WO2005016930A2

WO2005016930A2 - Methods for detecting morphinone

Info

Publication number: WO2005016930A2
Application number: PCT/US2004/023754
Authority: WO
Inventors: Saji K. Thomas; Dale M. Roberts
Original assignee: Euro-Celtique, S.A.
Priority date: 2003-08-08
Filing date: 2004-07-23
Publication date: 2005-02-24
Also published as: WO2005016930A3

Abstract

Disclosed is a method of detecting the presence of morphinone in a hydromorphone preparation.

Description

METHODS FOR DETECTING MORPHINONE

FIELD OF THE INVENTION

An improved method is disclosed for detecting the presence of morphinone in a hydromorphone preparation.

BACKGROUND OF THE INVENTION There is a continuing need for improved methods to determine the presence and amount of impurities in pharmaceutical preparations.

In the pharmaceutical industry, pharmaceutical compounds must be inspected to determine the presence of undesirable impurities. To be detected, the amount of impurity must usually be present above a certain threshold level which depends upon the assay technique utilized. For example, if a particular assay has a limit of quantitation of 100 ppm for a particular impurity in a sample, that assay would generally be unable to determine if the impurity were present in any amount less than 100 ppm. Such limitations can be especially problematic in assaying pharmaceutical preparations for the presence of toxic impurities.

Hydromorphone hydrochloride (commercialized as Dilaudid®, Laudicon®, Hydromorphan®) is a narcotic analgesic widely prescribed for the treatment of pain (Physicians Desk Reference, 55^th Ed., p. 1619 (2001); Merck Index, 12^th Ed., 4847). The precise mechanism of action of hydromorphone is not known, although it is believed to involve interaction with opiate receptors in the central nervous system. There is no intrinsic limit to the analgesic effect of hydromorphone; like morphine, even the most severe pain can be relieved given a sufficient amount of hydromorphone. Hydromorphone is also a centrally acting narcotic anti-tussive, which acts directly on the cough reflex center.

Typical HPLC detection and quantitation of impurities in hydromorphone, may be performed in accordance with Example 5 of U.S. Patent Nos. 6,512,117 and 6,589,960 to Harclerode et al. The '117 and '960 patents describe the detection and quantitation of impurities in hydromorphone compositions carried out using an elution gradient high performance liquid chromatography (HPLC) method. This method can be used to resolve and quantitate 8-hydroxyhydromorphone, morphine, dihydromorphine, hydromorphone N-oxide, hydromorphone, and 2,2-bishydromorphone, which are impurities of hydromorphone.

Morphinone, another impurity found in hydromorphone preparations, can be hepatotoxic. The limit of quantitation of morphinone in hydromorphone preparations utilizing HPLC according to art-recognized procedures is about 500 ppm. This is problematic in view of the hepatotoxic potential of morphinone.

There exists a need in the art to provide improved methods for detecting lower levels of morphinone in hydromorphone preparations.

OBJECTIONS AND SUMMARY OF THE INVENTION It is an object of the present invention to provide an improved method for detecting impurities in a hydromorphone preparation.

It is an object of certain embodiments of the present invention to provide an improved method for detecting morphinone in a hydromorphone preparation.

It is a further object of certain embodiments of the present invention to provide a method for detecting the presence of impurities in a hydromorphone preparation, which method exhibits increased sensitivity and higher cost efficiency.

It is a further object of certain embodiments of the present invention to provide a method for detecting morphinone in a hydromorphone preparation when the morpliinone is present in the preparation in an amount of less than 500 ppm. In accordance with the above objects, the present invention provides, in part, a method of determining the amount of morphinone contained in a hydromorphone preparation comprising: (a) preparing a first sample solution comprising hydromorphone from the hydromorphone preparation, with the hydromorphone in the first sample solution at a concentration sufficient to quantify an amount of morphinone contained therein utilizing an HPLC system; (b) preparing a second sample solution comprising hydromorphone from the hydromorphone preparation, with the hydromorphone in the second sample . solution at a concentration sufficient to quantify the hydromorphone contained therein utilizing the HPLC system, wherein the second sample solution has a concentration of the hydromorphone that is less than that of the first sample solution; (c) analyzing the first sample solution using the HPLC system to obtain a measurable peak area of morphinone; (d) analyzing the second sample solution using the HPLC system to obtain a measurable peak area of hydromorphone; and (e) determining the amount of morpliinone in the hydromorphone preparation based on the analysis of the first and second sample solutions.

In certain embodiments, the invention is directed to a method of determining the amount of morpliinone contained in hydromorphone preparation, comprising: (a) preparing a first sample solution comprising hydromorphone from the hydromorphone preparation, with the hydromorphone in the first sample solution at a concentration from about 10 mg/ l to about 50 mg/ml for analysis in an HPLC system; (b) preparing a second sample solution comprising hydromorphone from the hydromorphone preparation, with the hydromorphone in the second sample solution at a concentration sufficient to quantify the hydromorphone contained therein using the HPLC system, wherein the second sample solution has a concentration of hydromorphone less than that of the first sample solution; (c) analyzing the first sample solution using the HPLC system to obtain a measurable peak area of morphinone; (d) analyzing the second sample solution using the HPLC system to obtain a measurable peak area of hydromorphone; and (e) determimng the amount of morphinone present in the hydromorphone preparation based on the analysis of the first and second sample solutions.

