Method for detecting N-noranisodamine nitrosamine impurities

Abstract

The invention provides a detection method of N-noranisodamine nitrosamine impurities, and relates to a detection method of N-noranisodamine nitrosamine impurities. According to the method disclosed by the invention, the N-noranisodamine nitrosamine impurity in the anisodamine hydrobromide raw material medicine and the preparation can be effectively separated and identified by adopting a specific mobile phase combined gradient elution procedure and combining with optimized mass spectrum conditions; the method for detecting the N-noranisodamine nitrosamine impurities, provided by the invention, is good in separation degree, sensitivity and reproducibility. The detection method provided by the invention is suitable for quality control and finished product detection in the production process of anisodamine hydrobromide bulk drugs and injections, and has a wide application prospect.

Description

Technical Field

[0001] The present invention belongs to the field of pharmaceutical technology, and particularly relates to a method for detecting N-norhyoscyamine nitrosamine impurities. Background Art

[0002] During the storage process in the production of raw materials and preparations of anisodamine hydrobromide, NDSRI impurities may be produced, and the production mechanism is as follows:

[0003]

[0004] This impurity is a genotoxic impurity, which may induce gene mutations and chromosomal damage, and may even cause cancer, posing a significant safety risk. There is no reported method for detecting this impurity. When using the conventional LC-MS method, there are problems such as poor resolution, sensitivity, and reproducibility.

[0005] Therefore, it has become a problem to be solved to provide a method for detecting N-norhyoscyamine nitrosamine impurities with good resolution, sensitivity, and reproducibility. Summary of the Invention

[0006] In view of this, the technical problem to be solved by the present invention is to provide a method for detecting N-norhyoscyamine nitrosamine impurities, and the method for detecting N-norhyoscyamine nitrosamine impurities provided by the present invention has good resolution, sensitivity, and reproducibility.

[0007] The present invention provides a method for detecting N-norhyoscyamine nitrosamine impurities, comprising the following steps:

[0008] Dissolve the test sample containing N-norhyoscyamine nitrosamine impurities in a solvent to obtain a test sample solution;

[0009] Perform high performance liquid chromatography-mass spectrometry detection on the test sample solution;

[0010] The conditions of the high performance liquid chromatography include: the chromatographic column uses octadecylsilane chemically bonded silica gel as the filler; 0.1% formic acid water is used as mobile phase A, and 0.1% formic acid acetonitrile is used as mobile phase B; gradient elution is performed. In terms of volume percentage, the elution program of the gradient elution is as follows:

[0011] 0 - 3 min, mobile phase A 90%, mobile phase B 10%;

[0012] 3 - 12 min, mobile phase A decreases from 90% to 10%, and mobile phase B increases from 10% to 90%;

[0013] 12 - 18 min, mobile phase A 10%, mobile phase B 90%;

[0014] From 18 to 18.1 min, mobile phase A increases from 10% to 90%, and mobile phase B decreases from 90% to 10%.

[0015] From 18.1 to 25 min, mobile phase A is 90% and mobile phase B is 10%.

[0016] The conditions of the mass spectrometry include: detected by a triple quadrupole tandem mass spectrometer, the mass spectrometry ionization method is ESI, the positive ion scanning mode, and the monitoring mode is multiple reaction monitoring.

[0017] Preferably, the test sample includes anisodamine hydrobromide raw material drug and / or preparation.

[0018] Preferably, the solvent includes an acetonitrile aqueous solution with a volume fraction of 50%.

[0019] Preferably, the test sample solution includes anisodamine hydrobromide, and the concentration of anisodamine hydrobromide is 0.5 - 2 mg / ml.

[0020] Preferably, it also includes preparing a reference substance solution, and the preparation method of the reference substance solution includes the following steps:

[0021] Dissolve the N-norhyoscyamine nitrosamine impurity reference substance with a solvent to obtain a reference substance solution.

[0022] Preferably, the solvent is selected from an acetonitrile aqueous solution with a volume fraction of 50%;

[0023] The concentration of the reference substance solution is 1 - 32 ng / ml.

[0024] Preferably, the chromatographic column of the high performance liquid chromatography is Agilent Poroshell 120 EC-C18, 4.6 mm × 150 mm, 2.7 μm or a chromatographic column with equivalent efficiency.

