045] The analytical method disclosed herein comprises using an HPLC column containing porous silica-based particles comprising aryl groups.
[0046] For the purposes of the present specification the term “silica-based” particles comprising aryl groups refers to particles comprising moieties in which a Si (silicon) atom is linked to an aryl group.
[0047] Said silica-based particles may be also ethylene bridged hybrid silica-based particles (BEH), charged surface hybrid silica-based particles (CSH), high strength silica-based particles (HSS) or solid core silica-based particles. In this context, BEH silica-based particles comprising aryl groups refer to silica-based particles comprising aryl groups, where two silicon atoms of a silicon containing moiety are covalently linked by an ethylene bridge; CHS silica-based particles refer to surface charged ethylene bridged hybrid silica-based particles; HSS silica-based particles feature 100% silica containing particles; and solid-core silica-based particles include superficially porous particles.
[0048] In some embodiments, the HPLC column contains porous silica-based particles comprising aryl groups, preferably the HPLC column contains porous silica-based particles of 1 .5 to 10pm in diameter wherein phenyl groups are chemically bonded to said porous silica particles. In some embodiments, the HPLC column contains porous silica-based particles of 1 .5 to 10pm in diameter, wherein phenyl groups are chemically bonded to said porous silica particles. The silica-based particles may be superficially porous or fully porous particles and contain one or two phenyls, such as biphenyl, attached to one silicon atom. In some preferred embodiments, the HPLC column contains porous silica-based particles of 1 .5 to 10pm in diameter, wherein biphenyl groups are chemically bonded to said porous silica particles. [0049] In some preferred embodiments the silica-based particles comprise groups -O- Si(CH3)2(biphenyl):
[0050] In some embodiments, the HPLC column porous silica-based particles have a particle size of between 1 pm and 5 pm, for example between 2 pm and 4 pm, preferably 3 pm; a pore size of between 75 A to 120 A, preferably of between 85 A and 1 10 A, for example 90 A or 100 A; a carbon load of between 5% to 25%, preferably of about 15%; and a surface area of between 80 m2/g to 400 m2/g, for example between 100 m2/g to 350 m2/g, or for example between 280 m2/g to 320 m2/g. Preferably, the HPLC column contains fully porous particles (FPP) comprising biphenyl groups and have a particle size of between 2 pm and 5 pm, preferably 3 pm; a pore size of between 75 A to 150 A, preferably between 85 A and 1 10 A, for example 90 A or 100 A; a carbon load of between 5% to 25%, preferably of about 15%; and a surface area of between 200 m2/g to 400 m2/g, preferably of between 250 m2/g to 350 m2/g, more preferably of between 280 m2/g to 320 m2/g, even more preferably of 300 m2/g.
[0051] In some embodiments the column is used at a temperature between 10sC to 30sC, preferably between 12SC and at 25SC, more preferably at 15SC.
[0052] Some HPLC columns for use in the method disclosed herein include the Ultra Biphenyl column from Restek, Halo Biphenyl column from Advanced Materials Technologies, Allure Biphenyl column from Restek, Ultra II Biphenyl column from Restek, Viva Biphenyl column from Restek, Pinnacle II Biphenyl column from Restek, Shim-pack Velox Biphenyl column from Shimadzu, Accucore Biphenyl column from Thermo/Hypersyl, Cosmosil piNap column from Nacalai Tesque, Ascentis Express Biphenyl column from Supelco, Ultra II Aromax column from Restek, Pronto SIL 120 Phenyl column from Bischoff, Pinnacle DB Biphenyl column from Restek, Ultra Aromax column from Restek, ACE 5 Phenyl column from ACT, ACE Phenyl-300 column from ACT, Inertsil Ph-3 column from GL Sciences, Genesis Phenyl column from Grace/Jones, Ultimate XB-Phenyl from Welch, CHROMSHELL® Biphenyl HPLC column, Kinetex Core-Shell Biphenyl HPLC Columns, EC HPLC column, NUCLEOSHELL Biphenyl, 2.7 pm, LC Thermo Scientific™ Biphenyl Accucore™, and the Raptor™ Biphenyl column from Restek. Preferably the column used is a Ultra Biphenyl HPLC column from Restek.