In certain embodiments, the invention is directed to a method of determining the amount of morphinone contained in a hydromorphone preparation, comprising: (a) preparing a first sample solution comprising hydromorphone from the hydromorphone preparation, with the hydromorphone in the first sample solution at a concentration from about 10 mg/ml to about 50 mg/ml for analysis in an HPLC system; (b) preparing a second sample solution comprising hydromorphone from the hydromorphone preparation, with the hydromorphone in the second sample solution at a concentration from about 0.01 mg/ml to about 5 mg/ml for analysis in the HPLC system; (c) analyzing the first sample solution using the HPLC system to obtain a measurable peak area of morphinone; (d) analyzing the second sample solution using the HPLC system to obtain a measurable peak area of hydromorphone; (e) determining the amount of morpliinone in the hydromorphone preparation by dividing the peak area of morphinone in the first sample solution by (the peak area of hydromorphone in the second sample solution multiplied by (the concentration of hydromorphone in the first sample solution divided by the concentration of hydromorphone in the second sample solution) multiplied by the morphinone RRF) to obtain a quotient.

In certain embodiments, the invention is directed to a method of determining the amount of morphinone contained in a hydromorphone preparation, comprising: . (a) preparing a first sample solution comprising hydromorphone from the hydromorphone preparation, with the amount of hydromorphone in the first sample solution at a concentration sufficient to quantify an amount of morphinone contained therein using an HPLC system; (b) preparing a second sample solution comprising hydromorphone from the hydromorphone preparation, with the amount of hydromorphone in the second sample solution at a concentration sufficient to quantify an amount of hydromorphone contained therein using the HPLC system, wherein the second sample solution has a concentration of hydromorphone that is less than that of the first sample solution; (c) analyzing the first sample solution using the HPLC system comprising a first mobile phase adjusted to an apparent pH of about 6.5 to about 7.5 and an HPLC column maintained at a temperature of about 20 degrees C to about 60 degrees C, to obtain a measurable peak area of morphinone; (d) analyzing the second sample solution using the HPLC system to obtain a measurable peak area of hydromorphone; and (e) determining the amount of morphinone in the hydromorphone preparation based on the analysis of the first and second sample solutions.

In certain embodiments, the invention is directed to a method of determining the amount of morphinone contained in a hydromorphone preparation, comprising: (a) preparing a first sample solution comprising hydromorphone from the hydromorphone preparation, with the hydromorphone in the first sample solution at a concentration from about 10 mg/ml to about 50 mg/ml for analysis in an HPLC system; (b) preparing a second sample solution comprising hydromorphone from the hydromorphone preparation, with the hydromorphone in the second sample solution at a concentration from about 0.01 mg/ml to about 5 mg/ml for analysis in an HPLC system; (c) analyzing the first sample solution using an HPLC system comprising a first mobile phase adjusted to an apparent pH of about 6.5 to about 7.5 and an HPLC column maintained at a temperature of about 20 degrees C to about 60 degrees C to obtain a measurable peak area of morphinone; (d) analyzing the second sample solution using the HPLC system to obtain a measurable peak area of hydromorphone; and (e) determining the amount of morphinone in the hydromorphone preparation by dividing the peak area of morphinone in the first sample by (the peak area of hydromorphone in the second sample solution multiplied by (the concentration of hydromorphone in the first sample solution divided by the concentration of hydromorphone in the second sample solution) multiplied by the morphinone RRF) to obtain a quotient.

In certain embodiments, the invention is directed to a method of determining the amount of morphinone contained in a hydromorphone preparation, comprising: (a) preparing a first sample solution comprising hydromorphone from the hydromorphone preparation, with the hydromorphone in the first sample solution at a concentration sufficient to quantify an amount of morphinone contained therein using a first HPLC system; (b) preparing a second sample solution comprising hydromorphone from the hydromorphone preparation, with the hydromorphone in the second sample solution at a concentration sufficient to quantify the hydromorphone contained therein using a second HPLC system, wherein the second sample solution has a concentration of hydromorphone that is less than that of the first sample solution; . (c) analyzing the first sample solution using the first HPLC system to obtain a measurable peak area of morphinone; (d) analyzing the second sample solution using the second HPLC system to obtain a measurable peak area of hydromorphone, wherein the second HPLC system is the same or different than the first HPLC system; and (e) determining the amount of morphinone in the hydromorphone preparation based on the analysis of the first and second sample solutions. Unless otherwise indicated, the term "hydromorphone" includes both the hydromorphone free base and any salt form of hydromorphone that can be formed, including any pharmaceutically acceptable salt of hydromorphone, e.g., hydromorphone hydrochloride. Other pharmaceutically acceptable salts include, but are not limited to, metal salts such as sodium salt, potassium salt, secium salt and the like; alkaline earth metals such as calcium salt, magnesium salt and the like; organic a ine salts such as. triethylamine salt, pyridine salt, picoline salt, ethanolamine salt, triethanolamine salt, dicyclohexylamine salt, N,N'-dibenzylethylenediamine salt and the like; inorganic acid salts such as hydrobromide, sulfate, phosphate and the like; organic acid salts such as formate, acetate, trifluoro acetate, maleate, tartrate and the like; sulfonates such as methanesulfonate, benzenesulfonate, p-toluenesulfonate, and the like; amino acid salts such as arginate, asparginate, glutamate and the like. The pharmaceutically acceptable salts can include anhydrous forms and hydrous forms, e.g., monohydrates and dihydrates.

The term "hydromorphone preparation" means any composition of matter containing hydromorphone that can be quantified for morphinone and hydromorphone utilizing the methods of the present invention. The hydromorphone preparation can be, e.g., a hydromorphone active pharmaceutical ingredient e.g., hydromorphone hydrochloride U.S.P., which is utilized in the formulation of a pharmaceutical dosage form. Alternatively, the hydromorphone preparation can be a final dosage form, or an intermediate stage preparation for a final dosage form, that can be tested for the presence of morphinone for, e.g., quality assurance purposes.