[0025] Preferably, the column temperature of the high performance liquid chromatography is 30 - 40 °C; the flow rate is 0.5 - 1.2 ml per minute; the injection volume is 3 - 10 μL, and the injection tray temperature is 3 - 10 °C.

[0026] Preferably, the ion source parameters of the mass spectrometry are: collision gas (CAD) is 8 psi, curtain gas (CUR) is 20 psi, TEM is 500 °C, nebulizing gas (GS1) is 40 psi, nebulizing gas (GS2) is 40 psi, and voltage (IS) is 5000 V.

[0027] Preferably, the detection method includes the following steps:

[0028] Perform high performance liquid chromatography - mass spectrometry detection on reference substance solutions with different concentrations respectively to obtain chromatograms;

[0029] Taking the peak area corresponding to the N-norhyoscyamine nitrosamine impurity in the chromatograms obtained by high performance liquid chromatography-mass spectrometry (HPLC-MS) of reference substance solutions with different concentrations as the ordinate, and the concentration of the reference substance solution as the abscissa, a standard curve was plotted.

[0030] The test sample solution was subjected to HPLC-MS detection to obtain a chromatogram. The peak area corresponding to the N-norhyoscyamine nitrosamine impurity in the chromatogram was substituted into the standard curve, and the concentration of the N-norhyoscyamine nitrosamine impurity in the test sample solution was calculated through calculation.

[0031] Compared with the prior art, the present invention provides a method for detecting N-norhyoscyamine nitrosamine impurity, comprising the following steps: dissolving a test sample containing N-norhyoscyamine nitrosamine impurity in a solvent to obtain a test sample solution; subjecting the test sample solution to HPLC-MS detection; the conditions of the HPLC include: using octadecylsilane chemically bonded silica gel as the filler for the chromatographic column; using 0.1% formic acid water as mobile phase A and 0.1% formic acid acetonitrile as mobile phase B; performing gradient elution. In terms of volume percentage, the elution program of the gradient elution is as follows: 0 - 3 min, mobile phase A 90%, mobile phase B 10%; 3 - 12 min, mobile phase A decreases from 90% to 10%, and mobile phase B increases from 10% to 90%; 12 - 18 min, mobile phase A 10%, mobile phase B 90%; 18 - 18.1 min, mobile phase A increases from 10% to 90%, and mobile phase B decreases from 90% to 10%; 18.1 - 25 min, mobile phase A 90%, mobile phase B 10%; the conditions of the mass spectrometry include: detecting with a triple quadrupole tandem mass spectrometer, the mass spectrometry ionization mode is ESI, positive ion scanning mode, and the monitoring mode is multiple reaction monitoring. By adopting a specific mobile phase combined with a gradient elution program and jointly optimizing the mass spectrometry conditions, the present invention can effectively separate and identify the N-norhyoscyamine nitrosamine impurity in the raw material drug and preparation of anisodamine hydrobromide. The method for detecting N-norhyoscyamine nitrosamine impurity provided by the present invention has good resolution, sensitivity and reproducibility. The detection method provided by the present invention is applicable to the quality control and finished product detection in the production process of anisodamine hydrobromide raw material drug and injection, and has a wide application prospect. Description of the Drawings

[0032] Figure 1 It is the XIC diagram of the reference substance;

[0033] Figure 2 It is the standard curve diagram of the N-norhyoscyamine nitrosamine impurity reference substance;

[0034] Figure 3 It is the XIC diagram of the 100% spiked recovery solution;

[0035] Figure 4Liquid chromatography - mass spectrometry overlay map of 100% spiked recovery solution. Detailed implementation mode

[0036] The present invention provides a method for detecting N - norhyoscyamine nitrosamine impurities, comprising the following steps:

[0037] Dissolve the test sample containing N - norhyoscyamine nitrosamine impurities in a solvent to obtain a test sample solution;

[0038] Perform high - performance liquid chromatography - mass spectrometry detection on the test sample solution;

[0039] The conditions of the high - performance liquid chromatography include: the chromatographic column uses octadecylsilane - bonded silica gel as the filler; 0.1% formic acid in water is used as mobile phase A, and 0.1% formic acid in acetonitrile is used as mobile phase B; gradient elution is performed. In terms of volume percentage, the elution program of the gradient elution is as follows:

[0040] 0 - 3 min, mobile phase A 90%, mobile phase B 10%;

[0041] 3 - 12 min, mobile phase A decreases from 90% to 10%, mobile phase B increases from 10% to 90%;

[0042] 12 - 18 min, mobile phase A 10%, mobile phase B 90%;

[0043] 18 - 18.1 min, mobile phase A increases from 10% to 90%, mobile phase B decreases from 90% to 10%;

[0044] 18.1 - 25 min, mobile phase A 90%, mobile phase B 10%;

[0045] The conditions of the mass spectrometry include: detection is performed using a triple quadrupole tandem mass spectrometer, the mass spectrometry ionization method is ESI, the positive ion scanning mode, and the monitoring mode is multiple reaction monitoring.

