A method for detecting the content of a ticagrelor impurity

Abstract

The application discloses a detection method of the impurity content of ticagrelor, the impurity is N-nitroso-ticagrelor, and the detection method comprises the following steps: diluting an initial sample to prepare a detection sample; feeding the detection sample into a mobile phase through a sample injector; performing gradient elution treatment on the detection sample by using a chromatographic column with phenyl-hexyl bonded silica gel particles as filler to obtain an elution sample, the mobile phase comprises an organic phase and an aqueous phase, and the gradient elution conditions comprise the following steps: in the first stage, the ratio of the organic phase to the aqueous phase is directly proportional to the time length; in the second stage, the ratio of the organic phase to the aqueous phase is the ratio at the end of the first stage; in the third stage, the ratio of the organic phase to the aqueous phase in the mobile phase is inversely proportional to the time length; in the fourth stage, the ratio of the organic phase to the aqueous phase in the mobile phase is the ratio at the end of the third stage, and the ratio of the organic phase to the aqueous phase in the fourth stage is smaller than that in the second stage; and performing mass spectrometric analysis on the elution sample to determine the composition of the elution sample.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of analytical detection of ticagrelor, in particular to a method for detecting the content of N-nitroso-ticagrelor in ticagrelor bulk drug and preparation. BACKGROUND

[0002] Ticagrelor is a new type of potent oral anti-platelet aggregation drug, and the structure of ticagrelor contains a secondary amine (secondary amine), which provides an amino unit for the formation of nitrosamine, and the generated nitrosamine impurity is called drug matrix nitrosamine impurity (NDSRI).

[0003] At present, the NDSRI type nitrosamine impurity in ticagrelor is mainly N-nitroso-ticagrelor, but its structure is very similar to that of ticagrelor. The conventional chromatographic method cannot realize effective separation of the drug and the impurity, and the peak information of the impurity cannot be accurately detected.

[0004] Therefore, it is necessary to develop a rapid, sensitive, accurate and high-resolution detection method to determine the content of N-nitroso-ticagrelor in ticagrelor. SUMMARY

[0005] The present application aims to solve one of the problems in the related art to some extent. To this end, the present application provides a method.

[0006] In order to achieve the above-mentioned purpose, as a first aspect of the present application, a method for detecting the content of a ticagrelor impurity is disclosed, the ticagrelor impurity is N-nitroso-ticagrelor, and the detection method comprises:

[0007] An initial sample is provided, the initial sample is diluted to prepare a detection sample;

[0008] The detection sample is introduced into the mobile phase through a sample injector, and a chromatographic column with phenyl-hexyl bonded silica gel particles as the filler is used for gradient elution treatment of the detection sample at a set temperature to obtain an elution sample, the mobile phase comprises an organic phase and an aqueous phase, and the gradient elution conditions comprise:

[0009] In the first stage, the ratio of the organic phase to the aqueous phase is directly proportional to the time length;

[0010] In the second stage, the ratio of the organic phase to the aqueous phase is the ratio at the end of the first stage;

[0011] In the third stage, in the mobile phase, the ratio of the organic phase to the aqueous phase is inversely proportional to the time length;

[0012] In the fourth stage, the ratio of the organic phase to the aqueous phase in the mobile phase is the same as that at the end of the third stage, and the ratio of the organic phase to the aqueous phase in the fourth stage is less than the ratio of the organic phase to the aqueous phase in the second stage.

[0013] The eluted sample is subjected to mass spectrometry analysis to determine the components of the eluted sample.

[0014] Further, the pore size of the silica gel particles is between 8 nm and 12 nm, and the carbon loading is between 10% and 14%.

[0015] Further, the column length of the chromatographic column is between 90 mm and 110 mm, the column diameter of the chromatographic column is between 4.5 mm and 4.7 mm, the particle size of the packing is between 2 μm and 4 μm, and the column temperature is between 35°C and 45°C.

[0016] Further, in the mobile phase, the ratio of the organic phase to the aqueous phase is between 1:1 and 4:1.

[0017] Further, in the step of gradient elution treatment of the detection sample, the first stage is within 0 min to 7 min, the second stage is within 7 min to 11 min, the third stage is within 11 min to 11.1 min, and the fourth stage is within 11.1 min to 15 min.

[0018] Further, the flow rate of the organic phase and the aqueous phase is between 0.4 mL / min and 0.6 mL / min.

[0019] Further, the organic phase comprises acetonitrile or methanol, and the aqueous phase comprises 0.1% formic acid aqueous solution.

[0020] Further, in the step of providing an initial sample and diluting the initial sample,

[0021] A certain amount of initial sample is weighed;

[0022] The initial sample is diluted to a constant volume with a diluent, which comprises methanol or acetonitrile, and then mixed uniformly to obtain the detection sample, wherein the minimum concentration of the N-nitroso-ticagrelor impurity that can be detected in the detection sample is not less than 0.216 ng / mL, and the minimum concentration of the N-nitroso-ticagrelor impurity that can be quantitatively detected in the detection sample is not less than 0.721 ng / mL.

[0023] Further, in the step of passing the detection sample into the mobile phase through the injector, the sample disc temperature is between 8°C and 12°C, and the single injection volume is between 9 μL and 11 μL.

