Analytical methods for assessing cytisine purity

Abstract

Provided herein is a method for assessing the purity of cytisine using gradient chromatography at multiple wavelengths.

Description

[Technical field]

[0001] Claiming priority This application claims priority to U.S. Provisional Patent Application No. 63 / 241,829, filed September 8, 2021, the entire contents of which are incorporated herein by reference. [Background technology]

[0002] Nicotine is an addictive substance that is rapidly absorbed during cigarette smoking. The drug is rapidly distributed and is thought to interact with neuronal nicotinic acetylcholine receptors (nAChRs) in the central nervous system (CNS). Nicotine addiction results, at least in part, from this interaction. Many smokers attempt to quit smoking, but few are successful without supportive pharmacological treatment.

[0003] Tobacco smoking contributes to approximately 7 million premature deaths each year worldwide. Smoking is highly addictive, and more than 95% of unassisted quit attempts fail to last six months. It is estimated that for every year that a person continues to smoke beyond their mid-30s, they lose three months of life expectancy. The World Health Organization (WHO) Framework Convention on Tobacco Control identifies evidence-based approaches to promoting smoking cessation, including mass media campaigns, increased tobacco taxes, and support for smokers who want to quit.

[0004] (-)-Cytisine (cytisinicline, commonly referred to simply as cytisine) is a plant-derived alkaloid isolated from the seeds of Cytisus laburnum L. (king sedge) and other plants. References herein to cytisine refer to (-)-cytisine, cytisinicline.

[0005] Cytisine's mechanism of action has helped basic pharmacologists understand the complex pharmacology of the various subtypes of nicotinic acetylcholine receptors. These studies have shown that both nicotine and cytisine bind strongly and preferentially to alpha 4, beta 2 (α4β2) receptors, which mediate the release of dopamine in the shell of the nucleus accumbens and elsewhere. This receptor subtype has been implicated in the development and maintenance of nicotine addiction and has been the primary target of drugs such as varenicline.

[0006] There is a need for nicotine addiction treatments that are less expensive, more effective, have an improved safety profile, and / or have patient-friendly regimens that can more successfully treat individuals who have been unable to quit nicotine using known treatments. Summary of the Invention

[0007] Methods are provided for assessing the purity of cytisine.

[0008] In some aspects, the disclosure provides a chromatographic method for assessing the purity of cytisine, comprising: (a) introducing a cytisine sample into a column comprising a stationary phase comprising stearic acid (C18) and having a length of 150 mm, an internal diameter of 4.6 mm, and a particle size of 2.5 μm, where the cytisine sample contains one or more impurities; (b) applying a first mobile phase having a pH of about 10 to the column such that cytisine and the one or more impurities are retained on the column; (c) eluting cytisine and the one or more impurities by applying a second mobile phase having a pH of about 10 to the column; and (d) detecting cytisine and the one or more impurities.

[0009] In certain embodiments, cytisine and one or more impurities are directed to a mass spectrometer for detection in step (d). In some embodiments, the first and second mobile phases are adapted for injection into a mass spectrometer. In certain embodiments, the first and second mobile phases comprise a volatile component.

[0010] In some embodiments, the first and second mobile phases comprise buffers compatible with injection into a mass spectrometer. In some embodiments, the first mobile phase comprises 10 mM borate buffer and acetonitrile in a ratio of about 95 to about 5. In certain embodiments, the second mobile phase comprises 10 mM borate buffer and acetonitrile in a ratio of about 5 to about 95.

[0011] In some embodiments, the one or more impurities are selected from the group consisting of N-formylcytisine, N-methylcytisine, angustifoline, lupanine, anagyline, and sparteine. In yet another embodiment, cytisine is separated from each of N-formylcytisine, N-methylcytisine, angustifoline, lupanine, anagyline, sparteine, and ammodendrine. In some embodiments, each of N-formylcytisine, N-methylcytisine, angustifoline, lupanine, anagyline, sparteine, and ammodendrine is separated from cytisine and each other.

[0012] In another embodiment, the first mobile phase is applied to the column for at least about 2 minutes, hi some embodiments, the second mobile phase is applied to the column for about 20 minutes to about 24.5 minutes.

[0013] In some embodiments, the step of detecting cytisine and one or more impurities comprises UV / Vis absorption detection at one or more wavelengths. In some embodiments, the one or more wavelengths are 200 nm and 308 nm. In yet another embodiment, cytisine is detected at a wavelength of about 308 nm. In certain embodiments, the one or more impurities are selected from the group consisting of N-formylcytisine, N-methylcytisine, and anagyline, and the one or more impurities are detected at a wavelength of 308 nm. In another embodiment, the one or more impurities are angustifoline and / or lupanine, and the one or more impurities are detected at a wavelength of 200 nm. [Brief description of the drawings]

[0014] [Figure 1] 1 is a flow chart illustrating an exemplary process 100 for assessing the purity of cytisine according to an embodiment of the present disclosure.

[0015] [Figure 2A-2B] 2A-2B are exemplary chromatographs of a high performance liquid chromatography (HPLC) isocratic method that failed to separate cytisine and one or more impurities detected at wavelengths of 200 nm (FIG. 2A) and 310 nm (FIG. 2B) in accordance with embodiments of the present disclosure.

[0016] [Figure 3A-3G] 3A-3G are exemplary absorption spectra of cytisine (FIG. 3A) and known impurities including N-formylcytisine (FIG. 3B), N-methylcytisine (FIG. 3C), lupanine (FIG. 3D), angustifoline (FIG. 3E), anagyline (FIG. 3F), and sparteine ​​(FIG. 3G), in accordance with embodiments of the present disclosure.

[0017] [Figure 4]1 is an exemplary plot showing the respective retention times of cytisine and a known impurity with changing mobile phase conditions according to an embodiment of the present disclosure (legend: (1)-water:TFA(100:0.05) / acetonitrile:TFA(100:0.05), (2)-water:formic acid(100:0.1) / acetonitrile:formic acid(100:0.1), (3)-20 mM ammonium acetate(pH 5.8) / acetonitrile, (4) 10 mM ammonium bicarbonate(pH 8.0) / acetonitrile, and (5)-water:formic acid(100:0.1) / acetonitrile:formic acid(100:0.1) isocratic hold).

[0018] [Figure 5A-5C] 5A-5C are exemplary chromatographs of cytisine and N-formylcytisine (FIG. 5A), N-methylcytisine and anagyline (FIG. 5B), and angustifoline and lupanine (FIG. 5C) using a mobile phase including 2 mM ammonium formate (pH 8) and acetonitrile, according to embodiments of the present disclosure.

[0019] [Figures 6A-6C] 6A-6C are exemplary chromatographs of cytisine and N-formylcytisine (FIG. 6A), N-methylcytisine and anagyline (FIG. 6B), and angustifoline and lupanine (FIG. 6C) using a mobile phase containing 2 mM ammonium formate (pH 8) and methanol, according to embodiments of the present disclosure.

