Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D301/00—Preparation of oxiranes
  • C07D301/02—Synthesis of the oxirane ring
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
  • G01N30/02—Column chromatography
  • G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D303/00—Compounds containing three-membered rings having one oxygen atom as the only ring hetero atom
  • C07D303/02—Compounds containing oxirane rings
  • C07D303/12—Compounds containing oxirane rings with hydrocarbon radicals, substituted by singly or doubly bound oxygen atoms
  • C07D303/14—Compounds containing oxirane rings with hydrocarbon radicals, substituted by singly or doubly bound oxygen atoms by free hydroxyl radicals
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D307/00—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom
  • C07D307/02—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom not condensed with other rings
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
  • G01N30/02—Column chromatography
  • G01N30/62—Detectors specially adapted therefor
  • G01N30/72—Mass spectrometers
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
  • G01N30/02—Column chromatography
  • G01N30/62—Detectors specially adapted therefor
  • G01N30/74—Optical detectors
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
  • G01N30/02—Column chromatography
  • G01N30/84—Preparation of the fraction to be distributed
  • G01N2030/8447—Nebulising, aerosol formation or ionisation
  • G01N2030/8494—Desolvation chambers
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
  • G01N30/02—Column chromatography
  • G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
  • G01N2030/8809—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample
  • G01N2030/8872—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample impurities

Definitions

• This invention is directed to improved analytical methods for
• Dianhydrogalactitol (1 ,2:5,6 dianhydrogalactitol or DAG) is one of a number of hexitols or hexitol derivatives having significant pharmacological activity, including chemotherapeutic activity.
• dianhydrogalactitol has been suggested for use in chemotherapy, such as in United States Patent No. 7,157,059 to Nielsen et al., incorporated herein by this reference.
• Dianhydrogalactitol has activity against a number of neoplasms.
• dianhydrogalactitol is to be used successfully as a therapeutic agent, an extremely high degree of purity and the removal of impurities is essential. The presence of impurities can lead to undesirable side effects.
• impurities present in a batch of the amino acid tryptophan, a normal constituent of protein were responsible for a significant outbreak of eosinophilia- myalgia syndrome, which caused a large number of cases of permanent disability and at least 37 deaths.
• the therapeutic agent such as dianhydrogalactitol is to be employed in patients with compromised immune systems or liver or kidney dysfunction, or in elderly patients. Such patients may experience a greater incidence of undesirable side effects owing to their sensitivity to contaminants.
• dianhydrogalactitol is administered for therapeutic purposes.
• this analytical method employs high performance liquid chromatography (HPLC), in particular, HPLC with refractive index (Rl) detection.
• HPLC high performance liquid chromatography
• Rl refractive index
• an analytical method for analyzing the presence and quantity of impurities present in a preparation of dianhydrogalactitol comprises the steps of:
• the compounds other than dianhydrogalactitol itself can be at least one of: (1 ) dulcitol; (2) an impurity other than dulcitol; and (3) a degradation product of dianhydrogalactitol.
• elution is with a gradient of NaOH from about 2.5 mM to about 0.1 mM.
• elution is with a gradient of NaOH from about 1 .5 mM to about 0.1 mM. More preferably, elution is with a gradient of NaOH from about 1 mM to about 0.1 mM.
• elution is with 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 the ammonium formate and ammonium acetate is from about 2.5 mM to about 0.1 mM.
• the total concentration of the ammonium hydroxide and the volatile ammonium salt selected from the group consisting of ammonium formate and ammonium acetate is from about 1 .5 mM to about 0.1 mM.
• the total concentration of the ammonium hydroxide and the volatile ammonium salt selected from the group consisting of ammonium formate and ammonium acetate is from about 1 mM to about 0.1 mM.
• ammonium acetate is varied from about 100:1 at the beginning of elution to about 1 :100 at the end of elution.
• chromatography that represent compounds other than dianhydrogalactitol itself is performed by evaporative light scattering detection.