[0024] Further, in the step of performing mass spectrometric analysis on the eluted sample to determine the components of the eluted sample,

[0025] The eluted sample is subjected to electrospray ionization treatment to obtain gas phase ions, wherein the ionization voltage is between 3300V and 3700V, the sheath gas flow rate is between 40 Arb and 60 Arb, the auxiliary gas flow rate is between 5 Arb and 15 Arb, the backflush gas flow rate is between 0.5 Arb and 1.5 Arb, the vaporization chamber temperature is between 300℃ and 400℃, and the temperature of the ion transmission tube is between 250℃ and 350℃.

[0026] The gas phase ions are subjected to multi-stage ion channel scanning collection treatment to obtain target ions, wherein the collection mode includes a multiple reaction monitoring mode, the multi-stage ion channel includes a first stage ion channel, an intermediate channel, and a second stage ion channel, ions passing through the first stage ion channel enter the second stage ion channel after fragmentation through the intermediate channel, and the target ions are determined to be detected when the ions pass through the second stage ion channel, the mass-to-charge ratio of the first stage ion channel satisfies 552.2Da, the mass-to-charge ratio of the second stage ion channel satisfies 522.2Da, and the fragmentation energy of the intermediate channel is between 8V and 9V.

[0027] The technical solution provides a method for detecting the content of N-nitroso-ticagrelor impurities in ticagrelor drugs. The detection method can detect the impurity N-nitroso-ticagrelor which is very similar to the structure of ticagrelor, and has higher specificity and sensitivity. A liquid chromatograph is used in combination with a specific phenyl-hexyl bonded silica gel chromatographic column, which has stronger separation capacity between similar structure substances than a traditional chromatographic column, and reduces the influence of interfering substances. The optimized gradient elution condition improves the separation effect. Through four-stage gradient elution (change of the ratio of organic phase to aqueous phase), effective separation of each component in a complex sample can be realized. In particular, the gradual increase of the organic phase ratio in the first and second stages and the maintenance of the stability are helpful to the separation between the target sample and other interfering items, and the rapid elution of the target sample from other components. In the third and fourth stages, the ratio of the organic phase to the aqueous phase is changed back to the initial ratio and maintained, which restores the initial equilibrium state of the system, is conducive to the detection consistency of the next sample, and has high repeatability. The mass spectrometer is used as a detector, has high sensitivity and selectivity, and can effectively qualitatively and quantitatively detect trace amounts of N-nitroso-ticagrelor impurities.

[0028] The features and advantages of the present application will be more apparent from the following detailed description along with the accompanying drawings in which: like reference numerals refer to like elements or components throughout. The best mode for carrying out the present application or one of the best modes thereof will be described in detail in conjunction with the accompanying drawings, but the present application is not limited to the mode. In addition, the features, elements and components appearing in each of the following description and drawings are plural, and are marked with different symbols or numbers for the convenience of representation, but all represent the same or similar configuration or function. BRIEF DESCRIPTION OF DRAWINGS

[0029] The present application will be described in further detail with reference to the accompanying drawings.

[0030] Figure 1 is a flow chart of one embodiment of the detection method of N-nitroso-ticagrelor provided by the present application;

[0031] Figure 2(a) is the chemical structural formula of ticagrelor;

[0032] Figure 2(b) is the chemical structural formula of N-nitroso-ticagrelor;

[0033] Figure 3 is the mass chromatogram of the blank solution provided by the present application;

[0034] Figure 4 is the mass chromatogram of the reference solution provided by the present application;

[0035] Figure 5 is the mass chromatogram of the sample solution provided by the present application;

[0036] Figure 6 is the mass chromatogram of the spiked solution provided by the present application;

[0037] Figure 7 is the mass chromatogram of the limit of detection solution provided by the present application;

[0038] Figure 8 is the mass chromatogram of the limit of quantification solution provided by the present application. DETAILED DESCRIPTION

[0039] Embodiments of the present application are described in detail below, examples of which are shown in the accompanying drawings, wherein the same or similar reference numerals represent the same or similar elements or elements having the same or similar functions throughout. Based on the embodiments in the embodiments, it is intended to explain the present application, and cannot be understood as a limitation of the present application.

[0040] In this specification, "one embodiment" or "an example" or "an example" means that a particular feature, structure or characteristic described in connection with the embodiment itself can be included in at least one embodiment of the present disclosure. The appearance of the phrase "in one embodiment" at various places in the specification does not necessarily refer to the same embodiment.

[0041] Nitrosamines describe a class of compounds with the chemical structure nitroso group and amine (R1N(-R2)-N=0). These compounds can be formed by nitrosation reaction between amine (secondary, tertiary or quaternary amine) and nitrous acid (nitrite salt under acidic condition). FDA has identified 7 nitrosamine impurities that can potentially exist in drug products: dimethyl nitrosamine (NDMA), N-nitrosodiethylamine (NDEA), N-nitroso-N-methyl-4-aminobutyric acid (NMBA), N-nitrosoisopropylethylamine (NIPEA), N-nitrosodiisopropylamine (NDPA), N-nitrosodibutylamine (NDBA) and N-nitrosomethylaniline (NMPA). Five of them (NDMA, NDEA, NMBA, NIPEA and NMPA) have actually been detected in APIs or drug products. The above impurities belong to simple nitrosamine impurities, i.e. small molecule nitrosamine impurities resulted from the reaction between reagents, or between reagents and impurities during the manufacturing process of drug products. The carcinogenicity and teratogenicity of these simple nitrosamine impurities have been well studied, and their AI (Acceptable Intake) values have been clearly defined, for example, the AI value of NDMA is 96 ng / day, and the AI value of NDEA is 26.5 ng / day.

