Method for detecting related substances in penehyclidine hydrochloride injection by high performance liquid chromatography

Abstract

The invention discloses a method for detecting related substances of penehyclidine hydrochloride injection by high performance liquid chromatography. The method comprises the following steps: detecting a test solution by high performance liquid chromatography, adopting a C18 reverse phase chromatographic column, taking a mixed solution of dipotassium hydrogen phosphate solution: acetonitrile in a volume ratio of 70:30-90:10 as mobile phase A, wherein the pH value of the dipotassium hydrogen phosphate solution is 4.0-5.5 and the solution contains 0.5-1.0 vol% of triethylamine, taking a mixed solution of water: acetonitrile in a volume ratio of 10:90-30:70 as mobile phase B for elution, the temperature of the chromatographic column is 30-40 DEG C, the temperature of the injection plate is 4-8 DEG C, the detection wavelength is 200-270 nm, the flow rate is 0.8-1.5 ml / min, penehyclidine hydrochloride injection and six impurities thereof can be detected by one loading, the separation degree between peaks is guaranteed, and the quality control of penehyclidine hydrochloride injection is facilitated.

Description

Technical Field

[0001] The invention belongs to the technical field of drug analysis and detection methods, and specifically relates to a method for detecting related substances of penehyclidine hydrochloride injection by using a high performance liquid chromatography method. Background Art

[0002] Penehyclidine hydrochloride, the English name is Penehyclidine Hydrochloride, the chemical name is 3-(2-cyclopentyl-2-hydroxy-2-phenylethoxy)quinuclidine hydrochloride, the molecular formula is C 20 H 29 NO2·HCl, molecular weight is 351.92, CAS number is 87827-02-9, and the structural formula is as follows:

[0003]

[0004] Penehyclidine hydrochloride injection is an injection with the trade name Changtongning. It is a selective anticholinergic drug, mainly used for administration before anesthesia to inhibit the secretion of salivary glands and airway glands, for emergency treatment of organophosphorus poisoning (pesticides), and for maintaining atropine in the late stage of poisoning or after aging of cholinesterase (ChE).

[0005] The related substances in penehyclidine hydrochloride injection may come from two aspects, process impurities and degradation impurities. The structures of some process impurities and degradation impurities have been determined as shown in Table 1:

[0006] Table 1 Summary of impurity profile analysis of penehyclidine hydrochloride

[0007]

[0008]

[0009] After literature search, it was found that no major pharmacopoeias included this product. Only the new drug standard for penehyclidine hydrochloride raw material (WS1-(X-026)-2004Z) uses thin layer chromatography to control related substances. However, the accuracy of thin layer chromatography detection is affected by many factors such as temperature, humidity, and sampling technology. The operating error is large and the reproducibility is poor. In addition, this standard only controls two known impurities, and the impurity limit is 0.25%, which cannot meet the increasingly stringent requirements for the control of drug impurities.

[0010] At present, the impurity analysis and detection methods for penehyclidine hydrochloride mostly target the analysis of one or two impurities. The sources of the impurity spectrum of penehyclidine hydrochloride include process impurities and degradation impurities, and impurities A and B are process impurities. Penehyclidine hydrochloride is unstable under acidic and strong oxidizing conditions and is easily degraded. The main degradation impurities include impurity R, impurity C, impurity S and genotoxic impurity cyclopentylphenylacetaldehyde.

[0011] There is no literature report on the analysis method of impurities B, C and S. In the prior art, in the analysis of impurities A and R, the mobile phase is a potassium dihydrogen phosphate buffer and acetonitrile system, and all of the above impurities cannot be detected. Therefore, it is urgent to find an accurate and effective method for detecting related substances of penehyclidine hydrochloride that has high sensitivity, good reproducibility, and can detect multiple impurities simultaneously in one sample, so as to achieve precise control of product quality. Summary of the invention

[0012] The object of the present invention is to provide a method for detecting related substances of penehyclidine hydrochloride injection by high performance liquid chromatography, which is simple to operate, has a relative standard deviation of each impurity peak area of ​​less than 4.6%, good precision, a recovery rate of each impurity between 89.8% and 101.5%, high accuracy, a separation degree between each impurity of not less than 2.3, good separation, can simultaneously control 6 known impurities, effectively control the product quality of penehyclidine hydrochloride injection, ensure the safety and effectiveness of clinical medication, and solve the problems of low sensitivity in detecting related substances of penehyclidine hydrochloride injection and incomplete detection of impurities in the prior art.