[0046] The present invention first prepares a test sample solution. Specifically, dissolve the test sample containing N - norhyoscyamine nitrosamine impurities in a solvent to obtain a test sample solution.

[0047] Among them, the test article includes anisodamine hydrobromide raw material drug and / or preparation. In the present invention, regarding the limit of N-norhyoscyamine nitrosamine impurity, in the prior arts such as FDA and EMA, there is no clear toxicological data of the genotoxic impurity NDSRI in anisodamine hydrobromide injection; the limit of the genotoxic impurity is determined by combining the toxicological concern threshold (TTC) of the impurity NDSRI (Wang Ping, Xu Caihong, Chen Xian, etc. Research progress on the control of genotoxic impurities in raw material drugs [J]. Chinese Journal of Modern Applied Pharmacy, 2015, 32(1): 119-126.), that is, calculated according to the TTC value of 1.5 μg / day, and the maximum dosage of anisodamine hydrobromide injection is 100 mg; the limit of N-norhyoscyamine nitrosamine impurity is (1.5 μg / day) / 100 mg = 15 ppm, that is, the limit shall not exceed 15 ppm. The setting of this limit not only conforms to the scientific principle but also ensures the safety and effectiveness of the product.

[0048] In the present invention, the solvent for preparing the test article solution includes an acetonitrile aqueous solution with a volume fraction of 50%.

[0049] The test article solution includes anisodamine hydrobromide, and the concentration of anisodamine hydrobromide is 1 mg / ml.

[0050] The present invention also includes preparing a reference substance solution, and the preparation method of the reference substance solution includes the following steps:

[0051] Dissolve the N-norhyoscyamine nitrosamine impurity reference substance with a solvent to obtain a reference substance solution. Among them, the solvent is selected from an acetonitrile aqueous solution with a volume fraction of 50%;

[0052] The concentration of the reference substance solution is 7.5 - 30 ng / ml, and it can be 7.5, 10, 12.5, 15, 17.5, 20, 25, 30, or any value between 7.5 - 30 ng / ml.

[0053] Then, the test article solution and the reference substance solution are respectively subjected to high performance liquid chromatography - mass spectrometry detection.

[0054] Among them, the conditions of the high performance liquid chromatography include:

[0055] The chromatographic column of the high performance liquid chromatography uses octadecylsilane chemically bonded silica as the filler. Preferably, the chromatographic column of the high performance liquid chromatography is Agilent Poroshell120EC-C18, 4.6 mm × 150 mm, 2.7 μm or a chromatographic column with equivalent performance.

[0056] Using 0.1% formic acid water as mobile phase A and 0.1% formic acid acetonitrile as mobile phase B; performing gradient elution. In terms of volume percentage, the elution program of the gradient elution is as follows:

[0057] 0 - 3 min, mobile phase A: 90%, mobile phase B: 10%;

[0058] 3 - 12 min, mobile phase A decreases from 90% to 10%, mobile phase B increases from 10% to 90%;

[0059] 12 - 18 min, mobile phase A: 10%, mobile phase B: 90%;

[0060] 18 - 18.1 min, mobile phase A increases from 10% to 90%, mobile phase B decreases from 90% to 10%;

[0061] 18.1 - 25 min, mobile phase A: 90%, mobile phase B: 10%.

[0062] The column temperature of the high - performance liquid chromatography is 30 - 40 °C; the flow rate is 0.5 - 1.2 ml per minute; the injection volume is 3 - 10 μL, and the injection tray temperature is 3 - 10 °C. Preferably, the column temperature of the high - performance liquid chromatography is 35 °C; the flow rate is 0.8 ml per minute; the injection volume is 5 μL, and the injection tray temperature is 5 °C.

[0063] The conditions of the mass spectrometry include: detecting with a triple quadrupole tandem mass spectrometer, the mass spectrometry ionization method is ESI, the positive ion scanning mode, and the monitoring mode is multiple reaction monitoring.