[0053] In some embodiments the HPLC column is a Restek Ultra Biphenyl 3 pm, 100 x 2.1 mm HPLC column of 100 mm length and 2.1 mm of internal diameter, comprising fully porous silica particles of a particle size of 3.0 pm having a pore size of 100 A and a surface area of 300 m2/g. A skilled person will know how to modify column dimensions, particle size, flow rate and isocratic elution time, referring to the current version of the European Pharmacopoeia (Chapter 2.2.46). Mobile
[0054] The analytical methods disclosed herein comprise eluting with a mobile phase gradient, wherein the mobile phase comprises a mobile phase A and a mobile phase B; and wherein the mobile phase A comprises water, and the mobile phase B comprises a polar solvent not absorbing in the UV region (S2). In some embodiments the mobile phase further comprises a mobile phase C comprising water and an acid (a2).
[0055] Thus, in some embodiments, the method disclosed herein comprises eluting with a mobile phase gradient, wherein the mobile phase comprises a mobile phase A and a mobile phase B; and wherein the mobile phase A is water comprising an acid (a2), and the mobile phase B is a polar solvent not absorbing in the UV region (S2) comprising the acid (a2). In other embodiments the mobile phase further comprises a mobile phase C, and the method disclosed herein comprises eluting with a mobile phase gradient, wherein the mobile phase comprises a mobile phase A, a mobile phase B and a mobile phase C; and wherein the mobile phase A is water, the mobile phase B comprises a polar solvent not absorbing in the UV region (S2); and the mobile phase C is water comprising the acid (a2).
[0056] In some embodiments, the polar solvent not absorbing in the UV region (S2) may be an alcohol, for example, methanol, ethanol or isopropanol, or acetonitrile, or mixtures thereof, preferably the polar solvent not absorbing in the UV region (S2) is aprotic, more preferably the polar solvent not absorbing in the UV region (S2) is acetonitrile.
[0057] More preferably both the solvents (S1 ) and (S2) are both acetonitrile.
[0058] The acid (a2) may be any acid suitable to ensure dissolution of the potential by-products of formula (II) and (III) present in the sample. For example, the acid a2 may be a carboxylic acid R- C(O)OH, wherein R may be H, an alkyl group or a haloalkyl group, or other acids such as, organosulfonic acids, for example, methanesulfonic acid (MSA). In some embodiments the acid (a2) may be selected from fluoroacetic acid, methanesulfonic acid, or mixtures thereof. Preferably the acid (a2) is an acid not absorbing in the UV region; more preferably the acid (a2) is methanesulfonic acid,
[0059] In some embodiments the total amount of acid a2 (including the amount of acid a2 included in different the mobile phases) is a quantity adapted to obtain a pH below 5, more preferably a pH between 2 and 4. In some preferred embodiments the amount of acid (a2) in the mobile phase A and the amount of acid (a2) in the mobile phase B is the same and said amount is a quantity adapted to obtain a pH of the mobile phase A below 5, more preferably a pH between 2 and 4. Adjusting the pH to a value lower than 5 allows retaining (and detecting) the presence and quantity of the compounds of formula (II) and of formula (III).
[0060] In some particularly preferred embodiments the mobile phase A is an aqueous solution comprising methanesulfonic acid, and the mobile phase B is acetonitrile comprising methanesulfonic acid, wherein the amount of methanesulfonic acid in the mobile phase A and the amount of methanesulfonic acid in the mobile phase B is the same and is a quantity adapted to obtain a pH of the mobile phase A below 5, more preferably a pH between 1 and 3.
[0061] Preferably the mobile phase A and the mobile phase B contain between 0.01 % and 0.1 % (vol/vol) methanesulfonic acid, more preferably 0.05% methanesulfonic acid (vol/vol), said % referred to the mL of MSA in 100 mL of water for the mobile phase A or in 100 mL of acetonitrile for the mobile phase B.
[0062] In some embodiments the mobile phase A further comprises EDTA, preferably EDTA 1 -10 pM, more preferably EDTA 5 pM.