[0013] In order to achieve the above object, the present invention provides the following technical solutions:

[0014] A method for detecting related substances of penehyclidine hydrochloride injection by high performance liquid chromatography comprises the following steps:

[0015] The test solution was detected by high performance liquid chromatography, using a reverse phase C18 chromatographic column, with a mixed solution of potassium hydrogen phosphate solution and acetonitrile in a volume ratio of 70:30 to 90:10 as mobile phase A, wherein the pH of the potassium hydrogen phosphate solution was 4.0 to 5.5 and contained 0.5 to 1.0 vol% triethylamine, and a mixed solution of water and acetonitrile in a volume ratio of 10:90 to 30:70 as mobile phase B for gradient elution, the column temperature was 30 to 40° C., the detection wavelength was 200 to 270 nm, and the flow rate was 0.8 to 1.5 ml / min;

[0016] The related substances include: impurity A, impurity B, impurity C, impurity R and / or impurity S.

[0017] Preferably, the impurity A is 3[2-cyclopentyl-2-phenyl-2(2-cyclopentyl-2-hydroxy-2-phenylethoxy)ethoxy]quinuclidine hydrochloride, the impurity B is 3-(2-hydroxy-2,2-diphenylethoxy)quinuclidine hydrochloride, the impurity C is phenylcyclopentyl ketone, the impurity R is 1-cyclopentyl-1-phenyl-1,2-ethylene glycol, and the impurity S is 3-(2-cyclopentyl-2-hydroxy-2-phenylethoxy)quinuclidine-1-oxide.

[0018] Furthermore, the related substances include the impurity cyclopentylphenylacetaldehyde.

[0019] The gradient elution program is:

[0020] 0-5min, mobile phase A is 90%, and mobile phase B is 10%; 5-25min, mobile phase A is uniformly decreased from 90% to 70%, and mobile phase B is increased from 10% to 30%; 25-55min, mobile phase A is uniformly decreased from 70% to 0%, and mobile phase B is increased from 30% to 100%; 55-70min, mobile phase A is 0%, and mobile phase B is 100%; 70-75min, mobile phase A is increased from 0% to 90%, and mobile phase B is decreased from 100% to 10%, and then continue to equilibrate for 5 minutes with mobile phase A at 90% and mobile phase B at 10%.

[0021] Preferably, the reverse phase C18 chromatographic column has a length of 250 to 300 mm, the filler type is octadecylsilane bonded silica gel, and the filler particle size is 3 to 5 μm.

[0022] Preferably, the chromatographic column is selected from SHIMADZEU Inertsil series, Waters Xbridge series or Agilent ZORBAX series.

[0023] More preferably, the reverse phase C18 chromatographic column is SHIMADZE Inertsil ODS-3V, with an inner diameter of 4.6 mm×250 mm and a filler particle size of 5 μm.

[0024] Furthermore, the detector of the high performance liquid chromatograph is a diode array or an ultraviolet absorption detector, the temperature of the chromatographic column is 35° C., the detection wavelength is 206 nm, and the flow rate is 1.0 ml / min.

[0025] Furthermore, in the dipotassium hydrogen phosphate solution, the concentration range of dipotassium hydrogen phosphate is 0.015 mol / L to 0.035 mol / L; in the mobile phase B, the volume ratio of water to acetonitrile is 20:80.

[0026] Furthermore, the test solution is penehyclidine hydrochloride injection, or the test solution is a penehyclidine hydrochloride solution with a concentration of not less than 1 mg / ml, including penehyclidine hydrochloride raw material or its preparation.

[0027] Furthermore, the sample injection plate temperature of the high performance liquid chromatograph is 4-8°C.

[0028] In the present invention, by studying the properties of penehyclidine hydrochloride and its related substances, it is known that penehyclidine hydrochloride and its impurity S exist in the form of diastereoisomers, respectively. The similarity of the chemical properties of diastereoisomers leads to the difficulty of liquid phase separation, and the impurity S is similar to penehyclidine hydrochloride in structure, which further increases the difficulty of separation. In addition, the impurity C has poor stability in the test solution, which also affects the accurate quantitative detection of impurities.

