HPLC for analysis

Abstract

The present invention relates to a method for treating cancer, comprising administering to a patient a positron emission tomography (PET) tracer, 18 The present invention relates to an HPLC separation method useful for the synthesis of compounds labeled with [F]. 18 The present invention addresses the limitations of previous methods imposed by the need for [F] fluoride. 18 In the synthesis of compounds labeled with [F], 18 To provide a simplified method that allows for the rapid separation and analysis of [F] fluoride and chemical impurities.

Description

[Technical field]

[0001] The present invention relates generally to an improved method for analyzing the radiochemical purity and chemical impurities of radiopharmaceutical compositions using reversed-phase HPLC. [Background technology]

[0002] A radiopharmaceutical is a compound that is labeled with a radioactive element and is suitable for administration to a mammal in vivo for use in the fields of medical imaging, diagnosis or therapy. A radiopharmaceutical composition comprises a radiolabeled compound or a pharma- ceutical acceptable salt thereof, a solvent, and one or more stabilizers. A radiopharmaceutical composition, or "drug product (formulation)", must undergo a thorough quality control (QC) process before it can be administered to a patient. Quality control or quality assurance standards are published, for example, by the USP (usp.org). General Chapter <825> Radiopharmaceuticals-Preparation, Compounding, Dispensing, and Repackaging, originally published 1 June 2019 and General Chapter <1823> Positron Emission Tomography Drugs-Information. One of the important tests in the QC process is to quantify the chemical impurities and radiochemical purity (RCP) of the drug product (drug product). This analysis is routinely performed by high pressure liquid chromatography (HPLC).

[0003] Radiopharmaceuticals are manufactured within a short time before administration to patients. The reason is that the radioactivity of the radioactive elements in the radiopharmaceutical gradually decreases due to radioactive decay, thus causing a decrease in the radiopotency of the product. The radiopharmaceutical composition must undergo a QC process and pass all QC tests before it is released for administration to patients. Therefore, it is desirable that the time required to perform the QC process is as short as possible.

[0004] Fluorine-18 ([ 18 Fluorine-18 is a radioisotope commonly used in radiopharmaceuticals suitable for diagnostic use. The decay of fluorine-18 occurs by positron emission (97%) and electron capture (3%). 18 When [F] decays, the positrons released are utilized for positron emission tomography (PET) imaging. This in vivo imaging method is used, among others, for cardiac imaging, tumor imaging, and brain imaging.

[0005] [ 18 The half-life of [F] is 109.77 minutes. 18 The amount of radioactivity in a radiopharmaceutical sample containing [F] halves every 10 min. Therefore, the speed at which a radiopharmaceutical formulation sample can be analyzed and released for administration to a patient is crucial.

[0006] [ 18 F]HPLC analysis of radiopharmaceuticals requires the analysis of chemical and radioactive impurities in the presence of high concentrations of formulation matrix components. All radioactive impurities are [ 18 F] radionuclides. 18 F] HPLC analysis of radiopharmaceuticals is 18 Further complications may arise due to the nature of the radionuclide. 18 [F]nuclides are often incorporated into hydrophobic compounds. However, free [F]nuclides may be present and must be quantified in the formulation before it is released for administration to patients. 18 [F]fluoride is very hydrophilic. The analysis was performed by determining the free [F]fluoride on the stationary phase used for reversed-phase HPLC analysis of the more hydrophobic PET radiopharmaceuticals. 18 This is further complicated by the adsorption of [F] fluoride, and the free [ 18 It becomes difficult to quantify the free [F] fluoride from the reversed-phase HPLC column. 18Ensuring elution of [F]fluoride as a quantifiable peak has traditionally required a mobile phase with pH adjusted to near neutral pH. However, not all analysis of the more hydrophobic PET radiopharmaceuticals is best accomplished at neutral pH. Complex solvent systems are required to provide the mobile phase for the HPLC system. HPLC analysis of some radiopharmaceuticals is best accomplished with an acidic mobile phase. However, free [F]fluoride elution as a quantifiable peak has traditionally required a mobile phase with pH adjusted to near neutral pH. 18 [F]Fluoride cannot be quantified under acidic conditions.

[0007] Free [ 18 It would be highly advantageous to develop an HPLC separation method that does not have any of the limitations imposed by the need for [F] fluoride. The present invention makes it possible to achieve this, and provides a method for determining the amount of free [F] fluoride in radiopharmaceutical formulations. 18 To provide a simplified process that allows for rapid analysis of [F] fluoride and chemical impurities. Summary of the Invention

[0008] The present invention relates to 18 [F] in radiopharmaceuticals 18 The present invention relates to an improved analytical HPLC method for the quantification of [F]fluoride and the quantification of chemical impurities.

[0009] One aspect of the invention is a method for analyzing the radiochemical purity and chemical impurities in a radiopharmaceutical composition using reverse phase HPLC, the method comprising the steps of: 18 F] or a pharma- ceutically acceptable salt thereof, (a) applying a sample of the radiopharmaceutical composition to an HPLC column, wherein the mobile phase is water or water and an organic solvent, or a neutral pH buffer, or a neutral pH buffer and an organic solvent; followed by (b) supplying a different mobile phase to the HPLC column, the mobile phase comprising an acid denaturant, water and an organic solvent; The present invention relates to a method comprising the steps of:

[0010] This method allows the analysis of samples containing radiopharmaceutical compositions by reversed-phase HPLC, and the free [ 18 [F] fluoride is eluted during step (a) and the remaining components of the sample are separately 18 F] or a salt thereof and is eluted during step (b).