[0042] After the detection of simple nitrosamine impurities, with the development of detection technology and drug research, the detection of nitrosamine drug substance related impurities (NDSRI) is becoming more and more important. Unlike simple nitrosamine impurities, NDSRI refers to those types of drug molecules that participate in the formation of nitrosamine impurities. These drugs usually contain secondary amine (including tertiary amine) structures, which provide amino units for the formation of nitrosamine. For example, the nitrosamine impurities found in the drug rasagiline are N-nitrosorasagiline, and the N-nitrosotolperisone found in the drug orphenadrine, which proves that secondary amine (including tertiary amine) structures can also trigger the formation of nitrosamine. Compared with NDMA and NDEA, these product-related NDSRI nitrosamine impurities lack sufficient data support. FDA and EMA set AI for NDSRI impurities based on the activating structural features and deactivating structural features in the molecular structure of NDSRI, to predict the carcinogenicity of these molecules, and on this basis, set the corresponding AI value. According to the chemical structure of N-nitrosotagrel as NDSRI, the activity level of its impurities is determined to be level 5 by the above-mentioned activity level standard, and the AI value is determined to be 1.5 μg / day, and the upper limit of N-nitrosotagrel in tagrel is calculated to be 8 μg / g.

[0043] At present, the industry generally detects nitrosamine impurities by liquid chromatography-mass spectrometry method, mainly using a chromatographic column with octadecyl-bonded silica gel as the filling phase (referred to as C18 chromatographic column). The C18 chromatographic column has an alkyl chain of 18 carbon atoms, which has very small polarity. It is usually used to separate a variety of substances with large structural differences, and the polarity difference between them is very large, so the interaction with C18 is different, the retention time in the chromatographic column is different, and the elution time is also different, so the separation of multiple substance peaks is realized. However, as an NDSRI in tagrel, N-nitrosotagrel, its chemical structure is very similar to that of tagrel, as shown in FIG. 2(a) and FIG. 2(b), the difference in polarity between the two is very small. If the industry's general detection technology for nitrosamine impurities is used, it is impossible to effectively elute and separate the two. On the other hand, the acceptable value of N-nitrosotagrel is very low, which means the detection upper limit is low. In order to accurately detect the specific content, a more sensitive and accurate detection method needs to be developed. In addition, there is no detection method and related information developed for N-nitrosotagrel in the related art.

[0044] Based on the above problems, it is necessary to develop a detection method for N-nitroso-ticagrelor based on its unique structure and the special differences between drugs, so as to sensitively and accurately detect N-nitroso-ticagrelor and effectively separate N-nitroso-ticagrelor from ticagrelor.

[0045] It should be emphasized that the detection method for N-nitroso-ticagrelor in this application is a completely developed detection method. According to the acceptable standard amount and specific structure analysis, the actual detection situation is considered, and a large number of experimental verification is combined to develop a rapid and accurate detection method for determining the content of N-nitroso-ticagrelor.

[0046] Therefore, as a first aspect of the present application, a detection method for the content of ticagrelor impurities is disclosed, as shown in Figure 1 The ticagrelor impurity is N-nitroso-ticagrelor, and the detection method comprises:

[0047] S100, providing an initial sample, diluting the initial sample with a diluent to prepare a detection sample;

[0048] S200, introducing the detection sample into the mobile phase through a sample injector, and performing gradient elution treatment on the detection sample using a chromatographic column with phenyl-hexyl bonded silica gel as the filler at a set temperature to obtain an elution sample, wherein the mobile phase comprises an organic phase and an aqueous phase, and the gradient elution conditions comprise:

[0049] In the first stage, the ratio of the organic phase to the aqueous phase is directly proportional to the time length;

[0050] In the second stage, the ratio of the organic phase to the aqueous phase is the ratio at the end of the first stage;

[0051] In the third stage, the ratio of the organic phase to the aqueous phase in the mobile phase is inversely proportional to the time length;

[0052] In the fourth stage, the ratio of the organic phase to the aqueous phase in the mobile phase is the ratio at the end of the third stage, and the ratio of the organic phase to the aqueous phase in the fourth stage is less than the ratio of the organic phase to the aqueous phase in the second stage;

[0053] S300, determining the elution sample by mass spectrometer.

[0054] In step S100, the source of the initial sample is not particularly limited, and can be obtained by purchase or self-preparation. The detection method of the present application is suitable for any stage in the synthesis of ticagrelor, and can detect whether N-nitroso-ticagrelor exists. In some embodiments, the initial sample includes any one of the drug substance, intermediate or preparation of ticagrelor. Generally, the main generation stage of N-nitroso-ticagrelor is not in the synthesis of the drug substance, but is more likely to occur in the preparation stage. For example, some excipients and aluminum film are added in the preparation, and the printing ink may contain nitrite substances or impurities, including nitrite in water. Therefore, N-nitroso-ticagrelor may be generated in the preparation process, storage and packaging. Therefore, in some cases, in order to detect whether N-nitroso-ticagrelor exists, the preparation of ticagrelor is directly detected, and N-nitroso-ticagrelor can be more easily detected.