[0020] [Figure 7] 1 is an exemplary plot showing the retention time of cytisine and a known impurity with changing pH of the mobile phase according to an embodiment of the present disclosure. (Legend: (1)—20 mM ammonium formate (pH 8.0) / methanol, (2)—10 mM boric acid (pH 8.5) / methanol, (3)—10 mM boric acid (pH 9.0) / methanol, and (4)—ammonia solution (pH 9.2) / methanol.

[0021] [Figure 8]1 is an exemplary plot showing the retention time of cytisine and a known impurity with varying stationary phases according to embodiments of the present disclosure (Legend: (1)-C18, 150×4.6 mm, 3.5 μm particle size, (2)-Phenyl, 150×4.6 mm, 3.5 μm particle size, (3)-Phenyl-Hexyl, 150×4.6 mm, 3.5 μm particle size, (4)-YMC C18, 150×4.6 mm, 3.0 μm particle size, (5)-Phenyl, 150×4.6 mm, 3.0 μm particle size, (6)-Synergi MAX-RP, 150×4.6 mm, 4 μm particle size, and (7)-Gemini C6-Phenyl, 150×4.6 mm, 5 μm particle size).

[0022] [Figure 9A-9C] 9A-9C are exemplary chromatographs of cytisine and N-formylcytisine (FIG. 9A), N-methylcytisine and anagyline (FIG. 9B), and angustifoline and lupanine (FIG. 9C) using a mobile phase comprising 10 mM borate buffer (pH 8) and methanol, according to embodiments of the present disclosure.

[0023] [Figure 10A-10C] 10A-10C are exemplary chromatographs of cytisine and N-methylcytisine (FIG. 10A), N-formylcytisine and anagyline (FIG. 10B), and angustifoline, lupanine, and sparteine ​​(FIG. 10C) using stationary phase XBridge C18 3.5 μm and methanol according to embodiments of the present disclosure.

[0024] [Figure 11] 1 is an exemplary chromatograph showing that inclusion of methanol in the mobile phase causes a significant gradient increase in the chromatograph, according to an embodiment of the present disclosure.

[0025] [Figure 12]2 is an exemplary plot showing retention times of cytisine and one or more impurities with changes in gradient of the mobile phase according to an embodiment of the present disclosure (legend: (A)-gradient A, (D)-gradient D, (E)-gradient E, (F)-gradient F, and (G)-gradient F; gradient A provided in Table 22, and gradients D-G provided in Table 23).

[0026] [Fig. 13A-13F] 13A and 13B show exemplary chromatographs displaying an overlay of cytisine and one or more impurities at wavelengths of 200 nm (FIG. 13A) and 308 nm (FIG. 13B), an overlay of high concentrations of cytisine and known impurities at wavelengths of 200 nm (FIG. 13C) and 308 nm (FIG. 13D), and an overlay of low concentrations of cytisine and known impurities at wavelengths of 200 nm (FIG. 13E) and 308 nm (FIG. 13F), in accordance with embodiments of the present disclosure.

[0027] [Fig. 14A-14F] 14A-14C show exemplary chromatographs displaying overlays of cytisine, N-formylcytisine, N-methylcytisine, anagyline, angustifoline, and lupanine, including a blank (FIG. 14A), overlay (FIG. 14B), and a zoom-in of the overlay (FIG. 14C) at 308 nm, and a blank (FIG. 14D), overlay (FIG. 14E), and a zoom-in of the overlay (FIG. 14F) at 200 nm, in accordance with an embodiment of the present disclosure.

[0028] [Figure 15A-15B] 15A and 15B show exemplary plots confirming linear response using an HPLC method at 308 nm (FIG. 15A) and 200 nm (FIG. 15B) according to embodiments of the present disclosure. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0029] The present disclosure relates to a method for quantitatively determining the purity of cytisine. The method specifically involves the use of chromatography to resolve cytisine and known impurities. Cytisine is often associated with the following impurities: N-formylcytisine, N-methylcytisine, angustifoline, lupanine, anagyline, and sparteine, and the co-elution of these impurities with cytisine in chromatographic separation procedures presents a challenge for assessing cytisine purity. Provided herein is a gradient HPLC method capable of resolving cytisine and related impurities at multiple wavelengths.

[0030] The methods provided herein enable separation of cytisine and known impurities by utilizing a basic mobile phase (e.g., pH 10) in combination with a stationary phase (e.g., C18, 150×4.6 mm, 2.5 μm particle size), where the mobile phase is compatible with analytical techniques such as mass spectrometry, e.g., the mobile phase is suitable for injection into a mass spectrometer. The methods provided herein are selective for cytisine and known impurities and provide superior separation compared to previous analytical techniques used to assess cytisine purity.

[0031] While the present disclosure can be embodied in various forms, the following description of some embodiments is made with the understanding that the present disclosure should be considered as an example of the present invention and is not intended to limit the present invention to the specific embodiments shown. Headings are provided for convenience only and should not be construed as limiting the present invention in any manner. Embodiments shown under any heading may be combined with embodiments shown under any other heading.

[0032] definition Numerical values ​​used in the various quantitative values ​​specified in this application are described as approximations, unless expressly indicated otherwise, as if the minimum and maximum values ​​within the described ranges were both preceded by the word "about". It should be understood, although not necessarily expressly stated, that all numerical designations are preceded by the term "about". It should be understood that such range formats are used for convenience and brevity, and include numerical values ​​explicitly specified as limits of the ranges, but should be understood to be flexible to include all individual numerical values ​​or subranges subsumed within the ranges, as if each numerical value and subrange were explicitly specified. For example, a ratio in the range of about 1 to about 200 should be understood to include the explicitly recited limits of about 1 and about 200, but also include individual ratios such as about 2, about 3, and about 4, as well as subranges such as about 10 to about 50, about 20 to about 100, etc. It should also be understood, although not necessarily expressly stated, that the reagents described herein are merely exemplary, and that equivalents of such are known in the art.

[0033] As used herein, the term "about" when referring to a measurable value, such as an amount or concentration, is meant to encompass a variation of 20%, 10%, 5%, 1%, 0.5%, or even 0.1% of the specified amount.

[0034] As used herein, the terms "mass spectrometry" and "MS" refer to methods of filtering, detecting, and measuring ions based on their mass-to-charge ratio, or "m / z." Generally, one or more molecules of interest are ionized and the ions are then introduced into a mass analyzer, where a combination of magnetic and electric fields causes the ions to follow paths in space that depend on their mass ("m") and charge ("z").

[0035] Also, the disclosure of ranges is intended as a continuous range, including every value between the minimum and maximum values ​​recited, and any ranges that may be formed by such values. Also disclosed herein are any and all ratios (and any such ratio ranges) that may be formed by dividing a disclosed numerical value by any other disclosed numerical value. Thus, one of ordinary skill in the art will understand that many such ratios, ranges, and ratio ranges may be clearly derived from the numerical values ​​presented herein, and in all cases, such ratios, ranges, and ratio ranges represent various embodiments of the present disclosure.