[0011] When a sample of the radiopharmaceutical composition (or "drug product (formulation)") is applied to the HPLC column in step (a), the mobile phase ("mobile phase A") contains only organic solvent and water, or organic solvent and a neutral pH buffer. The radiopharmaceutical composition itself initially provides a local pH controlled environment within mobile phase A. That is, when step (a) is performed with water and organic solvent, the sample plug provides the pH control for fluoride recovery. When step (a) is performed with a neutral buffer and organic solvent, the pH control is provided by the mobile phase. The mobile phase provided by the formulation matrix provides the free [ 18 Ensure a pH environment (pH 5-7) that is favorable for the elution of [F]fluoride as an easily quantifiable chromatographic peak, thus ensuring the absence of free [F]fluoride. 18 F] fluoride recovery becomes possible.

[0012] In step (b), the free 18 After the [F] fluoride has eluted or has begun to elute the column, a different mobile phase ("mobile phase B") is dispensed into the column, comprising water, an organic solvent and an acid denaturant. Due to the acid denaturant, mobile phase B has an acidic pH. The acidic pH of mobile phase B can provide the advantage of controlled elution of the formulation matrix components and provide a more suitable mobile phase pH for elution of the remaining components of the sample through the column. The method of the present invention 18 The present invention allows for rapid and accurate quantification of [F] fluoride and good resolution of chemical and radioactive impurities.

[0013] The present invention relates to a method for producing a free 18This gives the freedom to choose a mobile phase that gives the best possible analysis of the radiopharmaceutical composition without being constrained by the need for quantitation of [F] fluoride. Optimized separation of radioactive impurities and drug-related chemical impurities is often achieved with acidic mobile phases. [Brief description of the drawings]

[0014] [Figure 1] FIG. 1 shows a comparison of HPLC chromatograms of the method of the present invention and a current HPLC solvent system specifically designed to allow quantification of free [18F]Fluoride. [Diagram 2] FIG. 1 shows a comparison of the resolution of radioactive impurities in HPLC chromatograms of a sample of a [18F]Fluoride labeled radiopharmaceutical composition by the current method and the method of the present invention. [Diagram 3] FIG. 1 shows a comparison of the resolution of radioactive impurities in HPLC chromatograms by the current method and the method of the present invention, which are based on the gradient of the mobile phase system. [Figure 4] FIG. 1 shows a comparison of the resolution of chemical impurities in HPLC chromatograms by the current method and the method of the present invention based on mobile phase systems. [Diagram 5] FIG. 1 shows an HPLC trace according to the method of the present invention and the gradient used in the mobile phase to separate chemical and radioactive impurities. [Figure 6] FIG. 2 shows the solvent concentration gradient used in the mobile phase in the method of the present invention. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0015] Radiopharmaceuticals and radiopharmaceutical compositions are typically prepared in a radiopharmacy or PET center, where they also undergo quality control testing. Once a formulation has passed the quality control (QC) process and is released for administration to a patient, it can be administered to the patient on-site where PET scanning equipment exists, or it can be transported to a PET scanning facility.

[0016] One of the important tests in the QC process is to quantify the chemical impurities and radiochemical purity (RCP) of a drug product. This analysis is routinely performed by high pressure liquid chromatography (HPLC). HPLC achieves separation of various components of a liquid composition by differential interactions of those components with a stationary phase (usually a silica-based column) and a mobile phase or eluent passing through the column. The chemical properties of the components determine their affinity for the stationary phase based on intermolecular interactions, and the time they spend in the column before eluting. The mobile phase composition during the separation process may be constant (i.e., isocratic elution) or may vary (i.e., gradient elution). To separate compositions containing components with very different chemical properties, additional solvent may be introduced into the column, usually by increasing the percentage of the solvent in the mobile phase. A detection unit (e.g., a UV detector) and computer processing unit record the time at which the test substance elutes and generate a chromatogram showing signal peaks for the separated components. The signal peaks provide qualitative and quantitative data and allow the calculation of the concentrations of the components. The retention times (t R ) refers to the time from injection of a component into a system to its elution (detection) from the column. Reversed-phase HPLC describes a mode of chromatography that uses a polar mobile phase and a non-polar stationary phase. Compounds are selectively eluted from the stationary phase as the mobile phase becomes increasingly non-polar, usually by the addition of increasing levels of organic solvent, i.e., by increasing the proportion of organic solvent in the mobile phase. Polymer-based columns can be used for reversed-phase chromatography. However, the most common stationary phase is prepared by the covalent attachment of an organic material to silica particles. The organic material can be a phenyl-type compound, or more commonly an alkyl chain. The alkyl chain length determines the hydrophobicity of the stationary phase, and HPLC columns are available with alkyl chain lengths ranging from C2 to C30. A common stationary phase is C18 bonded to silica, and typical mobile phases include aqueous mixtures of water and an organic solvent, such as acetonitrile or methanol.

[0017] Radiochemical purity (RCP) can be determined using HPLC and can be defined as the ratio of the (radiolabeled) drug substance peak to the total (radiolabeled) peak in the chromatogram.

[0018] The term radiopharmaceutical has its customary meaning and refers to a radioactive compound suitable for in vivo mammalian administration for use in diagnosis or therapy. 18 Any radiopharmaceutical composition containing a radiopharmaceutical labeled with [F] and pharma- ceutical acceptable excipients, stabilizers, solvents, etc. can be analyzed by the HPLC method of the present invention.

[0019] The term "comprising" has its conventional meaning throughout this application, implying that the composition must have the recited components, but that, in addition, other unspecified compounds or species may be present.