[0055] As a specific embodiment, in the step of providing an initial sample and diluting the initial sample with a diluent,

[0056] A certain amount of the initial sample is weighed by using an electronic balance;

[0057] The initial sample is placed in a volumetric flask of a certain volume, and is mixed uniformly after being diluted to volume with a diluent, which includes methanol. Generally, in order to avoid the interference of solvent peaks, the polarity of the diluent and the mobile phase is kept as consistent as possible. However, sometimes it is difficult to dissolve the sample with the mobile phase, and the solvent needs to be replaced. In this case, a diluent with a polarity close to that of the mobile phase is also selected as much as possible. If the polarity of the diluent and the mobile phase is greatly different, the retention time and peak shape will change. In the present application, HPLC-grade methanol is used as the diluent, and acetonitrile is selected as the organic phase of the mobile phase. The polarity of methanol is greater than that of acetonitrile. In reverse phase chromatographic analysis, the smaller the polarity, the stronger the elution ability, and the shorter the peak time of the sample. Therefore, when acetonitrile is used as the mobile phase, the retention time of the detected peak is smaller than that when methanol is used as the mobile phase, and the sample can be eluted and separated more quickly. In addition, the maximum injection volume of the diluent is several microliters, and the proportion of the mobile phase is not greatly changed.

[0058] In step S200, the application does not make special limitation on how the sample is introduced into the liquid chromatograph. In some embodiments, the liquid chromatograph is composed of a delivery system, a sample introduction system, a separation system, a detection system, and a data processing system. In the step of introducing the sample into the mobile phase through the sample injector, an automatic sample injector or a manual sample injector can be selected. The automatic sample injector has the function of sample chamber temperature control. Because in the liquid chromatographic detection, some temperature-sensitive samples require the sample chamber temperature to be controlled within a certain range. The automatic sample injector can accommodate an increasing number of samples in the sample chamber. After placing a batch of samples in the sample chamber, the instrument can work continuously for a long time. In this case, the temperature of the sample chamber must be controlled when the temperature-sensitive samples are stored in the sample chamber for a long time to prevent the samples from deteriorating. As an optional implementation, the temperature of the sample disc is between 8°C and 12°C. Within this range, ticagrelor and N-nitroso-ticagrelor are not easily deteriorated, have high chemical stability, and have less impact on the detection results.

[0059] The application does not make special limitation on the single injection volume. The selection of the injection volume needs to consider the capacity of the chromatographic column and the solvent effect. As a preferred embodiment, the single injection volume is between 9 μL and 11 μL. Too much injection volume can cause overload of the chromatographic column, resulting in broadening and diffusion of the chromatographic peak, deformation or peak fusion, affecting the accuracy of the peak area. Too little injection volume makes the detection sensitivity low and cannot accurately detect the corresponding substances.

[0060] The application does not make special limitation on the flow rate of the organic phase and the aqueous phase. Generally, the best flow rate should be used in the separation and elution process to obtain the best separation effect. The flow rate is determined according to the particle size and shape of the filler. According to the Van Deemter curve, the chromatographic column specifications used in the application, and the actual column efficiency, the flow rate of the organic phase and the aqueous phase is between 0.4 mL / min and 0.6 mL / min. The greater the flow rate, the higher the analysis speed, the smaller the plate height, the greater the column efficiency, i.e., the separation effect. When the flow rate exceeds 0.6 mL / min, the theoretical plate height increases, which makes the column efficiency worse. In addition, the flow rate also needs to consider the service life of the chromatographic column. Because the greater the flow rate, the less the silica gel matrix content of the chromatographic column, which cannot be recovered, and the surface-bonded phenyl-hexyl modification group is also taken away, which reduces the column efficiency.

[0061] This application does not impose special limitations on the selection of the mobile phase. The mobile phase solvent should have low viscosity and chemical stability, not react with the stationary phase and sample components, and not change any properties of the packing material. In reversed-phase liquid chromatography, the polarity of the mobile phase is greater than that of the chromatographic column. Preferably, the mobile phase is prepared by mixing an aqueous phase and a less polar organic phase in a certain proportion. Commonly used organic phases include acetonitrile and methanol. Pure water is generally not used alone as the aqueous phase because its pH is unstable and affects the peak time. An acidic aqueous phase is generally selected, such as a 0.1% formic acid aqueous solution. Formic acid can act as a pH adjuster to keep the pH of the mobile phase in a stable state and improve the peak shape.

[0062] In the development of reversed-phase liquid chromatography methods, it is common to change the organic phase ratio, aqueous phase pH, and elution gradient of the mobile phase. In this application, based on the use of a phenyl-hexyl-bonded silica column as the packing material, the above parameters were also adjusted accordingly. In addition, other stationary phases were added for verification, as shown in Table 1 below, to obtain the optimal resolution. The results in Table 1 demonstrate that, under the same conditions, the separation results of the phenyl-hexyl-bonded silica column are better than those of the C18 column. The recovery rate represents the proportion of N-nitrosoticagrelor detected to N-nitrosoticagrelor in the initial sample. The higher the recovery rate, the better the retention and separation effect of the column on the substance.