[0036] method The present disclosure provides a method for evaluating the purity of cytisine.In some aspects, the present disclosure provides a method for improving the resolution of cytisine and one or more impurities using chromatography.The impurities that are often associated with cytisine include N-formylcytisine, N-methylcytisine, angustifoline, lupanine, anagyline, and sparteine.To determine cytisine purity, a chromatography method needs to achieve optimal resolution of cytisine from known impurities.

[0037] In some embodiments, the chromatographic methods disclosed herein comprise high performance liquid chromatography (HPLC). In some embodiments, the methods comprise reverse phase high performance liquid chromatography (RP-HPLC).

[0038] 1 is a flow chart illustrating an exemplary process 100 that can achieve optimal resolution of cytisine and one or more impurities. In step 101, process 100 begins with preparing a solution containing a sample of cytisine, the sample containing one or more impurities associated with cytisine. In some embodiments, the one or more impurities include N-formylcytisine, N-methylcytisine, angustifoline, lupanine, anagyline, and / or sparteine.

[0039] In some embodiments, the sample of cytisine is prepared in a solution having the same composition of mobile phase A, which is the initial solution added to the process 100 in step 103a. In some embodiments, the solution includes an aqueous component and an organic component. In some embodiments, the solution is a basic solution (e.g., pH 10) that includes a volatile component. In some embodiments, the solution is a two-component system that includes only one aqueous component and only one organic component. In some embodiments, the aqueous component includes a buffer, which is compatible with mass spectrometry. In some embodiments, the aqueous component includes a borate buffer, the organic component includes acetonitrile, and the aqueous and organic components are present in a ratio of 95:5, 97:3, or 96:4. In some embodiments, the sample of cytisine is added to a solution that includes an aqueous component and an organic component, where the aqueous component is a borate buffer, the organic component is acetonitrile, the ratio of the aqueous component to the organic compounds is 95:5, and the pH of the solution is about 10. In some embodiments, the solution comprises 10 mM borate buffer and acetonitrile in a ratio of about 95 to about 5 at a pH of 10.

[0040] In some embodiments, the pH of the solution is about 8 to about 10. For example, in some embodiments, the pH of the solution is about 8, about 9, or about 10. In some embodiments, the pH of the solution is about 10. In some embodiments, the pH of the borate buffer is adjusted by the addition of ammonium hydroxide.

[0041] In some embodiments, the concentration of boric acid in the solution and / or in the aqueous component of the solution is about 5 mM to about 30 mM. For example, in some embodiments, the concentration of boric acid in the solution and / or in the aqueous component of the solution is about 5 mM, about 10 mM, about 15 mM, about 20 mM, about 25 mM, or about 30 mM. In some embodiments, the concentration of boric acid in the solution and / or in the aqueous component of the solution is about 10 mM.

[0042] In some embodiments, the organic component of the solution comprises acetonitrile. In some embodiments, the organic component of the solution comprises acetonitrile and methanol. In some embodiments, the organic component of the solution comprises 95% acetonitrile and 5% methanol by volume of the total volume of the organic component of the solution. In some embodiments, the organic component of the solution does not include any organic solvent other than acetonitrile. In some embodiments, the organic component of the solution does not include methanol.

[0043] The process 100 can proceed to step 102 where a solution containing a sample of cytisine is introduced into a column containing a stationary phase. In some embodiments, the stationary phase is contained in a separation column that functions as a separation channel or a chromatography column. In some embodiments, the stationary phase is a hydrophobic stationary phase. In some embodiments, the stationary phase is a C18 stationary phase. In some embodiments, the stationary phase is a C18 stationary phase in a column (C18, 150×4.6 mm, 3.0 μm) having a length of 150 mm, an inner diameter of 4.6 mm, and packed with particles having an average size of 3.0 μm. In another embodiment, the stationary phase is a C18 stationary phase in a column (C18, 150×4.6 mm, 2.7 μm) having a length of 150 mm, an inner diameter of 4.6 mm, and packed with particles having an average size of 2.7 μm. In another embodiment, the stationary phase is a C18 stationary phase in a column (C18, 150×4.6 mm, 3.5 μm) having a length of 150 mm, an internal diameter of 4.6 mm, and packed with particles having an average size of 3.5 μm. In one embodiment, the stationary phase is a C18 stationary phase in a column (C18, 150×4.6 mm, 2.5 μm) having a length of 150 mm, an internal diameter of 4.6 mm, and packed with particles having an average size of 2.5 μm.

[0044] Process 100 can proceed to step 103, where a mobile phase is passed through the column and the mobile phase is purposefully altered over the course of step 103 (e.g., gradient phase chromatography). In some embodiments, the mobile phase includes an aqueous component and an organic component that elute cytisine and one or more impurities, and the ratio of the aqueous component to the organic component is varied to provide a mobile phase gradient. In some embodiments, the mobile phase is altered during step 103 to affect retention of cytisine and known impurities to provide optimal resolution of cytisine and known impurities.

[0045] In certain embodiments, the one or more mobile phases are compatible with analytical techniques such as mass spectrometry, e.g., the one or more mobile phases are suitable for injection into a mass spectrometer. The mobile phases may be compatible with analytical techniques such as mass spectrometry because the mobile phases include volatile components. In some embodiments, the mobile phases are substantially free of "non-volatile components." As used herein, the term "non-volatile components" refers to components present in the one or more mobile phases that are substantially non-volatile under conditions used to remove mobile phase solvents when interfacing a liquid chromatography system with a mass spectrometer.

[0046] In some embodiments, step 103 includes a first step 103a in which a first mobile phase ("mobile phase A") is introduced into the column. In some embodiments, mobile phase A is a solution including an aqueous component and an organic component. In some embodiments, the solution is a basic solution (e.g., pH 10) including a volatile component. In some embodiments, mobile phase A is a two-component system including only one aqueous component and only one organic component. In some embodiments, the aqueous component includes a buffer, the buffer being compatible with mass spectrometry. In some embodiments, the aqueous component includes a borate buffer, the organic component includes acetonitrile, and the aqueous and organic components are present in a ratio of 95:5, 97:3, or 96:4. In some embodiments, mobile phase A includes an aqueous component and an organic component, the aqueous component is a borate buffer, the organic component is acetonitrile, the ratio of the aqueous component to the organic compounds is 95:5, and the pH of mobile phase A is about 10. In some embodiments, mobile phase A comprises 10 mM borate buffer and acetonitrile in a ratio of about 95 to about 5 at a pH of 10.

[0047] In some embodiments, the pH of mobile phase A is about 8 to about 10. For example, in some embodiments, the pH of mobile phase A is about 8, about 9, or about 10. In some embodiments, the pH of mobile phase A is about 10. In some embodiments, the pH of the borate buffer is adjusted by the addition of ammonium hydroxide.

[0048] In some embodiments, the concentration of boric acid in mobile phase A and / or in the aqueous component of mobile phase A is about 5 mM to about 30 mM. For example, in some embodiments, the concentration of boric acid in mobile phase A and / or in the aqueous component of mobile phase A is about 5 mM, about 10 mM, about 15 mM, about 20 mM, about 25 mM, or about 30 mM. In some embodiments, the concentration of boric acid in mobile phase A and / or in the aqueous component of mobile phase A is about 10 mM.

