JP2016538574A - HPLC analysis of impurities in dianhydrogalactitol - Google Patents

Abstract

An improved analytical method for the analysis of dianhydrogalactitol formulations is to determine the purity of dianhydrogalactitol, to detect impurities in dianhydrogalactitol formulations, as well as to identify any such impurities Provide a method. The method of the invention uses high performance liquid chromatography (HPLC), in particular HPLC with an evaporative light scattering detector (ELSD); the HPLC can be followed by tandem mass spectrometry. The method of the present invention may further comprise performing preparative HPLC recovery of at least one specific substance peak present in the dianhydrogalactitol formulation. [Selection] Figure 1

Description

CROSS-REFERENCE This application is due to Xiaoyun and Mike Li, entitled "Improved Analytical Methods for Analyzing and Determining Impurities in Dianhydrogalactitol ( improved analytical method for analyzing and determining impurities in dianhydro galactitol)", November 2013 Claims the benefit of U.S. patent application Ser. No. 14 / 083,135 filed on the 18th, which claims the benefit and, according to Xiaoun Lu, “Improved Analytical Methods for Analyzing and Determining Impr. In lactitol Improved Analytical Method for Analyzing and Determining Pure Products), which was filed on July 2, 2013, and is a continuation-in-part US patent application Ser. Xiaoun Lu, "Improved Analytical Methods for Analyzing and Determining Impurities in Dianhydroactitol, which was analyzed in the United States on the 26th year of the analysis and determination of impurities in the United States. PCT Application No. PCT / IB2013 / 000793, filed with the designation, in turn by Xiaoun Lu, “Improved Analytical Methods f”. US Provisional Application No. 61 / 603,464, filed Feb. 27, 2012, entitled “R Analyzing and Determining Impulses in Dianhydrogalactol”. Claimed the benefit of the issue. The entire contents of these applications are hereby incorporated by reference in their entirety.

  The present invention is directed to improved analytical methods for dianhydrogalactitol, particularly including high performance liquid chromatography (HPLC).

  Dianhydrogalactitol (1, 2: 5,6 dianhydrogalactitol or DAG) is one of many hexitols or hexitol derivatives that have significant pharmacological activity including chemotherapeutic activity. In particular, dianhydrogalactitol has been proposed for use in chemotherapy, such as US Pat. No. 7,157,059 to Nielsen, which is incorporated herein by this reference.

  Diahirodogalactitol has activity against many neoplasms. However, when dianhydrogalactitol is successfully used as a therapeutic agent, extremely high purity and removal of impurities is essential. The presence of impurities can lead to undesirable side effects. If an example occurred several years ago and the impurities are present in a batch of amino acid tryptophan, the normal constituents of the protein cause a significant pandemic of eosinophilia pain syndrome and are irreversible Caused so many cases of general disability and at least 37 deaths. This is particularly important when a therapeutic agent such as dianhydrogalactitol should be used in patients with immune deficiencies, liver or kidney dysfunction, or elderly patients. Such patients may experience a high incidence of undesirable side effects due to susceptibility to contaminants.

  One of the impurities found in dianhydrogalactitol formulations is dulcitol. Other impurities are present in dianhydrogalactitol formulations as well, depending on their method of preparation.

  Thus, when dianhydrogalactitol is administered for therapeutic purposes, it is intended to detect impurities and degradation products in the dianhydrogalactitol formulation to provide a high purity formulation that is less likely to induce side effects. There is a need for improved analytical methods.

  Described herein are methods for determining the purity of dianhydrogalactitol and detecting impurities and degradation products in formulations of dianhydrogalactitol that meet their needs.

  In general, this analytical method uses high performance liquid chromatography (HPLC), in particular HPLC with refractive index (RI) detection.

In one alternative, an analytical method for analyzing the presence and amount of impurities present in a dianhydrogalactitol formulation comprises the following steps:
(1) Analyzing said formulation by subjecting the dianhydrogalactitol formulation to high performance liquid chromatography using elution with a gradient mobile phase to separate dianhydrogalactitol from dulcitol and other contaminants of the formulation. And (2) determining the relative concentration of one or more peaks determined by high performance liquid chromatography representing compounds other than dianhydrogalactitol itself.

  The compound other than dianhydrogalactitol itself may be at least one of (1) dulcitol; (2) impurities other than dulcitol; and (3) degradation products of dianhydrogalactitol.

  In one alternative of this method, the elution is a gradient of about 2.5 mM to about 0.1 mM NaOH. Preferably, the elution is a gradient of about 1.5 mM to about 0.1 mM NaOH. More preferably, the elution is a gradient of about 1 mM to about 0.1 mM NaOH.

  In another alternative of this method, the elution is a gradient of a combination of ammonium hydroxide and a volatile ammonium salt selected from the group consisting of ammonium formate and ammonium acetate, and the total concentration of ammonium formate and ammonium acetate is about 2.5 mM to about 0.1 mM. Preferably, the total concentration of volatile ammonium salts selected from the group consisting of ammonium hydroxide and ammonium formate and ammonium acetate is from about 1.5 mM to about 0.1 mM. More preferably, the total concentration of volatile ammonium salts selected from the group consisting of ammonium hydroxide and ammonium formate and ammonium acetate is from about 1 mM to about 0.1 mM. Typically, the proportion of volatile ammonium salt selected from the group consisting of ammonium hydroxide and ammonium formate and ammonium acetate varies from about 100: 1 at the start of elution to 1: 100 at the end of elution.

  Typically in this method, the step of determining the relative concentration of one or more peaks as determined by high performance liquid chromatography representing compounds other than dianhydrogalactitol itself is performed by evaporative light scattering detection. Typically, evaporative light scattering detection corresponds to electrospray LC / MS. Typically, evaporative light scattering detection involves post-column addition of a volatile solvent that facilitates evaporation of a 100% aqueous mobile phase. Typically, the volatile solvent is selected from the group consisting of methanol, ethanol, isopropanol, and acetonitrile.

  In one alternative, an electrospray tandem mass spectrometer is installed and connected online to an HPLC system with ELSD. Typically in this alternative, mass spectral data is collected that provides chemical information for each impurity that may be present in the dianhydroxygalactitol formulation. Also, typically in this alternative, tandem mass spectral data is collected that provides structural information for each impurity that may be present in the dianhydroxygalactitol formulation.

  The method of the present invention may further comprise performing preparative HPLC of at least one particular substance peak present in the dianhydrogalactitol formulation. At least one substance peak present in the dianhydrogalactitol formulation can be an impurity.

In another alternative, isocratic elution can be used instead of gradient elution. When isocratic elution is used, in general, the method of the invention comprises the following steps:
(1) Analyzing the formulation by subjecting the dianhydrogalactitol formulation to high performance liquid chromatography using elution in an isocratic mobile phase to separate dianhydrogalactitol from dulcitol and other contaminants of the formulation. And (2) determining the relative concentration of one or more peaks determined by high performance liquid chromatography representing compounds other than dianhydrogalactitol itself.

