Qualitative and quantitative analysis methods for various organic impurities in hydroxydiphenylethane preparations

Abstract

The present invention discloses a method for simultaneously performing qualitative and / or quantitative analysis on two or more organic impurities in a 1-(3,5-dihydroxy-4-isopropylphenyl)-2-phenylethane pharmaceutical ointment preparation, characterized in that a liquid chromatography-mass spectrometry technique is used, wherein the chromatographic conditions are as follows: chromatographic column: C18; mobile phase A: water; mobile phase B: acetonitrile; detector: PDA / MS; gradient elution is used; mass spectrometry parameters are as follows: the ion source is APCI or ESI. The present invention adopts UPLC-Q-TOF technology, which can accurately determine the molecular weight of degraded organic impurities, thereby determining the elemental composition of organic impurities, and can quickly infer the potential structure of unknown organic impurities based on the molecular structure of known compounds.

Description

Technical Field

[0001] The present invention relates to a method for qualitative and / or quantitative analysis of multiple organic impurities in a hydroxydiphenylethane preparation, in particular, a method for qualitative and / or quantitative analysis of two or more organic impurities in 1-(3,5-dihydroxy-4-isopropylphenyl)-2-phenylethane by using liquid chromatography-mass spectrometry. Background Art

[0002] Phenolic compounds are hydroxyl derivatives of aromatic hydrocarbons. They are divided into volatile phenols and non-volatile phenols according to their volatility. Most of the phenolic compounds existing in nature are the result of plant life activities. The phenols contained in plants are called endogenous phenols, and the rest are called exogenous phenols. Phenolic compounds all have a special aromatic smell, are weakly acidic, and are easily oxidized in the environment. Among them, polyphenol compounds, composed of more than 40 chemical components, have the effects of anti-oxidation, strengthening blood vessel walls, promoting gastrointestinal digestion, reducing blood fat, increasing body resistance, and preventing arteriosclerosis and thrombosis; they can also act as diuretics, lower blood pressure, inhibit the growth of bacteria and cancer cells, and help digestion. They are widely used in the pharmaceutical, food, and cosmetics industries.

[0003] Phenol is a chemical poison of medium intensity, which reacts chemically with proteins in the cell plasma. At low concentrations, it denatures cells, and at high concentrations, it coagulates proteins. Phenolic compounds can enter the body through the skin, mucous membranes, respiratory tract, and digestive tract. Low concentrations can cause cumulative chronic poisoning, and high concentrations can cause acute poisoning leading to coma and death. Generally speaking, after phenol enters the human body, the body uses its own detoxification function to convert it into non-toxic substances and excrete it from the body. Only when the intake exceeds the detoxification function will there be accumulation and chronic poisoning. Phenolic compounds are widely used in the pharmaceutical, food, and cosmetic industries. Due to the oxidizable characteristics of phenolic compounds, they are unstable and easily affected by oxidants, light, and other factors, resulting in deterioration and some known or unknown components. Among these components, especially the unknown chemical components, their safety has not been evaluated. There are potential health risks when the human body is exposed to these unknown substances. Therefore, it is necessary to study these unknown components and control the content of this component in the product to ensure the health and safety of users.

[0004] The compound 1-(3,5-dihydroxy-4-isopropylphenyl)-2-phenylethane has the activity of treating immune diseases or inflammation. Chinese patent CN 103483158B discloses the following synthesis method:

[0005]

[0006] a). 1-(3,5-dihydroxy-4-isopropylphenyl)-2-phenylethylene was synthesized according to the methods described in patent documents CN1688535A, 2005; US7868047; US321,050; CA2393297; EP1490374; WO2004 / 031117; WO2002 / 057219 and WO01 / 42231.