[0049] In some embodiments, the organic component of mobile phase A comprises acetonitrile. In some embodiments, the organic component of mobile phase A comprises acetonitrile and methanol. In some embodiments, the organic component of mobile phase A comprises 95% acetonitrile by volume and 5% methanol by volume of the total volume of the organic component of mobile phase A. In some embodiments, the organic component of mobile phase A does not comprise any organic solvent other than acetonitrile. In some embodiments, the organic component of mobile phase A does not comprise methanol.

[0050] In some embodiments, in the first step 103a, mobile phase A is passed through the column for at least about 2 minutes. For example, in some embodiments, mobile phase A is passed through the column for at least about 2 minutes, at least about 3 minutes, at least about 4 minutes, at least about 5 minutes, at least about 6 minutes, at least about 7 minutes, at least about 8 minutes, or more. In some embodiments, passing mobile phase A through the column for at least about 2 minutes can increase the retention of cytisine on the stationary phase, thereby allowing selective separation of cytisine from one or more impurities and increasing the overall resolution of cytisine and one or more impurities. In some embodiments, passing mobile phase A through the column for at least about 2 minutes can increase the retention of cytisine, N-formylcytisine, and / or N-methylcytisine on the stationary phase, thereby providing optimal resolution from other impurities (e.g., angustifoline, lupanine, and anagyline) and from each other.

[0051] In some embodiments, step 103 includes a second step 103b in which a second mobile phase ("mobile phase B") is introduced into the column. In some embodiments, mobile phase B is a solution including an aqueous component and an organic component. In some embodiments, the solution is a basic solution (e.g., pH 10) including a volatile component. In some embodiments, mobile phase B is a two-component system including only one aqueous component and only one organic component. In some embodiments, the aqueous component includes a buffer, the buffer being compatible with mass spectrometry. In some embodiments, the aqueous component includes a borate buffer, the organic component includes acetonitrile, and the aqueous and organic components are present in a ratio of 5:95, 3:97, or 4:96. In some embodiments, mobile phase B includes an aqueous component and an organic component, the aqueous component is a borate buffer, the organic component is acetonitrile, the ratio of the aqueous component to the organic compounds is 5:95, and the pH of mobile phase B is about 10. In some embodiments, mobile phase B comprises 10 mM borate buffer and acetonitrile in a ratio of about 5 to about 95 at a pH of 10.

[0052] In some embodiments, the pH of mobile phase B is about 8 to about 10. For example, in some embodiments, the pH of mobile phase B is about 8, about 9, or about 10. In some embodiments, the pH of mobile phase B is about 10. In some embodiments, the pH of the borate buffer is adjusted by the addition of ammonium hydroxide.

[0053] In some embodiments, the concentration of boric acid in mobile phase B and / or in the aqueous component of mobile phase B is about 5 mM to about 30 mM. For example, in some embodiments, the concentration of boric acid in mobile phase B and / or in the aqueous component of mobile phase B is about 5 mM, about 10 mM, about 15 mM, about 20 mM, about 25 mM, or about 30 mM. In some embodiments, the concentration of boric acid in mobile phase B and / or in the aqueous component of mobile phase B is about 10 mM.

[0054] In some embodiments, the organic component of mobile phase B comprises acetonitrile. In some embodiments, the organic component of mobile phase B comprises acetonitrile and methanol. In some embodiments, the organic component of mobile phase B comprises 95% acetonitrile by volume and 5% methanol by volume of the total volume of the organic component of mobile phase B. In some embodiments, the organic component of mobile phase B does not comprise any other organic solvents other than acetonitrile. In some embodiments, the organic component of mobile phase B does not comprise methanol.

[0055] In some embodiments, in the second step 103b, mobile phase B is passed through the column for at least about 20 minutes. In some embodiments, mobile phase B is passed through the column for about 20 to about 24.5 minutes. In some embodiments, when mobile phase B is passed through the column for at least about 20 to about 24.5 minutes, cytisine and each of the one or more impurities are eluted from the column at different times.

[0056] In some embodiments, step 103 includes a third step 103c in which mobile phase A is reintroduced into the column to ensure elution of each of cytisine and the one or more impurities, hi some embodiments, mobile phase A is reintroduced into the column and allowed to run for at least about 0.5 minutes.

[0057] The process 100 can proceed to step 104 where cytisine and each of the one or more impurities are eluted from the column, with each of the cytisine and the one or more impurities being eluted from the column at different times to provide complete separation of each of the cytisine and the one or more impurities. In some embodiments, cytisine is separated from each of the one or more impurities. In some embodiments, cytisine is separated from one or more of N-formylcytisine, N-methylcytisine, angustifoline, lupanine, anagyline, and / or sparteine. In some embodiments, cytisine is separated from N-formylcytisine. In some embodiments, cytisine is separated from N-methylcytisine. In yet another embodiment, cytisine is separated from angustifoline. In some embodiments, cytisine is separated from lupanine. In some embodiments, cytisine is separated from anagyline. In some embodiments, cytisine is separated from sparteine.

[0058] In some embodiments, each of the one or more impurities is separated not only from cytisine, but also from each other. In some embodiments, N-formylcytisine is separated from one or more of cytisine, N-methylcytisine, angustifoline, lupanine, anagyline, and / or sparteine. In some embodiments, N-methylcytisine is separated from one or more of cytisine, N-formylcytisine, angustifoline, lupanine, anagyline, and / or sparteine. In some embodiments, angustifoline is separated from one or more of cytisine, N-formylcytisine, N-methylcytisine, lupanine, anagyline, and / or sparteine. In some embodiments, lupanine is separated from one or more of cytisine, N-formylcytisine, N-methylcytisine, angustifoline, anagyline, and / or sparteine. In some embodiments, anagyline is separated from one or more of cytisine, N-formylcytisine, N-methylcytisine, angustifoline, lupanine, and / or sparteine, hi some embodiments, sparteine ​​is separated from one or more of cytisine, N-formylcytisine, N-methylcytisine, angustifoline, lupanine, and / or anagyline.

[0059] Process 100 can proceed to step 105, where cytisine and one or more impurities are detected. In some embodiments, process 100 includes detecting cytisine and one or more impurities as they are eluted from the column by a detector.

[0060] In some embodiments, one or more of the mobile phases are compatible with a detection technique such as mass spectrometry. In some embodiments, the one or more mobile phases are compatible with a method of sample injection into a mass spectrometer. In some embodiments, the aqueous component of the one or more mobile phases comprises a buffer that is compatible with mass spectrometry. In some embodiments, the one or more mobile phases are compatible with direct injection into a mass spectrometer because the one or more mobile phases comprise a volatile component (e.g., the organic phase and / or the aqueous phase is volatile). In some embodiments, the buffer (e.g., a borate buffer) is a volatile buffer that is compatible with mass spectrometry.

[0061] In some embodiments, the method includes directing cytisine and one or more impurities to a mass spectrometer for detection. In some embodiments, cytisine and one or more impurities (contained in one or more mobile phases) are injected into a mass spectrometer after elution from the column. In some embodiments, analysis of cytisine and one or more impurities after elution from the column provides a "direct" method of analyzing the purity of cytisine.