[0020] Radiopharmaceutical Compositions The following description serves to illustrate the various possible components of a radiopharmaceutical composition. The radiopharmaceutical composition used in the method of the invention comprises a radiopharmaceutical, a solvent, a solubilizer and a radiostabilizer. The radiopharmaceutical composition used in the method of the invention can be analyzed by reversed-phase HPLC, 18 The radiolabeled compound may include a radiopharmaceutical labeled with [F]. 18 The present invention may include a radiopharmaceutical labeled with [F], or a pharma- ceutical acceptable salt thereof. 18 Examples of radiopharmaceuticals labeled with [F] include 18 F]FMAU(2'-deoxy-2'-[ 18 F]fluoro-5-methyl-1-beta-D-arabinofuranosyluracil), [ 18 F]FMISO (F18 Fluoromisonidazole), [ 18 F]FHBG (9-(4-18F-fluoro-3-[hydroxymethyl]butyl)guanine), 18 F]AV-45, [ 18 F]AV-19,18 F]AV-1, 18 F]Flutemetamol, 18 F]FES (fluoroestradiol F18), [ 18 F]Furupiridazu, [ 18 F]K5,[ 18 F]HX4,[ 18 F]W372,[ 18 F]VM4-037,[ 18 F]CP18, 18 F]ML-10, [ 18 F]T808,[ 18 F]T807, 2-[ 18 F]fluoromethyl-L-phenylalanine, or a combination thereof.

[0021] The radiolabeled compound can be a compound of formula (I):

[0022] [ka] In the formula, A is N(R 7 ), S, O, C(=O), C(=O)O, NHCH 2 CH 2 O, bond, or C(=O)N(R 7 ) are selected from; When present, B is hydrogen, alkoxyalkyl, alkyloxy, aryl, C optionally substituted with an imaging moiety. 1 -C 6 selected from alkyl, heteroaryl, and imaging moieties; When present, C is hydrogen, alkoxyalkyl, alkyloxy, aryl, C optionally substituted with an imaging moiety; 1 -C 6 selected from alkyl, heteroaryl, and imaging moieties; D is hydrogen, alkoxyalkyl, alkyloxy, aryl, optionally substituted with an imaging moiety, 1 -C 6 is selected from alkyl, heteroaryl, and imaging moieties; or C and D together with the atoms to which they are attached form a 3- or 4-membered carbocyclic ring; G is halo or haloalkyl; n is 0, 1, 2, or 3; R 1 , R 2 , R 3 , R 4 , R 5 , R 6 and R 7 are independently hydrogen, optionally substituted with an imaging moiety, C 1 -C 6 alkyl, and an imaging moiety; R 8 C is optionally substituted with an imaging moiety 1 -C 6 is alkyl; E is selected from a bond, carbon, and oxygen, with the proviso that when E is a bond, B and C are absent, D is selected from aryl and heteroaryl, and when E is oxygen, B and C are absent, and D is selected from hydrogen, alkoxyalkyl, aryl, C optionally substituted with an imaging moiety; 1 -C 6 alkyl, and heteroaryl; provided that at least one imaging moiety is present in formula (I).

[0023] The substituent A in formula (I) can be O. 8 can be tert-butyl. G can be chloro. The imaging moiety can be any radioisotope mentioned herein, for example 18 It's F.

[0024] The radiolabeled compound has the structure: 18 F] Flurpiridaz (also called flurpiridaz 18F):

[0025] [ka]

[0026] The radiolabeled compound is:

[0027] [ka]

[0028] [ka] The compound may be selected from:

[0029] The radiopharmaceutical composition may contain additional optional excipients. For example, such additional optional excipients include: antimicrobial preservatives, pH adjusters (buffers), bulking agents, solubilizers, or osmolality adjusters. Stabilizers are necessary in the radiopharmaceutical formulation to reduce the formation of radioactive impurities during its shelf life. Suitable stabilizers include ethanol, sodium ascorbate, ascorbic acid, maleic acid, para-aminobenzoic acid, polysorbates, polyethylene glycol, gentisic acid, and calcium chloride, among others.

[0030] The radiopharmaceutical composition may comprise a biocompatible carrier. A biocompatible carrier is a fluid, especially a liquid, in which the radiopharmaceutical can be suspended or preferably dissolved so that the composition is physiologically acceptable, i.e. can be administered to the mammalian body without toxicity or undue discomfort. The biocompatible carrier is suitably an injectable carrier liquid, such as sterile pyrogen-free water for injection; an aqueous solution, such as saline (which can advantageously be balanced so that the final product for injection is isotonic); an aqueous buffer containing a biocompatible buffering agent (e.g. phosphate buffer); an aqueous solution of one or more isotonic substances (e.g. salts of plasma cations and biocompatible counterions), sugars (e.g. glucose or sucrose), sugar alcohols (e.g. sorbitol or mannitol), glycols (e.g. glycerol) or other non-ionic polyol substances (e.g. polyethylene glycol, propylene glycol, etc.). Preferably, the biocompatible carrier is pyrogen-free water, isotonic saline or phosphate buffer for injection.

[0031] A radiopharmaceutical composition may include, for example, a radiolabeled compound, ascorbic acid, ethanol, and a cyclodextrin. An exemplary radiopharmaceutical composition includes ascorbic acid and ethanol, a cyclodextrin, such as hydroxypropyl-beta-cyclodextrin (HPbCD), and the radiolabeled compound [ 18 F] flurpiridaz. Ascorbic acid can be present in an amount of about 1 to about 100 mg / mL, ethanol can be present in an amount of about 2 to about 10% (v / v), and HPbCD can be present in an amount of about 1 to about 1000 mg / mL, such as about 1 to about 350 mg / mL, such as about 1 to about 100 mg / mL, such as about 30 mg / mL to about 70 mg / mL, such as about 50 mg / mL. Ethanol is typically less than 10% by volume of the composition.