[0064] The ion source parameters of the mass spectrometry are: collision gas (CAD) is 8 psi, curtain gas (CUR) is 20 psi, TEM is 500 °C, nebulizing gas (GS1) is 40 psi, nebulizing gas (GS2) is 40 psi, and voltage (IS) is 5000 V.

[0065] In the present invention, the test solution and the reference solution are respectively subjected to high - performance liquid chromatography - mass spectrometry detection to obtain chromatograms. Then, the content of N - noranisodamine nitrosamine impurities can be calculated by the external standard method. Further, according to the content of N - noranisodamine nitrosamine impurities in the anisodamine hydrobromide raw material drug and / or preparation, the quality control of the anisodamine hydrobromide raw material drug and preparation is carried out.

[0066] Among them, the external standard method specifically includes the following steps:

[0067] The reference solutions with different concentrations are respectively subjected to high - performance liquid chromatography - mass spectrometry detection to obtain chromatograms. Among them, the concentration range of the reference solutions with different concentrations is preferably 1 - 32 ng / mL.

[0068] In some specific embodiments of the present invention, the concentrations of the reference substance solutions with different concentrations include 1.0428 ng / mL, 3.1284 ng / mL, 7.8211 ng / mL, 15.6421 ng / mL, 23.4632 ng / mL, and 31.2842 ng / mL.

[0069] Then, taking the peak area corresponding to the N-norhyoscyamine nitrosamine impurity in the chromatogram obtained by high performance liquid chromatography-mass spectrometry detection of the reference substance solutions with different concentrations as the ordinate and the concentration of the reference substance solution as the abscissa, a standard curve is plotted.

[0070] Finally, the test sample solution is subjected to high performance liquid chromatography-mass spectrometry detection to obtain a chromatogram, and the peak area corresponding to the N-norhyoscyamine nitrosamine impurity in the chromatogram is brought into the standard curve, and then the concentration of the N-norhyoscyamine nitrosamine impurity in the test sample solution can be calculated.

[0071] By adopting a specific mobile phase combined with a gradient elution program and jointly optimizing the mass spectrometry conditions, the present invention can effectively separate and identify the N-norhyoscyamine nitrosamine impurity in the raw material drug and preparation of anisodamine hydrobromide. The detection method for the N-norhyoscyamine nitrosamine impurity provided by the present invention has good resolution, sensitivity and reproducibility. The detection method provided by the present invention is applicable to the quality control and finished product detection in the production process of the raw material drug and injection of anisodamine hydrobromide, and has broad application prospects.

[0072] To further understand the present invention, the following examples are used to illustrate the detection method for the N-norhyoscyamine nitrosamine impurity provided by the present invention. The protection scope of the present invention is not limited by the following examples.

[0073] Source of raw materials:

[0074] The raw material drug of anisodamine hydrobromide, Chengdu No. 1 Pharmaceutical Co., Ltd., batch number 230101;

[0075] Anisodamine hydrobromide injection (1 ml: 10 mg), Chengdu No. 1 Pharmaceutical Co., Ltd., batch number 29230101;

[0076] The reference substance of N-norhyoscyamine nitrosamine impurity, Chengdu No. 1 Pharmaceutical Co., Ltd., batch number 20240920DZ.

[0077] Example 1 Specificity experiment

[0078] Preparation of the test sample solution: Take an appropriate amount of anisodamine hydrobromide injection (batch number 29230101), and quantitatively dilute it with an acetonitrile aqueous solution with a solvent volume percentage of 50% to prepare a solution containing about 1 mg of anisodamine hydrobromide per 1 ml.

[0079] Preparation of reference stock solution: Weigh an appropriate amount of the reference substance of N-norhyoscyamine nitrosamine impurity accurately, dissolve it in an aqueous acetonitrile solution with a volume percentage of 50% and quantitatively dilute it to prepare a solution containing about 150 ng per 1 ml, and shake well.

[0080] Preparation of reference solution: Accurately measure an appropriate amount of the reference stock solution, quantitatively dilute it with an aqueous acetonitrile solution with a volume percentage of 50% to prepare a solution containing about 15 ng per 1 ml, and shake well.

[0081] The blank solvent is an aqueous acetonitrile solution with a volume percentage of 50%.