[0062] In some embodiments, the detection technique is mass spectrometry.The method of using mass spectrometry to analyze effluent is well known in the art.Any type of mass spectrometry that can directly analyze the components present in solution can be used, including, for example, electrospray mass spectrometry (ES-MS), atmospheric pressure chemical ionization (APCI), membrane introduction mass spectrometry (MIMS), continuous flow fast atom bombardment (cf-FAB), thermal spray technique, particle beam, moving belt interface, etc.

[0063] In some embodiments, mass spectrometry can include "tandem mass spectrometry" or "MS / MS" to enhance detection resolution. In this technique, a first or parent ion generated from a molecule of interest can be filtered in an MS instrument, and the parent ion can then be fragmented to generate one or more second or daughter ions, which are then analyzed in a second MS procedure. By careful selection of the parent ion, only ions generated by a particular analyte are passed through the fragmentation chamber, and collisions with atoms of an inert gas generate these daughter ions. Because both parent and daughter ions are generated in a reproducible manner under a given set of ionization / fragmentation conditions, the MS / MS technique can provide an extremely powerful analytical tool. For example, a combination of filtering / fragmentation can be used to eliminate interfering substances, which can be particularly useful in complex samples, such as biological samples.

[0064] In other embodiments, any of a variety of standard HPLC detectors can be used to detect the analytes as they elute from the analytical column. In this case, the elution of the compound from the column is detected as a peak in a chromatogram. The retention time of the peak is used to identify the compound, and the peak height (or area) is proportional to the amount of the compound in the sample. "Retention time" is the time required for an analyte to pass through a chromatographic system, measured from the time of injection to the time of detection. Ideally, each analyte of interest will have a characteristic retention time. Suitable detectors exhibit good sensitivity, good stability, reproducibility, linear response over several orders of magnitude, short response times, and ease of operation. Such detectors include, but are not limited to, UV / Vis absorption detectors, photodiode array detectors, fluorescence detectors, refractive index detectors, and conductivity detectors.

[0065] In some embodiments, a UV / Vis absorption detector including a scanning spectrophotometer with grafting optics can be used. The use of a deuterium source (UV range, 190-360 nm) either independently or in combination with a tungsten source (visible range, 360-800 nm) provides a simple means of detecting absorbing species emerging from the column.

[0066] In some embodiments, the detector is a variable wavelength detector. Cytisine and one or more impurities have different absorption maxima via UV / Vis detection, and a variable wavelength detector can maximize the sensitivity of the process by using multiple wavelengths to capture cytisine and one or more impurities in a single chromatogram. In some embodiments, cytisine and one or more impurities are detected at wavelengths of about 308 nm and about 200 nm.

[0067] In some embodiments, cytisine is detected at a wavelength of about 308 nm. In some embodiments, cytisine is detected at a wavelength of about 230 nm.

[0068] In some embodiments, N-formylcytisine is detected at a wavelength of about 308 nm. In some embodiments, N-methylcytisine is detected at a wavelength of about 308 nm. In some embodiments, anagyrine is detected at a wavelength of about 308 nm.

[0069] In some embodiments, angustifoline is detected at a wavelength of about 200 nm. In some embodiments, lupanine is detected at a wavelength of about 200 nm. In some embodiments, cytisine is detected at a wavelength of about 200 nm. In some embodiments, N-formylcytisine is detected at a wavelength of about 200 nm. In some embodiments, N-methylcytisine is detected at a wavelength of about 200 nm. In some embodiments, anagyline is detected at a wavelength of about 200 nm.

[0070] In some embodiments, cytisine and one or more impurities are sequentially eluted from the column in the following order: N-formylcytisine, cytisine, N-methylcytisine, and anagyline, detected at a wavelength of 308 nm, hi other embodiments, cytisine and one or more impurities are sequentially eluted from the column in the following order: N-formylcytisine, cytisine, N-methylcytisine, angustifoline, lupanine, and anagyline, detected at a wavelength of 200 nm.

[0071] In some embodiments, the retention times (a measure of the time it takes for a solute, e.g., cytisine, to pass through a column) of cytisine and one or more impurities differ to result in degradation of cytisine from the one or more impurities, in some embodiments, the retention times of the one or more impurities differ from each other and from cytisine to result in degradation of the one or more impurities from each other and from cytisine.

[0072] In some embodiments, cytisine has a retention time of about 6.5 minutes. In some embodiments, N-formylcytisine has a retention time of about 5.5 minutes. In some embodiments, N-methylcytisine has a retention time of about 8.2 minutes. In some embodiments, anagyline has a retention time of about 10.9 minutes. In some embodiments, angustifoline has a retention time of about 10 minutes. In some embodiments, lupanine has a retention time of about 10.2 minutes.

[0073] In some embodiments, cytisine has a relative retention time of about 1 minute. In some embodiments, N-formylcytisine has a relative retention time of about 0.8 minutes. In some embodiments, N-methylcytisine has a relative retention time of about 1.3 minutes. In some embodiments, anagyline has a relative retention time of about 1.7 minutes. In some embodiments, angustifoline has a relative retention time of about 1.53 minutes. In some embodiments, lupanine has a relative retention time of about 1.56 minutes.

[0074] As can be appreciated from the above disclosure, the present invention has a wide variety of applications. The present invention is further illustrated by the following examples, which are merely illustrative and are not intended to limit the definition and scope of the invention in any way.

[0075] Example: Analytical methods for assessing the purity of cytisine The goal of this study was to develop an analytical HPLC method for assessing cytisine purity. A summary of the parameters screened and the final system parameters optimized for the HPLC method are provided below.

[0076] One of the challenges associated with developing an HPLC method to assess cytisine purity is the ability to resolve cytisine from known impurities, including: N-formylcytisine, N-methylcytisine, angustifoline, lupanine, anagyline / thermopsin (enantiomers), and sparteine ​​(Table 1). [Table 1]

[0077] Previous methods for analyzing cytisine used an isocratic HPLC method in which the mobile phase used remained constant throughout the analysis. However, the isocratic method was limited because the method failed to resolve cytisine from known impurities as shown in Figure 2A (wavelength 200 nm) and Figure 2B (wavelength 310 nm).

[0078] Furthermore, previous reports on analyzing cytisine that relied on reversed-phase chromatography using a C18 column used only one wavelength at 310 nm. This is problematic because some of the expected impurities, angustifoline, lupanine, and sparteine, have no absorption at all at 310 nm wavelength (see Figures 3A-3G for UV absorption spectra for cytisine and known impurities). This demonstrates the need for a dual wavelength method to achieve optimal resolution of cytisine from known impurities.

[0079] This study was designed in an effort to yield a gradient HPLC method (as opposed to an isocratic method) capable of resolving cytisine and expected related impurities at multiple wavelengths. During the development process, sparteine ​​and ammodendrine were removed from the screening process. This was because ammodendrine could not be sourced and sparteine ​​would be difficult to analyze using a stand-alone HPLC method. In addition, thermopsin was also removed from the study on the basis that if a peak was detected in the sample corresponding to anagyrine, a chiral method would also be developed.