[0032] HPLC method [ 18 HPLC analysis of [F]fluoride is generally difficult because many factors must be considered.18 [F] fluoride is absorbed into the stationary phase of the column and is difficult to elute: free [ 18 [F]fluoride does not elute as a separate chromatographic peak that can be quantified.

[0033] The present invention provides a method for analyzing the radiochemical purity and chemical impurities in a sample containing a radiopharmaceutical composition using reversed-phase HPLC, the method comprising the steps of: 18 F] or a pharma- ceutical acceptable salt thereof, the method comprising: (a) applying a sample comprising the radiopharmaceutical composition to an HPLC column, wherein the mobile phase is water, or water and an organic solvent, or a neutral pH buffer, or a neutral pH buffer and an organic solvent; and subsequently, (b) applying a different mobile phase comprising an acid denaturant, water and an organic solvent to the HPLC column.

[0034] Typically, a radiopharmaceutical composition contains water, ethanol, a buffer or stabilizer such as ascorbic acid, and a cyclodextrin. When a sample of the radiopharmaceutical composition (formulation) is applied to the HPLC column, the mobile phase ("Mobile Phase A") contains only organic solvent and water. Alternatively, Mobile Phase A may contain a neutral pH buffer, or a neutral pH buffer and an organic solvent.

[0035] When "mobile phase A" contains only organic solvents and water, this mobile phase does not provide pH control. When a sample of a radiopharmaceutical composition is injected into the HPLC column, the matrix of the sample establishes pH control. That is, the matrix of the sample dissolves fluoride ions (free [ 18 The injected sample provides a pH environment of 5-7 for its matrix components, ensuring that free [F] fluoride is eluted from the column. 18 F] fluoride is eluted from the column. This is the free [ 18F] creates a favorable environment for the elution of fluoride, resulting in an easily quantifiable chromatographic peak.

[0036] The "matrix" of a formulation is the molecular weight of the compound present in the formulation (e.g., a radiopharmaceutical composition). 18 The matrix is ​​defined as any component other than the chemical compound radiolabeled with [F] fluoride. The matrix may include, for example, ethanol, buffer salts (formate, phosphate, citrate, acetate, and / or one or more amino acids), radioactive stabilizers, solubilizers, as described above with respect to the radiopharmaceutical compositions.

[0037] Free [ 18 After [F] fluoride has eluted from or begins to elute the column, a different mobile phase ("mobile phase B") is dispensed into the column. Mobile phase B comprises an acid modifier, water and an organic solvent. Any acid modifier can be used in mobile phase B, making the pH of mobile phase B acidic. An acidic pH is defined as less than 7.0. Preferably, the pH of mobile phase B is less than 5.0, more preferably less than 4.5, between 0 and 5.0, between 0 and 4.0, between 1.0 and 3.0, between 1.5 and 2.5, for example about 2.0. Examples of suitable acid modifiers in the mobile phase include, among others, trifluoroacetic acid (TFA), formic acid, acetic acid, phosphoric acid, citric acid and acidic phosphate buffers. The proportion of acid modifier in mobile phase B may be constant or may be gradually increased. If a mixture of different components is used in mobile phase B, their respective proportions may increase or decrease with a gradient.

[0038] A pH modifier is any substance added to the mobile phase that aids in controlling or regulating the pH. An acid modifier is any substance added to the mobile phase that establishes an acidic pH, usually defined as a pH less than 7.0. For the purposes of this invention, an acid modifier is defined as any substance added to the mobile phase that establishes an acidic pH of 5.0 or less. A basic modifier is any substance added to the mobile phase that establishes a basic pH, usually defined as a pH greater than 7.0 in polymer-based columns.

[0039] In general, trifluoroacetic acid (TFA) is a very useful pH modifier (acid modifier) ​​for the mobile phase, or eluent, in HPLC, because it aids in better separation of sample components, including chemical and radiochemical impurities. 18 In quality control testing of PET products radiolabeled with [F] 18 HPLC methods for the analysis of [F]radiopharmaceuticals do not typically use TFA as a mobile phase acid modifier because TFA is a free [ 18 The inventors have determined that the radiopharmaceutical composition sample plug itself provides an initial, temporary pH adjustment of mobile phase A (containing no pH modifiers) to allow for the quantification of free [F] fluoride present in the sample. 18 It has been found that a simple analysis of [F] fluoride can be achieved.

[0040] The mobile phase can then be adjusted to optimize the separation of the remaining components of the sample. 18 Once [F] fluoride) begins to elute from the column, step (b) partitions only the acid modifier into the mobile phase (as a component of "mobile phase B"). The acid modifier partitions into the mobile phase with an increasing gradient and can provide precise pH control, which helps control the elution of the remaining components from the column.

[0041] Hydrophilic free [ 18Unlike [F] fluoride, the remaining components are more hydrophobic and the mobile phase needs to become increasingly non-polar in order for these components to elute through the column. This can be achieved, for example, by increasing the ratio of organic solvent to water in the mobile phase to elute the more hydrophobic components. The mobile phase is fed to the column from two or more solvent tanks. The composition of the mobile phase can be changed by varying the ratio of contributions from the tanks, for example from the first and second tanks, to the column. The HPLC apparatus may include at least two tanks, for example two, three or four tanks. The tanks may, for example, contain different components or different concentrations of certain components. The composition of each tank remains constant, but the composition of the mobile phase can be changed by changing the ratio of contributions from each tank to the mobile phase at a given time.

[0042] Mobile phase A is defined herein as the mobile phase that runs through the column without any acid modifier. Mobile phase B is defined herein as the mobile phase that runs through the column with an amount of acid modifier. The composition of mobile phase B can be varied by changing the ratio of components, for example by varying the contribution from a tank with a gradient.