[0063] Table 1

[0064]

[0065] In Table 1, the phenyl groups bonded to the silica gel surface of stationary phase 1 enhance the intermolecular interactions with aromatic analytes and also exhibit some interaction with N-containing heterocycles. Furthermore, the phenyl groups provide steric hindrance, further enhancing retention. This results in stronger retention of N-nitrosotic ticagrelor compared to the C18 alkane-bonded phase, ultimately leading to significantly different resolutions and recoveries. It is worth noting that stationary phases 2 and 3 in Table 1 are both C18 bonded packing materials. The recoveries of both stationary phases are not high, even with smaller column inner diameters (2.1 mm) and particle sizes (3.5 μm). Generally, with a column length of 100 mm, smaller inner diameters result in higher column efficiency, but even with smaller inner diameters, stationary phases 2 and 3 still show lower recoveries than stationary phase 1. This demonstrates the superior retention of N-nitrosotic ticagrelor by the phenyl-hexyl bonded column described in this application. In addition, the selectivity differences between different C18 columns are more obvious. Stationary phase 2 and stationary phase 3 have the same column specifications, but come from different manufacturers, resulting in a large difference in their recovery rates. Using C18 makes it more difficult to predict the selectivity of different stationary phases, leading to poor consistency.

[0066] This application does not impose specific limitations on the specifications of the chromatographic column. Preferably, a column length between 90 mm and 110 mm, a column diameter between 4.5 mm and 4.7 mm, a packing particle size between 2 μm and 4 μm, and a column temperature between 35°C and 45°C can achieve better separation results. Preferably, the silica gel particles have a pore size between 8 nm and 12 nm and a carbon loading between 10% and 14%.

[0067] Based on the aforementioned phenyl-hexyl chromatographic column, this application employs gradient elution, which can increase the separation of multiple substances. By adding different ratios of organic and aqueous phases at different time points, the concentration of impurities eluted by the organic phase varies at different times, thereby reducing problems such as peak broadening, distortion, tailing, and fusion. Furthermore, since the concentration of the added organic phase can be adjusted, the analysis time can be shortened compared to isocratic elution, and several substances that cannot be separated by isocratic elution can be separated in one step, thus allowing the analysis of multiple substances using a single analytical method and reducing costs. As an optional implementation, the gradient elution conditions in this application are as follows:

[0068] In the first stage, the ratio of organic phase to aqueous phase is increased from a first set ratio to a second set ratio. Since this application uses reversed-phase liquid chromatography adsorption, substances with stronger polarity usually elute first, which is usually some polar impurities in the solvent. However, the chromatographic column of this application has a certain retention capacity for N-nitrosoticagrelor. In the initial stage, the proportion of organic phase is low, and polar impurities are eluted first. Then, the proportion of organic phase is gradually increased and the proportion of aqueous phase is decreased, so that the more strongly retained N-nitrosoticagrelor is eluted. N-nitrosoticagrelor is a compound with a large molecular weight, and its retention behavior is very sensitive to small changes in the mobile phase. Therefore, the gradually increasing gradient can significantly change the retention factor, thereby rapidly eluting out a large amount of N-nitrosoticagrelor.

[0069] In the second stage, the ratio of organic phase to aqueous phase is consistent with the second set ratio. This stage can be regarded as the system equilibrium and stability stage. The ratio of mobile phase remains stable and does not change gradient, so as to ensure that all N-nitrosotic ticagrelor impurities are eluted and that they do not interfere with the detection in the next injection elution.

[0070] In the third stage, the ratio of organic phase to aqueous phase is reduced from the second set ratio to the first set ratio, and adjusted to the initial ratio to prepare for the next injection elution.

[0071] In the fourth stage, the ratio of organic phase to aqueous phase is consistent with the first set ratio, restoring the mobile phase ratio in the chromatographic column to the initial ratio (equilibration column) to ensure the stability of the next injection detection.

[0072] As a specific implementation method, gradient elution conditions include:

[0073] The first stage is from 0 min to 7 min, the ratio of organic phase to water phase is increased from 1:1 to 4:1, preferably, is increased in a linear gradient, so that the test results are more stable and controllable. The gradient of the present application is sufficient to separate N-nitroso-ticagrelor in unit time, and the rate and efficiency of the gradient meet the requirements of determining impurities. The target impurities are completely eluted in the gradient change stage.

[0074] The second stage is from 7 min to 11 min, the ratio of organic phase to water phase is kept at 4:1; the third stage is from 11 min to 11.1 min, the ratio of organic phase to water phase is decreased from 4:1 to 1:1; the fourth stage is from 11.1 min to 15 min, the ratio of organic phase to water phase is kept at 1:1.

[0075] As a specific embodiment, the gradient elution conditions are:

[0076] From 0 min to 7 min, the content of organic phase is increased from 50% to 80%, and the content of water phase is decreased from 50% to 20%;

[0077] From 7 min to 11 min, the content of organic phase is 80%, and the content of water phase is 20%;

[0078] From 11 min to 11.1 min, the content of organic phase is decreased from 80% to 50%, and the content of water phase is increased from 20% to 50%;

[0079] From 11.1 min to 15 min, the content of organic phase and water phase is 50%.

[0080] In step S300, as a specific embodiment, the eluted sample is subjected to electrospray ionization treatment to obtain gas phase ions, wherein the ionization voltage is between 3300V and 3700V, the sheath gas flow rate is between 40 Arb and 60 Arb, the auxiliary gas flow rate is between 5 Arb and 15 Arb, the backflush gas flow rate is between 0.5 Arb and 1.5 Arb, the vaporization chamber temperature is between 300℃ and 400℃, and the temperature of the ion transmission tube is between 250℃ and 350℃;

[0081] The gas phase ions are scanned and collected in multiple ion channel stages to obtain target ions, wherein the collection mode includes a multiple reaction monitoring mode, the multiple ion channel stages include a first ion channel stage, an intermediate channel stage and a second ion channel stage, the ions passing through the first ion channel stage enter the second ion channel stage after fragmentation in the intermediate channel stage, and the target ions are determined when the ions pass through the second ion channel stage. The mass-to-charge ratio of the first ion channel stage satisfies 552.2 Da, the mass-to-charge ratio of the second ion channel stage satisfies 522.2 Da, and the fragmentation energy of the intermediate channel stage is between 8 V and 9 V.