[0082] Perform high performance liquid chromatography-mass spectrometry detection on the test solution, reference solution and blank solvent respectively to obtain chromatograms.

[0083] Among them, the chromatographic conditions of high performance liquid chromatography are as follows:

[0084] The chromatographic column uses octadecylsilane chemically bonded silica as the filler (Agilent Poroshell 120 EC-C18, 4.6 mm × 150 mm, 2.7 μm);

[0085] Use 0.1% formic acid water as mobile phase A and acetonitrile (containing 0.1% formic acid) as mobile phase B, and perform gradient elution according to Table 1; the column temperature is 35 °C; the flow rate is 0.8 ml per minute; the injection volume is 5 μl, and the injection tray temperature is 5 °C.

[0086] Table 1 Gradient elution program

[0087]

[0088] Mass spectrometry detection conditions: Detect with a triple quadrupole tandem mass spectrometer, the mass spectrometry ionization method is ESI, the positive ion scanning mode, the monitoring mode is multiple reaction monitoring (MRM), and the monitored ion pairs of the compound are shown in Table 2:

[0089] Table 2 Mass spectrometry detection conditions

[0090]

[0091] Ion source parameters: The collision gas (CAD) is 8 psi, the curtain gas (CUR) is 20 psi, the TEM is 500 °C, the nebulizing gas (GS1) is 40 psi, the nebulizing gas (GS2) is 40 psi, and the voltage (IS) is 5000 V. The MS end is equipped with a six-way valve, which enters the MS from 7.5 minutes to 9.5 minutes, and enters the waste liquid at other times.

[0092] The liquid chromatography-mass spectrometry combined chromatograph corresponding to the above mass spectrometry parameters is AB Sciex 5500+.

[0093] See Figure 1 , Figure 1 , Figure 1 is the XIC chart of the reference substance.

[0094] The results show that: the blank solvent does not interfere with the determination of the components to be measured, there are no other impurity peaks interfering in the test solution, and the resolution is 5.3; the peak shape of the NDSRI in the reference solution is good, and the sensitivity meets the detection requirements, proving that the method has good specificity.

[0095] Example 2 Sensitivity Test (Quantitation Limit and Detection Limit)

[0096] Prepare NDSRI solutions with gradient concentrations and detect the signal-to-noise ratio. The results show that the quantitation limit is 1.0428 ng / ml and the detection limit is 0.3325 ng / ml, far lower than the limit concentration of 15 ng / ml, indicating that the method has good sensitivity.

[0097] Example 3 Linearity and Range

[0098] Preparation of the reference stock solution: Weigh an appropriate amount of the N-desmethylanisodamine nitrosamine impurity reference substance accurately, dissolve it with an aqueous acetonitrile solution with a volume percentage of 50%, and quantitatively dilute it to prepare an NDSRI reference stock solution with a concentration of 10 μg / ml.

[0099] Linear stock solution: Accurately measure 1.5 ml of the NDSRI reference stock solution (10 μg / ml), place it in a 100-ml volumetric flask, dilute it to the mark with an aqueous acetonitrile solution with a volume percentage of 50%, and shake well to obtain a linear stock solution with a concentration of 0.15 μg / ml.

[0100] Linearity 1: Accurately measure 0.2 ml of the linear stock solution, place it in a 10-ml volumetric flask, dilute it to the mark with an aqueous acetonitrile solution with a volume percentage of 50%, and shake well to obtain it.

[0101] Linearity 2: Accurately measure 0.5 ml of the linear stock solution, place it in a 10-ml volumetric flask, dilute it to the mark with an aqueous acetonitrile solution with a volume percentage of 50%, and shake well to obtain it.

[0102] Linearity 3: Accurately measure 1 ml of the linear stock solution, place it in a 10-ml volumetric flask, dilute it to the mark with an aqueous acetonitrile solution with a volume percentage of 50%, and shake well to obtain it.

[0103] Linearity 4: Accurately measure 1.5 ml of the linear stock solution, place it in a 10-ml volumetric flask, dilute it to the mark with an aqueous acetonitrile solution with a volume percentage of 50%, and shake well to obtain it.

[0104] Linearity 5: Accurately measure 2 ml of the linear stock solution, place it in a 10-ml volumetric flask, dilute it to the mark with an aqueous acetonitrile solution with a volume percentage of 50%, and shake well to obtain it.