[0043] Mobile phase A is water and an amount of organic solvent. For example, mobile phase A may contain 0-60% organic solvent in water, such as 10-60% organic solvent, such as 30% organic solvent to 50% organic solvent. Mobile phase A may contain one or more organic solvents.

[0044] In an alternative embodiment of the invention, one or more neutral pH buffers can be used as mobile phase A. A suitable neutral pH buffer is any buffer that maintains a pH in the range of approximately 4.0 to 8.0. Suitable buffers include, but are not limited to, phosphate, ammonium acetate, ammonium formate, sodium acetate, tartrate, citric acid-phosphate, citrate, pyridine-formate, pyridine-acetate, dimethyl glutarate, succinate, phthalate, picoline acetate, cacodylate buffer, tris-maleate, sodium hydrogen maleate buffer, sodium arsenate, and imidazole. Preferred neutral pH buffers include phosphate, ammonium acetate, and ammonium formate. The preferred concentration range of the neutral pH buffer is 0.1 to 100 mM.

[0045] In mobile phase B, an acid denaturant is introduced into the column along with an organic solvent and water. The percentage of organic solvent in mobile phase B can be 1% to 100% organic solvent, for example, 30% to 100% organic solvent. If the percentage of organic solvent is less than 100%, the remainder is water. The amount of organic solvent in mobile phase B can be increased in a gradient to aid elution from the column, for example, 1% to 95%, 1% to 50%, 30% to 95%, 30% to 80%, 50% to 80% or 50% to 70% organic solvent in water. Mobile phase B can contain one or more organic solvents.

[0046] The one or more organic solvents may be the same or different in mobile phase A and mobile phase B. Preferably, the one or more organic solvents are the same in mobile phase A and mobile phase B. The percentage of acid modifier may be between 0.05% and 1%, for example between 0.1% and 0.25%. The percentage of acid modifier may remain constant, for example 0.1%, or may increase proportionately with increasing percentage of the solvent it is in. This may depend on the HPLC equipment used.

[0047] The organic solvent of mobile phase A and / or mobile phase B may be selected, for example, from acetonitrile or methanol. Mobile phase A may, for example, initially contain 30% MeCN and 70% water. The percentage of MeCN in the mobile phase passing through the column may be increased in a gradient by adjusting the contribution from the solvent tank. Mobile phase B may contain MeCN, water and an acid modifier. The steepness of the gradient (increase in the percentage of organic solvent over time) depends on the properties of the sample components. The final percentage of mobile phase passing through the column may, for example, be 70% MeCN, up to 29.9% water and at least 0.1% acid modifier. In another example, mobile phases A and B may contain MeOH and water. For example, mobile phase A may contain 50% MeOH and 50% water. The percentage of MeOH in mobile phase B may be increased in a gradient by adjusting the contribution from the solvent tank. Mobile phase B further contains an acid modifier. The acid modifier may, for example, be trifluoroacetic acid, formic acid, acetic acid, phosphoric acid, citric acid and acidic phosphate buffer or phosphate buffer.

[0048] The control of pH is considered crucial in chromatography. The reason is that ionizable functional groups behave differently depending on their ionized state in reversed-phase chromatography. The charge of ionizable groups changes depending on the pH. Therefore, the control of pH is often crucial when performing analyses such as HPLC. The control of pH can be achieved by pH modifiers, e.g., acid modifiers to achieve an acidic pH (and base modifiers to achieve a basic pH in polymer-based columns). Buffers can also be used to achieve robust pH control and to compensate for fluctuations.

[0049] In contrast to standard procedures in the art, for the analysis of analytes that require pH adjustment (e.g., fluoride), no pH modifiers are included in mobile phase A. Rather, the pH adjustment near fluoride (approximately pH 5 to pH 7) comes from the formulation matrix itself. This allows for the free 18This allows for the elution and accurate quantification of [F] fluoride. When mobile phase A is a neutral buffer, pH control is provided by the neutral buffer. The introduction of an acid denaturant in mobile phase B then provides an acidic environment, which elutes the free [ 18 The method of the present invention allows for good separation and quantification of the remaining components, including chemical impurities, of the sample without interfering with the elution and quantification of [F] fluoride. 18 This allows for easy quantification of [F] fluoride as well as good resolution of chemical impurities in the sample. As described in more detail below, the addition of an acid denaturant is very important for the adjustment of large peaks in the chromatograms due to the matrix material of the formulation.

[0050] The time between the introduction of the formulation sample to the column and the introduction of mobile phase B to the column can be as short as 1 second. Preferably, the time between the introduction of the formulation sample to the column and the introduction of mobile phase B to the column is from 1 second to 5 minutes, preferably from 5 seconds to 60 seconds, more preferably from 5 seconds to 30 seconds, for example about 20 seconds. In another embodiment, the time between the introduction of the formulation sample to the column and the introduction of mobile phase B to the column is zero seconds.

[0051] Another advantage of the method of the present invention is that HPLC analysis of a formulation sample can be performed within approximately 10 minutes. This is in contrast to current methods which typically take approximately 20 minutes. As previously explained, radiopharmaceutical compositions are prepared and subjected to quality control testing within a short time frame prior to administration to a patient, in view of the radioactive decay of the radionuclide. Reducing the time required to perform HPLC analysis in half would be highly advantageous. The method of the present invention allows for the subsequent determination of non-free [ 18 [F] fluoride-component separation can be carried out in the most optimal way possible and thus make it possible to significantly reduce the analysis time, which is further explained with reference to the figures.