[0080] 1. Sample Preparation Over the course of development, several diluents were tried in an attempt to optimize the cytisine peak shape and impurities screened. The diluents screened are listed in Table 2 below. [Table 2]

[0081] Acetonitrile (100%) proved to be unsuitable for peak resolution across the various mobile phases. A 1:1 mixture of acetonitrile:water resulted in a single broad peak. Significant improvement was observed when using the selected diluent, mobile phase A, which resulted in favorable peak shapes as shown in Table 3. [Table 3]

[0082] 2. Mobile Phase Screening: Stage 1 The mobile phases listed in Table 4 were screened during method development to cover a selection of different pH levels. Table 4 shows the mobile phases screened in the first stage of the screening process with the aqueous phase and its corresponding organic phase detailed in each row. Mobile phase system number 2 was also run using a different gradient (Table 5) that mimics an isocratic method. At this stage of mobile phase screening, a reference material for sparteine ​​was not available and is therefore not included in the data. [Table 4] [Table 5]

[0083] Screening was performed by injecting samples at 0.5 mg / ml cytisine and all available impurities on an HPLC system with a standard set of parameters shown in Tables 6 and 7 below. [Table 6] [Table 7]

[0084] For each set of injections, the mobile phase was changed to investigate the effect that each mobile phase system had on the chromatography. The main objective of the mobile phase screening was to find a mobile phase that delivered adequate retention of all impurities (e.g., N-formylcytisine, N-methylcytisine, anagyline, angustifoline, lupanine) and good peak shape.

[0085] FIG. 4 and Table 8 show how the retention time of each available impurity changes with mobile phase conditions. [Table 8]

[0086] Due to the lack of retention for cytisine in the acidic gradient, further screening was performed using different buffer systems. 20 mM ammonium formate (pH 8.0) was prepared and screened separately with both acetonitrile and methanol as the organic phase. Ammonium formate was preferred over ammonium bicarbonate due to fewer gradient impurities found at 200 nm. Methanol proved to have a large effect on the retention of cytisine and the screened impurities. Retention was increased by at least 2 minutes for each impurity. This screening was performed before the diluent was modified, so the peak shapes are still very poor at this point, but distinguishable peaks can still be identified in each chromatogram. Chromatograms of this stage of screening are shown in Figures 5A-5C (2 mM ammonium formate (pH 8) and acetonitrile) and Figures 6A-6C (2 mM ammonium formate (pH 8) and methanol).

[0087] 3. Mobile Phase Screening: Stage 2 The increase in retention observed using methanol as the organic phase meant that methanol was carried over to the next stage of mobile phase screening. Additional buffers were screened at different pH levels. Figure 7 and Tables 9 and 10 show how retention time changes with pH. Results were plotted against 20 mM ammonium formate (pH 8.0) with methanol to show the retention time comparison (Figure 7). [Table 9] [Table 10]

[0088] Findings from this stage of mobile phase screening were used for further mobile phase optimization, detailed in Section 5.

[0089] 4. Stationary Phase Screening Stationary phase screening was started in parallel with the second stage of mobile phase screening. This screening was performed in a similar manner to the mobile phase screening, but with the change of column instead of mobile phase. To address back pressure issues observed with the smaller particle size column, the temperature was increased from 30° C. to 40° C. The parameters used for screening are listed in Tables 11 and 12. [Table 11] [Table 12]

[0090] The range of stationary phases screened incorporate different reversed-phase chemistries as well as varying particle sizes and column dimensions. Selection of the best stationary phase was based on good resolution of all impurities and comparable peak shapes. A good window around the cytisine peak was also desired to allow for high sample loading, if necessary. The columns screened are listed in Table 13 below. A report demonstrating the separation of the impurities is shown in Figure 8. [Table 13]

[0091] The Synergi Polar-RP results have been omitted from this report as only two compounds were eluted using this column. The Synergi MAX-RP showed very poor peak shape for cytisine. Both the phenyl and phenyl-hexyl stationary phases struggled to give sufficient separation between cytisine and N-formylcytisine. The C18 stationary phase gave the best separation around the cytidine peak, showed good peak shape, and resolved all impurities. Additional C18 stationary phases listed in Table 14 below were screened to explore more particle sizes. [Table 14]

[0092] The YMC 3.0 μm and 2.7 μm C18 stationary phases provided poor separation of N-methylcytisine, angustifoline, and lupanine. The YMC 3.0 μm column also provided a split peak for N-formylcytisine. The 2.7 μm C18 column also showed very poor peak shape for cytisine. The 2.5 μm C18, ACE 3.0 μm C18, and 3.5 μm C18 stationary phases provided good separation of cytisine and expected impurities. These columns were carried forward to the next stage and screened against additional mobile phases.

[0093] 5. Further Mobile Phase and Stationary Phase Screening For an additional, much more targeted development step, the most promising columns and mobile phases were combined: 10 mM borate buffer (pH 8.0) was used as opposed to 20 mM ammonium formate (pH 8.0) because the borate buffer mobile phase increased the resolution around the cytisine peak, as shown in Figures 9A-9C (20 mM ammonium formate (pH 8.0) and methanol) and 10A-10C (XBridge C18 3.5 μm and methanol).

[0094] The mobile and stationary phases screened at this stage are listed below in Tables 15 and 16, respectively, with the system parameters in Tables 17 and 18. The combination of 10 mM borate buffer (pH 8.0) and methanol could not be screened together for the smallest particle size due to the high pressure on the column. Table 19 shows the mobile and stationary phase combinations with peak height, peak width, and USP tailing of the cytisine peak. [Table 15] [Table 16] [Table 17] [Table 18] [Table 19]

[0095] The absorption of methanol resulted in a much smaller peak height for cytisine and therefore a lower method sensitivity. The peak width was also narrower for cytisine with acetonitrile as the organic phase. There is also a correlation between smaller particle size and increased peak height / decreased peak width. However, increased USP tailing was observed. Further runs were performed with borate buffer adjusted to pH 10.0 using a 2.5 μm C18 column. The mobile phase also contained 5% v / v methanol. The results from this experiment are shown in Table 20 below. This study was performed on a different instrument with a different detector. Therefore, the peak heights are not directly comparable to the results above. [Table 20]

[0096] Another test was performed with increasing buffer concentration to 25 mM boric acid, also using a 2.5 μm C18 column. The results of this run are included in Table 21 below for the cytisine peak. [Table 21]

[0097] These results indicate that there is no significant effect on peak shape with increasing buffer concentration. In accordance with these results, 10 mM boric acid (pH 10.0) was deemed suitable for the aqueous mobile phase. It was also noted that changing the pH modifier of boric acid to ammonia, as opposed to sodium hydroxide, increased compatibility with liquid chromatography mass spectrometry (LC-MS).