The term "ppm" as used herein with respect to morphinone is parts per million to a sample of hydromorphone active pharmaceutical ingredient. The hydromorphone active pharmaceutical ingredient can be uncombmed with other ingredients, or combined with other ingredients, e.g., in a dosage form.

For purposes of the present invention, the term "relative response factor" or "RRF" is a correction factor applied in HPLC to correct for the difference in response of different compounds, e.g., impurities, to the response of the reference standard. BRIEF DESCRIPTION OF THE DRAWINGS Fig. 1 is an HPLC chromatogram of a sample of hydromorphone active pharmaceutical ingredient.

DETAILED DESCRIPTION OF THE INVENTION Using HPLC techniques known in the art, chromatographic peaks representing morphinone at levels below 500 ppm cannot be accurately quantified. The present invention provides an HPLC method to detect the presence of morphinone in an amount less than 500 ppm. This method involves overloading the concentration of hydromorphone in a first sample solution in order to increase the peak area of morphinone to a level that can be accurately determined. Certain adjustments in HPLC conditions can also be implemented to "sharpen" the morphinone HPLC peak.

In certain embodiments of the present invention, the increased concentration of the overloaded hydromorphone in the first sample solution results in an increase in the peak area of hydromorphone beyond the upper threshold that can be measured accurately. Accordingly, a second sample solution is prepared at a lower concentration of hydromorphone, to produce a chromatographic peak for hydromorphone that is on-scale, and the area of which can be accurately quantified. This second solution can be prepared by diluting a portion of the first sample solution, by preparing a less concentrated solution, or by any other method, as long as the differences in concentration are known.

The peak area of morphinone from the high concentration first sample solution, and the peak area of hydromorphone from the lower concentration second sample solution can be used to calculate the amount (e.g., percentage) of morphinone in the hydromorphone preparation being analyzed. An example of making such a calculation is by dividing the peak area of morphinone by (the peak area of hydromorphone multiplied by (the concentration of hydromorphone in the first sample solution divided by the concentration of morpliinone in the second sample solution) multiplied by the morphinone RRF) to obtain a quotient. The quotient can then be optionally multiplied by 100 to obtain a percentage.

The method of the present invention can provide for the detection of morphinone levels in a hydromorphone preparation where the morphinone is present in an amount of less than 500 ppm, more preferably less than 400 ppm, more preferably less than 300 ppm, more preferably less than 200 ppm, more preferably less than 100 ppm, more preferably less than 50 ppm, more preferably less than 25 ppm, and more preferably less than 10 ppm. In certain embodiments, the method of the present invention provides for the detection of morphinone levels in a hydromorphone preparation where the morphinone is present in an amount of from about 1 ppm to 499 ppm or from about 1 ppm to about 100 ppm; preferably from about 1 ppm to about 10 ppm, or from about 1 ppm to about 5 ppm; more preferably from about 5 ppm to about 25 ppm or from about 5 ppm to about 10 ppm.

An HPLC system typically includes at least the following components: an HPLC column packed with a suitable stationary phase; a mobile phase; a pump for directing the mobile phase through the column; and a detector for detecting the presence of compounds eluting from the column.

Routine methods and apparatus for carrying out HPLC separations are well known in the art, and are described, for example, in J. Chromatography, 192:222-227 (1980); J. Liquid Chromatography 4:661-680 (1981); and J. Chromatography, 249:193- 198 (1982), among many other publications.

In certain embodiments, the HPLC system is an adsorption chromatography system, an ion-exchange chromatography system, a size exclusion chromatography system, or the like. Preferably the HPLC system is an adsorption chromatography system such as, for example, a normal phase chromatography system or a reverse phase chromatography system. In a more preferred embodiment, the system is a reverse, phase chromatography system. Reverse-phase chromatography involves contacting a solution of a desired compound with a solid, hydrophobic support, or stationary phase, under conditions whereby the compound is adsorbed to the support. The compound is then eluted, after washing, by rinsing the support with an apolar organic solvent (i.e., the mobile phase). The stationary phase can comprise a support such as alumina, or can be a silica-based support, to which is bonded various non-polar organic groups. Such bonded phases may be prepared, for example, by reacting surface silanol groups on the silica with an organo- chlorosilane, as known in the art. Silica-based supports include, for example, spherical silica particles, irregular silica particles or particulate substrates coated with silica. The particle size and porosity should be appropriately selected for separation of the specific components in the assay.

The mobile phase selected for reverse-phase HPLC should have low toxicity and viscosity and be readily available in pure form. The mobile phase may be selected from the group consisting of water, miscible lower alcohols (e.g. methanol, n-propanol, or isopropanol), tetrahydrofuran, dioxane, acetonitrile, and mixtures thereof.

In order to resolve the peak of mo hinone provided by the first sample solution, the temperature and pH of the system, among other factors, may be modified to preferred parameters, such as those discussed below. Preferably, the resolution between hydromorphone and morphinone according to U.S. P. methodology is preferably at least 3, preferably at least 4, more preferably at least 5, and most preferably at least 6.

In certain embodiments of the present invention, the mobile phase of the HPLC system is modified in order to control the pH on the column. In certain embodiments, the mobile phase is adjusted to an apparent pH of from about 6.5 to about 7.5, preferably to an apparent pH of from about 6.8 to about 7.2 and most preferably to an apparent pH of about 7.0. In certain embodiments of the present invention, the temperature of the column of the HPLC system is controlled. In certain embodiments, the temperature of the column is maintained from about 20 degrees C to about 60 degrees C, preferably from about 30 degrees C to about 50 degrees C, more preferably from about 40 degrees C to about 45 degrees C, and most preferably at about 45 degrees C.