[0029] The inventors found during the development of a detection method for penehyclidine hydrochloride that the pH value of the mobile phase affects the separation of impurities. The present invention adjusts the elution gradient under appropriate pH conditions, maintains a relatively slow gradient change during the elution time period of penehyclidine hydrochloride and impurity S, and ensures good separation between the peaks; for impurity C, the stability of impurity C during sample detection is ensured by controlling the temperature of the injection plate, thereby ensuring the accuracy of the detection result; in addition, since the nitrogen atoms in penehyclidine hydrochloride and its multiple impurity structures will react with the residual silanol groups in the chromatographic column to cause peak tailing, in order to solve this problem, the present invention uses relatively stable dipotassium hydrogen phosphate in mobile phase A to improve the peak shape, and on the other hand, adds an appropriate amount of triethylamine to further suppress peak tailing. Under the coordinated action of the two aspects, the separation degree is further improved, the system pH is stable, and the accurate quantification of each impurity is ensured.

[0030] In the present invention, suitable HPLC detection conditions are screened, a mixed solution of a dipotassium hydrogen phosphate solution: acetonitrile in a volume ratio of 70:30 to 90:10 is used as a mobile phase A, wherein the pH of the dipotassium hydrogen phosphate solution is 4.0 to 5.5 and contains 0.5 to 1.0 vol% of triethylamine, and a mobile phase B of water: acetonitrile in a volume ratio of 10:90 to 30:70 is used for gradient elution, and in combination with the chromatographic column conditions of the present invention, penehyclidine hydrochloride and its impurity S in the form of diastereoisomers, as well as impurities A, B, C and R can be separated, with good separation and precision, and the recovery rate of each impurity is between 89.8% and 101.5%, with high accuracy.

[0031] Compared with the prior art, the present invention has the following beneficial effects:

[0032] The analytical method for the related substances of penehyclidine hydrochloride injection of the present invention is simple to operate, and 6 known impurities can be controlled at the same time, wherein impurities A, impurity B, impurity C, impurity R and impurity S can be qualitatively and quantitatively detected, and the specific content of each impurity is calculated by the principal component self-control method with a correction factor added, and the detection result has good repeatability, thereby ensuring the controllability of the quality of the finished product of penehyclidine hydrochloride injection, and qualitatively detecting the genotoxic impurity cyclopentylphenylacetaldehyde.

[0033] The detection method of the invention has high accuracy and good separation. Through the sample addition recovery test, the recovery rate of each impurity is between 89.8% and 101.5%, the relative standard deviation of the peak area of ​​each impurity is less than 4.6%, the precision is good (two people, a total of 12 samples), the three concentrations of low, medium and high are all accurately controllable, the repeatability is good, the sensitivity is high, the separation between each impurity is good, the separation between each peak is not less than 2.3, the result has high credibility, and a reliable inspection basis is provided for strictly controlling the quality of penehyclidine hydrochloride injection and improving the quality standard. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1-3 This is a structural characterization spectrum of impurity A in the embodiment of the present invention.

[0035] Figure 4-5 This is a structural characterization spectrum of impurity B in the embodiment of the present invention.

[0036] Figure 6-7 This is a structural characterization spectrum of impurity R in an embodiment of the present invention.

[0037] Figure 8-9 This is a structural characterization spectrum of impurity S in an embodiment of the present invention.

[0038] Fig.10 This is the solution spectrum of the system applicability in Example 1 of the present invention.

[0039] Fig.11 This is the reference solution spectrum in Example 1 of the present invention.

[0040] Fig.12 This is the sample solution spectrum in Example 6 of the present invention.

[0041] Fig.13 This is the sample solution spectrum in Example 7 of the present invention.

[0042] Fig.14 This is the sample solution spectrum in Example 8 of the present invention.

[0043] Fig.15 This is the sample solution spectrum in Comparative Example 1 of the present invention.