  In one alternative, when isocratic elution is used, the isocratic mobile phase is NaOH and the NaOH concentration is from about 5 mM to about 0.1 mM. Preferably, the concentration of NaOH is about 2.5 mM to about 0.1 mM. More preferably, the concentration of NaOH is about 1 mM.

  In another alternative, when isocratic elution is used, the isocratic mobile phase is a combination of ammonium hydroxide and a volatile ammonium salt selected from the group consisting of ammonium formate and ammonium acetate, And the total concentration of the volatile ammonium salt selected from the group consisting of ammonium formate and ammonium acetate is about 5 mM to 0.1 mM. Preferably, the total concentration of ammonium hydroxide and volatile ammonium acetate is from about 2.5 mM to about 0.1 mM. More preferably, the total concentration of volatile ammonium salts selected from the group consisting of ammonium hydroxide and ammonium formate and ammonium acetate is about 1 mM. Typically, the ratio of volatile ammonium salts selected from the group consisting of ammonium hydroxide and ammonium formate and ammonium acetate is about 50:50.

  Typically in this alternative, determining the relative concentration of one or more peaks as determined by high performance liquid chromatography representing compounds other than dianhydrogalactitol itself comprises evaporative light scattering detection (as described above). ELSD). Typically, evaporative light scattering detection is compatible with electrospray LC / MS. Typically, evaporative light scattering detection involves post-column addition of a volatile solvent that facilitates evaporation of a 100% aqueous mobile phase. Typically, the volatile solvent is selected from the group consisting of methanol, ethanol, isopropanol, and acetonitrile.

  Similarly in this alternative, an electrospray tandem mass spectrometer is installed and connected online to an HPLC system with ELSD. Typically in this alternative, mass spectral data is collected that provides chemical information for each impurity that may be present in the dianhydroxygalactitol formulation. Also typically in this alternative, tandem mass spectral data is collected that provides structural information for each impurity that may be present in the dianhydroxygalactitol formulation.

  This alternative of the method according to the invention further comprises performing a HPLC recovery of at least one specific substance peak present in the dianhydrogalactitol formulation. At least one substance peak present in the dianhydrogalactitol formulation can be an impurity.

In yet another alternative, an analytical method for analyzing the presence and amount of impurities present in a dianhydrogalactitol formulation is a gradient mobile phase that separates dianhydrogalactitol from dulcitol and other contaminants of the formulation. Analyzing said formulation by subjecting the formulation of dianhydrogalactitol to high performance liquid chromatography (HPLC) using HPLC elution with HPLC;
The high performance liquid chromatography uses evaporation light scattering detection (ELSD).

  Typically, the HPLC column is a silica gel column coupled to a C18 compound and is finished with an end-capping approach using Lewis acid-Lewis base chemistry.

  Typically, elution is performed with a gradient of 95% water / 5% acetonitrile to 70% water / 30% acetonitrile and back to 95% water / 5% acetonitrile. Typically, the time schedule for changing the eluent is as follows: 0 minutes, 95% water / 5% acetonitrile; 15 minutes, 95% water / 5% acetonitrile; 15.1 minutes, 70% Water / 30% acetonitrile; 20 minutes, 70% water / 30% acetonitrile; 20.1-35 minutes, 95% water / 5% acetonitrile. Typically, the method of the present invention detects the degradation products of dianhydrogalactitol monoepoxide, monoepoxide dimer, and dulcitol. Preferably, the method of the present invention also detects dian hydrogalactitol dimers and enriched products.

  Typically, the peak due to HPLC is analyzed by LC-MS.

  Typically, the methods of the invention further comprise determining the relative concentration of one or more peaks determined by high performance liquid chromatography representing compounds other than dianhydrogalactitol itself.

  In an alternative to detection by ELSD, the column temperature is typically about 30 ° C.

  Typically, the flow rate is about 0.5 mL / min. Typically, the ELSD detector is operated in a 35 ° C. drift tube temperature and a gain 400, 2 pps, 45 PSI cooling mode. Typically in this alternative, mobile phase A and mobile phase B are used together with mobile phase A which is 0.05% formic acid / water and mobile phase B which is 100% methanol. Typically, with these mobile phases, elution is 0% to 25 minutes with 100% 0.05% formic acid / water, 90% 0.05% formic acid / water and 10% 100% methanol. For 25 minutes to 25.1 minutes, 10% 0.05% formic acid / water and 90% 100% methanol for 25.1 minutes to 35 minutes, and 100% 0.05% formic acid / water For 35.1 to 50 minutes.

  The method of the present invention further includes the creation of an external calibration standard curve for impurities. The impurities are selected from the group consisting of dulcitol, the degradation product of dianhydrogalactitol monoepoxide, and the dimer of dianhydrogalactitol. The method of the present invention can estimate the content of unknown impurities using a calibration standard curve established by chromatography of dianhydrogalactitol reference material.

  Another alternative using HPLC and ELSD uses a dual elution sequence. The dual elution sequence is as follows: In the first part of the elution sequence, elution is 0% to 25 minutes with 100% 0.05% formic acid / water, 90% 0.05% formic acid. For 25 minutes to 25.1 minutes using 10% 100% methanol / water and 25.1 minutes to 35 minutes using 10% 0.05% formic acid / water and 90% methanol, and 100% Performed 35.1-50 minutes with 0.05% formic acid / water, and in the second part of the elution sequence, elution is performed as follows: 0 using 100% 0.05% formic acid. Min-7.5 min; 7.5 min-7.6 min with 97% 0.05% formic acid and 3% methanol, and 7.6 min with 100% 0.05% formic acid- 20 minutes. In this alternative, the HPLC column temperature is typically about 30 ° C., the HPLC sample temperature is about 5 ° C., the HPLC flow rate is about 0.5 mL / min, and the injection volume is about 10 ° C. ~ 100 μL. In this alternative, typically for ELSD, the gain is about 400, the drift tube temperature is about 45 ° C., the gas pressure is about 35 PSI of nitrogen, the nebulizer is set to cool, and the data transfer rate is 2 points per second, and the Rayleigh coefficient is about 6.0. Typically in this alternative, 0.1, 0.08, 0.05, 0.03, 0.01, 0.005 mg / mL dulcitol standards are used to determine system sensitivity and linearity. Is done. Typically in this alternative, the retention time of dulcitol is about 6.4 minutes and the retention time of dianhydrogalactitol is about 12.1 minutes. In this alternative, the amount and percentage of the dulcitol impurity can be determined from the HPLC and ELSD results. Also in this alternative, the amount and percentage of unknown impurities other than dulcitol can be determined from HPLC and ELSD results.