In certain embodiments, the signahnoise ratio of the system is preferably at least 5:1, preferably at least 10:1, more preferably at least 15.T and most preferably at least 20:1.

The methods of the present invention include the use of at least one mobile phase, which acts as a carrier for the sample solution. The chemical interactions of the mobile phase and sample with the column determine the degree of migration and separation of the components of the sample. In certain preferred embodiments, the methods of the present invention include the use of a first mobile phase and a second mobile phase.

In certain embodiments, the methods of the present invention include performing HPLC through the use of isocratic elution (isocratic mobile phase) or gradient elution (gradient mobile phase). In isocratic elution, for example, compounds are eluted using a mobile phase having a constant composition. The compounds migrate through the column at onset, with each compound migrating at a different rate, resulting in separation of the compounds. In gradient elution, for example, different compounds may be eluted as the composition of the mobile phase changes, e.g., by increasing the strength and/or the concentration of the organic solvent. For example, the sample may be injected during application of a "weaker" mobile phase through the system. The mobile phase may be gradually or incrementally changed by, e.g., increasing the fraction of the mobile phase comprising the organic solvent, resulting in elution of retained compounds.

The mobile phase(s) of the present invention preferably includes an eluent comprising, for example, acetonitrile, dioxane, ethanol, methanol, isopropanol, tetrahydrofuran, water, or a mixture thereof. In one embodiment, the mobile phase is acetonitrile. In another embodiment, the mobile phase is methanol. In certain preferred embodiments, wherein the methods of the present invention utilize a first mobile phase and a second mobile phase, the first mobile phase may comprise one solvent or a combination of solvents, and the second mobile phase consists of an organic solvent.

Typical adsorbents in the HPLC column for use in the present invention include, for example and without limitation, EB-Sil C18, Prodigy ODS, Selectosil C18, Ultracarb ODS, Zorbax ODS, Kromasil C18, LiChrospher RP-18, Inertsil ODS-2, Nucleosil C18, Spherisorb ODS, Hypersil C18, Novapak C18, and Symmetry C18. Preferably the adsorbent is Waters Symmetry C18.

In one embodiment of the present invention, the eluent for use in the HPLC methods of the present invention comprises acetonitrile, sodium phosphate monobasic monohydrate, sodium dodecyl sulfate, water, or a mixture thereof.

In certain embodiments, the mobile phase(s) utilized in the present invention for isocratic elution comprises from about 50% (v/v) aqueous medium to about 85% aqueous medium, and preferably from about 60% aqueous medium to about 75% aqueous medium. In certain embodiments, the mobile phase comprises from about 50% acetonitrile to about 15% acetonitrile, preferably from about 40% acetonitrile to about 25% acetonitrile. In certain embodiments, the mobile phase comprises about 50% aqueous medium and about 50% acetonitrile, preferably about 60% aqueous medium and about 40% acetonitrile, or about 75% aqueous medium and about 25% acetonitrile. In certain of the above ranges for acetonitrile, methanol can be substituted for all of, or a portion of the acetonitrile. Alternatively, an appropriate gradient elution profile may be selected to carry out the methods of the present invention.

In certain embodiments, the mobile phase(s) of the present invention is (are) delivered at a rate of from about 1.0 to about 2.0 ml per minute, preferably at a rate of about 1.5 ml per minute. As used herein, an "HPLC compatible detector" is any detector capable of generating a measurable or detectable signal when a compound elutes from the column of an HPLC. Where component absorbance varies widely, it may be necessary to utilize more than one detector. A detector capable of detecting a desired component is not "incompatible" simply due to its inability to detect a non-desirbd component. The detector can be a refractive index detector, an ultra-violet detector, a fluorescent detector, a radiochemical detector, an electrochemical detector, a near-infrared detector, a mass spectroscopy detector, a nuclear magnetic resonance detector, a light scattering detector, or any other detector known in the art.

In certain embodiments, the detector is an ultra-violet detector. In certain embodiments, the ultra-violet detector is selected from the group consisting of a fixed wavelength detector, a variable wavelength detector and a diode array detector. Preferably the ultra-violet detector is a fixed wavelength detector. In certain embodiments the fixed wavelength detector measures at a wavelength of from about 200 n to about 275 nm, preferably at a wavelength of about 220 run.

The present invention comprises the use of two sample solutions, a first, more concentrated sample solution used to detect the morphinone and a second, less concentrated sample solution used to detect the hydromorphone. In certain embodiments of the present invention, the concentration of the sample of hydromorphone for use in the first sample solution is from about 10 mg/ml to about 50 mg/ml; preferably, from about 15 mg/ml to about 35 mg/ml; and more preferably about 25 mg/ml.

In certain embodiments, the concentration of hydromorphone in the second sample solution is from about 2 times to about 500 times less than the concentration of hydromorphone in the first sample solution; preferably from about 10 times to about 250 times less than the concentration of hydromorphone in the first sample solution; more preferably from about 50 times to about 100 times less than the concentration of hydromorphone in the first sample solution. Li a preferred embodiment, the concentration of hydromorphone in the second sample solution is from about 0.01 mg/ml to about 10 mg/ml, preferably about 0.10 mg/ml to about 2 mg/ml, and more preferably about 0.25 mg/ml.

In certain embodiments, the HPLC apparatus comprises an auto injector with a preferable injection volume of from about 10 microliters to about 100 microliters, from about 25 microliters to about 75 microliters, or about 50 microliters.