[0007] b). Dissolve 1-(3,5-dimethoxy-4-isopropylphenyl)-2-phenylethylene (0.3 g) in 50 ml methanol, add Pd / C (25 mg, 10% W / W), and put into a hydrogenation reactor. Pass hydrogen into the reaction solution at room temperature. After the reaction is completed, reduce the pressure, open the hydrogenation reactor, filter the reaction solution through Celite, and evaporate to dryness to obtain 1-(3,5-dimethoxy-4-isopropylphenyl)-2-phenylethane.

[0008] c). A mixture of 1-(3,5-dimethoxy-4-isopropylphenyl)-2-phenylethane (3.0 g) and pyridine hydrochloride (6.7 g) was heated at 200°C for 2 hours under nitrogen flow protection. After the reaction mixture was cooled to room temperature, 100 ml of 2N hydrochloric acid and 300 ml of ethyl acetate were added. After the organic phase and the aqueous phase were separated, the aqueous phase was extracted three times with 150 ml of ethyl acetate, the extracts were combined, washed with saturated brine, and dried over sodium sulfate. After the ethyl acetate was evaporated, the residue was separated and purified by column chromatography, MeOH / CH2Cl2 (V / V=1:4), to obtain 1-(3,5-dihydroxy-4-isopropylphenyl)-2-phenylethane.

[0009] UPLC-Q-TOF consists of three parts: UPLC (ultra-performance liquid chromatography), Q (quadrupole), and TOF (time-of-flight mass spectrometry). The UPLC experiment is the initial efficient separation of multiple components in the sample, and the quadrupole realizes the focusing of the ion beam. The principle of TOF is that ions are accelerated through the flight tube under the action of an electric field, and the mass-to-charge ratio of the measured ions is determined based on the flight time to the detector (the mass-to-charge ratio (M / Z) of the ion is proportional to the flight time of the ion).

[0010] UPLC-Q-TOF is widely used in the research of metabolism-related topics in medicine, food science, biology, environment and other related disciplines, especially for macromolecules (M w >1000) is the only choice. In the study of small molecule compounds, M w <500, the molecular formula can be directly inferred from the precise molecular weight, 500<M W <1000. The molecular formula can be inferred from the precise molecular weight in combination with the elemental analysis results. It is suitable for qualitative and quantitative analysis of trace unknown components. Summary of the invention

[0011] Technical Problems to be Solved by the Invention

[0012] During the research process of developing 1-(3,5-dihydroxy-4-isopropylphenyl)-2-phenylethane into a pharmaceutical preparation, the inventors found that, possibly because the compound contains two hydroxyl groups, some unreported impurities of unknown structure may be produced due to degradation during the storage process. Therefore, it is necessary to perform qualitative and quantitative analysis on these known and unknown impurities in order to ensure the safety and controllability of the drug.

[0013] In view of the above technical problems, the object of the present invention is to provide a method for simultaneously performing qualitative and quantitative analysis on multiple organic impurities in 1-(3,5-dihydroxy-4-isopropylphenyl)-2-phenylethane drugs.

[0014] Technical solutions to technical issues

[0015] The present invention provides a method for simultaneously performing qualitative and / or quantitative analysis on two or more organic impurities in a 1-(3,5-dihydroxy-4-isopropylphenyl)-2-phenylethane pharmaceutical preparation, characterized in that liquid chromatography-mass spectrometry is used, wherein the chromatographic conditions are as follows:

[0016] Chromatographic column: C18; Mobile phase A: water; Mobile phase B: acetonitrile; Detector: PDA / MS;

[0017] The gradient elution program was:

[0018] Time / min Mobile phase A (%) Mobile phase B (%) 0 65±3 35±3 8±2 35±3 65±3 13±2 5±1 95±1 23±2 5±1 95±1 23.1±2 65±3 35±3 26±2 65±3 35±3

[0019] Mass spectrometry parameters: ion source is APCI or ESI.

[0020] In the qualitative and quantitative analysis method of the present invention, the gradient elution procedure is preferably as follows:

[0021] Time / min Mobile phase A (%) Mobile phase B (%) 0 65 35 8 35 65 13 5 95 23 5 95 23.1 65 35 26 65 35

[0022] In the qualitative and quantitative analysis method of the present invention, the organic impurities are three or more.