[0082] In some embodiments, the present application adopts a liquid chromatograph-mass spectrometer to perform separation determination, selects a triple quadrupole mass spectrometer, and can qualitatively and quantitatively determine target impurities.

[0083] The detection method of the present application can detect N-nitroso ticagrelor at a minimum concentration of not less than 0.216 ng / mL, and can quantitatively detect a sample at a minimum concentration of not less than 0.721 ng / mL, which fully meets the FDA standard of 8 μg.

[0084] As a second aspect of the present application, an application of a detection method of a ticagrelor impurity content is disclosed, which adopts the above-mentioned detection method of a ticagrelor impurity content to detect impurities generated in any one of a raw material, an intermediate or a preparation of ticagrelor.

[0085] The present application will be further described below in combination with preparation examples and embodiments.

[0086] Preparation Example

[0087] Preparation Example 1

[0088] The method for preparing a detection sample of the present application is shown in Table 2. N-nitroso ticagrelor is used as a control sample with a purity of 92.02%. Methanol, acetonitrile and formic acid are all HPLC grade;

[0089] Sample solution: ticagrelor is taken as a sample, 20 mg of the sample is taken and placed in a 20 mL volumetric flask, and methanol is used as a diluent to make up the volume, and the sample solution is prepared by shaking. The concentration of ticagrelor in the sample solution is 1 mg / mL;

[0090] Control sample stock solution: N-nitroso ticagrelor is taken as a control sample, 4 mg of the control sample is taken and placed in a 20 mL volumetric flask, and methanol is used as a diluent to make up the volume. After shaking, 0.2 mL is accurately taken from the volumetric flask and placed in a 10 mL volumetric flask, and methanol is used as a diluent to make up the volume. After shaking, 1 mL is accurately taken from the volumetric flask and placed in a 25 mL volumetric flask, and the diluent is used to make up the volume, and the control sample stock solution is prepared by shaking. The concentration of N-nitroso ticagrelor in the control sample stock solution is 0.16 ug / mL.

[0091] The control solution was prepared by taking 1 mL of the control stock solution, placing it in a 20 mL volumetric flask, diluting to volume with the methanol diluent, and shaking to homogeneity, to give a control solution with an N-nitroso-ticagrelor concentration of 0.008 ug / mL.

[0092] The control solution was prepared by taking 1 mL of the control stock solution, placing it in a 20 mL volumetric flask, diluting to volume with the methanol diluent, and shaking to homogeneity, to give a control solution with an N-nitroso-ticagrelor concentration of 0.008 ug / mL.

[0093] The detection limit solution was prepared by taking 0.3 mL of the control solution, placing it in a 10 mL volumetric flask, diluting to volume with the methanol diluent, and shaking to homogeneity.

[0094] The limit of quantitation solution was prepared by taking 1 mL of the control solution, placing it in a 10 mL volumetric flask, diluting to volume with the methanol diluent, and shaking to homogeneity.

[0095] The LOQ spike solution was prepared by taking 20 mg of the ticagrelor sample, placing it in a 20 mL volumetric flask, adding 0.1 mL of the control stock solution, diluting to volume with the methanol diluent, and shaking to homogeneity.

[0096] The 100% spike solution was prepared by taking 20 mg of the ticagrelor sample, placing it in a 20 mL volumetric flask, adding 1 mL of the control stock solution, diluting to volume with the methanol diluent, and shaking to homogeneity.

[0097] The 150% spike solution was prepared by taking 20 mg of the ticagrelor sample, placing it in a 20 mL volumetric flask, adding 1.5 mL of the control stock solution, diluting to volume with the methanol diluent, and shaking to homogeneity.

[0098] Table 2

[0099]

[0100] The model number of the balance used to prepare the solutions and the model number of the associated instrument used in this application are shown in Table 3.

[0101] Table 3

[0102]

[0103] Example

[0104] Example 1

[0105] Selection of the chromatographic column:

[0106] Three different chromatographic columns were used to perform gradient elution under the same conditions on three portions of the control solution prepared in Preparation Example 1. The gradient elution conditions are shown in Table 5. In the mobile phase, 0.1% formic acid aqueous solution was used as the water phase and acetonitrile was used as the organic phase, the flow rate was 0.5 mL / min, the column temperature was 40°C, the single injection volume was 10 μL, and the sample tray temperature was 10°C.

[0107] Table 4

[0108]

[0109] The phenyl-hexyl bonded silica gel of stationary phase 1 in Table 4 is a column of PerkinElmer brand under the Epic Phenyl Hexyl series, with column specifications of length 100 mm, inner diameter 4.6 mm, particle size 3 μm, and column temperature 40℃.

[0110] Table 5

[0111]

[0112] The triple quadrupole mass spectrometer detector was used to detect, using the multiple reaction monitoring mode (MRM), the triple quadrupole mass spectrometer detector ion source was an electrospray ion source (H-ESI), the source voltage was 3500V, the sheath gas flow rate was 50Arb, the auxiliary gas flow rate was 10Arb, the backflush gas flow rate was 1Arb, the ion transmission tube temperature was 300℃, the gasification chamber temperature was 350℃, the multiple reaction monitoring mode (MRM) quantitative ion channel was 552.2→522.2Da, the fragmentation energy was 8.58V, the above process parameters are shown in Table 6, and the test results obtained are shown in Table 7.