[0044] Fig.16 This is the solution spectrum for locating impurity B in Comparative Example 1 of the present invention. DETAILED DESCRIPTION

[0045] The present invention is further described below in conjunction with specific embodiments, but the present invention is not limited to the scope of the embodiments.

[0046] The experimental methods in the following examples without specifying specific conditions are selected according to conventional methods and conditions, or according to the product instructions; in the chromatograms of the examples and comparative examples, the unit of peak time is minute (min).

[0047] The test samples and impurity reference substances used in the examples of the present invention are as follows:

[0048] Impurity A reference substance: self-made by Shanghai Xudong Haipu Pharmaceutical Co., Ltd., the structural characterization spectrum is shown in Figure 1-3 , the structural formula and hydrogen atom numbering are as follows:

[0049]

[0050] in, Figure 1 It is the H NMR spectrum of the impurity A reference substance, and the H assignments are as follows: δ12.57ppm, assigned to the active hydrogen signal of the hydroxyl group; δ7.13~7.48ppm, assigned to the two benzene ring proton peak signals; δ3.73~3.89ppm, assigned to the methylene proton peaks at positions 11 and 22; δ3.60~3.62ppm, assigned to the quinuclidine proton peak at position 23; δ3.44ppm, assigned to one of the quinuclidine proton peaks at position 29; δ3.15~3.16ppm, assigned to the quinuclidine proton peaks at positions 29, 26, and 27; δ2.40~2.50ppm, assigned to the quinuclidine proton peak at position 24; δ2.33ppm, assigned to the quinuclidine proton peak at position 28; δ1.90~2.10ppm, assigned to the quinuclidine proton peak at position 25; δ1.90~2.10ppm, assigned to the quinuclidine proton peak at position 25. The hydrogen spectrum is consistent with the structure of impurity A of penehyclidine hydrochloride: 3[2-cyclopentyl-2-phenyl-2(2-cyclopentyl-2-hydroxy-2-phenylethoxy)ethoxy]quinuclidine hydrochloride.

[0051] Figure 2 and 3 These are the mass spectra of the impurity A reference substance in positive ion detection mode and negative ion detection mode, respectively, and the [M+H-HCl] of impurity A is obtained. + Positive ion peak 505 and [MH]-negative ion peak 538; the molecular weight of the impurity A reference substance was determined to be 539, which is consistent with the molecular weight of impurity A 3[2-cyclopentyl-2-phenyl-2(2-cyclopentyl-2-hydroxy-2-phenylethoxy)ethoxy]quinuclidine hydrochloride C33H46ClNO3.

[0052] Impurity B reference substance: self-made by Shanghai Xudong Haipu Pharmaceutical Co., Ltd., structural characterization spectrum is shown in Figure 4-5 , the structural formula and hydrogen atom numbering are as follows:

[0053]

[0054] in, Figure 4 It is the H NMR spectrum of the impurity B reference substance, and the H assignments are as follows: δ11.90ppm, assigned to the active hydrogen signal of the hydroxyl group; δ7.25~7.42ppm, assigned to the proton peak signals of the benzene rings at positions 1-10; δ3.99~4.09ppm, assigned to the methylene proton peak at position 18; δ3.94~3.96ppm, assigned to the quinuclidine proton peak at position 11; δ3.45~3.49ppm, assigned to the quinuclidine proton peak at position 17; δ3.06~3.22ppm, assigned to the quinuclidine proton peaks at positions 14 and 16; δ2.36~2.37ppm, assigned to the quinuclidine proton peak at position 12; δ1.79~2.01ppm, assigned to the quinuclidine proton peaks at positions 13 and 15. It can be seen that the hydrogen spectrum of the impurity B reference substance is consistent with the structure of the impurity B 3-(2-hydroxy-2,2-diphenylethoxy)quinuclidine hydrochloride of penehyclidine hydrochloride.

[0055] Figure 5 The mass spectrum of the impurity B reference substance in positive ion detection mode is obtained, and the [M+H-HCl] of the impurity B reference substance is obtained. + The positive ion peak is 324, and the molecular weight of the impurity B reference substance is determined to be 323, which is the same as the impurity B 3-(2-hydroxy-2,2-diphenylethoxy)quinuclidine hydrochloride C 21 H 26 The molecular weight of ClNO2 is consistent.