• the evaporative light scattering detection is compatible with electrospray LC/MS.
• the evaporative light scattering detection comprises post-column addition of a volatile solvent to enhance evaporation of the 100% aqueous mobile phase.
• the volatile solvent is selected from the group consisting of methanol, ethanol, isopropanol, and acetonitrile.
• an electrospray tandem mass spectrometer is installed and connected on-line to an HPLC system with ELSD.
• mass spectral data providing chemical information for each of the impurities that may be present in a preparation of dianhydrogalactitol is collected.
• tandem mass spectral data providing structural information for each of the impurities that may be present in a preparation of dianhydrogalactitol is collected.
• the method can further comprise the step of performing preparative HPLC collection of at least one specific substance peak present in a preparation of dianhydrogalactitol.
• the at last one substance peak present in the preparation of dianhydrogalactitol can be an impurity.
• isocratic elution can be used.
• the method comprises the steps of:
• the isocratic mobile phase is NaOH, and the concentration of NaOH is from about 5 mM to 0.1 mM.
• the concentration of NaOH is from about 2.5 mM to about 0.1 mM. More preferably, the concentration of NaOH is about 1 mM.
• 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 ammonium hydroxide and the volatile ammonium salt selected from the group consisting of ammonium formate and ammonium acetate is from about 5 mM to 0.1 mM.
• the total concentration of the ammonium hydroxide and the volatile ammonium acetate is from about 2.5 mM to about 0.1 mM.
• the total concentration of the ammonium hydroxide and the volatile ammonium salt selected from the group consisting of ammonium formate and ammonium acetate is about 1 mM.
• the proportion of ammonium hydroxide and the volatile ammonium salt selected from the group consisting of ammonium formate and ammonium acetate is about 50:50.
• chromatography that represent compounds other than dianhydrogalactitol itself is performed by evaporative light scattering detection (ELSD), as described above.
• ELSD evaporative light scattering detection
• the evaporative light scattering detection is compatible with electrospray LC/MS.
• the evaporative light scattering detection comprises post-column addition of a volatile solvent to enhance evaporation of the 100% aqueous mobile phase.
• the volatile solvent is selected from the group consisting of methanol, ethanol, isopropanol, and acetonitrile.
• an electrospray tandem mass spectrometer can be installed and connected on-line to an HPLC system with ELSD.
• mass spectral data providing chemical information for each of the impurities that may be present in a preparation of dianhydrogalactitol is collected.
• tandem mass spectral data providing structural information for each of the impurities that may be present in a preparation of dianhydrogalactitol is collected.
• This alternative of a method according to the present invention can further comprise the step of performing HPLC collection of at least one specific substance peak present in a preparation of dianhydrogalactitol.
• the at last one substance peak present in the preparation of dianhydrogalactitol can be an impurity.
• an analytical method for analyzing the presence and quantity of impurities present in a preparation of dianhydrogalactitol comprises the step of analyzing a preparation of dianhydrogalactitol by subjecting the preparation to high performance liquid chromatography (HPLC) on an HPLC column using elution with a mobile phase gradient to separate dianhydrogalactitol from dulcitol and other contaminants of the preparation; wherein the high performance liquid chromatography employs evaporative light scattering detection (ELSD).
• HPLC high performance liquid chromatography
• ELSD evaporative light scattering detection
• the HPLC column is a silica gel column bonded to C18 compounds and finished with an endcapping procedure employing Lewis acid-Lewis base chemistry.
• the time schedule for varying the eluant 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 to 35 minutes, 95% water/5% acetonitrile.
• the method detects a monoepoxide degradation product of dianhydrogalactitol, a monoepoxide dimer, and dulcitol.
• the method also detects a dimer of dianhydrogalactitol and condensed products.
• the method further comprises a step of determining the relative concentration of one or more peaks resolved by high performance liquid
• the column temperature is about 30° C.
• the flow rate is about 0.5 mL/min.