EXAMPLE 1 A preparation of hydromorphone HC1 was dissolved in 0.85% phosphoric acid solution and analyzed for morphinone content by HPLC using a 5 μm reverse-phase, Waters Symmetry C18 column (3.9 x 150 mm) at 45 °C and a mobile phase (prepared as described below) consisting of acetonitrile, water, sodium phosphate monobasic monohydrate and sodium dodecyl sulfate, the mobile phase adjusted to an apparent pH of 7.0. Quantitation was achieved by measuring the peak area response at 220nm. Calculation was based on area normalization and a relative response factor of 1.44 for morphinone to hydromorphone HC1.

None of the known related substances of hydromorphone HC1 eluting earlier than hydromorphone (e.g., morphine N-oxide, hydromorphone N-oxide, morphine and dihydromorphine) interfere with the analysis of morphinone, and none of these were quantitated using this method. Typical retention times and relative retention times for all early eluting related substances of hydromorphone HC1, as well as the relative response factor for morphinone are listed in Table 1 below. Table 1

* The relative response factor is relative to hydromorphone HC1.

The reagents used for Example 1 were as follows:

(Equivalent reagents may be used in place of those specified). 1. Sodium phosphate, monobasic, monohydrate, crystal analytical reagent grade; 2. Water, HPLC grade or equivalent, 16 megaohm-cm or greater, or purified water; 3. Phosphoric acid 85%, analytical reagent grade; 4. Acetonitrile, HPLC grade; 5. Sodium dodecyl sulfate, 99% + reagent grade; 6. Sodium hydroxide pellets, commercial 50% w/v NaOH or 2 N NaOH, ACS reagent grade; 7. Hydromorphone HC1 working reference standard (WRS), USP reference standard or any raw material that contains more than 0.05% peak area response of morphinone. Equipment used for Example 1 The equipment used for Example 1 was as follows:

A. HPLC System 1. HPLC pump capable of delivering mobile phase at 1.5 mL/minute; 2. Multiwavelength detector set at 220 nm; 3. Autoinjector capable of 50 μL injections for standard and sample solutions; 4. Integrator or suitable data recording system; 5. Waters Symmetry C column (3.9 x 150 mm, 5 microns); 6. Column heater capable of maintaining a constant temperature of 45 °C. Pre- column plumbing is preferably heated to column oven temperature to minimize peak fronting of the hydromorphone.

B. Mobile Phase Filtration System HPLC filtration Assembly, 47 mm ULTRA-WARE all glass, from Kimble and Nylon-66 membrane filter (0.45 μm) from Rainin.

C. Ultrasonication Bransonic model 8510, input power 320 watts, Branson Corp.

Solutions for Example 1 The solutions used for Example 1 were prepared as follows:

A. 50% NaOH Solution

Carefully and slowly, 50 ± 2 g of sodium hydroxide pellets were transferred into a 250-mL beaker containing 100 mL of water, and sonicated for about 5 minutes until the pellets were completely dissolved, and then mixed well.Commercially available 50% w/v NaOH or 2 N NaOH solutions can also be used for adjusting the mobile phase pH. B. 0.85% Phosphoric Acid

1 mL of 85% phosphoric acid was diluted to a volume of 100 mL with water and mixed well.

C. Mobile Phase A

2.76 g ± 0.10 g of sodium phosphate monobasic monohydrate was added to a 2-L beaker, followed by 800 mL of water, and the solution was stirred with a magnetic stirrer until dissolved. 200 mL of acetonitrile and 4.33 g ± 0.10 g of sodium dodecyl sulfate were added and the solution was mixed well. The pH was adjusted with 50% NaOH solution to a final pH of 7.0 ± 0.1. The mobile phase was filtered using the filtration system described in the equipment section above, and degassed using a vacuum, ultrasonication_. or another suitable means.

D. Mobile Phase B 100% Acetonitrile

E. Resolution Solution for System Suitability

32 ± 5 mg to the nearest 0.1 mg of Hydromorphone HC1 WRS, USP reference standard was weighed and transferred into a 100-mL amber glass light-sensitive volumetric flask. The material was dissolved and diluted to volume with 0.85% phosphoric acid.

F. Sample Preparation

The sample solutions were prepared as follows: 1. First Sample Solution (High Concentration) (Equivalent to 25 mg/mD 50 ± 5 mg of hydromorphone HC1 API sample material was weighed to the nearest 0. Img and placed into a suitable container. The sample material was dissolved with 2.0 mL of 0.85% phosphoric acid and mixed well.

2. Second Sample Solution (Low Concentration) (Equivalent to 0.25 mg/mL) 1.0 mL of the high sample solution was pipetted into a 100-mL amber glass light- sensitive volumetric flask and diluted to volume with 0.85% phosphoric acid and mixed well.

The HPLC conditions for Example 1 The HPLC conditions for Example 1 were as follows:

1. HPLC Conditions Column: Waters Symmetry C18 (3.9 mm X 15 cm), 5 micron particle size Gradient Composition A: mobile phase A Gradient Composition B: mobile phase B Gradient Program* Time (minutes) A (%) B (%) Initial 100 0 - 25 100 0 Flow Rate: 1.5 mL/minute Column Temperature: 45 °C Injection Volume: 50 μL Detection Wavelength: 220 nm Injection vial: 1 or as required

To wash the column after injecting the first (high concentration) sample solution, a gradient method with increased organic content was applied after 25 minutes run time. The gradient program presented in Table 2 for the running the first sample solution worked adequately: Table 2

Fifty μL of the resolution solution (the working reference standard) was injected five times and the blank solution (0.85% phosphoric acid) once After confirming that there were no unidentified peaks in the solvent and no contamination of the auto-injector, a system suitability test was performed in order to determine the resolution between hydromorphone and morphinone using U.S.P methodology For purposes of the present invention, the resolution between hydromorphone and morphinone according to U.S.P methodology is preferably at least 3, preferably at least 4, more preferably at least 5, and most preferably at least 6.