  These and other features, aspects, and advantages of the present invention will be better understood with reference to the following description, appended claims, and accompanying drawings:

FIG. 1 is a representative HPLC / RI chromatogram of a preparation of dianhydrogalactitol showing the degradation of dulcitol and unknown related substances at an RRT of about 0.6 in bulk drugs and drug products.

FIG. 2 shows a representative HPLC chromatogram showing the degradation of dianhydrogalactitol and dulcitol in the standard for the comparative, water blank; in FIG. 2, the dianhydrogalactitol-dulcitol standard is shown in the top panel; The water blank is shown in the lower panel.

FIG. 3 shows the HPLC of a clinical sample of dianhydrogalactitol using detection evaporative scattering detection showing the presence of possible dianhydrogalactitol dimers and possible concentrated products with monoepoxide and dulcitol as degradation products. It is a chromatogram.

FIG. 4 is a mass spectrometric profile of an impurity peak occurring at 22.6 minutes in the HPLC chromatogram of FIG.

FIG. 5 is a chromatogram of a sample of dianhydrogalactitol as performed in Example 3 using 0.05% formic acid / water as mobile phase A and 100% methanol as mobile phase B.

FIG. 6 is a chromatogram of an example of a blank solution as performed in Example 4.

FIG. 7 is a chromatogram of an example of a 0.10% dulcitol solution as performed in Example 4.

FIG. 8 is a chromatogram of an example test solution as performed in Example 4.

Detailed Description of the Invention

  The present invention is directed to an improved analytical method for determining the purity of dianhydrogalactitol and determining the presence and concentration of impurities present in a dianhydrgalactitol formulation.

The structure of dianhydrogalactitol is shown below in formula (I).

One of the important impurities present in the dianhydrogalactitol formulation is dulcitol. The structure of dulcitol is shown in the following formula (II). Other impurities are known to be present in the preparation of dianhydroxygalactitol.

  An improved method for analyzing dianhydrogalactitol formulations is based on HPLC (High Performance Liquid Chromatography) with evaporative light scattering detection (ELDS). In one alternative, HPLC is combined with mass spectrometry (MS) to detect and identify all important components present in such dianhydrogalactitol formulations.

  The theory and practice of HPLC is described in LR Snyder et al., "Introduction to Modern Liquid Chromatography" (3rd edition, John Wiley & Sons, New York, 2009). The theory and practice of MS is described in E. de Hoffmann & V. Strobant, “Mass Spectroscopy: Principles and Applications” (3rd edition, John Wiley & Sons, 7th, New York, Sons Y Is done.

  The HPLC method showed the degradation of the synthetic intermediate, dulcitol, in the dianhydrogalactitol formulation, in addition to the degradation of unknown related substances observed at RRT 0.6 (FIG. 1). FIG. 1 is a representative HPLC / RI chromatogram of a dianhydrogalactitol formulation showing the degradation of dulcitol and unknown related substances at RRT of about 0.6 in bulk drugs and drug products. A representative HLPC chromatogram showing the degradation of dianhydrogalactitol and dulcitol by default for a comparative, water blank is shown in FIG. In FIG. 2, the dianhydrogalactitol-dulcitol standard is shown in the top panel and the water blank is shown in the bottom panel.

  This application demonstrates improved chromatographic conditions for the degradation of potentially co-eluting materials. Thermal stressed dianhydrogalactitol product samples are evaluated to provide confirmation of proper chromatographic conditions for the degradation of dulcitol and other related impurities and degradation products. LC / MS and LC / MS / MS are then performed to characterize the unknown DAG related material with an RRT of about 0.6, providing mass spectral characterization and chemical structure determination of this unidentified component.

  Previously used HPLC conditions include isocratic elution of dianhydrogalactitol and its related substances using 50 mM NaOH mobile phase. In improving these conditions, a gradient mobile phase is used, as used as part of the inventive method disclosed herein. One alternative is the use of NaOH in a concentration gradient. When NaOH is used in a concentration gradient, typically the elution is a gradient of about 2.5 mM to about 0.1 mM NaOH. Preferably, the elution is a gradient of about 1.5 mM to about 0.1 mM NaOH. More preferably, the elution is a gradient of about 1 mM to about 0.1 mM NaOH.

  In another alternative, a combination of ammonium hydroxide and a volatile ammonium salt selected from the group consisting of ammonium formate and ammonium acetate can be used as the eluent. In this alternative, the total concentration of ammonium formate and ammonium acetate is about 2.5 mM to about 0.1 mM. Preferably, the total concentration of volatile ammonium salts selected from the group consisting of ammonium hydroxide and ammonium formate and ammonium acetate is from about 1.5 mM to about 0.1 mM. More preferably, the total concentration of volatile ammonium salts selected from the group consisting of ammonium hydroxide and ammonium formate and ammonium acetate is from about 1 mM to about 0.1 mM. Typically, the ratio of volatile ammonium salts selected from the group consisting of ammonium hydroxide and ammonium formate and ammonium acetate varies from 100: 1 at the start of elution to 1: 100 at the end of elution.

  Other gradient elution schemes are known in the art.

  Typically, in the HPLC analysis method according to the present invention, the detection is by evaporative light scattering (ELSD). The evaporative light scattering detector (ELSD) atomizes the column eluate, irradiates the obtained particle component with light, and detects the obtained scattered light. In theory, ELSD can detect any non-volatile component. Evaporative light scattering detection of non-chromogenic compounds is based on spraying of HPLC eluent and evaporation of mobile phase solvent to produce sprayed solute particles for light scattering detection. This evaporation and solvent evaporation method to produce atomized solute particles is equivalent to the electrospray LC / MS method. Typically, ELSD detection is compatible with electrospray LC / MS.

  Implementation of the HPC method with ELSD detection compatible with electrospray LC / MS applications involves the post-column addition of a volatile solvent that facilitates the evaporation of a 100% aqueous mobile phase. The volatile solvent is typically selected from the group consisting of methanol, ethanol, isopropanol, and acetonitrile.

  Thus, in the method according to the invention, an electrotandem mass spectrometer is installed and online connected to an HPLC system with ELSD. Mass spectral data providing molecular information and tandem mass spectral data providing chemical structure information for each impurity that may be present in the dianhydrogalactitol formulation can be collected. Mass spectrometry in tandem using HPLC will provide molecular ion information and possible chemical structures with molecular weights consistent with the molecular ion information for each observed impurity and degradation product.

  In another alternative, preparative HPLC recovery of specific DAG-related substance peaks can be performed, including impurities present in the formulation of DAG.

Thus, an analytical method for analyzing the presence and amount of impurities present in a dianhydrogalactitol formulation comprises the following steps:
(1) Analyzing said formulation by subjecting the dianhydrogalactitol formulation to high performance liquid chromatography using elution with a gradient mobile phase to separate dianhydrogalactitol from dulcitol and other contaminants of the formulation. And (2) determining the relative concentration of one or more peaks determined by high performance liquid chromatography representing compounds other than dianhydrogalactitol itself.