• the ELSD detector is operated in cooling mode with the drift tube temperature of 35° C and gain 400, 2 pps, 45 PSI.
• Mobile Phase A and Mobile Phase B are employed, with Mobile Phase A being 0.05% formic acid in water and Mobile Phase B being 100% methanol.
• elution is performed from 0 minutes to 25 minutes with 100% of 0.05% formic acid in water, from 25 minutes to 25.1 minutes with 90% of 0.05% formic acid in water and 10% of 100% methanol, from 25.1 minutes to 35 minutes with 10% of 0.05% formic acid in water and 90% of 100% methanol, and from 35.1 minutes to 50 minutes with 100% of 0.05% formic acid in water.
• the method can further comprise the preparation of an external calibration standard curve for an impurity.
• the impurity can be selected from the group consisting of dulcitol, a monoepoxide degradation product of dianhydrogalactitol, and a dimer of dianhydrogalactitol.
• the method can estimate the content of an unknown impurity by using a calibration standard curve established by chromatography of dianhydrogalactitol reference material.
• the dual elution sequence is as follows: a first part of the elution sequence in which elution is performed from 0 minutes to 25 minutes with 100% of 0.05% formic acid in water, from 25 minutes to 25.1 minutes with 90% of 0.05% formic acid in water and 10% of 100% methanol, from 25.1 minutes to 35 minutes with 10% of 0.05% formic acid in water and 90% of methanol, and from 35.1 minutes to 50 minutes with 100% of 0.05% formic acid in water, and a second part of the elution sequence in which elution is performed as follows: from 0 minutes to 7.5 minutes with 100% of 0.05% formic acid; from 7.5 minutes to 7.6 minutes with 97% of 0.05% formic acid and 3% of methanol; and from 7.6 minutes to 20 minutes with 100% of 0.05% formic acid.
• the column temperature for HPLC is about 30° C
• the sample temperature for HPLC is about 5° C
• the flow rate for HPLC is about 0.5 mL/min
• the injection volume is about 10-100 ⁇ .
• 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 cooling
• the data rate is 2 points per second
• the Rayleigh factor is about 6.0.
• standards of dulcitol at 0.1 , 0.08, 0.05, 0.03, 0.01 , 0.005 mg/mL are employed to determine the sensitivity and linearity of the system.
• the retention time for dulcitol is about 6.4 minutes and the retention time for dianhydrogalactitol is about 12.1 minutes.
• the amount and percentage of a dulcitol impurity can be determined from the results of HPLC and ELSD. Also, in this alternative, the amount and
• Figure 1 is a representative HPLC/RI chromatogram of a preparation of dianhydrogalactitol, showing resolution of dulcitol and an unknown related substance at RRT -0.6 in the bulk drug and drug product.
• Figure 2 shows representative HPLC chromatograms showing resolution of dianhydrogalactitol and dulcitol in a standard, and, for comparison, a water blank; in Figure 2, the dianhydrogalactitol-dulcitol standard is shown in the top panel, and the water blank is shown in the bottom panel.
• Figure 3 is a HPLC chromatogram of a dianhydrogalactitol clinical sample using an evaporative light scattering detector for detection showing the existence of a possible dianhydrogalactitol dimer and possible condensed products, along with the monoepoxide and dulcitol as degradation products.
• Figure 4 is a mass spectroscopy profile of the impurity peak occurring at 22.6 minutes of the HPLC chromatogram of Figure 3.
• Figure 5 is a chromatogram of a sample of dianhydrogalactitol as performed in Example 3 employing 0.05% formic acid in water as Mobile Phase A and 100% methanol as Mobile Phase B.
• Figure 6 is an example chromatogram of a blank solution as performed in Example 4.
• Figure 7 is an example chromatogram of an 0.10% dulcitol solution as performed in Example 4.
• Figure 8 is an example chromatogram of a test solution as performed in Example 4.
• This invention is directed to improved analytical methods for determining the purity of dianhydrogalactitol and determining the existence and concentration of impurities present in preparations of dianhydrogalactitol.