[0052] The HPLC method of the present invention is 18The method is applicable to any reversed-phase HPLC analysis for radiopharmaceutical preparations radiolabeled with [F] fluoride. Any silica-based reversed-phase HPLC column (C2-C30) of certain column dimensions, particle size and pore size is suitable for use in the method of the present invention. Exemplary columns that can be used in the present invention include Zorbax SB-C18 P / N 822975-902 (Agilent, USA), and Kinetex C18, P / N 00B-4462-E0 (Phenomenex Inc.).

[0053] Figure 1 shows the HPLC chromatogram. The traces are numbered from top to bottom as trace 1, trace 2, trace 3 and trace 4.

[0054] Trace 1 shows the free [ 18 This figure shows the chromatogram generated by HPLC of a sample of [F]fluoride. The mobile phase initially contains no acid. After sample injection, TFA is added to the mobile phase, which results in a drop in pH to approximately 2.0. However, as can be seen in trace 1, when the sample matrix is ​​water only, there is a large amount of free [ 18 [F]Fluoride elutes from the column as a broad peak at approximately 2.3 min to 3.8 min. The broad peak is not quantifiable.

[0055] Trace 2 shows the free [ 18 The chromatogram generated by HPLC of a sample of [F] fluoride is shown. The mobile phase was the buffer salt ammonium acetate (NH 4 Acetate salts) are included. 18 [F] fluoride elutes easily and gives a quantifiable peak in the chromatogram. However, this mobile phase is more difficult to handle in radiopharmacy or PET centers. The use of a buffered mobile phase imposes an additional burden on radiopharmacy performing the analysis prior to release of the formulation for administration to patients. Preparation and pH modification of the mobile phase with buffer salts is more labor intensive, and the equipment must be rigorously cleaned after using a buffered mobile phase to avoid precipitation of buffer salts in the equipment.

[0056] Trace 3 shows the current standard method using a C18 column [ 18 The chromatograms shown are generated by HPLC on a sample of a radiopharmaceutical formulation labeled with [F]fluoride. 18 F] flurpiridaz (GE Healthcare, USA), ascorbic acid, ethanol and cyclodextrin (HP-b-cyclodextrin, Cyclodextrin HPB EMPROVE, Merck P / N 1.42020). The mobile phase was buffered at pH 6.0 with the salt ammonium acetate (NH 4 Acetate salts) are included. 18 [F]Fluoride elutes around 0.5 min. The radiopharmaceutical elutes as a large sharp peak around 13 min, and the radioactive impurities elute between 12.5 and 17.5 min (smaller peaks). Although the chromatogram shows sharp peaks, the method requires a 20 min runtime because the mobile phase contains ammonium acetate. The reason the method takes 10 min longer is because the method is a multidimensional analysis. The radioactive impurities (all [ 18 Some of the components are radiolabeled (labeled with [F] fluoride) and some are not radiolabeled. For example, the product matrix, which contains large amounts of ascorbic acid and cyclodextrin, gives a large response in HPLC analysis (detected using UV light absorption). An acidic mobile phase allows these matrix components to elute quickly from the column. However, at the neutral pH provided by ammonium acetate, the matrix components take about 10 minutes to elute from the column and form large peaks in the chromatogram.

[0057] Trace 4 shows the results of the present invention. 18 HPLC chromatograms of a sample of the formulation labeled with [F] fluoride are shown. 18 F] flurpiridaz (GE Healthcare, USA), ascorbic acid, ethanol and cyclodextrin. The sample was the same as that analyzed in trace 4. 18The formulation is labeled with [F] fluoride. The formulation sample is first injected onto the C18 column, which itself provides the temporary mobile phase A. The pH of the sample plug is 5.0-7.0, determined by the pH of the formulation. This is due to the free [ 18 This allows [F]fluoride to elute rapidly as a sharp, quantifiable peak around 1.0-1.4 min. Following injection of the formulation sample, TFA is distributed to the column as a component of mobile phase B, thereby lowering the pH to approximately pH 2.0. The radiopharmaceutical elutes as a strong, sharp peak around 5-6.3 min, and the radioactive impurities elute (smaller peaks) at 5.0-7.5 min. The total run time is approximately 10 min.

[0058] Figure 2 compares traces 3 and 4 in Figure 1. HPLC analysis showed 18 A sample of the same radiopharmaceutical formulation labeled with [F]fluoride is performed. In trace 4, the formulation sample is injected onto the column, which itself provides a temporary pH adjustment to mobile phase A. Free [F]fluoride is then added to the column. 18 [F]fluoride elutes quickly as a sharp, quantifiable peak (peak 1) around 1.0-1.4 min. Following injection of the formulation sample, TFA is distributed to the column as a component of mobile phase B, thereby lowering the pH level to 2.0. The radiopharmaceutical elutes as a sharp peak around 4.5-5 min (peak 3), and additional radioactive impurities elute between 4.0-6.5 min (peaks 2 and 4). The overall run time is approximately 10 min. In contrast, in trace 3, the same formulation sample is injected onto the column and ammonium acetate provides a pH of 6.0 to the mobile phase. Free [ 18 [F]Fluoride elutes quickly as a sharp quantifiable peak (peak 1), while the radiopharmaceutical (peak 3) and chemical impurities (peaks 2 and 4) have a long retention time and come off the column at 12.5-14.5 min. A closer look at this figure shows that with TFA additional radioactive impurities are found in the tail of the main peak. In other words, being able to use TFA results in better separation of the radioactive impurities and more accurate analysis of the radiopharmaceutical. Having the flexibility to choose the optimal mobile phase is a great benefit.