  The compound other than dianhydrogalactitol itself can be at least: (1) dulcitol; (2) impurities other than dulcitol; and (3) degradation products of dianhydrogalactitol.

  Typically, in one alternative, in this method, the mobile phase gradient is a sodium hydroxide gradient.

  In another alternative, in this method, the mobile phase gradient is a gradient of a combination of volatile ammonium salts selected from the group consisting of ammonium hydroxide and ammonium formate and ammonium acetate.

  Typically in this method, detection is by evaporative light scattering. Typically, when using evaporative light scattering, the method of the present invention further includes a post-column addition of a volatile solvent that facilitates evaporation of the components of the mobile phase.

  Typically, the invention further comprises analyzing one or more peaks eluting from high performance liquid chromatography by electrospray tandem mass spectrometry.

  In one alternative, the present invention further comprises the step of preparative HPLC recovery of at least one particular DAG-related substance peak.

  If impurities or degradation products (other than dulcitol) are present, unknown impurities or degradation products can be identified by column chromatography separation, followed by nuclear magnetic resonance (NMR), mass spectrometry (MS), Fourier Characterized for identification by at least one standard analytical technique selected from the group consisting of transform infrared spectroscopy (FT-IR), elemental analysis, determination of purity by HPLC, and determination of moisture content by Karl Fischer titration Following at least one purification procedure to obtain a solid unknown sample that can be applied. These methods are well known in the art.

In another alternative, the method of the invention comprises:
(1) Analyzing the formulation by subjecting the dianhydrogalactitol formulation to high performance liquid chromatography using elution in an isocratic mobile phase to separate dianhydrogalactitol from dulcitol and other contaminants of the formulation. And (2) determining the relative concentration of one or more peaks determined by high performance liquid chromatography representing compounds other than dianhydrogalactitol itself.

  As in the method using gradient elution, compounds other than dianhydrogalactitol itself include: (1) dulcitol; (2) impurities other than dulcitol; and (3) at least one degradation product of dianhydrogalactitol. Can be one.

  In this alternative, elution with an isocratic mobile phase is either elution with sodium hydroxide or elution with a combination of volatile ammonium salts selected from the group consisting of ammonium hydroxide and ammonium formate and ammonium acetate. Can be. When the isocratic mobile phase is sodium hydroxide, typically the NaOH concentration is from about 5 mM to about 0.1 mM. Preferably, the concentration of NaOH is about 2.5 mM to about 0.1 mM. More preferably, the concentration of NaOH is about 1 mM. When the isocratic mobile phase is a combination of volatile ammonium salts selected from the group consisting of ammonium hydroxide and ammonium formate and ammonium acetate, volatilization selected from the group consisting of ammonium hydroxide, ammonium formate and ammonium acetate The total concentration of the sex ammonium salt combination is about 5 mM to about 0.1 mM. Preferably, the total concentration of ammonium hydroxide and volatile ammonium acetate is from about 2.5 mM to about 0.1 mM. More preferably, the total concentration of volatile ammonium salts selected from the group consisting of ammonium hydroxide and ammonium formate and ammonium acetate is about 1 mM. Typically, the ratio of volatile ammonium salts selected from the group consisting of ammonium hydroxide and ammonium formate and ammonium acetate is about 50:50.

  In an alternative method to improve resolution, an evaporative light scattering detector (ELSD) is used with modified elution conditions. Typically, in this method, the HPLC column is a silica gel column coupled to a C18 compound and is finished with an end-capping technique using Lewis acid-Lewis base chemistry such as a YMC C18 column. Typically, elution is performed with a gradient of 95% water / 5% acetonitrile to 70% water / 30% acetonitrile and back to 95% water / 5% acetonitrile. Preferably, the time schedule for changing the eluent is as follows: 0 minutes, 95% water / 5% acetonitrile; 15 minutes, 95% water / 5% acetonitrile; 15.1 minutes, 70% water / 30% acetonitrile; 20 minutes, 70% water / 30% acetonitrile; 20.1-35 minutes, 95% water / 5% acetonitrile. Preferably, the HPLC method detects monoepoxide degradation products, monoepoxide dimers, and dulcitol of dianhydrogalactitol. More preferably, the HPLC method also detects dianhydrogalactitol dimers and concentrated products.

  Preferably, in this alternative of the method of the invention, the peak obtained from HPLC is analyzed by LC-MS.

  In another alternative, an Atlantis HPLC column is used, as shown in Example 3. Typically in this method, the column temperature is about 30 ° C. Typically in this method, the flow rate is about 0.5 mL / min. Typically in this method, the injection volume is from about 10 μL to about 100 μL. Typically in this method an ELSD detector is used. Typically in this method, the ELSD detector is operated at a drift tube temperature of 35 ° C. and a cooling mode of gain 400, 2 pps, 45 PSI. Typically in this process, mobile phase A and mobile phase B are used together with mobile phase A which is 0.05% formic acid / water and mobile phase B which is 100% methanol. Typically, in this method, elution is from 0 minutes to 25 minutes with 100% 0.05% formic acid / water, 25 minutes with 90% 0.05% formic acid / water and 10% 100% methanol. ~ 25.1 minutes, 25.1 minutes to 35 minutes with 10% 0.05% formic acid / water and 90% 100% methanol, and 35% with 100% 0.05% formic acid / water It takes 1 to 50 minutes.

  Typically, the alternative means of the present invention further includes the creation of an external calibration standard curve for impurities. The impurities can be selected from the group consisting of, but not limited to, dulcitol, the degradation product of dianhydrogalactitol monoepoxide and the dimer of dianhydrogalactitol. In this way, for unknown impurities, the content of unknown impurities can be estimated using a calibration standard curve established by chromatography of a dianhydrogalactitol reference material.

  In another alternative, as shown in Example 4, after the above elution sequence, i.e., elution is 0-25 minutes with 100% 0.05% formic acid / water, 90% 0.05%. 25% to 25.1 minutes with 10% formic acid / water and 10% 100% methanol, 25.1 minutes to 35 minutes with 10% 0.05% formic acid / water and 90% 100% methanol, A 35.1 to 50 minute run with 100% 0.05% formic acid / water is performed and a further elution sequence is performed as follows: 0 to 7.5 with 100% 0.05% formic acid. Minutes; 7.5 minutes to 7.6 minutes with 97% 0.05% formic acid and 3% methanol; and 7.6 minutes to 20 minutes with 100% 0.05% formic acid. In this alternative, the HPLC column temperature is typically about 30 ° C., the HPLC sample temperature is about 5 ° C., the HPLC flow rate is about 0.5 mL / min, and the injection volume is about 100 μL. It is. Typically in this alternative, for ELSD, the gain is about 400, the drift tube temperature is about 45 ° C., the gas pressure is about 35 PSI of nitrogen, the nebulizer is set to cool, and the data transfer rate is per second. 2 points, and the Rayleigh coefficient is about 6.0. Typically in this alternative, the criteria for dulcitol from 0.005 to 0.1 mg / mL are used to determine the sensitivity and linearity of the system. Typically in this alternative, the retention time of dulcitol is about 6.4 minutes and the retention time of dianhydrogalactitol is about 12.1 minutes. In this alternative, the amount and percentage of the dulcitol impurity can be determined from the HPLC and ELSD results. Also in this alternative, the amount and percentage of unknown impurities other than dulcitol can be determined from HPLC and ELSD results.