[0098] 6. Gradient Screening Methanol has a different effect on the retention of the cytisine peak. Therefore, a simple test of the gradient was performed using a combination of methanol and acetonitrile as the organic phase. The following conditions listed in Table 22 (A=10 mM borate buffer (pH 8.0), B=methanol, and C=acetonitrile) were screened with the aim of optimizing the separation between the impurities and reducing the absorption of the gradient. [Table 22]

[0099] Methanol has been demonstrated to affect the resolution of all screened impurities, with 5% v / v (gradient A) methanol being sufficient to affect the resolution. However, including methanol introduces additional complications and may affect method robustness. The chromatogram in Figure 11 also shows the significant gradient increase observed with inclusion of methanol.

[0100] The development proceeded with further investigations to find an improved gradient that used 100% acetonitrile as the organic phase, thus removing the complication of adding methanol. Longer gradients and gradients with a 2 minute isocratic hold at the beginning were screened, as listed in Table 23. This gradient screening was done after fine tuning of the mobile phase, meaning that the borate buffer was at pH 10, as opposed to the previous gradient that was screened at pH 8. The mobile phase screening showed that the change from pH 8 to pH 10 improved peak shape and had negligible impact on retention time. Therefore, the results of the previous gradient screening and this further screening were considered to still be comparable. The results of the gradient screening were tabulated to show the retention time of each screened impurity. The gradient was compared to gradient A (Table 22), which was the only gradient using methanol with an acceptable gradient rise (Figure 12). [Table 23]

[0101] The gradient selected gave optimal separation and peak shape for cytisine and related process impurities. By introducing a 2 min hold, retention of cytisine, N-formylcytisine, and N-methylcytisine was increased sufficiently to show that suitable chromatography could be obtained without the use of methanol. This allows for a binary mobile phase system with lower absorption, which is also less susceptible to column overpressure. The finalized method parameters are listed in Tables 24-26 below. [Table 24] [Table 25] [Table 26]

[0102] 7. Method performance Cytisine and all impurities screened in the development had different response levels with a λ of approximately 200 nm. max Cytisine, N-formylcytisine, N-methylcytisine, and anagyrine have separate λ maxThe signal to noise ratio proved to be about 10 times higher with the method at 308 nm due to the significantly lower gradient absorption. As a result, development moved to a method where samples were primarily analyzed at 308 nm, but data was also collected at 200 nm to still detect lupanine, angustifoline, and any other impurities that absorbed UV at 200 nm but not at 308 nm. As shown in Figures 13A-13F, exemplary overlays at 200 nm and 308 nm are included for relevant impurities. Specifically, Figures 13A and 13B show all of the impurities screened at 200 nm and 308 nm, respectively, and Figures 13C and 13D show typical chromatograms of samples collected at high concentration samples at 200 nm and 308 nm, respectively. Finally, Figures 13E and 13F show typical chromatograms of samples collected at low concentration samples at 200 nm and 308 nm, respectively.

[0103] 8. Experimental Verification Test Preliminary validation has been carried out in terms of system suitability, specificity, detection limit, quantification limit, linearity, precision, and accuracy at the working concentration for cytisine (200 μg / mL).

[0104] specificity Chromatograms from the specificity test are shown in Figures 14A-14F, which include a chromatogram of the diluent along with overlaid chromatograms of cytisine, N-formylcytisine, N-methylcytisine, anagyline, angustifoline (200 nm only), and lupanine (200 nm only) at 308 nm (Figures 14A-14C) and 200 nm (Figures 14D-14F). No interference with cytisine was detected. A diluent blank injection injected prior to the impurities showed no peaks above the limit of quantitation (LOQ) at the retention times of all impurities.

[0105] System suitability test System suitability testing was performed by six injections of cytisine at the working concentration (200 μg / mL). System suitability results are shown in Table 27. [Table 27]

[0106] linearity Linearity solutions were prepared by diluting the stock standard solution to yield a concentration range of cytisine from 20 to 300 μg / ml in diluent. The peak area results of the linearity solutions at concentrations from 20 to 300 μg / ml are shown graphically in Figures 15A-15B. The peak area response was linear over the concentration range tested. The correlation coefficients of the slope of the linear regression line of peak area versus nominal concentration were 0.9997 at 308 nm and 0.9962 at 200 nm.

[0107] sensitivity Duplicate injections of cytisine and all screened impurities were performed to obtain the theoretical limits of detection and quantitation (at a working concentration of 0.2 mg / ml) listed in Tables 28 and 29 below. [Table 28] [Table 29]

[0108] Sensitivity was significantly improved at 308 nm, but the limit of detection (LOD) and LOQ were still above typical acceptance criteria (LT 0.02% and LT 0.05%, respectively). To combat sensitivity issues, a high / low analytical methodology was required at concentrations as high as 10 mg / mL.

[0109] Assay repeatability Six cytisine standards were individually prepared at a high concentration of 10 mg / ml, then diluted to a working concentration of 0.2 mg / ml, injected in triplicate, and assayed against the system preferred standard. Each standard was spiked with approximately 0.15% N-formylcytisine and N-methylcytisine. The assay results and impurity profile results are shown in Table 30. [Table 30]

[0110] Assay repeatability solution results yielded a mean assay of 99.59%, with an accuracy ranging from 99.37% to 99.90% and a precision of 0.23% RSD. Impurities were spiked at 0.15%, yielding averages of 0.16% and 0.14% for N-formylcytisine and N-methylcytisine, respectively. Precision ranged from 0.15% to 0.16% for N-formylcytisine and 0.137% to 0.138% for N-methylcytisine. Impurity precision is well within ±20.0%.

[0111] 9. Summary of Cytisine Analysis Procedure Based on the above findings, an analytical procedure was developed as described below.

[0112] Mobile phase preparation Dissolve 0.62 g of boric acid in deionized water. Adjust the pH to 10.0 by adding ammonium hydroxide (35% w / w) to the solution. Filter the buffer through a 0.22 micron filter. Add 50 ml of buffer to 950 ml of acetonitrile to make mobile phase B. Add 50 ml of acetonitrile to the remaining 950 ml of buffer to make mobile phase A.

[0113] Blank / Diluent Preparation The blank / diluent consisted of mobile phase A (10 mM borate buffer, pH 10.0:acetonitrile (95:5)).

[0114] RT-ID standard preparation Accurately weigh 20 mg of cytisine and 10 mg of the related substance of interest into a 100 ml volumetric flask. Dissolve and bring to volume in diluent.

[0115] Stock sample solution preparation Accurately weigh 100 mg of sample into a suitable container. Dissolve in 10 ml of diluent, using sonication if necessary. Label the solution as Stock Sample Solution.

[0116] Dilute sample solution preparation Accurately pipette 1 ml of stock sample solution into a 50 ml volumetric flask and dilute to volume with diluent. Label as the diluted sample solution.