[0059] Figure 3 compares the degradation of radioactive impurities in trace 3 and trace 4 of Figure 1. Overlaid on the chromatograms are the mobile phase gradient profiles depicted as dashed lines. Tank A contains ammonium acetate and water and provides the initial mobile phase. Tank B contains 100% MeCN. The contributions from the tanks are adjusted to achieve the gradient of the mobile phase in the column. For the analysis in ammonium acetate, the dashed lines represent the change in the organic solvent content of the mobile phase during the analysis, the amount increased linearly over a period of 6.0 to 14.5 min. The level of organic solvent is then kept constant for a further 1.5 min to ensure that all compounds are eluted from the column. At 17.5 min the organic solvent is returned to a level suitable for starting the next analysis.

[0060] Figure 4 compares the resolution of chemical impurities in traces 3 and 4 of Figure 1. The mobile phase gradient profile is drawn as a dotted line overlaid on the chromatogram. As can be seen in trace 4 (mobile phase B contains TFA), the matrix components give rise to large peaks in the chromatogram that elute within 2 minutes. In trace 3 (ammonium acetate), the matrix components (here: ascorbic acid and cyclodextrin) give rise to large broad peaks that elute in approximately 10 minutes. This is due to the neutral pH of the mobile phase buffered with ammonium acetate. Other compounds cannot be analyzed until the matrix components have completely eluted from the column. Thus, the chemical impurity peaks do not elute until approximately 12-16 minutes. It can also be seen that the peak (numbered 1) is resolved in the trace with TFA in mobile phase B (trace 4) but is not visible in trace 3. Furthermore, peaks 2 and 3 are resolved as two separate peaks in the trace with TFA in mobile phase B. In trace 3, peaks 2 and 3 are not resolved. This comparison clearly shows the role that acid modifiers, such as TFA, in the mobile phase play both in modulating the elution of matrix material (resulting in a sharper peak that elutes within 2 minutes compared to a broad peak that takes 10 minutes to elute and affects the subsequent elution profile) and in improving the resolution of other components in the sample.

[0061] Figure 5 shows the multidimensional nature of the analysis. 18 FIG. 1 shows the decomposition of chemical and radiochemical impurities in a sample of a radiopharmaceutical composition (referring to trace 4 of FIG. 1) labeled with [F]. 18 F] fluoride is eluted and the free [ 18 The gradient profile of mobile phase B is depicted (dashed line) overlaid on the chromatogram. The gradient was achieved with a dual-pump HPLC system - i.e., the HPLC system has two solvent tanks, here Tank A and Tank B. The mobile phase gradient profile is shown in Table 1 below. Tank A is 30% acetonitrile in water. Tank B is 95% v / v acetonitrile, water (5% v / v) and 0.1% v / v TFA.

[0062] [Table 1]

[0063] The gradient means that the concentration of TFA ranges from 0.00 to 0.075% v / v during the analysis (the gradient profile is shown by the dotted line in Figure 5 ), but there is enough acid present to impose a pH adjustment (as indicated by the focusing of peaks due to matrix components) even as the concentration increases from 0.00 to 0.025% in the first part of the gradient.

[0064] The above solvent gradients are performed in a binary pump HPLC system. However, the invention can also be performed in a ternary (three tank) or quaternary (four tank) pump system utilizing three or four separate components of the mobile phase from tanks A, B, C, and D.

[0065] Table 2, for example, shows a mobile phase gradient protocol performed in a quaternary pump system using three eluent channels (i.e., tanks A, B, and C). Tank A is 100% water, tank B is 100% acetonitrile, and tank C is 0.15% v / v TFA in water (the amount of TFA can be varied if necessary).

[0066] [Table 2]

[0067] When the method of the invention is carried out on an HPLC system with three available eluent channels, the preparation of the mobile phase is greatly simplified. In the example described, the concentration of TFA is kept constant throughout the region of the chromatogram where the compounds related to the radiopharmaceutical elute. The concentration of TFA on the column can be increased, if necessary, by adding more TFA when preparing mobile phase C.

[0068] In FIG. 6, the gradient profiles of TFA and MeCN are superimposed on the chromatogram as dashed lines predicted from the gradients shown in Table 2.

[0069] The method of the present invention uses a single solvent system to 18 The inventors have demonstrated that the free [F] fluoride can be analyzed by HPLC by providing a temporary pH adjustment in the vicinity of the fluoride in mobile phase A using the formulation sample itself. 18 We have found that it is possible to achieve quantification of [F] fluoride and achieve well resolved peaks of radiopharmaceuticals and chemical impurities by skill in using an acidic denaturant as a component of mobile phase B. The run time of HPLC analysis by the method of the present invention is less than half compared to current methods, which is a significant advantage.

[0070] The invention will now be described with reference to the following non-limiting examples. EXAMPLES

[0071] Using the method of the present invention, 18 Quantification of [F]fluoride and [ 18 [F] Fluoride recovery accuracy

[0072] The data in Table 4 show that the present method (ammonium acetate) and the method of the present invention using the composition itself to provide an initial temporary pH adjustment in mobile phase A followed by TFA as an acid denaturant in mobile phase B (TFA) were used. 18 In the same radiopharmaceutical composition containing [F]flurpiridaz, 18 HPLC analysis of [F] fluoride. Known amounts of free [F] fluoride were used to demonstrate the accuracy of the determination. 18 [F] fluoride was added to the sample.

[0073] From the activity measurement, the free 18 [F] Fluoride level (background corrected): 0.7%

[0074] [Table 3]

[0075] The radiochromatograms are analyzed according to the QC procedure applying a reporting threshold of 0.3% (ie impurities present at levels below 0.3% are not reported). 1) Analysis performed on a Zorbax C18 (50 x 4.6 mm) 1.8 μm column. The mobile phase was near neutral pH, ammonium acetate (pH 6). 2) Analysis performed on a Kinetex C18 (50 x 4.6 mm) 2.6 μm column. The mobile phase was initially aqueous MeCN (no pH modifiers) progressing to 0.1% TFA in the gradient. Both methods are 18 F] giving a relative area of ​​0.7% relative to the fluoride peak (peak 1).