[0056] Impurity C reference substance: purchased from San Chemical Technology Co., Ltd.

[0057] Impurity R reference substance: self-made by Shanghai Xudong Haipu Pharmaceutical Co., Ltd., the structural characterization spectrum is shown in Figure 6-7 , the structural formula and hydrogen atom numbering are as follows:

[0058]

[0059] in, Figure 6It is the H NMR spectrum of the impurity R reference substance, and the H assignments are as follows: δ7.459-7.441ppm, multiple peaks, assigned to the 1, 5, proton signal peaks of the benzene ring; δ7.279-7.241ppm, multiple peaks, assigned to the 2, 4 proton signal peaks of the benzene ring; δ7.166-7.148ppm, multiple peaks, assigned to the 3 proton signal peak of the benzene ring; δ4.477-4.433ppm, multiple peaks, assigned to the hydroxyl proton signal peak; δ3.657-3.616ppm, multiple peaks, assigned to the 15, 16 proton signal peaks; δ2.340-2.318ppm, multiple peaks, assigned to the 14 proton signal peak; δ1.652-1.063ppm, multiple peaks, assigned to the 6, 7, 8, 9, 10, 11, 12, 13 proton signal peaks. It can be seen that the hydrogen spectrum of the impurity R reference substance is consistent with the structure of the impurity R1-cyclopentyl-1-phenyl-1,2-ethylene glycol in penehyclidine hydrochloride.

[0060] Figure 7 The mass spectrum of the impurity R reference substance in positive ion detection mode is shown in Figure 1. + The positive ion peak is 212.26, and the molecular weight is determined to be 206, which is consistent with the structure of the impurity R1-cyclopentyl-1-phenyl-1,2-ethylene glycol.

[0061] Impurity S reference substance: self-made by Shanghai Xudong Haipu Pharmaceutical Co., Ltd., structural characterization spectrum see Figure 8-9 , the structural formula and hydrogen atom numbering are as follows:

[0062]

[0063] in, Figure 8 It is the H NMR spectrum of the impurity S reference substance, and the H assignments are as follows: δ7.502-7.483ppm, multiple peaks, assigned to the 2nd and 4th proton signal peaks of the benzene ring; δ7.268-7.249ppm, multiple peaks, assigned to the 1st and 5th proton signal peaks of the benzene ring; δ7.180-7.162ppm, multiple peaks, assigned to the 6th proton signal peak of the benzene ring; δ5.268ppm, single peak, assigned to the hydroxyl proton signal peak; δ3.68 3-3.379ppm, multiple peaks, attributed to the proton signal peaks at positions 20 and 23; δ2.505-2.498ppm, multiple peaks, attributed to a proton signal peak at position 13; δ1.973ppm, single peak, attributed to a proton signal peak at position 23; δ1.706-1.224ppm, multiple peaks, attributed to the proton signal peaks at positions 22, 17, 16, 11, 12, 10, 8, 23 (1H), and 13 (1H).

[0064] The hydrogen spectrum of the impurity S reference substance is consistent with the structure of the impurity S of penehyclidine hydrochloride 3-(2-cyclopentyl-2-hydroxy-2-phenylethoxy)quinuclidine-1-oxide.

[0065] Fig. 9 The mass spectrum of the impurity S reference substance in positive ion detection mode is shown in Figure 1. + The positive ion peak is 332.23, and the molecular weight is determined to be 331, which is consistent with the impurity SC 20 H 29 The structure of NO3 is consistent.

[0066] Test solution: Penehyclidine hydrochloride injection prepared by Shanghai Xudong Haipu Pharmaceutical Co., Ltd., with a specification of 1 ml: 1 mg.