Sample analysis for Example 1 The sample analysis for Example 1 was performed as follows:

Fifty μL of the hydromorphone HC1 API first sample solution and second sample solution were each subjected to HPLC analysis , and the resulting chromatograms were examined by changing the Y-scale of area count (Y-axis) to observe and quantify the morphinone and hydromorphone HC1 peaks. These chromatograms could be reintegrated if necessary. The morphinone peak was identified using the relative retention time for morphinone (see Table 1 above), and the amount of morphinone was quantified according to the calculation below. EXAMPLE 2 In Example 2, the following calculations were performed using the results obtained in Example 1, above.

Using Millennium software, the processing method parameters (see "Suitability" tab) for calculating the signal-to-noise ratio were entered in the "Baseline Noise and Drift Measurements" section. The following parameters are suggested for signal-to-noise calculations:

Signal-to-noise calculations Baseline Start Time (min) = [approximate Morphinone retention time (min)] - B/2 Baseline End Time (min) = [approximate Morphinone retention time (min)] + B/2

Make "B" equal to the length of the baseline used, in minutes. A baseline section of at least 2 minutes is suggested, which should be centered on the morphinone retention time.

% Run Time over Which to Average = Baseline End Time - Baseline Start Time 100% 2 x Total Run Time

Make sure that no peaks or eluting components are present in the baseline section.

After the baseline was stable, the baseline noise in microvolts (note that Millennium will calculate peak height in v and the conversion to μv is necessary) was deteraiined for a blank solution (0.85% phosphoric acid) at the approximate two-minute window for morphinone. Using the signal response (μv) and the area percent of morphinone obtained from the initial system suitability injections, the signal to noise ratio for morphinone at 0.05% level was calculated according to the following equation.

, „ ,- . _, .. SiαnalResponseof Morphinone 0.05% Sιgnalto NoιseRatιo= — ^Ξ — _{: :} — - x BaselineNoise Area Percent of Morphinone

Percent of Morphinone

Percent of Morphinone = Peak Area of Morphinone of High Sample Solution x 100 % Peak Area of Hydromorphone HCl of Low Sample Solution x 100 x Morphinone RRF

Set B = 4, Total Rim Time = 25 minutes, and Morphinone retention time = 10.9 minutes

Baseline Start Time (min) « 10.9 - (4/2) « 8.9 Baseline End Time (min) - 10.9 + (4/2) ~ 12.9

% RunTimeover Whichto Averages (¹²-^{9" 8}- __ _x oo« 8% 50 Siqna il t _.o _M No •ise R _Dat _.-io = 1213.4 μ ^•—v- x 0.05% = 3 _ „1 ^ϋ 17.163 μv 0.1 15%

Where: Signal response of the morphinone peak = 1213.4 μv Baseline Noise of the blank (0.85% H₃P04) injection = 17.163 μv Area percent for morphinone of the system suitability injections = 0.115% Typical Sample Calculation

Percent of Morphinone = x 100 = 0.0005% ^w 14625724 x 100 x 1.44

Where: Peak area of morphinone of high sample solution = 10353.5 Peak area of hydromorphone HCl of low sample solution = 14625724 Morphinone RRF = 1.44 Percent of morphinone in the sample = 0.0005%

Report the morphinone percent determined using the calculation above relating to percent morphinone Report as < LOQ (less than limit of quantitation) for result below the LOQ

(0.0005%). Report as calculated percent for result greater than or equal to the LOQ.

Claims

What is claimed is:

1. A method of determining the amount of morphinone contained in a hydromorphone preparation comprising: (a) preparing a first sample solution comprising hydromorphone from the hydromorphone preparation, with the hydromorphone in the first sample solution at a concentration sufficient to quantify an amount of morphinone contained therein utilizing an HPLC system; (b) preparing a second sample solution comprising hydromorphone from the hydromorphone preparation, with the hydromorphone in the second sample solution at a concentration sufficient to quantify the hydromorphone contained therein utilizing the HPLC system, the second sample solution having a concentration less than that of the first sample solution; (c) analyzing the first sample solution using the HPLC system to obtain a measurable peak area of morphinone; (d) analyzing the second sample solution using the HPLC system to obtain a measurable peak area of hydromorphone; and (e) determining the amount of morphinone in the hydromorphone preparation based on the analysis of the first and second sample solutions.

2. A method of determining the amount of morphinone contained in a hydromorphone preparation comprising: (a) preparing a first sample solution comprising hydromorphone from the hydromoφhone preparation, with the hydromorphone in the first sample solution at a concentration from about 10 mg/ml to about 50 mg/ml for analysis in an HPLC system; (b) preparing a second sample solution comprising hydromorphone from the hydromorphone preparation, with the hydromorphone in the second sample solution at a concentration sufficient to quantify the hydromorphone contained therein utilizing the HPLC system, the second sample solution having a concentration less than that of the first sample solution; (c) analyzing the first sample solution using the HPLC system to obtain a measurable peak area of morphinone; (d) analyzing the second sample solution using the HPLC system to obtain a measurable peak area of hydromoφhone; and (e) determining the amount of moφhinone in the hydromoφhone preparation based on the analysis of the first and second sample solutions.