• dulcitol The structure of dulcitol is shown below in Formula (II). Other impurities are known to exist in dianhydrogalactitol preparations.
• An improved method of analyzing dianhydrogalactitol preparations is based on HPLC (high performance liquid chromatography) with evaporative light scattering detection (ELSD).
• HPLC high performance liquid chromatography
• ELSD evaporative light scattering detection
• MS mass spectroscopy
• Figure 1 is a
• the present application describes improved HPLC chromatographic conditions for resolution of potentially co-eluting substances.
• a thermally stressed dianhydrogalactitol product sample is evaluated to provide confirmation of the
• LC/MS and LC/MS/MS is performed to characterize the unknown DAG-related substance at RRT ⁇ 0.6 to provide mass spectral characterization and determination of the chemical structure of this unidentified component.
• Previously employed HPLC conditions involve isocratic elution of dianhydrogalactitol and its related substances using a 50 mM NaOH mobile phase.
• a gradient mobile phase is employed.
• NaOH in a concentration gradient.
• elution is with a gradient of NaOH from about 2.5 mM to about 0.1 mM.
• elution is with a gradient of NaOH from about 1 .5 mM to about 0.1 mM.
• elution is with a gradient of NaOH from about 1 mM to about 0.1 mM.
• 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 eluant.
• the total concentration of the ammonium formate and ammonium acetate is from about 2.5 mM to about 0.1 mM.
• the total concentration of the ammonium hydroxide and the volatile ammonium salt selected from the group consisting of ammonium formate and ammonium acetate is from about 1 .5 mM to about 0.1 mM.
• the total concentration of the ammonium hydroxide and the volatile ammonium salt selected from the group consisting of ammonium formate and ammonium acetate is from about 1 mM to about 0.1 mM.
• the proportion of ammonium hydroxide and the volatile ammonium salt selected from the group consisting of ammonium formate and ammonium acetate is varied from about 100:1 at the beginning of elution to about 1 :100 at the end of elution.
• ELSD evaporative light scattering detector
• HPLC analytical methods detection is by means of evaporative light scattering (ELSD).
• ELSD evaporative light scattering detector
• An ELSD atomizes the column eluate, shines light on the resulting particulate components, and detects the resulting scattered light.
• an ELSD can detect any nonvolatile component.
• the evaporative light scattering detection of a non-chromogenic compound is based on nebulization of the HPLC eluant and evaporation of mobile-phase solvents to produce atomizing solute particles for light scattering detection. This nebulization and solvent evaporation process to produce atomizing analyte solute particles is comparable to the electrospray LC/MS procedure.
• the ELSD detection is compatible with electrospray LC/MS.
• the volatile solvent is typically selected from the group consisting of methanol, ethanol, isopropanol, and acetonitrile.
• electrospray tandem mass spectrometer is installed and connected on-line to an HPLC system with ELSD.
• Mass spectral data providing molecular information and tandem mass spectral data providing chemical structural information for each of the impurities that may be present in a preparation of dianhydrogalactitol can be collected.
• Mass spectroscopy in tandem with HPLC will provide molecular ion information and possible chemical structures having a molecular weight consistent with the molecular ion information for each of the observed impurities and degradation products.
• one analytical method for analyzing the presence and quantity of impurities present in a preparation of dianhydrogalactitol comprises the steps of:
• the compounds other than dianhydrogalactitol itself can be at least one of: (1 ) dulcitol; (2) an impurity other than dulcitol; and (3) a degradation product of dianhydrogalactitol.
• the mobile phase gradient is a gradient of sodium hydroxide.
• the mobile phase gradient 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.
• detection is by evaporative light scattering.
• the method further comprises the step of post-column addition of a volatile solvent to enhance evaporation of components of the mobile phase.
• the present invention further comprises the step of analyzing one or more peaks eluting from the high performance liquid chromatography by electrospray tandem mass spectroscopy.