HPLC process of the method to determine the purity of the compound of formula (I)
[0063] The analytical method to determine the purity, in a sample, of a compound of formula (I) disclosed herein comprises subjecting the sample to a high liquid performance chromatography (HPLC) process using a sandwich injection system, the HPLC process comprising: injecting in an HPLC column a volume (1 ) of water and a volume (2) containing the sample dissolved in a water-free polar solvent not absorbing in the UV region (S1 ) containing an acid (a1 ); eluting with a mobile phase gradient, wherein the mobile phase comprises a mobile phase A and a mobile phase B; and determining the purity of the compound of formula (I) in the sample obtaining a chromatogram with an UV detector or with a Charged Aerosol Detector (CAD); wherein the compound of formula (I), the volume (1 ), volume (2), solvent (S1 ), acid (a1 ), the HPLC column, the mobile phase gradient and the mobile phase A and mobile phase B, are as described and defined in the present specification.
[0064] In some preferred embodiments the determining the purity of the compound of formula (I) in the sample obtaining a chromatogram with an UV detector.
[0065] In some embodiments, the volume (1 ) of water and the volume (2) containing the sample are included as layers in a syringe, wherein the syringe comprises, sequentially, a first layer of a volume (1 ) of water, a second layer of a volume (2) containing the sample dissolved in a water-free polar solvent not absorbing in the UV region (S1 ) containing an acid (a1 ), and preferably a third layer of a second volume (1 ) of water.
[0066] In some embodiments the step of eluting is carried out immediately after the step of injecting the contents of the syringe containing the sample.
[0067] For the purposes of the present specification one step is carried out “immediately after” another step when there is no other steps carried out between said steps and the time between said steps is the usual time for a skilled person to carry out said steps sequentially.
[0068] In some embodiments the eluting comprises a gradient of from 98% of a mobile phase A:2% of a mobile phase B, to 2% of a mobile phase A:98% of a mobile phase B, and returning to 98% of a mobile phase A:2% of a mobile phase B, being each % referred to the volume of a mobile phase in relation to the total volume of mobile phase A and mobile phase B. If the composition of the mobile phases used to make a gradient are adjusted (e.g. ratio of aqueous to organic solvent), the gradient must be adapted to ensure that the mobile phase composition at the head of the column remains equivalent according to the gradient described above herein.
[0069] In some preferred embodiments the eluting comprises conducting an elution sequence of 0 minutes, 98% mobile phase A and 2% mobile phase B; 1 .5 minutes, 98% mobile phase A and 2% mobile phase B; 15 minutes, 2% mobile phase A and 98% mobile phase B; 17 minutes, 2% mobile phase A and 98% mobile phase B; 17.1 minutes, 98% mobile phase A and 2% mobile phase B; and 20 minutes 98% mobile phase A and 2% mobile phase B, being each % referred to the volume of a mobile phase in relation to the total volume of mobile phase A and mobile phase B.
[0070] In some embodiments the method further comprises conducting an isocratic elution phase after injecting and prior to eluting with a mobile phase gradient. In particular, the isocratic elution phase is carried out using 98% mobile phase A and 2% mobile phase B, being each % referred to the volume of a mobile phase in relation to the total volume of mobile phase A and mobile phase B.
[0071] In some embodiments, the HPLC column is a Ultra Biphenyl 3.0 pm, 100 x 2.1 mm HPLC column of 100 mm length and 2.1 mm of internal diameter, having a dwell volume of 0.44 mL and comprising fully porous silica particles of a particle size of 3.0 pm having a pore size of 100 A and a surface area of 300 m2/g, the isocratic elution phase is carried out for 15 minutes; and, the eluting is carried out at a flow rate lower than 1 mL/min, preferably a flow rate of between 0.2 mL/min to 0.7 mL/min, more preferably a flow rate of 0.5 mL/min. The skilled person will know how to modify column dimensions, particle size, flow rate and isocratic elution time, referring to the current version of the European Pharmacopoeia (Chapter 2.2.46).