3. A method of determining the amount of moφhinone contained in a hydromoφhone preparation, comprising: (a) preparing a first sample solution comprising hydromoφhone from the hydromoφhone preparation, with the hydromoφhone in the first sample solution at a concentration from about 10 mg/ml to about 50 mg/ml for analysis in an HPLC system; (b) preparing a second sample solution comprising hydromoφhone from the hydromoφhone preparation, with the hydromoφhone in the second sample solution at a concentration from about 0.01 mg/ml to about 5 mg/ml for analysis in the HPLC system; (c) analyzing the first sample solution using the HPLC system to obtain a measurable peak area of moφhinone; (d) analyzing the second sample solution using the HPLC system to obtain a measurable peak area of hydromoφhone; and (e) determining the amount of moφhinone in the hydromoφhone preparation by dividing the peak area of moφhinone in the first sample solution by (the peak area of hydromoφhone in the second sample solution multiplied by (the concentration of hydromoφhone in the first sample solution divided by the concentration of hydromoφhone in the second sample solution) multiplied by the moφhinone RRF) to obtain a quotient.

4. A method of determining the amount of moφhinone contained in a hydromoφhone preparation, comprising: (a) preparing a first sample solution comprising hydromoφhone from the hydromoφhone preparation, with the hydromoφhone in the first sample solution at a concentration sufficient to quantify an amount of moφhinone contained therein utilizing an HPLC system; (b) preparing a second sample solution comprising hydromoφhone from the hydromoφhone preparation, with the hydromoφhone in the second sample solution at a concentration sufficient to quantify an amount of hydromoφhone contained therein utilizing an HPLC system, the second sample solution having a concentration less than that of the first sample solution; (c) analyzing the first sample solution using an HPLC system comprising a first mobile phase adjusted to an apparent pH of about 6.5 to about 7.5 and an HPLC column maintained at a temperature of about 20 degrees C to about 60 degrees C, to obtain a measurable peak area of moφhinone; (d) analyzing the second sample solution using the HPLC system of (c) to obtain a measurable peak area of hydromoφhone; and (e) determining the amount of moφhinone in the hydromoφhone preparation based on the analysis of the first and second sample solutions.

5. A method of deterrnining the amount of moφliinone contained in a hydromoφhone preparation, comprising: (a) preparing a first sample solution comprising hydromoφhone from the hydromoφhone preparation, with the hydromoφhone in the first sample solution at a concentration from about 10 mg/ml to about 50 mg/ml for analysis in an HPLC system; (b) preparing a second sample solution comprising hydromoφhone from the hydromoφhone preparation, with the hydromoφhone in the second sample solution at a concentration from about 0.01 mg/ml to about 5 mg/ml for analysis in an HPLC system; (c) analyzing the first sample solution using an HPLC system comprising a first mobile phase adjusted to an apparent pH of about 6.5 to about 7.5 and an HPLC column maintained at a temperature of about 20 degrees C to about 60 degrees C, to obtain a measurable peak area of moφhinone; (d) analyzing the second sample solution using the HPLC system of (c) to obtain a measurable peak area of hydromoφhone; and (e) determining the amount of moφhinone in the hydromoφhone preparation by dividing the peak area of moφhinone in the first sample by (the peak area of hydromoφhone in the second sample multiplied by (the concentration of hydromoφhone in the first sample solution divided by the concentration of hydromoφhone in the second sample solution) multiplied by the moφhinone RRF) to obtain a quotient.

6. A method of determining the amount of moφhinone contained in a hydromoφhone preparation, comprising: (a) preparing a first sample solution comprising hydromoφhone from the hydromoφhone preparation, with the hydromoφhone in the first sample solution at a concentration sufficient to quantify an amount of moφhinone contained therein using a first HPLC system; (b) preparing a second sample solution comprising hydromoφhone from the hydromoφhone preparation, with the hydromoφhone in the second sample solution at a concentration sufficient to quantify the hydromoφhone contained therein using a second HPLC system, the second sample solution having a concentration less than that of the first sample solution; (c) analyzing the first sample solution using the first HPLC system to obtain a measurable peak area of moφhinone; (d) analyzing the second sample solution using the second HPLC system to obtain a measurable peak area of hydromoφhone, the second HPLC system being the same or different than the first HPLC system; and (e) determining the amount of moφhinone in the hydromoφhone preparation based on the analysis of the first and second sample solutions.

7. The method of claim 1 , which can provide for the detection of moφhinone from about 1 ppm to 499 ppm.

8. The method of claim 7, which can provide for the detection of moφhinone from about 1 ppm to about 100 ppm.

9. The method of claim 1 , which can provide for the detection of moφhinone from about 1 ppm to about 10 ppm.

10. The method of claim 1, which can provide for the detection of moφhinone from about 1 ppm to about 5 ppm.

11. The method of claim 1 , which can provide for the detection of moφhinone from about 5 ppm to about 25 ppm.

12. The method of claim 1, which can provide for the detection of moφhinone from about 5 ppm to about 10 ppm.

13. The method of claim 1, wherein the HPLC system comprises a first mobile phase adjusted to an apparent pH of about 6.5 to about 7.5.

14. The method of claim 13, wherein the first mobile phase is adjusted to an apparent pH of about 7.0. ^'

15. The method of claim 1, wherein the HPLC system is selected from the group consisting of an adsoφtion chromatography system, an ion-exchange chromatography system and a size exclusion chromatography system.

16. The method of claim 15, wherein the HPLC system is an adsoφtion . chromatography system.

17. The method of claim 16, wherein the adsoφtion chromatography system is selected from the group consisting of a normal phase chromatography system and a reverse phase chromatography system.

18. The method of claim 17, wherein the adsoφtion chromatography system is a reverse phase chromatography system.

19. The method of claim 1, wherein the HPLC system comprises a column maintained at a temperature from about 20 degrees C to about 60 degrees C.

20. The method of claim 19, wherein the column is maintained at a temperature of about 30 degrees C to about 50 degrees C.