[0023] In the qualitative and quantitative analysis method of the present invention, the organic impurities include 17 H 22 O3 compound, the compound having one of the following structural formulas:

[0024]

[0025] In the qualitative and quantitative analysis method of the present invention, the organic impurities include 17 H18 O3, a compound having the following structural formula

[0026]

[0027] In the qualitative and quantitative analysis method of the present invention, the organic impurities include 17 H 21 O3, a compound selected from one of the following structures:

[0028]

[0029] In the qualitative and quantitative analysis method of the present invention, the impurities also include compounds with a retention time of approximately 9.76.

[0030] In the qualitative and quantitative analysis method of the present invention, the chromatographic conditions also include:

[0031] Detection wavelength: 210±5nm; and / or,

[0032] Column temperature 40 ± 2 °C, and / or,

[0033] Injection volume: 3±1μL.

[0034] In the qualitative and quantitative analysis method of the present invention, the mass spectrometry conditions also include:

[0035] Scan mode: MSe; and / or,

[0036] Scan range: 50-1200 m / z; and / or,

[0037] Capillary voltage: 3kV; and / or,

[0038] Cone voltage: 40V; and / or,

[0039] Source temperature: 100°C; and / or,

[0040] Cone gas flow rate: 50L / h; and / or,

[0041] Collision energy: low energy channel: 6eV, high energy channel: 25-30eV.

[0042] In the qualitative and quantitative analysis method of the present invention, the mass spectrometry conditions also include:

[0043] Scan time: 0.3s; and / or,

[0044] Desolvation temperature: 600°C; and / or,

[0045] Desolventizing gas flow rate: 800 L / h; and / or,

[0046] Schedule: 0 min to scrap, 1 min to MS.

[0047] The qualitative and quantitative analysis method of the present invention also includes:

[0048] Preparation steps of blank preparation solution: accurately weigh the blank preparation, add 90 volume % acetonitrile aqueous solution, heat and stir to extract, cool, filter, and obtain; and

[0049] Sample solution preparation steps: accurately weigh the preparation sample, add 90 volume % acetonitrile aqueous solution, heat and stir to extract, cool, filter, and obtain.

[0050] Beneficial technical effects

[0051] Using UPLC-Q-TOF technology, the molecular weight of degradation impurities can be accurately determined (to four decimal places), thereby determining the impurity element composition.

[0052] Based on the molecular structure of known compounds, the potential structure of unknown impurities can be quickly inferred.

[0053] This method requires a small amount of sample and is suitable for the qualitative analysis of unknown chemical components that are difficult to obtain.

[0054] The qualitative analysis of this method has a clear direction and can provide clear instructions for subsequent work. BRIEF DESCRIPTION OF THE DRAWINGS

[0055] Figure 1 It is the UV chromatogram of the sample and blank preparation (Placebo) at 210nm.

[0056] Figure 2 This is a comparison of the sample collected under ESI ion source conditions and the Placebo TIC image.

[0057] Figure 3 This is a comparison of samples collected under APCI ion source conditions and Placebo TIC mirror images.

[0058] Figure 4 This is the primary mass spectrum of API under APCI ion source conditions.

[0059] Figure 5 It is the fragmentation attribution diagram and secondary mass spectrum of API under APCI ion source conditions.

[0060] Figure 6 This is the primary mass spectrum of impurity A under APCI ion source conditions.

[0061] Figure 7 It is the fragmentation attribution diagram and secondary mass spectrum of impurity A under APCI source conditions.

[0062] Figure 8 This is the primary mass spectrum of impurity B under APCI source conditions.

[0063] Fig. 9 It is the fragmentation attribution diagram and secondary mass spectrum of impurity B under APCI source conditions.

[0064] Fig.10 This is the primary mass spectrum of impurity C under APCI source conditions.