  The invention is illustrated by the following examples. These examples are for illustrative purposes only and are not intended to limit the invention.

Example 1 HPLC analysis of a dianhydrogalactitol formulation using elution of isocratic sodium hydroxide The procedure described in this example was performed by using ion exchange high performance liquid chromatography with refractive index detection in a dianhydrogalactitol drug formulation. And used to determine related impurities.

  In this procedure, a sample is prepared with dianhydrogalactitol at a target concentration of 5 mg / mL. Dulcitol, dianhydrogalactitol, and related impurities use an anion exchange column (Hamilton RCX-10, 250 × 4.1 mm, 7 μm) using 50 mM NaOH as an isocratic mobile phase with refractive index detection. Separated. The dulcitol concentration is determined using an external reference standard and the content of related substances is estimated using the DAG reference standard.

  One suitable HPLC system and data collection system is an Agilent Technologies 1200 Series HPLC system or equivalent equipped with: Quat pump, Model G1311A or equivalent; Autosampler, Model 1329A or equivalent; RID detector, Model 1362A Or equivalent; possible column temperature controller of 30 ± 3 ° C .; and deaerator, model G1322 or equivalent. The column is a Hamilton RCX anion exchange column 250 × 4.1 mm, 7 μm, P / N 79440, or equivalent. Data collection is performed by ChemStation and ChemStore Client / Server or equivalent data systems.

  The following chemicals are used. The water is Milli-Q water or deionized water. Sodium hydroxide is a standard purified grade. Dulcitol and DAG reference standards are> 98.0% pure.

  For the mobile phase (50 mM NaOH), 2.0 g NaOH is dissolved in 1 liter of water. The solution is filtered through a 0.45 μm filter. The mobile phase can be stored at room temperature for up to 1 month. For a dulcitol reference stock solution (nominal 500 μg / mL), accurately weigh 25 mg dulcitol reference standard into a 50 mL volumetric flask. Dilute dulcitol in deionized water and mixed wells. The prepared stock solution can be stored at 2-8 ° C. for up to 3 days. For the DAG reference stock solution (nominal 500 μg / mL), accurately weigh 25 mg into a 50 mL volumetric flask. The DAG is diluted with deionized water and mixed wells. The prepared stock solution can be stored at 2-8 ° C. for up to 3 days. For the dulcitol-DAG standard solution (dulcitol 50 μg / mL + DAG 50 μg / mL; 1% of each 5 mg / mL DAG), 1.0 mL dulcitol stock and 1.0 mL DAG stock were quantitatively transferred into a 10 mL volumetric flask, respectively. And dilute to volume with water and mixing wells.

  For formulations of DAG from API samples (nominal 1 mg / mL), weigh accurately about 25 mg of DAG API sample in a clean 25 mL volumetric flask. DAG API sample is dissolved in about 5 mL of ionized water, diluted with deionized water and mixed. Transfer a 1-2 mL portion of the test sample to an HPLC vial. The prepared sample can be stored at 2-8 ° C. for up to 2 days.

  For the DAG sample formulation (nominal 5 mg / mL) against the API sample, accurately weigh approximately 50 mg of the API sample into a clean 10 mL volumetric flask. Dissolve the DAG API sample in approximately 5 mL of water, dilute to volume with water and mix.

  For formulation of DAG samples from lyophilized (40 mg / vial) samples, the samples are removed from the refrigerator where the samples are stored and the seal is removed. Quantitatively transfer the volume of 5.0 mL of water and mix the solution to dissolve the DAG to obtain an 8 mg / mL solution. A 1.0 g portion of the reconstituted solution is diluted to 8.0 g with deionized water and mixed. Transfer an additional 1-2 mL of the test sample to the HPLC vial. The prepared sample can be stored at 2-8 ° C. for up to 2 days.

  For drug product DAG sample formulations (nominal 5 mg / mL) using lyophilized powder (40 mg / vial), the vial seal is washed and removed. Reconstitute the lyophilized vial with 8.0 mL of water to obtain a 5 mg / mL solution. Transfer a portion of 1-2 mL to an HPLC vial. Samples are prepared in duplicate (using two vials). The prepared sample can be stored at 2-8 ° C. for up to 24 hours.

For HPLC analysis, the HPLC system can be warmed up to at least 20 minutes. If necessary, place the HPLC mobile phase prepared as described above at the appropriate solvent inlet. Prime the solvent line with the mobile phase. Equilibrate the system and column with HPLC mobile phase for at least 30 minutes at a flow rate of 1.5 mL / min. Create a sample analysis sequence. After system suitability has been verified, a water blank is injected by standard and then sample injection. The dulcitol-DAG standard is inserted after all 10 injections of the sample, and then the final section (bracketing) standard is inserted at the end of the run. An appropriate sample analysis sequence is shown in Table 1.

  Samples are analyzed using RID. As indicated above, one suitable column is a Hamilton RCX ion exchange column (250 × 4.1 mm, 7 μm), P / N 79440 or equivalent. The mobile phase is 50 mM NaOH in deionized water (isocratic elution). The flow rate is 1.5 mL / min. The column temperature is 30 ° C. The injection volume is 50 μL. Detection is by RID at 35 ° C. The execution time is 8 minutes.

  HPLC software is used for chromatogram analysis and integration. Chromatograms for blanks, samples, and test standards are reviewed and compared. Some peak manual integration and assignment may be required. The slope sensitivity, peak width, peak height threshold for rejection, shoulder peak, baseline, and split peak integration types, as well as other parameters are adjusted to obtain proper integration, and the values for these parameters are: Recorded and applied to all samples and standards.

Assess system suitability as follows: Six repeated injections of dulcitol-DAG standard solution are evaluated using the chromatographic performance requirements in Table 2.

  The dulcitol and DAG peak areas in the bracketing standard solution injection should be 80% to 120% of each average peak area in the previous SST injection. If one bracketing standard does not meet the criteria, samples analyzed after the final pass bracketing standard should be re-analyzed.

  In analysis of data, relative peak area = (peak area / total peak area) × 100, where “peak area” is an individual peak area, and “total peak area” is the sum of peak areas.

  The dulcitol concentration is calculated as shown: dulcitol (Cu, μg / mL) = Cs × average sample peak area / average dulcitol peak area of Dul-DAG standard injection, where Cs is dulcitol in μg / mL Concentration.