• the present invention further comprises the step of preparative HPLC collection of at least one specific DAG-related substance peak.
• the unknown impurity or degradation product can be identified by separation by column chromatography followed by at least one purification procedure to yield a solid unknown sample which can then be characterized for identification by at least one standard analytical procedure selected from the group consisting of nuclear magnetic resonance (NMR), mass spectroscopy (MS), Fourier transform infrared spectroscopy (FT-IR), elemental analysis, determination of purity by HPLC, and determination of water content by the Karl Fischer titration method. These methods are well known in the art.
• the method comprises:
• the compounds other than dianhydrogalactitol itself can be at least one of: (1 ) dulcitol; (2) an impurity other than dulcitol; and (3) a degradation product of dianhydrogalactitol.
• the elution with the isocratic mobile phase can either be elution with sodium hydroxide or elution with a combination of ammonium hydroxide and a volatile ammonium salt selected from the group consisting of ammonium formate and ammonium acetate.
• the isocratic mobile phase is sodium hydroxide
• the concentration of NaOH is from about 5 mM to 0.1 mM.
• the concentration of NaOH is from about 2.5 mM to about 0.1 mM. More preferably, the concentration of NaOH is about 1 mM.
• 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
• the total concentration of the ammonium hydroxide and the volatile ammonium salt selected from the group consisting of ammonium formate and ammonium acetate is from about 5 mM to 0.1 mM.
• the total concentration of the ammonium hydroxide and the volatile ammonium acetate is from about 2.5 mM to about 0.1 mM. More preferably, the total concentration of the ammonium hydroxide and the volatile ammonium salt selected from the group consisting of ammonium formate and ammonium acetate is about 1 mM.
• the proportion of ammonium hydroxide and the volatile ammonium salt selected from the group consisting of ammonium formate and ammonium acetate is about 50:50.
• an evaporative light scattering detector employing altered elution conditions.
• the HPLC column is a silica gel column bonded to C18 compounds and finished with an endcapping procedure employing Lewis acid-Lewis base chemistry such as the YMC C18 column.
• elution is performed with a gradient of 95% water/5% acetonitrile to 70% water/30% acetonitrile, returning to 95% water/5% acetonitrile.
• the time schedule for varying the eluant 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 to 35 minutes, 95% water/5% acetonitrile.
• the HPLC method detects a monoepoxide degradation product of dianhydrogalactitol, a monoepoxide dimer, and dulcitol. More preferably, the HPLC method also detects a dimer of dianhydrogalactitol and condensed products.
• the peaks resulting from HPLC are analyzed by LC-MS.
• an Atlantis HPLC column is employed. 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 about 10 ⁇ _ to about 100 ⁇ _.
• an ELSD detector is used. Typically, in this method, the ELSD detector is operated in cooling mode with the drift tube temperature of 35° C and gain 400, 2 pps, 45 PSI.
• Mobile Phase A and Mobile Phase B are employed, with Mobile Phase A being 0.05% formic acid in water and Mobile Phase B being 100% methanol.
• elution is performed from 0 minutes to 25 minutes with 100% of 0.05% formic acid in water, from 25 minutes to 25.1 minutes with 90% of 0.05% formic acid in water and 10% of 100% methanol, from 25.1 minutes to 35 minutes with 10% of 0.05% formic acid in water and 90% of 100% methanol, and from 35.1 minutes to 50 minutes with 100% of 0.05% formic acid in water.
• this alternative of the method further comprises the preparation of an external calibration standard curve for an impurity.
• the impurity can be, but is not limited to, an impurity selected from the group consisting of dulcitol, a monoepoxide degradation product of dianhydrogalactitol, and a dimer of dianhydrogalactitol.
• the content of the unknown impurity can be estimated using a calibration standard curve established by chromatography of dianhydrogalactitol reference material.