[0065] Fig.11 This is the fragmentation attribution diagram and secondary mass spectrum of impurity C under APCI source conditions. DETAILED DESCRIPTION

[0066] This method is based on UPLC-Q-TOF technology to qualitatively analyze the degradation products of the active ingredient in 1-(3,5-dihydroxy-4-isopropylphenyl)-2-phenylethane preparations.

[0067] The sample used in the present invention is an ointment preparation of 1-(3,5-dihydroxy-4-isopropylphenyl)-2-phenylethane, and the main components of the ointment preparation are: active ingredient, white vaseline, light liquid paraffin, medium chain triglyceride, isopropyl myristate. The inventor found in the process of studying the ointment preparation that 1-(3,5-dihydroxy-4-isopropylphenyl)-2-phenylethane produced some unreported impurities (related substances) of unknown structure due to slow oxidation and degradation during the stability placement process (for example, 30°C / 65%RH). We believe that the impurities produced by slow oxidation and degradation are due to the fact that the compound itself contains two hydroxyl groups. Oxidation and degradation should be common in 1-(3,5-dihydroxy-4-isopropylphenyl)-2-phenylethane preparations of other types and formulas. Therefore, the analysis method of the present invention should have universal applicability.

[0068] The specific steps are as follows:

[0069] 1. Sample preparation

[0070] 1) Diluent (blank solvent): acetonitrile: water 85-95:15-5 (v / v) as the diluent;

[0071] 2) Preparation of blank preparation solution: accurately weigh the blank preparation, add the diluent, heat and stir to extract, cool, filter, and obtain;

[0072] 3) Sample solution preparation: accurately weigh the preparation sample, add the diluent, heat and stir to extract, cool, filter, and obtain the solution.

[0073] 2. Testing conditions

[0074] 1. Chromatographic parameters:

[0075]

[0076]

[0077] 2. Mass spectrometry parameters:

[0078]

[0079] Example

[0080] The present invention is described in detail below with reference to the examples. The examples illustrate detailed implementation methods and processes to fully disclose and demonstrate that the invention can be implemented. The examples do not represent limitations on the invention, and the protection scope of the present invention is not limited to the following examples. Based on the specific examples in the present invention, all other embodiments obtained by ordinary technicians in the field without creative work belong to the protection scope of the present invention.

[0081] In the examples of the present invention, unless otherwise specified, all raw material components are commercially available products well known to those skilled in the art; in the examples of the present invention, unless otherwise specified, the technical means used are conventional means well known to those skilled in the art. The progress of the reaction of the present invention can be monitored by conventional monitoring methods in the art (such as TLC, LCMS or NMR), and the reaction endpoint is generally taken as the disappearance of the reaction substrate.

[0082] The experimental methods in the embodiments of the specification that do not specify specific conditions are usually carried out according to conventional conditions in the art or according to the conditions recommended by the manufacturer. In the present invention, unless otherwise specified, "parts" represent parts by weight and "%" represents percentage by weight. In the present invention, unless otherwise specified, "above", "below" and "within" represent the number itself. In the present invention, "related substances" refer to organic impurities related to the active ingredient (1-(3,5-dihydroxy-4-isopropylphenyl)-2-phenylethane), which mainly come from the preparation and storage process of the drug, may be the product of self-degradation, or may be the product of reaction with other excipients, which may be harmful to human health or produce serious adverse reactions. "Blank preparation (placebo)" refers to an ointment that does not contain 1-(3,5-dihydroxy-4-isopropylphenyl)-2-phenylethane, and its main components are white vaseline, light liquid paraffin, medium-chain triglycerides, and isopropyl myristate.