  The dulcitol content (% by weight) in the DAG drug substance or drug product is calculated as indicated: dulcitol weight% = Cu (μg / mL) / 1000 / SC (mg / mL) × 100%, where Cu is The dulcitol concentration (μg / mL) calculated as above, and SC is the sample concentration (mg / mL) as prepared for the drug substance or drug product. If dulcitol is present, the weight percent of dulcitol is reported if it is equal to or greater than 0.05% and is reported to the nearest 0.01%.

  If an unknown or previously identified impurity other than dulcitol is present in the DAG formulation, its concentration is calculated as follows: unknown impurity concentration (μg / mL) = Cs × average sample peak area / Dul -Average DAG peak area of DAG standard injection. If present, the unknown impurity weight percent is calculated as follows: Cu (μg / mL) / 1000 / SC (mg / mL) × 100%, where Cu = calculated as above. Unknown concentration (μg / mL) and SC = sample concentration (mg / mL) as prepared in [0077] for drug substance or [0079] for drug product. If unknown impurities are present, each unknown impurity is reported at the nearest 0.01% if equal to or greater than 0.05%.

  The assay results in weight percent are calculated for each sample and the average of duplicate samples.

Example 2 HPLC analysis using an evaporative light scattering detector using a water / acetonitrile gradient An evaporative light scattering detector (ELSD) with a water / acetonitrile gradient as described below was used to facilitate the degradation of impurities. Another method of HPLC analysis was used.

  Due to refractive index (RI) detector limitations, the HPLC / RI method does not have sufficient specificity to obtain reliable impurity profile data, which is an unacceptable level of unknown or incompletely characterized impurities Exposure to patient exposure risk. To address this concern, more sensitive detectors, such as the Evaporative Light Scattering Detector (ELSD) manufactured by Agilent, are used to determine impurities found in dianhydrogalactitol drug substances or drug products. Used in combination with the system.

For example, DAG samples were analyzed by HPLC / ELSD method using a YMC C18 column with the gradient shown in Table 3:

As shown in the chromatogram (FIG. 3), dulcitol was eluted with a retention time of 4.5 minutes. As supported by the LC-MS data summarized in Table 4, the peak eluted immediately after dulcitol was identified as a monoepoxide related compound. The peak observed at 11.46 minutes is likely a DAG dimer and contributes to multiple peaks with m / z of 271, 357, 417, 512, and other peaks 22.6 minutes (Fig. 4). These data are consistent with the impurity profile expected by previous studies. As expected, monoepoxide and dulcitol are the two major degradation products obtained by this method.

Example 3 HPLC analysis using formic acid in a water / methanol gradient to improve monoepoxide peak resolution A new method was developed to improve monoepoxide peak resolution. This new method employs the following parameters: The column was Atlantis C18, 250 × 4.6 mm, 5 μm. The column temperature was 30 ° C. The flow rate was 0.5 mL / min. The injection volume was 100 μL. The ELSD detector was operated in a cooling mode with a gain of 400, 2 pps, 45 PSI at a drift tube temperature of 35 ° C. Mobile phase A was 0.05% formic acid / water. Mobile phase B was 100% methanol. The gradient is shown in Table 5:

  A better resolution of the early eluting impurities was observed (see chromatogram of the following DAG sample in FIG. 5). Dulcitol labeled peak 2 was eluted with a retention time of 6.26 minutes or a relative retention time (RRT) of 0.59. Dianhydrogalactitol was eluted at 10.86 minutes.

  Since the ELSD response is not linear, an external calibration standard curve is required for known impurities such as dulcitol and determines the content of impurities in a sample of dianhydrogalactitol tested. For unknown impurities contained in a sample of dianhydrogalactitol, the unknown impurity content can be estimated using a calibration standard curve established by chromatography of a dianhydrogalactitol reference material.

Example 4 Further Improved Method for Detection or Determination of Impurities Using Double Gradient HPLC Elution A further improved analytical method for the detection or determination of impurities in dianhydrogalactitol uses double gradient elution on HPLC. Employ the HPLC and ELSD used. This method is described below.

  In this analytical method, the following materials and equipment are used: Atlantis C18, 250 × 4.6 mm, 5 μm HPLC column; quadruple or two-component HPLC pump; evaporative light scattering detector (ELSD); integrator Or a computer-based analytical system; a calibration chemical balance; and a class A volumetric flask and pipette. The following reagents and standards are used: dulcitol reference standard as described above; HPLC grade water or equivalent formic acid (FA); HPLC grade or equivalent acetonitrile (ACN); and HPLC grade or equivalent methanol (MeOH).

  For the solution used in the method of the invention, the volume can be measured to suit the analytical needs. It is important that all mobile phases are filtered. The sintered glass in the filtration device may be a source of buffer that can interfere with sensitivity in ELSD. All filtration equipment should be thoroughly washed with Milli-Q grade water. To do this, about 500 mL of Milli-Q grade water is filtered through a filtration device. The water is discarded and the mobile phase is then filtered.

  The preparation of the test solution should be done in a draft using appropriate PPE (gloves, laboratory coat, and safety glasses). The preparation of the test solution should be stored in a draft for disposal and should be appropriately labeled.

  Mobile phase A is prepared by pipetting 0.5 mL formic acid into 1000 mL water. Mobile phase A is filtered and degassed.

  Mobile phase B is MeOH. Mobile phase B is filtered and degassed.

  Diluent A is water. Diluent B is prepared by mixing 180 mL water and 20 mL ACN with mixing wells.

  The preparation of standard solutions and sample solutions is described below. The blank solution is water. A dulcitol stock solution is prepared by accurately transferring 100 mg dulcitol reference standard to a 20 mL volumetric flask. Add about 15 mL of Diluent B and sonicate for dissolution. Cool the solution to room temperature and dilute to volume with Diluent B and mixing wells (5 mg / mL). The following standard solutions are prepared: 0.2, 0.1, 0.08, 0.05, 0.03, 0.01 and 0.005 mg / mL (system sensitivity solution). A 4.0% standard solution is prepared by pipetting 2.0 mL of dulcitol stock solution into a 50 mL volumetric flask. Dilute the solution with water and mixing wells (0.2 mg / mL). A 2.0% standard solution is prepared by pipetting 5.0 mL of 4.0% standard solution into a 10 mL volumetric flask. The solution is diluted with water and mixed wells (0.10 mg / mL). A 1.6% standard solution is prepared by pipetting 4.0 mL of 4.0% standard solution into a 10 mL volumetric flask. The solution is diluted with water and mixed wells (0.08 mg / mL). A 1.0% standard solution is prepared by pipetting 2.5 mL of 4.0% standard solution into a 10 mL volumetric flask. The solution is diluted with water and mixed wells (0.05 mg / mL). A 0.60% standard solution is prepared by pipetting 3.0 mL of 4.0% standard solution into a 20 mL volumetric flask. The solution is diluted with water and mixed wells (0.30 mg / mL). A 0.20% standard solution is prepared by pipetting 1.0 mL of 4.0% standard solution into a 20 mL volumetric flask. Dilute the solution in water and mixed wells (0.01 mg / mL). A 0.10% standard solution (system sensitivity solution) is prepared by pipetting 5.0 mL of 0.20% standard solution into a 10 mL volumetric flask. Dilute the solution with water and mix wells (0.005 mg / mL).