[0067] Example 1 is an example of system adaptability and detection of related substances in penehyclidine hydrochloride injection, and the experimental conditions are as follows:

[0068] Instrument: Waters e2695 / 2489 high performance liquid chromatograph;

[0069] Chromatographic column: SHIMADZU Inertsil ODS-3V (4.6 mm × 250 mm, 5 μm);

[0070] Mobile phase A: dipotassium hydrogen phosphate solution-acetonitrile, wherein the pH of the dipotassium hydrogen phosphate solution is 4.0-5.5 and contains 1 vol% triethylamine, dipotassium hydrogen phosphate solution: acetonitrile = 80:20, v / v, and the concentration of dipotassium hydrogen phosphate in mobile phase A is 0.02 mol / L;

[0071] Mobile phase B: water-acetonitrile (water:acetonitrile=20:80, v / v);

[0072] Flow rate: 1.0 ml / min;

[0073] The column temperature was 35°C;

[0074] The detection wavelength is 206nm;

[0075] The injection volume was 35 μl;

[0076] Sample plate temperature: 4-8°C;

[0077] Blank solution: water.

[0078] Test solution: penehyclidine hydrochloride injection.

[0079] Preparation of system suitability solution: Accurately weigh appropriate amounts of impurity A reference substance, impurity B reference substance, impurity C reference substance, impurity R reference substance, and impurity S reference substance, first dissolve them respectively in 80% acetonitrile, then take appropriate amounts of each solution and place them in the same volumetric flask, and dilute with the test solution to make a mixed solution containing approximately 1 mg of penehyclidine hydrochloride, 1 μg of impurity A, 1 μg of impurity B, 1 μg of impurity C, 1 μg of impurity R, and approximately 1 μg of impurity S per 1 ml, as the system suitability test solution.

[0080] Control solution: Accurately measure 1 ml of the test solution, place it in a 200 ml volumetric flask, dissolve it with mobile phase A and dilute it to the scale, shake well, and use it as the control solution.

[0081] Determination: Take 35 μl of blank solvent, system suitability solution, control solution and test solution respectively, inject them into high performance liquid chromatography for sampling and detection, perform gradient elution according to the gradient shown in Table 2, record the chromatogram, and the system suitability spectrum is shown in Fig.10 The results are shown in Table 3. It can be seen that the impurities can be well separated from each other; the spectrum of the control solution is shown in Fig.11 .

[0082] Table 2 Gradient elution program

[0083]

[0084]

[0085] Table 3 Separation degree of penehyclidine hydrochloride and related substances

[0086]

[0087] It can be seen that the blank solvent has no interference with the determination. The elution order is impurity B peak, penehyclidine hydrochloride racemate I peak and racemate II peak, impurity S racemate I peak and racemate II peak, impurity R peak, impurity A peak, and impurity C peak. The separation degrees between the peaks are between 2.3 and 19.6, all satisfying the separation degree ≥1.5. It can be seen that the results meet the system suitability requirements.

[0088] In addition, penehyclidine hydrochloride and its impurity S exist in the form of diastereomers (racemate I and racemate II). The similarity of their chemical properties makes it difficult to separate diastereomers in liquid phase, and the similar structures between penehyclidine hydrochloride and impurity S increase the difficulty of separation. By trying to adjust the elution gradient and procedure, the separation of two pairs of diastereomers and between penehyclidine hydrochloride and impurity S was achieved under the screened chromatographic column and elution conditions, laying the foundation for accurate quantitative detection of impurities.

[0089] Example 2 Detection Limit and Quantification Limit

[0090] The experimental conditions and liquid chromatography method are as shown in Example 1, and the solution preparation is as shown below, or can be prepared according to methods well known to those skilled in the art.

[0091] For known impurities: prepare each known impurity into a solution of appropriate concentration (about 0.50 μg / ml) and dilute it, compare the measured signal with the baseline signal (baseline noise), and the concentration corresponding to the signal-to-noise ratio s / n ≥ 10 of the known impurity response on the instrument is the quantitative limit of the method; the concentration corresponding to the signal-to-noise ratio s / n ≥ 3 of the lowest response of the known impurity on the instrument is the detection limit of the method.

[0092] The main component penehyclidine hydrochloride was used to replace the quantitative limit concentration and detection limit concentration of a single unknown impurity. The test results are shown in Table 4.

[0093] Table 4 Results of detection limit and quantification limit of each impurity Concentration unit: μg / ml

[0094]

[0095] Note: Penehyclidine hydrochloride and impurity S are calculated based on the one with the lowest response in the racemate.