[0072] Differences in instrument dwell volumes between different HPLCs columns used in the method herein disclosed can be compensated for. In some embodiments, if the HPLC column has a dwell volume lower than 0.44 mL, a compensating (additional) isocratic step can be added before the gradient to maintain comparable selectivity. To compensate for columns with a dwell volume larger than 0.44 mL, if an isocratic step is already part of the method, the time of said isocratic step can be reduced accordingly.
[0073] In some embodiments the method does not comprise an initial isocratic step and the step of eluting with the gradient elution sequence is started before the step of injecting the sample, i.e. using a so-called injection delay or pre-injection volume. This compensation ensures consistent gradient profiles independent of the instrument type and is therefore not considered to be a deviation from the control procedure.
[0074] The sample comprises the compound of formula (I) in a theoretical concentration. In that connection, determining the purity of the compound of formula (I) in the sample refers to the ratio between the real concentration of the compound of formula (I) determined with the method to determine the purity of the compound of formula (I) herein disclosed and the theoretical concentration of the sample, said ratio expressed as a %, being 100% the theoretical concentration of the sample.
[0075] In some embodiments the step of determining the purity of the compound of formula (I) in a sample containing a theoretical concentration of the compound of formula (I) is carried out obtaining a chromatogram with an UV detector; and comprises: determining if the sample contains the compound of formula (I) if the difference between the retention time (RTss) of the main peak in the chromatogram of the sample and the retention time (RTcs) of the main peak in a reference sample containing the same theoretical concentration of the compound of formula (I) in the sample is below a certain threshold value; calculating the real concentration of the compound of formula (I) in the sample by calculating the ratio between the area of the main peak of the sample and the area of the main peak of a reference sample containing the same theoretical concentration of the compound of formula (I); and determining the % purity of the compound of formula (I) in the sample by obtaining the ratio between the real concentration of the compound of formula (I) and the theoretical concentration of the sample, said ratio expressed as a %, being 100% the theoretical concentration of the sample.
[0076] In some embodiments the step of determining the potential presence and quantity of the byproducts of formula (II) and (III) comprises determining the concentration of the compounds of formula (II) and (III) present in the sample by: o calculating the concentration of the compound of formula (II) in the sample by calculating the ratio between the area of the peak belonging to the compound of formula (II) in the chromatogram of the sample and the area of the peak of a reference sample containing a known concentration of the compound of formula (II); and o calculating the concentration of the compound of formula (III) in the sample by calculating the ratio between the area of the peak belonging to the compound of formula (III) in the chromatogram of the sample and the area of the peak of a reference sample containing a known concentration of the compound of formula (III).
[0077] In some embodiments the steps of determining the purity of the compound of formula (I), determining the presence and quantity of the potential by-products of formula (II) and (III), and determining the concentration of formula (I), are carried out with a diode array detector at a wavelength of 201 nm.
[0078] This invention will be further illustrated by the following non-limiting examples which are given for illustrative purposes only and should not restrict the scope of the appended claims. Examples
[0079] A sample of DOTA-NHS (compound of formula (I), 2,2’,2”-(10-{2-[(2,5-dioxopyrrolidin-1 - yl)oxy]-2-oxoethyl}-1 ,4,7,10-tetraazacyclododecane-1 ,4,7-triyl)triethanoic acid; CAS Nr: 170908-81 - 3) from ChemMatech was used to exemplify the method disclosed in the present specification.
[0080] Reagents
Water e.g. Milli-Q-equipment.
Acetonitrile HPLC grade, e.g. Merck 1 .00030.
Methane sulfonic acid (MSA) HPLC grade, e.g. Sigma-Aldrich 59510
Trifluoro acetic acid (TFA) HPLC grade, e.g. Sigma-Aldrich 91707
Acetonitrile anhydrous HPLC grade, e.g. Sigma-Aldrich 271004
Solvent Acetonitrile anhydrous/TFA (100:1 , v/v).
Reference standard(s) Current reference standard batches DOTA-NHS. (CheMatech).
[0081] Equipment
Apparatus e.g. Agilent 1290 HPLC with high pressure mixing and compatible with sandwich injection.