  The test sample working solution should be prepared in duplicate (A and B). The sample solution should be prepared immediately before analysis. Sample injection must occur within 15 minutes of sample solution preparation. In some cases, sample dilution may be required to quantify any impurities that are overloaded.

  For sample preparation, approximately 50 mg of test sample is accurately transferred to a 10 mL volumetric flask. The test sample is dissolved in water and diluted to volume and mixing well (5 mg / mL).

The HPLC operating conditions are as follows: The column is Atlantis C18 250 × 4.6 mm, 5 μm. Mobile phase A is 0.05% FA / water. Mobile phase B is MeOH. The gradients A and B are shown in Table 6 below. The column temperature is 30 ° C. The sample temperature is 5 ° C. The flow rate is 0.5 mL / min. The injection volume is 100 μL. The execution time is 50 minutes for gradient A and 20 minutes for gradient B.

  The operating conditions for SLSD are as follows: the gain is 400. The drift tube temperature is 45 ° C. The gas pressure (nitrogen) is 35 PSI. Set the nebulizer to cool. The data transfer rate is 2 seconds per second. The Rayleigh coefficient is set directly at the detector and is 6.0.

The injection sequence is shown in Table 7. The blank injection should be repeated until a stable and reproducible baseline is obtained.

  Additional test sample injections may be added as needed. Less than 6 test sample injections should be made before repeating the 0.20% standard solution check injection.

For evaluation, all peaks should be integrated in relation to the unknown impurities that are detected. If baseline noise is a problem, make sure that the waste is flowing properly from the detector. It may be necessary to ensure that there is no accumulated liquid inside the tube that drains the waste from the ELSD. If necessary, the tube position should be corrected. If the waste is flowing properly, baseline noise is a problem and the detector may be cleaned. If necessary, the following washing method may be used prior to analysis. For washing, the HPLC conditions are as follows: the column should be removed from the instrument and a union should be used. The mobile phase is 100% H ₂ O (isocratic 100%). The flow rate is 1.0 mL per minute. The column temperature is the ambient temperature. The execution time is 60 minutes. The ELSD operating conditions are as follows: the gain is 50. The drift tube temperature is 100 ° C. The gas pressure (nitrogen) is 50 PSI. The nebulizer is set to heat at 75%.

Typical retention times are shown in Table 8. In Table 8, “DAG” is dianhydrogalactitol. DAG in the test sample is not quantified by this method. DAG is observed as a broad peak depending on the concentration of DAG required. The retention time of DAG in the test solution is between about 10 minutes and 13 minutes.

  FIG. 6 is a chromatogram of an example of a blank solution.

  FIG. 7 is a chromatogram of an example of a 0.10% standard solution (system sensitivity solution).

  FIG. 8 is a chromatogram of an example test solution.

  The system suitability requirements are as follows. For blank solution injection, a non-interfering peak should be observed at the retention time of dulcitol peak or any known impurity. A stable and reproducible baseline should be observed and continue to inject the blank solution until this condition is met. For system sensitivity, a dulcitol peak following injection of a 0.10% standard solution should be observed. The signal to noise ratio for the dulcitol peak should be reported. If contamination is suspected in the mobile phase (ie, baseline noise is greater than 1.0 LSU) or no dulcitol peak is observed, the mobile phase should be reconstituted or the above cleanup procedure is performed. Should be. The USP tailing factor for dulcitol peak for the 0.20% first and last injections is less than 2.0. For accuracy,% RSD is calculated for the log of peak area in 5 injections. The% RSD must be less than 15%.

  For the calculation, all peaks that are not attributed to blanks should be integrated. The logarithm of the response to the logarithm of 0.10% concentration is plotted through a 2.0% standard solution of dulcitol (including 5 correct injections of the 0.20 standard). The correlation coefficient (r) must be greater than 0.98 for the linearity curve. The slope and intercept are determined from the curve. The linearity curve for dulcitol is used to determine the concentration in mg / mL of unknown and dulcitol impurities in the sample.

The slope and intercept from the linearity curve for dulcitol as described above is used to determine individual unknown impurities in the sample. Using the unknown Log [area response], the unknown Log [concentration] is determined using equation (1) as follows:

The amount of unknown impurities (mg / mL) is determined using equation (2) as follows:

The percentage of each unknown impurity is determined using equation (3) as follows:

  Alternatively, quantification using dulcitol standards may be formed using the log-log linear function in Empower (Waters Corp.).

  Similar equations, in particular equations (4)-(6), are used for the determination of the impurities of dulcitol in the sample.

Using log [area response] to dulcitol, the log [concentration] of dulcitol is determined using equation (4):

The concentration of dulcitol impurity (mg / mL) is determined using equation (5):

The percentage of dulcitol impurity is determined using equation (6):

[Effect of the invention]
The present invention provides an improved analytical method for the detection and quantification of impurities present in dianhydrogalactitol formulations, including dulcitol and unknown impurities, as well as a simple description of unknown impurities present in dianhydrogalactitol formulations. A method for separation and identification is provided. The method of the present invention allows for the large-scale preparation of high purity dianhydrogalactitol suitable for pharmaceutical use and the potential for serious side effects caused by the presence of impurities in the preparation of dianhydrogalactitol intended for pharmaceutical use Reduce.

  The method according to the invention has industrial applicability for the determination and quantitative analysis of impurities in dianhydrogalactitol and dianhydrogalactitol formulations.

  With respect to a range of values, the present invention includes each intermediate value between the upper and lower limits of the range of at least one tenth of the lower limit unit, unless the context clearly indicates otherwise. Also, the invention includes any other described intermediate values and ranges that include either or both of the upper and lower limits of the range, unless specifically excluded from the stated range.

  Unless defined otherwise, the meaning of all technical and scientific terms used herein are generally understood by those of ordinary skill in the art to which this invention belongs. Those skilled in the art will also understand that any methods and materials similar or equivalent to the invention described herein can also be used to practice or test the present invention.

  The publications and patents discussed herein are provided solely for their disclosure prior to the filing date of the present invention. In this description, the invention is to be construed as an admission that it is not entitled to antedate like a publication because of the prior invention. Further, the dates of publication provided may be different from the actual publication dates that may need to be independently confirmed.