[0083] Example 1: Qualitative analysis of organic impurities in 1-(3,5-dihydroxy-4-isopropylphenyl)-2-phenylethane ointment

[0084] (1) Solution preparation

[0085] Diluent: acetonitrile: water 90:10 (v / v);

[0086] Preparation of blank preparation solution: accurately pipette 1 g of blank preparation (whose main ingredients are white vaseline, light liquid paraffin, medium-chain triglycerides, and isopropyl myristate), add 30 mL of diluent, heat and stir to extract for 30 minutes, let stand to cool, and filter to obtain;

[0087] Preparation of 1-(3,5-dihydroxy-4-isopropylphenyl)-2-phenylethane drug preparation sample solution: accurately pipette 1 g of the preparation product, add 30 mL of diluent, heat and stir to extract for 30 minutes, let stand to cool, filter, and take the filtrate to obtain the solution.

[0088] (2) Testing conditions

[0089] 1. Chromatographic parameters:

[0090]

[0091] 2. Mass spectrometry parameters:

[0092]

[0093] Test results such as Figure 1 and Figure 2 As shown, the ESI ion source was used to collect data for the sample and blank preparation Placebo. The mass spectrometry total ion current (TIC) of the sample and Placebo was compared with the mirror image. The results of the liquid chromatogram using the UV detector for the impurity to be identified (RRT 0.9) and impurity B (RRT 1.47) were very reproducible. In the ESI ion source analysis results, a new impurity (RRT 1.94) was found at 9.76min in the TIC diagram. Its response on the UV detector was very low, see Figure 2 , indicating that its content is very low.

[0094] Embodiment 2:

[0095] Based on the analysis method provided in Example 1, the ESI ion source was replaced with an APCI ion source for testing.

[0096] Solution preparation

[0097] Diluent: acetonitrile: water 90:10 (v / v) was used as the diluent;

[0098] Preparation of blank preparation solution: accurately pipette 1g of blank preparation, add 30mL of diluent, heat and stir to extract for 30 minutes, let stand and cool, filter, and obtain;

[0099] To prepare the drug preparation solution, accurately pipette 1 g of the preparation product, add 30 mL of diluent, heat and stir to extract for 30 minutes, let it stand and cool, and filter to obtain the solution.

[0100] 1) Test conditions

[0101] Chromatographic parameters: same as in Example 1.

[0102] Mass spectrometry parameters: Except for changing the ion source to APCI, the other parameters are the same as those of Example 1.

[0103] Test results such as Figure 3 and Figure 4 As shown in the figure, the APCI ion source was used to collect data for the sample and Placebo. The total ion current (TIC) of the mass spectra of the sample and Placebo were compared with the mirror image. The impurity to be identified (RRT 0.9) and impurity B (RRT1.47) had very good reproducibility with the results of the liquid chromatogram using the UV detector. In the ESI ion source identification results, TIC Fig. 9 A new impurity (RRT 1.94) was discovered at .76min, and the peak was barely visible in the APCI source.

[0104] From the TIC graph of the data collected by the ESI and APCI ion sources, it can be seen that the data collected under the APCI ion source condition has less noise interference and the spectrum is cleaner than the data collected under the ESI ion source. Therefore, the present invention uses the data collected under the APCI ion source condition for analysis in the subsequent primary and secondary mass spectra.

[0105] Embodiment 3:

[0106] Primary and secondary mass spectrometry analysis of the data collected under APCI ion source conditions in Example 2.

[0107] 1) API analysis

[0108] The API 1-(3,5-dihydroxy-4-isopropylphenyl)-2-phenylethane has an ion peak of m / z 255.1528 in the primary mass spectrum in negative ion mode, and the precise molecular weight can match the API theoretical molecular weight. The secondary fragments were matched according to the metabolite identification software of Waters Company, and the identification of the fragment ions is shown below. The fragment ions identified by the API are 77, 91, 135, 150, 151, 164, 213, 239 and 241.