[0105] Inject the above solutions respectively according to the chromatographic parameters of Example 1, and record the mass spectra. Using the NDSRI concentration (ng / ml) as the abscissa and the peak area as the ordinate, draw the standard curve. The results are shown in Table 3, and the standard curve graph is shown in Figure 2 , Figure 2 which is the standard curve graph of the N-desmethyl anisodamine nitrosamine impurity reference substance.

[0106] Table 3 Investigation table of NDSRI linearity and range

[0107] Name Concentration ng / ml Peak area LOQ 1.0428 11425 Linearity 1 3.1284 21759 Linearity 2 7.8211 53195 Linearity 3 15.6421 100400 Linearity 4 23.4632 152530 Linearity 5 31.2842 208600

[0108] The results show that: the linear equation of NDSRI is y = 6502.7466x + 2033.4794, and the correlation coefficient r is 0.9994. It shows that NDSRI has a good linear relationship in the concentration range of 1.0428 ng / ml to 31.2842 ng / ml.

[0109] Example 4 Repeatability experiment

[0110] Take anisodamine hydrobromide injection, prepare 6 test solutions, and detect them in the same way. The results show that the NDSRI contents of the 6 samples are all lower than the detection limit, and the method has good repeatability.

[0111] Example 5 Standard addition recovery experiment

[0112] Determination of sample background value: Take the repeatability determination result as the background value determination result.

[0113] Reference solution: See under injection precision.

[0114] 20% standard addition recovery solution: Accurately measure 1 ml of anisodamine hydrobromide injection (batch number 29230101), place it in a 10-ml volumetric flask, add 0.2 ml of the above-mentioned reference stock solution, dilute to the mark with the solvent, and shake well (prepare 3 parallels).

[0115] 50% standard addition recovery solution: Accurately measure 1 ml of anisodamine hydrobromide injection (batch number 29230101), place it in a 10-ml volumetric flask, add 0.5 ml of the reference stock solution, dilute to the mark with the solvent, and shake well (prepare 3 parallels).

[0116] 100% standard addition recovery solution: Accurately measure 1 ml of anisodamine hydrobromide injection (batch number 29230101), place it in a 10-ml volumetric flask, add 1 ml of the reference stock solution, dilute to the mark with the solvent, and shake well (prepare 6 parallels).

[0117] 150% spiked recovery solution: Accurately measure 1 ml of anisodamine hydrobromide injection (batch number 29230101), place it in a 10-ml volumetric flask, add 1.5 ml of the reference stock solution, dilute to the mark with the solvent, and shake well to obtain (prepare 3 portions in parallel).

[0118] Inject the above solutions respectively according to the chromatographic conditions of Example 1 for analysis, record the mass spectra, see Figure 3 , Figure 3 is the XIC diagram of the 100% spiked recovery solution, Figure 4 is the liquid chromatography-mass spectrometry overlay diagram of the 100% spiked recovery solution. The results are shown in Table 4.

[0119] Table 4 Results of the accuracy test of NDSRI

[0120]

[0121]

[0122] The results show that: the recovery rate of NDSRI is between 92.68% and 102.77%, the average recovery rate is 96.87%, within 80% - 115%; RSD% (n = 15) is 3.17%, less than 10%. It shows that this method has high accuracy and can accurately detect the residual amount of N-norhyoscyamine nitrosamine impurities in the sample.

[0123] Example 5

[0124] Preparation of the test solution: Take an appropriate amount of anisodamine hydrobromide raw material (batch number 230101), quantitatively dilute it with an acetonitrile aqueous solution with a solvent volume percentage of 50% to prepare a solution containing about 1 mg of anisodamine hydrobromide per 1 ml.

[0125] The preparation of the reference solution is the same as that in Example 1, and the detection is carried out according to the chromatographic and mass spectrometric conditions in Example 1. The results show that: N-norhyoscyamine nitrosamine impurities were not detected in the anisodamine hydrobromide raw material with batch number 230101.

[0126] Comparative Example 1:

[0127] 1. Take an appropriate amount of NDSRI reference substance, quantitatively dilute it with pure water to prepare a solution containing about 15 ng per 1 ml, and filter for later use.

[0128] 2. Separate according to the liquid chromatography conditions disclosed in Example 1 and detect according to the mass spectrometric conditions.

[0129] 3. It was found that due to the poor compatibility between pure water as the solvent and NDSRI, the separation effect of the sample in the chromatographic column was not good, the impurity peaks overlapped seriously with the main peak, and the types and contents of the impurities could not be accurately identified.