[0096] The detection limit of impurity B is 0.008μg / ml, and the quantification limit is 0.024μg / ml; the detection limit of penehyclidine hydrochloride is 0.193μg / ml, and the quantification limit is 0.200μg / ml; the detection limit of impurity S is 0.068μg / ml, and the quantification limit is 0.1956μg / ml; the detection limit of impurity R is 0.070μg / ml, and the quantification limit is 0.200μg / ml; the detection limit of impurity A is 0.070μg / ml, and the quantification limit is 0.201μg / ml; the detection limit of impurity C is 0.012μg / ml, and the quantification limit is 0.025μg / ml.

[0097] Example 3 Linear situation

[0098] The experimental conditions, liquid chromatography method and solution preparation are the same as in Example 1. The solution is prepared according to methods well known to those skilled in the art.

[0099] For the known impurity impurity A reference substance, impurity B reference substance, impurity C reference substance, impurity R reference substance and impurity S reference substance, 6 concentration points were taken in the range of LOQ (limit of quantitation) concentration to not less than 150% of the index concentration for study. The linear series concentration includes 6 concentrations: limit of quantitation (LOQ) concentration L0, 50% impurity limit concentration L1, 100% impurity limit concentration L2, 150% impurity limit concentration L3, 200% impurity limit concentration L4, 500% impurity limit concentration L5.

[0100] Linear relationship: Plot the measured response signal (peak area) as a function of the analyte concentration and perform linear regression using the least squares method. At least the correlation coefficient r is reported to confirm a good linear relationship, which should be no less than 0.990.

[0101] Penehyclidine hydrochloride was used instead to investigate the linearity and range of a single unknown impurity.

[0102] The linear relationship of each substance was determined using this analytical method. The results are shown in Tables 5-10.

[0103] 1. Impurity B

[0104] Table 5 Linear determination results of impurity B

[0105]

[0106] The linear equation was y=119824.70x+425.06; within the range of 0.024 μg / ml to 4.830 μg / ml, the linear regression coefficient r was 1.000, indicating a good linear relationship.

[0107] 2. Penehyclidine hydrochloride

[0108] Table 6 Linear determination results of penehyclidine hydrochloride

[0109]

[0110] The linear equation is y=48747.66x+138.88; in the range of 0.200 μg / ml to 4.998 μg / ml, the linear regression coefficient r is 1.000, and the linear relationship is good.

[0111] 3. Impurity S

[0112] Table 7 Linear determination results of impurity S

[0113]

[0114] The linear equation is y=50215.58x-2424.85; in the range of 0.193 μg / ml to 4.835 μg / ml, the linear regression coefficient r is 1.000, and the linear relationship is good.

[0115] 4. Impurity R

[0116] Table 8 Linear determination results of impurity R

[0117]

[0118] The linear equation is y=70838.31x+632.09; in the range of 0.200 μg / ml to 4.991 μg / ml, the linear regression coefficient r is 1.000, and the linear relationship is good.

[0119] 5. Impurity A

[0120] Table 9 Linear determination results of impurity A

[0121]

[0122] The linear equation was y=62036.04x+5805.94; within the range of 0.201 μg / ml to 5.024 μg / ml, the linear regression coefficient r was 0.997, indicating a good linear relationship.

[0123] 6. Impurity C

[0124] Table 10 Linear determination results of impurity C

[0125]

[0126] The linear equation is y=155380.01x+1742.75; in the range of 0.025 μg / ml to 4.905 μg / ml, the linear regression coefficient r is 1.000, and the linear relationship is good.

[0127] Example 4 Accuracy

[0128] The experimental conditions and liquid chromatography method are the same as in Example 1, and the solution is prepared as shown below or according to methods well known to those skilled in the art.

[0129] Accuracy refers to the degree to which the result measured by the method of the present invention is close to the true value or reference value, and is generally expressed as recovery rate (%).

[0130] Since only the known impurity B was detected in the homemade product penehyclidine hydrochloride injection, in order to investigate the accuracy of the detection results of the method of the present invention for each impurity, a series of impurities with known contents were added to the penehyclidine hydrochloride injection, and then the method of the present invention was used for detection, so as to judge the accuracy of the method of the present invention based on the comparison between the actual detection results and the known addition amounts.

[0131] Solution A-50% preparation: Add 0.05% of impurities A, B, C, R and S to the homemade product penehyclidine hydrochloride injection.