• Example 4 In another alternative, as shown in Example 4, following the elution sequence described above, namely where elution is performed from 0 minutes to 25 minutes with 100% of 0.05% formic acid in water, from 25 minutes to 25.1 minutes with 90% of 0.05% formic acid in water and 10% of 100% methanol, from 25.1 minutes to 35 minutes with 10% of 0.05% formic acid in water and 90% of 100% methanol, and from 35.1 minutes to 50 minutes with 100% of 0.05% formic acid in water, an additional elution sequence is performed as follows: from 0 minutes to 7.5 minutes with 100% of 0.05% formic acid; from 7.5 minutes to 7.6 minutes with 97% of 0.05% formic acid and 3% of methanol; and from 7.6 minutes to 20 minutes with 100% of 0.05% formic acid.
• the column temperature for HPLC is about 30° C
• the sample temperature for HPLC is about 5° C
• the flow rate for HPLC is about 0.5 mL/min
• the injection volume is about 100 ⁇ .
• 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 cooling
• the data rate is 2 points per second
• the Rayleigh factor is about 6.0.
• standards of dulcitol at 0.005 to 0.1 mg/mL are employed to determine the sensitivity and linearity of the system.
• the retention time for dulcitol is about 6.4 minutes and the retention time for dianhydrogalactitol is about 12.1 minutes.
• the amount and percentage of a dulcitol impurity can be determined from the results of HPLC and ELSD.
• the amount and percentage of an unknown impurity other than dulcitol can be determined from the results of HPLC and ELSD.
• a suitable HPLC system and data acquisition system is an Agilent Technologies 1200 Series HPLC system or equivalent equipped with the following: Quat pump, Model G131 1A or equivalent; auto sampler, Model 1329A or equivalent; RID detector, Model 1362A or equivalent; column temperature controller capable of 30 ⁇ 3° C; and degasser, Model G1322 or equivalent.
• the column is a Hamilton RCX anion exchange column 250 ⁇ 4.1 -mm, 7 ⁇ , P/N 79440, or equivalent.
• Data acquisition is performed by a ChemStation and ChemStore Client/Server or an equivalent data system.
• Water is Milli-Q water or deionized water.
• Sodium hydroxide is standard purified grade.
• Dulcitol and DAG reference standards are of purity > 98.0%.
• the mobile phase 50 mM NaOH
• 2.0 g NaOH is dissolved in 1 liter of water.
• the solution is filtered through an 0.45- ⁇ filter.
• the mobile phase can be stored up to 1 month at room temperature.
• 25 mg of dulcitol reference standard is accurately weighed into a 50-mL volumetric flask.
• the dulcitol is diluted to volume with deionized water and mixed well.
• the prepared stock solution can be stored up to 3 days at 2-8° C.
• the DAG reference stock solution nominal 500 g/mL
• 25 mg of DAG reference standard is accurately weighed into a 50-mL volumetric flask.
• the DAG is diluted to volume with deionized water and mixed well.
• the prepared stock solution can be stored up to 3 days at 2-8° C.
• dulcitol-DAG standard solution dulcitol 50 Mg/mL + DAG 50 Mg/mL; each at 1 % of 5 mg/mL DAG
• 1 .0 ml of dulcitol stock and 1 .0 ml of DAG stock are each quantitatively transferred into a 10-mL volumetric flask, diluted to volume with water and mixed well.
• DAG sample preparation (nominal 5 mg/mL) for an API sample, about 50 mg of the API sample is accurately weighed into a clean 10-mL volumetric flask. The DAG API sample is dissolved in approximately 5 mL of water, diluted to volume with water, and mixed.
• DAG sample preparation from a lyophilized (40 mg/vial) sample
• the sample is removed from the refrigerator in which the sample is stored and the seal removed.
• a volume of water of 5.0 mL is quantitatively transferred and the solution is mixed to dissolve the DAG, yielding an 8 mg/mL solution.
• An aliquot of 1 .0 g of the reconstituted solution is diluted to 8.0 g with deionized water and mixed.