21. The method of claim 1, wherein the HPLC system comprises a first mobile phase and a second mobile phase.

22. The method of claim 21, wherein the first mobile phase and/or the second mobile phase comprise acetonitrile.

23. The method of claim 21, wherein the first mobile phase and/or the second mobile phase comprise sodium phosphate monobasic monohydrate.

24. The method of claim 21, wherein the first mobile phase and/or the second mobile phase comprise sodium dodecyl sulfate.

25. The method of claim 21, wherein the first mobile phase comprises acetonitrile, water, sodium phosphate monobasic monohydrate and sodium dodecyl sulfate.

26. The method of claim 21, wherein the second mobile phase consists of acetonitrile.

27. The method of claim 21, wherein the delivery of the first mobile phase and/or the second mobile phase is from about 1.0 to about 2.0 ml per minute.

28. The method of claim 27, wherein the delivery of the first mobile phase and/or the second mobile phase is about 1.5 ml per minute.

29. The method of claim 1, wherein the HPLC system comprises a detector selected from the group consisting of a refractive index detector, an ultra-violet detector, a fluorescent detector, a radiochemical detector, an electrochemical detector, a near- infra red detector, a mass spectroscopy detector, a nuclear magnetic resonance detector and a light scattering detector.

30. The method of claim 29, wherein the detector is an ultra-violet detector.

31. The method of claim 30, wherein the ultra-violet detector is selected from the group consisting of a fixed wavelength detector, a variable wavelength detector and a diode array detector.

32. The method of claim 31, wherein the ultra-violet detector is a fixed wavelength detector.

33. The method of claim 32, wherein the fixed wavelength detector measures at a wavelength of from about 200 nm to about 275 nm.

34. The method of claim 33, wherein the fixed wavelength detector measures at a wavelength of about 220 nm.

35. The method of claim 1, wherein the concentration of hydromoφhone in the first sample solution is from about 10 mg/ml to about 50 mg/ml.

36. The method of claim 35 wherein the concentration of hydromoφhone in the first sample solution is from about 15 mg/ml to about 35 mg/ml.

37. The method of claim 35, wherein the concentration of hydromoφhone in the first sample solution is about 25 mg/ml. ,

38. The method of claim 1, wherein the concentration of hydromoφhone in the second sample solution is from about 2 times to about 500 times less than the concentration of hydromoφhone in the first sample solution.

39. The method of claim 38, wherein the concentration of hydromoφhone in the second sample solution is from about 10 times to about 100 times less than the concentration of hydromoφhone in the first sample solution.

40. The method of claim 38, wherein the concentration of hydromoφhone in the second sample solution is from about 50 times to about 250 times less than the concentration of hydromoφhone in the first sample solution.

41. The method of claim 1, wherein the concentration of hydromoφhone in the second sample solution is from about 0.01 mg/ml to about 10 mg/ml.

42. The method of claim 41, wherein the concentration of hydromoφhone in the second sample solution is from about 0.10 mg/ml to about 2 mg/ml.

43. The method of claim 41, wherein the concentration of hydromoφhone in the second sample solution is about 0.25 mg/ml.

44. The method of claim 1, wherein the HPLC system comprises an autoinjector with an injection volume of from about 10 microliters to about 100 microliters.

45. The method of claim 44, wherein the injection volume is from about 25 microliters to about 75 microliters.

46. The method of claim 44, wherein the injection volume is about 50 microliters.

47. The method of claim 1, wherein the amount moφhinone is obtained by dividing the peak area of moφhinone in the first sample solution by (the peak area of hydromoφhone in the second sample solution multiplied by (the concentration of hydromoφhone in the first sample solution divided by the concentration of hydromoφhone in the second sample solution) multiplied by the moφhinone RRF) to obtain a quotient.

48. The method of claim 1, wherein the second sample solution is obtained by diluting a portion of the first sample solution.

49. The method of claim 1, wherein the HPLC system utilizes isocratic elution.

50. The method of claim 1, wherein the HPLC system utilizes gradient elution.

51. The method of claim 21 , wherein the first mobile phase and/or the second mobile phase comprise from about 50% aqueous medium to about 85% aqueous medium.

52. The method of claim 21, wherein the first mobile phase and/or the second mobile phase comprise from about 60% aqueous medium to about 75% aqueous medium.

53. The method of claim 21, wherein the first mobile phase and/or the second mobile phase comprise from about 50% acetonitrile to about about 15% acetonitrile.

54. The method of claim 21 , wherein the first mobile phase and/or the second mobile phase comprise from about 40% acetonitrile to about about 25% acetonitrile.

55. The method of claim 21, wherein the first mobile phase and/or the second mobile phase comprise about 50% aqueous medium and about 50% acetonitrile.

56. The method of claim 21, wherein the first mobile phase and/or the second mobile phase comprise from about 60% aqueous medium and about 40% acetonitrile.

57. The method of claim 21, wherein the first mobile phase and/or the second mobile phase comprise from about 75% aqueous medium to about 25% acetonitrile.

58. The method of any of claims 1-6, wherein the hydromoφhone preparation is hydromoφhone active pharmaceutical ingredient.

59. The method of claim 58, wherein the hydromoφhone active pharmaceutical ingredient is hydromoφhone hydrochloride active pharmaceutical ingredient.

60. The method of claim 13, wherein the HPLC system comprises a first mobile phase adjusted to an apparent pH of about 6.8 to about 7.2.

61. The method of claim 19, wherein the column is maintained at a temperature of about 40 degrees C to, about 45 degrees C.

62. The method of claim 19, wherein the column is maintained at a temperature of about 45 degrees C.