[0117] HPLC parameters In Tables 31 and 32 below, the HPLC parameters used to analyze the purity of cytisine are listed. [Table 31] [Table 32]

[0118] Chromatogram integration and calculation of impurity % and purity % Any peaks resulting from the blank solution should not be integrated in the stock sample chromatogram. At 308 nm, all peaks should be integrated in the stock sample solution. At 200 nm, integrate the peaks corresponding to lupanine and angustifoline. In addition, integrate any peaks in the stock sample at 200 nm that do not have a corresponding impurity in the 308 nm stock sample chromatogram.

number

[0119] Relative response factors were calculated relative to cytisine at 308 nm. The normalization factors contained in Table 33 below are then applied to the peak areas obtained from the stock sample solutions. These normalized areas are what are used for the % impurities calculations. [Table 34]

[0120] 10. Conclusion In conclusion, the following example provides a methodology that allows for the quantification of cytisine and related substances / impurities. The work specifically provides a gradient HPLC method capable of resolving cytisine and potential related impurities at multiple wavelengths.

[0121] It is specifically contemplated that the various features of the invention described herein may be used in any combination unless the context dictates otherwise. Moreover, the present disclosure also contemplates that in some embodiments, any feature or combination of features described herein may be excluded or omitted. To illustrate, if the specification states that a complex comprises components A, B, and C, it is specifically contemplated that any of A, B, or C, or combinations thereof, alone or in any combination, may be omitted or discarded.

[0122] Section A. A chromatographic method for assessing the purity of cytisine, comprising: (a) introducing a cytisine sample into a column comprising a stationary phase comprising stearic acid (C18) and having a length of 150 mm, an internal diameter of 4.6 mm, and a particle size of 2.5 μm, wherein the cytisine sample contains one or more impurities; (b) applying a first mobile phase having a pH of about 10 to the column such that cytisine and the one or more impurities are retained on the column; (c) eluting cytisine and the one or more impurities by applying a second mobile phase having a pH of about 10 to the column; and (d) detecting cytisine and the one or more impurities.

[0123] Item B. The chromatographic method of item A, wherein the cytisine and one or more impurities are directed to a mass spectrometer for detection in step (d).

[0124] Item C. The chromatographic method of items A or B, wherein the first and second mobile phases are compatible for injection into a mass spectrometer.

[0125] Item D. The chromatography method according to any one of Items A to C, wherein the first and second mobile phases contain a volatile component.

[0126] Item E. The chromatography method of any one of items A-D, wherein the first and second mobile phases comprise a buffer that is compatible with injection into a mass spectrometer.

[0127] Item F. The chromatography method of any one of items A-E, wherein the first mobile phase comprises 10 mM borate buffer and acetonitrile in a ratio of about 95 to about 5.

[0128] Item G. The chromatographic method of any one of items A-F, wherein the second mobile phase comprises 10 mM borate buffer and acetonitrile in a ratio of about 5 to about 95.

[0129] Item H. The chromatography method of any one of items A-G, wherein the one or more impurities are selected from the group consisting of N-formylcytisine, N-methylcytisine, angustifoline, lupanine, anagyline, sparteine, and ammodendrine.

[0130] Item I. The chromatographic method of item H, wherein cytisine is separated from each of N-formylcytisine, N-methylcytisine, angustifoline, lupanine, anagyline, sparteine, and ammodendrine.

[0131] The chromatographic method of paragraph H, wherein each of N-formylcytisine, N-methylcytisine, angustifoline, lupanine, anagyline, sparteine, and ammodendrine is separated from cytisine and from each other.

[0132] Item K. The chromatography method of any one of items A-J, wherein the first mobile phase is applied to the column for at least about 2 minutes.

[0133] Item L. The chromatography method of any one of items A to K, wherein the second mobile phase is applied to the column for about 20 minutes to about 24.5 minutes.

[0134] Item M. The chromatography method of any one of items A-L, wherein the step of detecting cytisine and one or more impurities comprises UV / Vis absorbance detection at one or more wavelengths.

[0135] Item N. The chromatography method of item M, wherein one or more wavelengths are 200 nm and 308 nm.

[0136] Item O. The chromatographic method of item M, wherein cytisine is detected at a wavelength of about 308 nm.

[0137] Item P. The chromatographic method of item M, wherein the one or more impurities are selected from the group consisting of N-formylcytisine, N-methylcytisine, and anagyline, and the one or more impurities are detected at a wavelength of 308 nm.

[0138] Item Q. The chromatographic method of item M, wherein the one or more impurities are angustifoline and / or lupanine, and the one or more impurities are detected at a wavelength of 200 nm.

Claims

1. 1. A chromatographic method for assessing the purity of cytisine, comprising: (a) introducing a cytisine sample into a column comprising a stationary phase comprising stearic acid (C18) and having a length of 150 mm, an internal diameter of 4.6 mm, and a particle size of 2.5 μm, wherein the cytisine sample contains one or more impurities; (b) applying a first mobile phase having a pH of about 10 to the column such that cytisine and the one or more impurities are retained on the column; (c) eluting cytisine and the one or more impurities by applying a second mobile phase having a pH of about 10 to the column; (d) detecting cytisine and said one or more impurities.

2. 2. The chromatography method of claim 1, wherein cytisine and the one or more impurities are directed to a mass spectrometer for detection in step (d).

3. 10. The chromatography method of claim 1, wherein the first and second mobile phases are adapted for injection into a mass spectrometer.

4. The chromatography method of claim 3 , wherein the first and second mobile phases comprise a volatile component.

5. 4. The chromatography method of claim 3, wherein the first and second mobile phases comprise buffers compatible with injection into a mass spectrometer.

6. 2. The chromatography method of claim 1, wherein the first mobile phase comprises 10 mM borate buffer and acetonitrile in a ratio of about 95 to about 5.

7. 2. The chromatography method of claim 1, wherein the second mobile phase comprises 10 mM borate buffer and acetonitrile in a ratio of about 5 to about 95.

8. 2. The chromatography method of claim 1, wherein the one or more impurities are selected from the group consisting of N-formylcytisine, N-methylcytisine, angustifoline, lupanine, anagyline, sparteine, and ammodendrine.

9. 9. The chromatographic method of claim 8, wherein cytisine is separated from each of N-formylcytisine, N-methylcytisine, angustifoline, lupanine, anagyline, and sparteine.

10. 9. The chromatographic method of claim 8, wherein each of N-formylcytisine, N-methylcytisine, angustifoline, lupanine, anagyline, sparteine, and ammodendrine is separated from cytisine and from each other.

11. 2. The chromatography method of claim 1, wherein the first mobile phase is applied to the column for at least about 2 minutes.

12. 10. The chromatography method of claim 1, wherein the second mobile phase is applied to the column for about 20 minutes to about 24.5 minutes.

13. 2. The chromatography method of claim 1, wherein the step of detecting cytisine and the one or more impurities comprises UV / Vis absorbance detection at one or more wavelengths.

14. 14. The chromatography method of claim 13, wherein the one or more wavelengths are 200 nm and 308 nm.

15. 14. The chromatography method of claim 13, wherein cytisine is detected at a wavelength of about 308 nm.

16. 14. The chromatographic method of claim 13, wherein the one or more impurities are selected from the group consisting of N-formylcytisine, N-methylcytisine, and anagyrine, and the one or more impurities are detected at a wavelength of 308 nm.

17. 14. The chromatography method of claim 13, wherein the one or more impurities are angustifoline and / or lupanine, and the one or more impurities are detected at a wavelength of 200 nm.

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