• a further aliquot of 1 to 2 mL of the test sample is transferred into an HPLC vial. Prepared samples can be stored for up to 2 days at 2-8° C.
• the system and the column are equilibrated with HPLC mobile phase at a flow rate of 1 .5 mL/min for at least 30 minutes.
• a sample analysis sequence is created. Once system suitability has been confirmed, a water blank is injected followed by injections of the standards and then the samples. A dulcitol-DAG standard is inserted after every 10 injections of samples and then a last bracketing standard at the end of the run.
• a suitable sample analysis sequence is shown in Table 1 .
• the samples are analyzed using RID.
• a suitable column is a Hamilton RCX ion exchange column (250 ⁇ 4.1 mm, 7 ⁇ ), 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 ⁇ _. Detection is by RID at 35° C.
• the run time is 8 minutes.
• the HPLC software For analysis and integration of the chromatograms, the HPLC software is used. The chromatograms for the blank, the samples, and the test standards are reviewed and compared. Manual integration and assignment of some peaks may be necessary. Integration parameters such as slope sensitivity, peak width, peak height threshold value for rejection, integration type of shoulder peak, baseline, and split peak, as well as other parameters, are adjusted to obtain appropriate integration and values for these parameters are recorded and applied to all samples and standards.
• ⁇ concentration mg/mL
• concentration mg/mL
• the HPLC/RI method does not have sufficient specificity to obtain reliable impurity profile data, which pose the risks of exposure of patients to unacceptable levels of impurities that are unknown or are incompletely characterized.
• a more sensitive detector such as the evaporative light scattering detector (ELSD) manufactured by Agilent, is used in conjunction with HPLC system for determination of impurities found in dianhydrogalactitol drug substance or drug product.
• ELSD evaporative light scattering detector
• a DAG sample was analyzed by HPLC/ELSD method using a YMC C18 column with the gradient shown in Table 3:
• Dianhydrogalactitol was eluted at 10.86 minutes.
• ELSD response is not linear
• an external calibration standard curve is required for a known impurity, such as dulcitol, to determine the impurity content in a dianhydrogalactitol sample tested.
• the unknown impurity content can be estimated using a calibration standard curve established by chromatography of dianhydrogalactitol reference material.
• a further improved analytical method for the detection or determination of impurities in dianhydrogalactitol employs HPLC and ELSD with dual-gradient elution in HPLC. This method is described below.
• the following materials and equipment are used: an Atlantis C18, 250 ⁇ 4.6-mm, 5- ⁇ HPLC column; a quaternary or binary HPLC pump; an Evaporative Light Scattering Detector (ELSD); an integrator or computer- based analytical system; a calibrated analytical balance; and Class A volumetric flasks and pipettes.
• the following reagents and standards are used: a dulcitol reference standard as described above; HPLC grade water; HPLC grade or equivalent fornnic acid (FA); HPLC grade or equivalent acetonitrile (ACN); and HPLC grade or equivalent methanol (MeOH).
• the volume may be scaled to suit the needs of the analysis. It is important that all mobile phases are filtered.
• the sintered glass in the filtration apparatus may be a source of buffers that may interfere with sensitivity in the ELSD. All filtration apparatus should be rinsed thoroughly with Milli-Q grade water. To perform this, approximately 500 mL of Milli-Q grade water is filtered through the filtration apparatus. The water is discarded and the mobile phase is then filtered.
• Test solution preparations are to be made in a fume hood using appropriate PPE (gloves, lab coat, and safety glasses). Test solution preparations are to be stored in a fume hood for disposal and are to be labelled appropriately.
• PPE gloss, lab coat, and safety glasses
• Mobile Phase A is prepared by pipetting 0.5 mL of formic acid into 1000 mL of water and mixing well. The Mobile Phase A is filtered and degassed.
• Mobile Phase B is MeOH.
• the Mobile Phase B is filtered and degassed.
• Diluent A is water.
• Diluent B is prepared by mixing 20 mL of ACN with 180 mL of water and mixing well.