Column e. g. Restek Ultra Biphenyl , Part No: 9109312 or equivalent column.
Length 100 mm, internal diameter 2.1 mm, particle size:
3 pm.
[0082] Test replicates
Single measurement per sample. [0083] Chromatographic conditions
Mobile phase A 0.05% MSA in water
Mobile phase B 0.05% MSA in Acetonitrile
Gradient Time (min.) Phase B (%)
0 2
1.5 2
15 98
17 98
17.1 2
20 2 End of
Comment on gradient Dwell volume of approximately 0.44 mL.
Flow rate 0.5 mL/min
Detection UV 201 nm
Reference wavelength Off
Bandwidth 4 nm
Cell type Standard cell 10 mm Column temperature 15 °C
Autosampler temperature 5 °C
Injection principle Sandwich injection with three aliquots drawn into the autosampler syringe from multiple vials and injected into the system.
Injection volume sample 0.2 pL
Sandwich solvent Water
Injection Program 4 pL Sandwich solvent + 0.2 pL sample + 4 pL Sandwich solvent
Needle wash solvent Acetonitrile
Pressure at initial conditions 300 bar
Data acquisition rate 20 Hz
0.25 seconds. Adjusted depending on the observed peak
Response time width (response time < 25% of the peak width at half height).
[0084] Test Procedu e
Note: Sample solution was injected within 3 hr of storage in amber vial at 5SC
Sample solution Weigh about 30 - 34 mg of the sample accurately to 0.1 mg into a 2 mL volumetric flask, dissolve in and dilute to volume with solvent.
Evaluation By peak area ratio (area %).
See sample solution chromatogram of figure 2.
[0085] Results are shown in the table 1 below: [0086] Table 1 (Channel: A / Reporting limit (RL): 0.100%)

Claims

Claims
[Claim 1] A method for determining, in a sample, the purity of a compound of formula (I): said method comprising subjecting the sample to a high-performance liquid chromatography (HPLC) process comprising: injecting in an HPLC column a volume (1 ) of water and a volume (2) containing the sample dissolved in a water-free polar solvent not absorbing in the UV region (S1 ) containing an acid (a1 ); eluting with a mobile phase gradient; and determining the purity of the compound of formula (I) in the sample obtaining a chromatogram with an UV detector or with a Charged Aerosol Detector (CAD), preferably with an UV detector; wherein the HPLC column contains porous silica-based particles comprising aryl groups, preferably the HPLC column contains porous silica-based particles of 1 .5 to 10pm in diameter wherein phenyl groups are chemically bonded to said porous silica particles; and the mobile phase comprises a mobile phase A and a mobile phase B; and wherein the mobile phase A comprises water, and the mobile phase B comprises a polar solvent not absorbing in the UV region (S2).
[Claim 2] The method according to claim 1 , further comprising determining the presence and quantity of potential by-products selected from the group consisting of a compound of formula (II) and a compound of formula (III), or a mixture thereof:
[Claim 3] The method according to any of claims 1 or 2, wherein the mobile phase comprises a mobile phase A and a mobile phase B, wherein the mobile phase A is water comprising an acid (a2), and the mobile phase B is a polar solvent not absorbing in the UV region (S2) comprising the acid (a2).
[Claim 4] The method according to any of claims 1 or 2, wherein the mobile phase comprises a mobile phase A, a mobile phase B and a mobile phase C, wherein the mobile phase A is water, the mobile phase B comprises a polar solvent not absorbing in the UV region (S2); and the mobile phase C is water comprising the acid (a2).
[Claim 5] The method according to any of claims 1 to 4, wherein the amount of acid (a2) in the mobile phase is a quantity adapted to obtain a pH below 5, more preferably a pH between 2 and 4.
[Claim 6] The method according to any of claims 1 to 5, wherein the injecting step comprises injecting the contents of a syringe comprising, sequentially, a first layer of a volume (1 ) of water and a second layer of a volume (2) containing the sample dissolved in a water-free polar solvent not absorbing in the UV region (S1 ) containing an acid (a1 ).