[0113] Table 6

[0114]

[0115] Table 7

[0116]

[0117] The recovery rate here is the ratio of the N-nitroso-ticagrelor content obtained by the liquid chromatograph-mass spectrometer quantitative detection used in the application to the N-nitroso-ticagrelor content in the initial control solution prepared.

[0118] Table 7 shows that the chromatographic column will directly affect the separation of ticagrelor and N-nitroso-ticagrelor, and then affect the ionization efficiency of N-nitroso-ticagrelor, which is ultimately manifested as the recovery rate. The recovery rates of the two chromatographic columns of Agilent and Waters are low, and the recovery rate of the chromatographic column of PerkinElmer is close to 100%, indicating that the separation effect of N-nitroso-ticagrelor is the best, and finally the chromatographic column PerkinElmer Epic Phenyl Hexyl (4.6mm 100mm 3μm) is selected.

[0119] Example 2

[0120] Selection of column temperature

[0121] The control sample solution was detected by the same detection method as in Example 1, and stationary phase 1 was selected as the chromatographic column, except that different column temperatures were set, as shown in Table 8, and the test results obtained are shown in Table 9.

[0122] Table 8

[0123]

[0124] Table 9

[0125]

[0126] Table 9 shows that the column temperature directly affects the elution bandwidth of the compound in the chromatographic column, thereby affecting the peak width of the target peak. When the column temperature is 40°C, the theoretical plate number of the target peak is the largest, the column efficiency is the best, and different temperatures have almost no effect on the recovery rate of the target substance, so the column temperature of 40°C is selected.

[0127] Example 3

[0128] Setting of source voltage

[0129] The control sample solution was detected by the same detection method as in Example 1, and stationary phase 1 was selected as the chromatographic column, except that when detected by a triple quadrupole mass spectrometer detector, the eluted sample was detected by using different source voltages, as shown in Table 10, and the test results obtained are shown in Table 11.

[0130] Table 10

[0131]

[0132] Table 11

[0133]

[0134] Table 11 shows that the source voltage directly affects the ionization of the precursor ion of N-nitrosoticagrelor, thereby affecting the peak area of the target peak. When the source voltage is 3500V, the peak area is the largest, so the source voltage of 3500V is selected.

[0135] Example 4

[0136] System applicability and specificity

[0137] The same detection method as in Example 1 was used, except that different detection samples were used, and the number of injections was different, and the specific injection parameters are shown in Table 12. According to different injection samples, the calculation method of the detection results is shown in Table 13, and the calculation results are shown in Table 14.

[0138] Table 12

[0139]

[0140] Table 13

[0141]

[0142] Table 14

[0143]

[0144] Table 14 shows that the response value of the blank solution is 0, there is no interference, the peak area RSD (relative standard deviation) of the control solution is 1.7% for 6 consecutive injections, the deviation is small, the applicability of the method is strong, the spiked solution has no interference, and the specificity is strong.

[0145] Example 5

[0146] Limit of detection and limit of quantification

[0147] The same detection method as in Example 1 was used, except that different detection samples were used, and the number of injections was different. The injection parameters are shown in Table 15. The calculation method of the detection results according to different injection samples is shown in Table 16, and the calculation results are shown in Table 17.

[0148] Table 15

[0149]

[0150] Table 16

[0151]

[0152] Table 17

[0153]

[0154] Table 17 shows that the concentration of the limit of detection solution is 0.216 ng / mL, the signal-to-noise ratio is 32.3, the concentration of the limit of quantification solution is 0.721 ng / mL, the peak area RSD for 6 consecutive injections is 2.4%, and the signal-to-noise ratio is 90.8~308.6. The detection limit and the limit of quantification of the detection method of the present application both reach an acceptable level.

[0155] Example 6

[0156] Accuracy and repeatability

[0157] The same detection method as in Example 1 was used, except that different detection samples were used, and the number of injections was different. The injection parameters are shown in Table 18. The calculation method of the detection results according to different injection samples is shown in Table 19, and the calculation results are shown in Table 20.

[0158] Table 18

[0159]

[0160] Table 19

[0161]

[0162] Table 20

[0163]

[0164] Table 20 shows that the recovery rate of 9 spiked solutions at 3 spiked levels is 91.3%~97.4%, the RSD is 2.5%, the recovery rate RSD of each spiked level is 1.6%~3.0%, the accuracy and repeatability of the detection method meet the requirements.

[0165] Example 7

[0166] Solution stability

[0167] The same detection method as in Example 1 was used, except that different detection samples were used, and the injection method was different. The injection parameters are shown in Table 21. According to different injection samples, the calculation method of the detection results is shown in Table 22, and the calculation results are shown in Table 23.

[0168] Table 21

[0169]

[0170] Table 22

[0171]

[0172] Table 23

[0173]

[0174] Table 23 shows that the detection results of the reference solution are stable within 11.3 hours, the RSD is 7.0%, and the detection results of the spiked solution are stable within 11.3 hours, the RD is 7.0%.