• the standard and sample solution preparation is described below.
• the blank solution is water.
• the dulcitol stock solution is prepared by accurately transferring 100 mg of dulcitol reference standard to a 20-mL volumetric flask. About 15 mL of Diluent B is added and sonicated to dissolve. The solution is allowed to cool down to room temperature and diluted to volume with Diluent B and mixed well (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.
• the solution is diluted to volume with water and mixed well (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 to volume with water and mixed well (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 to volume with water and mixed well (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 to volume with water and mixed well (0.05 mg/mL).
• An 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 to volume with water and mixed well (0.30 mg/mL).
• An 0.20% standard solution is prepared by pipetting 1 .0 mL of 4.0% standard solution into a 20-mL volumetric flask. The solution is diluted to volume with water and mixed well (0.01 mg/mL).
• An 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. The solution is diluted to volume with water and mixed well (0.005 mg/mL).
• Test sample working solutions are to be prepared in duplicate (A and B). Sample solutions must be prepared just before analysis. Sample injections must be performed within 15 minutes of sample solution preparation. Sample dilution may be required, in some cases, to quantitate any impurities that are overloaded.
• test sample preparation approximately 50 mg of test sample is accurately transferred into a 10-mL volumetric flask. The test sample is dissolved in water and diluted to volume and mixed well (5 mg/mL).
• the HPLC operating conditions are as follows: The column is the Atlantis C18 250 ⁇ 4.6-mm, 5- ⁇ HPLC column. Mobile Phase A is 0.05% FA in water. Mobile Phase B is MeOH. Gradients A and B are described below in Table 6. 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 ⁇ _. The run time is 50 minutes for Gradient A and 20 minutes for Gradient B.
• the ELSD operating conditions are as follows: The gain is 400. The drift tube temperature is 45° C. The gas pressure (nitrogen) is 35 PSI. The nebulizer is set to cooling. The data rate is 2 points per second. The Rayleigh factor, set directly in the detector, is 6.0.
• test sample injections may be added as required. No more than 6 test sample solution injections are to be performed before repeating the 0.20% standard solution check injection.
• the HPLC conditions are as follows: The column is to be removed from the instrument and a union is to 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 ambient temperature. The run 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 heating at 75%.
• Typical retention times are shown in Table 8.
• DAG is dianhydrogalactitol. DAG in the test sample is not quantitated in this method. DAG is observed as a wide peak due to the concentration of DAG required. Retention time for DAG in sample solution is approximately between 10 and 13 minutes.
• Figure 6 is an example chromatogram of a blank solution.
• Figure 7 is an example chromatogram of 0.10% standard solution (system sensitivity solution).
• Figure 8 is an example chromatogram of a test solution.
• the USP tailing factor for the dulcitol peak for the first and last injections of the 0.20% standard solution is no more than 2.0.
• the % RSD for the log of peak area in the five injections is calculated. The % RSD must be no more than 15%.
• quantitation using dulcitol standards may be formed using log-log linear function in Empower (Waters Corp.) [0122] Similar equations, specifically Equations (4)-(6), are used for the determination of the dulcitol impurity in the sample.
• % Dulcitol Dulcitol concentration (mg/mL) ⁇ 100
• the present invention provides an improved analytical method for the detection and quantitation of impurities present in dianhydrogalactitol preparations, including dulcitol and unknown impurities, as well as methods for isolation and identification of unknown impurities present in dianhydrogalactitol preparations.
• the methods of the present invention allow the large-scale preparation of dianhydrogalactitol of high purity suitable for pharmaceutical use and reduce the possibility of significant side effects caused by the presence of impurities in dianhydrogalactitol preparations intended for pharmaceutical use.
• the invention encompasses each intervening value between the upper and lower limits of the range to at least a tenth of the lower limit's unit, unless the context clearly indicates otherwise. Moreover, the invention encompasses any other stated intervening values and ranges including either or both of the upper and lower limits of the range, unless specifically excluded from the stated range.

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