[Claim 7] The method according to claim 6, wherein the syringe comprises, sequentially, a first layer of a volume (1 ) of water, a second layer of a volume (2) containing the sample dissolved in a water- free polar solvent not absorbing in the UV region (S1 ) containing an acid (a1 ), and a third layer of a volume (1 ) of water.
[Claim 8] The method according to any of claims 1 to 7, wherein the volume (1 ) is 10 times to 30 times the volume (2).
[Claim 9] The method according to any of claims 1 to 8, wherein the HPLC column particles are fully porous silica-based particles comprising biphenyl groups.
[Claim 10] The method according to any of claims 1 to 9, wherein the eluting comprises a gradient of from 98% of a mobile phase A:2% of a mobile phase B; to 2% of a mobile phase A:98% of a mobile phase B; and returning to 98% of a mobile phase A:2% of a mobile phase B; being each % referred to the volume of a mobile phase in relation to the total volume of mobile phase A and mobile phase B.
[Claim 11] The method according to any of claims 1 to 10, wherein the method further comprises conducting an isocratic elution phase after injecting and prior to eluting with a mobile phase gradient; preferably wherein the isocratic elution phase is carried out using 98% mobile phase A and 2% mobile phase B.
[Claim 12] The method according to any of claims 1 to 1 1 , wherein the acid (a1 ) is trifluoroacetic acid.
[Claim 13] The method according to any of claims 1 to 12, wherein the polar solvent (S1 ) and the polar solvent (S2) are both acetonitrile.
[Claim 14] The method according to any of claims 1 to 13, wherein the acid (a2) is methanesulfonic acid. [Claim 15] The method according to any of claims 1 to 14, wherein the mobile phase A further comprises EDTA.
PCT/EP2025/051804 2024-01-26 2025-01-24 Method for determining the purity of a compound by high-performance liquid chromatography Pending WO2025157997A1 (en)

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EP2740491A1 (en) * 2012-12-05 2014-06-11 Institut Curie Conjugates of the B-subunit of shiga toxin for use as contrasting agents for imaging and therapy
CN113582975A (en) * 2021-07-03 2021-11-02 上海蓝纳成生物技术有限公司 Truncated Evans blue modified fibroblast activation protein inhibitor and preparation method and application thereof

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Publication number Priority date Publication date Assignee Title
EP2740491A1 (en) * 2012-12-05 2014-06-11 Institut Curie Conjugates of the B-subunit of shiga toxin for use as contrasting agents for imaging and therapy
CN113582975A (en) * 2021-07-03 2021-11-02 上海蓝纳成生物技术有限公司 Truncated Evans blue modified fibroblast activation protein inhibitor and preparation method and application thereof

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LU SHARON X. ET AL: "Mass Spectral Analyses of Labile DOTA-NHS and Heterogeneity Determination of DOTA or DM1 Conjugated Anti-PSMA Antibody for Prostate Cancer Therapy", JOURNAL OF PHARMACEUTICAL SCIENCES, vol. 94, no. 4, 1 April 2005 (2005-04-01), US, pages 788 - 797, XP055976500, ISSN: 0022-3549, DOI: 10.1002/jps.20289 *
MOJARRAD PARIA ET AL: "Novel radiopharmaceutical (Technetium-99m)-(DOTA-NHS-ester)-Methionine as a SPECT-CT tumor imaging agent", vol. 141, 17 October 2019 (2019-10-17), NL, pages 105112, XP093173783, ISSN: 0928-0987, Retrieved from the Internet <URL:https://pdf.sciencedirectassets.com/271051/1-s2.0-S0928098719X00147/1-s2.0-S0928098719303859/main.pdf?X-Amz-Security-Token=IQoJb3JpZ2luX2VjENv//////////wEaCXVzLWVhc3QtMSJHMEUCIQC/S9iiCqMI1G9axXQsB5DCu1/uDBf2ZH71/tdCv8AopQIgAcHU/nHftcSyRRAv5eQ6iBD2o7QXZKLcF4IiGNg6RRUqswUIdBAFGgwwNTkwMDM1NDY4NjUiDM/UR> DOI: 10.1016/j.ejps.2019.105112 *
no. 170908-81-3

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