[0175] Test example

[0176] The detection results in Examples 1 to 7 above were extracted from the mass chromatogram, as shown in Figures 3 to 8 It can be seen that Figure 3 the mass chromatogram of the blank solution in has no obvious peak shape, and the peak intensity is also clear, which proves that the detection method of the application is less affected by the interference of the blank solution, Figure 4 the mass chromatogram of the reference solution in has a clear target peak, and no other impurity peak appears around it, the peak intensity is in the order of 10 to the fourth power, and the specificity and sensitivity are higher; Figure 5The mass chromatogram of the sample solution in the above proves that the sample solution of ticagrelor cannot be effectively separated and detected by the detection method of the present application, and therefore can be clearly distinguished from N-nitroso-ticagrelor. In addition, it can also be shown that the N-nitroso-ticagrelor impurity in the sample solution does not exceed the acceptable amount standard, and therefore cannot be detected. Figure 6 The mass chromatogram of the spiked solution in the above shows that only 1 ml of the control sample solution containing 8 ng / mL of N-nitroso-ticagrelor impurity is added to the ticagrelor sample 1, which can be clearly detected, and the specificity of the detection method is very strong. Figure 7 and Figure 8 The mass chromatograms of the detection limit solution and the quantification limit solution, respectively, show that at such low concentrations of N-nitroso-ticagrelor impurities at the detection limit and quantification limit, the detection method of the present application can still be clearly detected, with obvious peak shape and high peak strength, indicating higher sensitivity.

[0177] The above is only a specific embodiment of the present application, but the protection scope of the present application is not limited thereto. Those skilled in the art should understand that the present application includes but is not limited to the contents described in the above specific embodiments and the accompanying drawings. Any modification that does not deviate from the functional and structural principles of the present application shall be included in the scope of the claims.

Claims

1. A method for detecting the content of ticagrelor impurities, characterized in that, The ticagrelor impurity is N-nitrosoticagrelor, and the detection method includes: An initial sample is provided, and the initial sample is diluted to prepare a test sample, wherein the initial sample includes a ticagrelor formulation; The test sample is introduced into the mobile phase through an injector. At a set temperature, the test sample is subjected to gradient elution using a chromatographic column packed with phenyl-hexyl-bonded silica particles to obtain the eluted sample. The mobile phase comprises an organic phase and an aqueous phase. The organic phase is acetonitrile, and the aqueous phase is a 0.1% formic acid aqueous solution. The gradient elution conditions include: In the first stage, from 0 min to 7 min, the content of the organic phase increases from 50% to 80%, while the content of the aqueous phase decreases from 50% to 20%. In the second stage, from 7 to 11 minutes, the content of the organic phase is 80% and the content of the aqueous phase is 20%. In the third stage, from 11 min to 11.1 min, the content of the organic phase decreased from 80% to 50%, while the content of the aqueous phase increased from 20% to 50%. In the fourth stage, from 11.1 min to 15 min, the content of both the organic phase and the aqueous phase was 50%. The eluted sample was analyzed by mass spectrometry to determine its composition.

2. The detection method according to claim 1, characterized in that, The silica particles have a pore size between 8 nm and 12 nm and a carbon loading between 10% and 14%.

3. The detection method according to claim 2, characterized in that, The column length is between 90 mm and 110 mm, the column diameter is between 4.5 mm and 4.7 mm, the packing material particle size is between 2 μm and 4 μm, and the column temperature is between 35°C and 45°C.

4. The detection method according to claim 1, characterized in that, The flow rates of the organic phase and the aqueous phase are between 0.4 mL / min and 0.6 mL / min.

5. The detection method according to any one of claims 1 to 4, characterized in that, In the steps of providing an initial sample and diluting the initial sample... Weigh the set amount of initial sample; The initial sample is diluted and brought to a constant volume using a diluent, and then mixed thoroughly to obtain the test sample. The diluent includes methanol or acetonitrile. In the test sample, the lowest detectable concentration of N-nitrosoticagrelor impurity is not less than 0.216 ng / mL, and the lowest quantitatively detectable concentration of N-nitrosoticagrelor impurity is not less than 0.721 ng / mL.

6. The detection method according to any one of claims 1 to 4, characterized in that, In the step of introducing the test sample into the mobile phase through the injector, the sample pan temperature is between 8°C and 12°C, and the single injection volume is between 9 μL and 11 μL.

7. The detection method according to any one of claims 1 to 4, characterized in that, In the step of performing mass spectrometry analysis on the eluted sample to determine the composition of the eluted sample, The eluted sample is subjected to electrospray ionization treatment to obtain gaseous ions, wherein the ionization voltage is between 3300V and 3700V, the sheath gas flow rate is between 40 Arb and 60 Arb, the auxiliary gas flow rate is between 5 Arb and 15 Arb, the backflush gas flow rate is between 0.5 Arb and 1.5 Arb, the vaporization chamber temperature is between 300°C and 400°C, and the ion transmission tube temperature is between 250°C and 350°C. The gaseous ions are scanned and acquired using a multi-stage ion channel to obtain target ions. The acquisition mode includes a multi-reaction monitoring mode. The multi-stage ion channel includes a first-stage ion channel, an intermediate channel, and a second-stage ion channel. Ions passing through the first-stage ion channel are fragmented through the intermediate channel and then enter the second-stage ion channel. When the ions pass through the second-stage ion channel, the target ion is detected. The mass-to-charge ratio of the first-stage ion channel is 552.2 Da, the mass-to-charge ratio of the second-stage ion channel is 522.2 Da, and the fragmentation energy of the intermediate channel is between 8 V and 9 V.

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