[0130] Comparative Example 2:

[0131] 1. In the liquid chromatography conditions, the mobile phase was changed to pure water and acetonitrile, and formic acid was not added.

[0132] 2. Separation was carried out according to the liquid chromatography conditions disclosed in Example 1 above, and detection was carried out under the mass spectrometry conditions.

[0133] 3. It was found that since formic acid was not added to the mobile phase, the ionization efficiency of its impurities was low, the mass spectrometry signal was weak, and trace impurities could not be effectively detected.

[0134] Comparative Example 3:

[0135] 1. In the liquid chromatography conditions, acetonitrile in the mobile phase was changed to methanol, and other conditions remained unchanged.

[0136] 2. Separation was carried out according to the liquid chromatography conditions disclosed in Example 1 above, and detection was carried out under the mass spectrometry conditions.

[0137] 3. It was found that since the elution of the mobile phase became slower, the target substance and the main component could not be effectively separated, and the resolution was 0.96, which affected the accuracy of the detection result.

[0138] Comparative Example 4:

[0139] 1. In the liquid chromatography conditions, the chromatographic column was changed to a C8 reversed-phase chromatographic column (250 mm × 4.6 mm, 5 μm).

[0140] 2. Separation was carried out according to the liquid chromatography conditions disclosed in Example 1 above, and detection was carried out under the mass spectrometry conditions.

[0141] 3. It was found that since the C8 chromatographic column had a weak retention ability for the main component and its impurities, the separation effect between the impurity peak and the main peak was poor, the resolution was 0.94, and the types and contents of impurities could not be accurately identified.

[0142] Comparative Example 5:

[0143] 1. In the liquid chromatography conditions, the chromatographic column was changed to an amino column (250 mm × 4.6 mm, 5 μm).

[0144] 2. Separation was carried out according to the liquid chromatography conditions disclosed in Example 1 above, and detection was carried out under the mass spectrometry conditions.

[0145] 3. It was found that since the retention mechanism of the amino column for the main component and its impurities was different from that of the C18 column, the separation effect between the impurity peak and the main peak was not ideal, the resolution was 0.32, and some impurities could not be effectively eluted.

[0146] Comparative Example 6:

[0147] 1. In the liquid chromatography conditions, the chromatographic column is changed to a silica gel normal-phase chromatographic column (250 mm × 4.6 mm, 5 μm).

[0148] 2. The mobile phase is changed to n-hexane and isopropanol with a volume percentage of 90:10 (90:10, v / v), and isocratic elution is performed.

[0149] 3. Separation is carried out according to the liquid chromatography conditions disclosed in Example 1 above, and detection is performed under the mass spectrometry conditions.

[0150] 4. It is found that due to the polarity mismatch between the normal-phase chromatographic column and the main component and its impurities, the separation effect is poor, the impurity peaks overlap severely with the main peak, and the types and contents of impurities cannot be accurately identified.

[0151] Comparative Example 7:

[0152] 1. Separation is carried out according to the liquid chromatography conditions disclosed in Example 1 above.

[0153] 2. In the mass spectrometry conditions, the ion source is changed to an atmospheric pressure chemical ionization source (APCI), positive ion mode, and other conditions remain unchanged.

[0154] 3. It is found that due to the low ionization efficiency of APCI for the main component and its impurities, the mass spectrometry signal is weak, and trace impurities cannot be effectively detected.

[0155] Comparative Example 8:

[0156] 1. Separation is carried out according to the liquid chromatography conditions disclosed in Example 1 above.

[0157] 2. In the mass spectrometry conditions, the scanning mode is changed to only selected ion monitoring (SIM), and full scan is not performed.

[0158] 3. It is found that due to the lack of full scan, some unknown impurities cannot be detected, resulting in incomplete detection results.

[0159] Comparative Example 9:

[0160] 1. Separation is carried out according to the liquid chromatography conditions disclosed in Example 1 above.

[0161] 2. Mass spectrometry parameters are modified:

[0162] Ion source parameters: The collision gas (CAD) is 8 psi, the curtain gas (CUR) is 20 psi, the TEM is 200 °C, the nebulizing gas (GS1) is 40 psi, the nebulizing gas (GS2) is 40 psi, and the voltage (IS) is 5000 V. A six-port valve is equipped at the MS end, and it enters the MS from 7.5 minutes to 9.5 minutes, and enters the waste liquid at other times.