[0109]

[0110] 2) Unknown impurity A with RRT (relative retention time) 0.90

[0111] The primary mass spectrum of unknown impurity A (RRT = 0.90) contains an adduct ion peak of m / z 273.1316, which is [MH] -peak, indicating that its exact molecular weight is 274, which differs by 18 from the API molecular weight (256). Based on the molecular weight, it is inferred that its molecular formula is C 17 H 22 O3, compared with API, has an additional H2O molecule. Based on the identified fragment ions 91, 139 and 168 of the impurity to be identified, it is inferred that 2 H and 1 O are added to the dashed part of the fragment attribution diagram. Since the key fragments cannot be hit, the specific structure of the impurity to be identified cannot be confirmed based on the mass spectrometry data.

[0112]

[0113] 2) Analysis of impurity B

[0114] The primary mass spectrum of impurity B (RRT = 1.47) contains an adduct ion peak of m / z 269.1409, which is [MH] - peak, indicating that its exact molecular weight is 270, which differs by 14 from the API molecular weight (256). Based on the molecular weight, its molecular formula is estimated to be C 17 H 18 O3. The fragment ions of impurity B are shown in the figure below. The fragment ions of impurity B are 91, 135, 150, 151, 178, 179 and 241.

[0115]

[0116] 3) Structural prediction of unknown impurity A with RRT 0.90

[0117] It is speculated that the RRT 0.90 impurity may have the structure shown below.

[0118]

[0119] Changes in compound structure may lead to changes in UV absorption wavelength. From the inferred structural formulas of the API, impurity B, and impurity A, there are large differences in conjugated structures, which in turn lead to large differences in UV absorption coefficients. The sensitivity of conventional UV detection methods cannot be guaranteed. The mass spectrometry detection method has greatly improved the sensitivity of the method, and can achieve quantitative detection of multiple impurities at the same time.

[0120] The protection content of the present invention is not limited to the above embodiments. Without departing from the spirit and scope of the inventive concept, changes and substitutions that can be thought of by those skilled in the art are included in the present invention, and the protection scope is defined by the attached claims.

Claims

1. A method for simultaneously performing qualitative and / or quantitative analysis on two or more organic impurities in a 1-(3,5-dihydroxy-4-isopropylphenyl)-2-phenylethane pharmaceutical preparation, characterized in that: Liquid chromatography-mass spectrometry was used, wherein the chromatographic conditions were as follows: Chromatographic column: C18; Mobile phase A: water; Mobile phase B: acetonitrile; Detector: PDA / MS; The gradient elution program was: Mass spectrometry parameters: ion source is APCI or ESI; The organic impurities are selected from any two or more of the following (1), (2), and (3): (1) Molecular weight is 274 and molecular formula is C 17 H 22 O3, and the structural formula is Compounds of (2) Molecular weight is 270 and molecular formula is C 17 H 18 O3, and the structural formula is Compounds of (3) Molecular weight is 273 and molecular formula is C 17 H 21 O3, and the structural formula is selected from of compounds.

2. The analytical method as claimed in claim 1, wherein the gradient elution procedure is as follows: 。 3. The analysis method according to claim 1 or 2, wherein the organic impurities are three or more types.

4. The analytical method according to claim 1 or 2, wherein the chromatographic conditions further comprise: Detection wavelength: 210±5nm; and / or, Column temperature 40 ± 2 °C, and / or Injection volume: 3±1μL.

5. The analysis method according to claim 1 or 2, wherein the mass spectrometry conditions further include: Scan mode: MSe; and / or, Scan range: 50-1200 m / z; and / or, Capillary voltage: 3kV; and / or, Cone voltage: 40V; and / or, Source temperature: 100°C; and / or, Cone gas flow rate: 50L / h; and / or, Collision energy: low energy channel: 6eV, high energy channel: 25-30eV.

6. The analysis method according to claim 1 or 2, further comprising: Preparation steps of blank preparation solution: accurately weigh the blank preparation, add 90±5 volume % acetonitrile aqueous solution, heat and stir to extract, cool, filter, and obtain; and Sample solution preparation steps: accurately weigh the preparation sample, add 90±5 volume % acetonitrile aqueous solution, heat and stir to extract, cool, filter, and obtain.

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