[0076] The following examples and Table 5 relate to methods of incorporating neutral buffers based on a quaternary pump HPLC system. Table 5, for example, describes a mobile phase gradient protocol performed in a quaternary pump system using four eluent channels (i.e., tanks A, B, C and D). Tank A is 30% acetonitrile in water, tank B is 0.10% v / v TFA in 95% acetonitrile (the amount of TFA can be varied if necessary), tank C is 10 mM ammonium acetate in water (the amount of ammonium acetate can be varied if necessary or an alternative neutral buffer can be chosen), and tank D is 100% acetonitrile. Tank A: 30% MeCN Tank B: 0.10% TFA in 95% MeCN Tank C: 10 mM ammonium acetate (NH) in water 4 Ac) Tank D: 100% MeCN

[0077] [Table 4]

[0078] As will be readily understood by those skilled in the art, the embodiments of the present invention described herein are widely useful and applicable. Thus, although the present invention has been described in detail herein in connection with exemplary embodiments, it should be understood that the disclosure is exemplary of the embodiments and is intended to provide an enabling disclosure of the exemplary embodiments. The disclosure is not intended to be construed as limiting the embodiments of the present invention or excluding all other such embodiments, adaptations, variations, modifications, and equivalent arrangements. The scope of the present invention is defined by the appended claims.

Claims

1. A method for analyzing the radiochemical purity and chemical impurities of a radiopharmaceutical composition using reverse phase HPLC, comprising: １８ F] or a pharma- ceutically acceptable salt thereof, wherein the method comprises: (a) applying a sample of the radiopharmaceutical composition to an HPLC column, wherein the mobile phase is water, or water and an organic solvent, or a neutral pH buffer, or a neutral pH buffer and an organic solvent; followed by (b) supplying a different mobile phase to the HPLC column, the mobile phase comprising an acid denaturant, water and an organic solvent; A method comprising:

2. The method of claim 1 , wherein the mobile phase in step (a) is water and an organic solvent.

3. 2. The method of claim 1, wherein the organic solvent is selected from acetonitrile or methanol.

4. 10. The method of claim 1 , wherein the mobile phase in step (a) and / or step (b) comprises one or more organic solvents.

5. 2. The method of claim 1, wherein in step (a) and step (b), the one or more organic solvents are the same.

6. 2. The method according to claim 1, wherein the proportion of organic solvent in step (a) is between 10% and 60%.

7. 2. The method according to claim 1, wherein the proportion of organic solvent in step (b) is from 1% to 100%.

8. 2. The method of claim 1, wherein the proportion of organic solvent is increased gradiently in step (b).

9. 9. The method of claim 8, wherein the increase in the proportion of organic solvent in step (b) is carried out in a stepwise or continuous increment.

10. 2. The method of claim 1, wherein the proportion of acid modifier in step (b) is at least 0.1%.

11. 2. The method of claim 1, wherein the acid denaturant in step (b) is selected from trifluoroacetic acid (TFA), formic acid, acetic acid, phosphoric acid, citric acid, and one or more acidic phosphate buffers, or a combination thereof.

12. The method of claim 1, wherein the acid denaturant in step (b) is trifluoroacetic acid (TFA).

13. The method of claim 3, wherein the acid denaturant in step (b) is trifluoroacetic acid (TFA).

14. 10. The method of claim 1, wherein the radiopharmaceutical composition further comprises ethanol and a cyclodextrin.

15. 10. The method of claim 1, wherein the radiopharmaceutical composition further comprises ascorbic acid.

16. 2. The method of claim 1, wherein the time between steps (a) and (b) is at least 1 second.

17. 2. The method of claim 1, wherein the time between steps (a) and (b) is from 1 second to 5 minutes.

18. 2. The method of claim 1, wherein the time between steps (a) and (b) is between 5 seconds and 60 seconds.

19. 2. The method of claim 1, wherein the time between steps (a) and (b) is 0 seconds.

20. The method according to claim 1, wherein the concentration of the buffer in mobile phase A is 0.1 to 100 mM.

21. 10. The method of claim 1, wherein the radiopharmaceutical composition is used for positron emission tomography (PET).

22. Radioactive drugs are １８ F]FMAU (2'-deoxy-2'-[ １８ F]fluoro-5-methyl-1-beta-D-arabinofuranosyluracil), [ １８ F]FMISO ([ １８ F]fluoromisonidazole), [ １８ F]FHBG(9-(4-[ １８ F]-fluoro-3-[hydroxymethyl]butyl)guanine), [ １８ F] AV-45, [ １８ F] AV-19, [ １８ F]AV-1, [ １８ F]Flutemetamol, １８ F]FES (fluoroestradiol F18), [ １８ F] Fururupiridazu, [ １８ F]K5, [ １８ F]HX4, [ １８ F]W372, [ １８ F] VM4-037, [ １８ F]CP18,[ １８ F]ML-10, １８ F] T808, [ １８ F] T807, 2-[ １８ F] fluoromethyl-L-phenylalanine, 【Chemistry 1-1】 【Chemistry 1-2】 or a combination thereof.

23. The radiopharmaceutical is １８ F] Fururupiridazu 【Chemistry 2】 The method of claim 1, wherein

24. The radiopharmaceutical is [18F]flurpiridazol. 【Chemistry 2】 The method of claim 12, wherein 25. The radiopharmaceutical composition further comprising ethanol and a cyclodextrin; and / or the radiopharmaceutical composition further comprising ascorbic acid; 24. The method of claim 23.

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