[0163] 3. It was found that due to the low temperature of the ion source, the ionization efficiency of the main component and its impurities decreased, the mass spectrometry signal was weak, and trace impurities could not be effectively detected.

[0164] Comparative Example 10:

[0165] 1. On the basis of Example 1, the gradient elution program was modified. For the specific program, see Table 5.

[0166] Table 5 Gradient Elution Program

[0167]

[0168] 2. Separation was carried out according to the liquid chromatography conditions disclosed in Example 1 above, and detection was carried out under the mass spectrometry conditions.

[0169] 3. It was found that the resolution of the main component and its impurities was poor, and the resolution was 0.6, so effective separation could not be achieved.

[0170] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. A method for detecting N-norhyoscyamine nitrosamine impurities, characterized in that, It includes the following steps: Dissolve the test sample containing N-norhyoscyamine nitrosamine impurities in a solvent to obtain a test sample solution; Perform high performance liquid chromatography-mass spectrometry (HPLC-MS) detection on the test sample solution; The conditions of the HPLC include: using an octadecylsilane-bonded silica gel column as the filler; using 0.1% formic acid in water as mobile phase A and 0.1% formic acid in acetonitrile as mobile phase B; performing gradient elution. In terms of volume percentage, the elution program of the gradient elution is as follows: From 0 to 3 min, mobile phase A is 90% and mobile phase B is 10%; From 3 to 12 min, mobile phase A decreases from 90% to 10%, and mobile phase B increases from 10% to 90%; From 12 to 18 min, mobile phase A is 10% and mobile phase B is 90%; From 18 to 18.1 min, mobile phase A increases from 10% to 90%, and mobile phase B decreases from 90% to 10%; From 18.1 to 25 min, mobile phase A is 90% and mobile phase B is 10%; The conditions of the mass spectrometry include: detecting with a triple quadrupole tandem mass spectrometer, the mass spectrometry ionization method is ESI, positive ion scan mode, and the monitoring mode is multiple reaction monitoring.

2. The detection method according to claim 1, characterized in that, The test sample includes anisodamine hydrobromide raw material drug and / or preparation.

3. The detection method according to claim 1, wherein The solvent includes an acetonitrile aqueous solution with a volume fraction of 50%.

4. The detection method according to claim 1, wherein The test sample solution contains anisodamine hydrobromide, and the concentration of anisodamine hydrobromide is 0.5 - 2 mg / ml.

5. The detection method according to claim 1, wherein It also includes preparing a reference solution, and the preparation method of the reference solution includes the following steps: Dissolve the N-norhyoscyamine nitrosamine impurity reference substance in a solvent to obtain a reference solution.

6. The detection method according to claim 5, wherein The solvent is selected from an acetonitrile aqueous solution with a volume fraction of 50%; The concentration of the reference solution is 1 - 32 ng / ml.

7. The detection method according to claim 1, characterized in that The HPLC column is Agilent Poroshell120EC-C18, 4.6 mm × 150 mm, 2.7 μm or a chromatographic column with equivalent efficiency.

8. The detection method according to claim 1, wherein The column temperature of the HPLC is 30 - 40 °C; the flow rate is 0.5 - 1.2 ml per minute; the injection volume is 3 - 10 μL, and the injection tray temperature is 3 - 10 °C.

9. The detection method according to claim 1, wherein The ion source parameters of the mass spectrometry are: collision gas (CAD) is 8 psi, curtain gas (CUR) is 20 psi, TEM is 500 °C, nebulizing gas (GS1) is 40 psi, nebulizing gas (GS2) is 40 psi, and voltage (IS) is 5000 V.

10. The detection method according to claim 1, characterized in that, It includes the following steps: Perform HPLC-MS detection on reference solutions with different concentrations respectively to obtain spectra; Use the peak area corresponding to the N-norhyoscyamine nitrosamine impurity in the spectra obtained by performing HPLC-MS detection on reference solutions with different concentrations as the ordinate, and the concentration of the reference solution as the abscissa to draw a standard curve; Perform HPLC-MS detection on the test sample solution to obtain a spectrum, substitute the peak area corresponding to the N-norhyoscyamine nitrosamine impurity in the spectrum into the standard curve, and calculate the concentration of the N-norhyoscyamine nitrosamine impurity in the test sample solution.

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