  All cited publications are hereby incorporated by reference in their entirety, including all published patents, patent applications, and references, as well as those publications incorporated into their published documents. Incorporated into. However, as long as any publication incorporated herein by reference refers to the published information, the applicant shall publish any such information after the filing date of this application, which is prior art. Will not admit.

  As used in this specification and the appended claims, the singular forms include the plural forms. For example, the terms “a”, “an”, and “the” include multiple references unless the content clearly dictates otherwise. It should also be understood that the term “at least” preceding a series of elements refers to all the elements in the series. The invention described by way of example in the specification can be suitably practiced without any element, limitation or limitation. It is not specifically disclosed in this specification. Thus, for example, the terms “comprising”, “including”, “containing” and the like are intended to be read in an expanded and non-limiting manner. Also, the terms and expressions used herein are used as descriptive terms, and are not limiting, such as excluding any equivalent in the future shown or described or at any position thereof. It will be appreciated that there is no intent in the use of the terms and expressions and that various modifications are possible within the scope of the claimed invention. Thus, although the present invention has been specifically disclosed by means of preferred embodiments and optional features, modifications and alterations of the invention disclosed herein can be addressed by those skilled in the art. Such modifications and changes are considered to be within the scope of the invention disclosed herein. The invention has been described broadly and generically herein. Each of the narrower types and sub-concept groups that fall within the general disclosure also form part of their invention. This includes a general description of each invention with proviso or negative limitations excluding any issues from the type, whether or not the deleted material is present therein. Also, where features or aspects of the present invention are described in the Markush group terminology, those skilled in the art will also recognize that the present invention will also thereby enable any individual member or sub-member of the Markush group members. You will recognize that it is written in group terms. It is also to be understood that the above description is intended to be illustrative and not restrictive. Many aspects will be apparent to those of skill in the art upon reviewing the above description. Accordingly, the scope of the invention should be determined without reference to the above description, but instead should be determined with reference to the appended claims, and such claims should Rights are granted along equal full scope. One of ordinary skill in the art will recognize or be able to ascertain with many uses for the specific embodiments of the invention described, less than routine experimentation. Such equivalents are intended to be encompassed by the following claims.

Claims (26)

Analytical method for analyzing the presence and amount of impurities present in a dianhydrogalactitol formulation, using elution with a gradient mobile phase to separate dianhydrogalactitol from dulcitol and other contaminants of the formulation. Analyzing said formulation by subjecting the formulation of dianhydrogalactitol to HPLC on a liquid chromatography (HPLC) column;
An analytical method using evaporative light scattering detection (ELDS) in the high performance liquid chromatography.   The analytical method according to claim 1, wherein the HPLC column is a silica gel column bonded to a C18 compound and is finished by an end-capping technique using Lewis acid-Lewis base chemistry.   The analytical method according to claim 1, wherein elution is performed with a gradient of 95% water / 5% acetonitrile to 70% water / 30% acetonitrile and back to 95% water / 5% acetonitrile.   The analytical method according to claim 3, wherein the time schedule for changing the eluent is as follows: 0 minutes, 95% water / 5% acetonitrile; 15 minutes, 95% water / 5% acetonitrile; 15.1. Min, 70% water / 30% acetonitrile; 20 min, 70% water / 30% acetonitrile; 20.1-35 min, 95% water / 5% acetonitrile.   The analytical method according to claim 1, wherein the method detects a degradation product of dianhydrogalactitol monoepoxide, a dimer of monoepoxide, and dulcitol.   6. The method of claim 5, wherein the method further detects dian hydrogalactitol dimer and concentrated product.   The method according to claim 1, wherein peaks due to HPLC are analyzed by LC-MS.   The method of claim 1, further comprising determining the relative concentration of one or more peaks determined by high performance liquid chromatography representing compounds other than dianhydrogalactitol itself.   The method of claim 1, wherein the column temperature is about 30 ° C.   The method of claim 1, wherein the flow rate is about 0.5 mL / min.   The method of claim 1, wherein the ELSD detector is operated in a 35 ° C. drift tube temperature and a cooling mode of gain 400, 2 pps, 45 PSI.   The process of claim 1 wherein mobile phase A and mobile phase B are used together with mobile phase A being 0.05% formic acid / water and mobile phase B being 100% methanol.   Elution 0% to 25 minutes with 100% 0.05% formic acid / water, 25% to 25.1 minutes with 90% 0.05% formic acid / water and 10% 100% methanol, 10 In 25.1 minutes to 35 minutes using 1% 0.05% formic acid / water and 90% 100% methanol and 35.1 minutes to 50 minutes using 100% 0.05% formic acid / water. 13. The method according to claim 12, wherein:   The method of claim 1, further comprising generating an external calibration standard curve for impurities.   The method of claim 12, further comprising creating an external calibration standard curve for impurities.   15. The method of claim 14, wherein the impurity is selected from the group consisting of dulcitol, a degradation product of dianhydrogalactitol monoepoxide, and a dimer of dianhydrogalactitol.   16. The method of claim 15, wherein the impurity is selected from the group consisting of dulcitol, a degradation product of dianhydrogalactitol monoepoxide, and a dimer of dianhydrogalactitol.   2. The method of claim 1, wherein for unknown impurities, the content of the unknown impurities is estimated using a calibration standard curve established by chromatography of a dianhydrogalactitol reference material.   13. The method of claim 12, wherein for unknown impurities, the content of the unknown impurities is estimated using a calibration standard curve established by chromatography of a dianhydrogalactitol reference material.   14. The method of claim 13, wherein after the elution sequence, an additional elution sequence is performed as follows: 0-7.5 minutes with 100% 0.05% formic acid; 97% 0.05 7.5-7.6 minutes with% formic acid and 3% methanol; and 7.6-20 minutes with 100% 0.05% formic acid.   21. The HPLC column temperature is about 30 ° C., the HPLC sample temperature is about 5 ° C., the HPLC flow rate is about 0.5 mL / min, and the injection volume is about 10-100 μL. The method described.   For ELSD, the gain is about 400, the drift tube temperature is about 45 ° C., the gas pressure is about 35 PSI of nitrogen, the nebulizer is set to cool, the data transfer rate is 2 points per second, and Rayleigh ( 21. The method of claim 20, wherein the Rayleigh) factor is about 6.0.   Standards of 2.0% dulcitol, 1.6% dulcitol, 1.0% dulcitol, 0.60% dulcitol, 0.20% dulcitol, and 0.10% dulcitol to determine system sensitivity and linearity 21. The method according to claim 20, wherein the method is used.   21. The method of claim 20, wherein the retention time of dulcitol is about 6.4 minutes and the retention time of dianhydrogalactitol is about 12.1 minutes.   21. The method of claim 20, wherein the amount and percentage of the dulcitol impurity is determined from HPLC and ELSD results.   21. The method of claim 20, wherein the amount and percentage of unknown impurities other than dulcitol are determined from HPLC and ELSD results.