[0132] Solution A-100% preparation: 0.1% of impurities A, B, C, R and S were added to the homemade product penehyclidine hydrochloride injection.

[0133] Solution A-150% preparation: 0.15% of impurities A, B, C, R and S were added to the homemade product penehyclidine hydrochloride injection.

[0134] The accuracy of the test results for each impurity is shown in Table 11:

[0135] Table 11 Accuracy of the impurity detection results of penehyclidine hydrochloride

[0136]

[0137]

[0138] The recovery rate of each impurity is between 89.8% and 101.5%, and the measurement accuracy is high.

[0139] Example 5

[0140] The difference between this comparative example and Example 1 lies in the preparation of the test solution. Based on the incomplete types of impurities in the actual sample, in order to investigate the detection of each impurity by the analytical method, about 0.1% of each known impurity is added to the test solution to prepare a spiked test solution for detection.

[0141] The results of the tests showed that the detection results of impurity A, impurity B, impurity C, impurity R and impurity S in the spiked test solution were 0.08%, 0.11%, 0.10%, 0.10% and 0.09%, respectively, which were all close to the known addition amount of 0.1%. It can be seen that the method of the present invention can accurately detect each impurity.

[0142] Example 6

[0143] The difference between this embodiment and embodiment 5 is that the column temperature in the chromatographic conditions is different. The chromatographic column temperature of this embodiment is 33°C.

[0144] The results of the tests showed that the detection results of impurity A, impurity B, impurity C, impurity R and impurity S in the spiked test solution were 0.07%, 0.11%, 0.11%, 0.11% and 0.09% respectively. The detection spectrum is shown in Fig.12 It can be seen that each impurity can still be accurately detected under this condition.

[0145] Example 7

[0146] The difference between this embodiment and embodiment 5 is that the flow rate in the chromatographic conditions is different, and the flow rate in this embodiment is 0.9 ml / min.

[0147] The results of the tests showed that the detection results of impurity A, impurity B, impurity C, impurity R and impurity S in the spiked test solution were 0.07%, 0.11%, 0.11%, 0.11% and 0.09% respectively. The detection spectrum is shown in Fig.13It can be seen that each impurity can still be accurately detected under this condition.

[0148] Example 8

[0149] The only difference between this embodiment and embodiment 5 is that the mobile phase pH value in the chromatographic conditions is different, which is pH 4.8 in this embodiment.

[0150] The results of the tests showed that the detection results of impurity A, impurity B, impurity C, impurity R and impurity S in the spiked test solution were 0.08%, 0.11%, 0.10%, 0.10% and 0.09% respectively. The detection spectrum is shown in Fig.14 It can be seen that each impurity can still be accurately detected under this condition.

[0151] Comparative Example 1

[0152] This comparative example detects the test solution in Example 1, and the high performance liquid phase detection method adopted comprises:

[0153] Instrument: Waters e2695 / 2489 high performance liquid chromatograph;

[0154] Chromatographic column: SHIMADZU Inertsil ODS-3V (4.6 mm × 250 mm, 5 μm);

[0155] Mobile phase A: 0.02 mol / L potassium dihydrogen phosphate solution (containing 0.5% triethylamine) at pH 3.0;

[0156] Mobile phase B: acetonitrile;

[0157] Flow rate: 1.0 ml / min;

[0158] The column temperature was 35°C;

[0159] The detection wavelength is 210nm;

[0160] The injection volume was 20 μl; the test results of the test solution and the impurity B localization solution were shown in Figure 15-16 It can be seen that it cannot effectively detect the known impurity B.

[0161] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these changes and modifications of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these changes and modifications.

Claims

1. A method for detecting related substances of penehyclidine hydrochloride injection by high performance liquid chromatography, comprising the following steps: The test solution was detected by high performance liquid chromatography, using a reverse phase C18 chromatographic column, with a mixed solution of potassium hydrogen phosphate solution and acetonitrile = 70:30~90:10 by volume as mobile phase A, wherein the pH of the potassium hydrogen phosphate solution was 4.0~5.5 and contained 0.5~1.0vol% triethylamine, and a mixed solution of water: acetonitrile = 10:90~30:70 by volume as mobile phase B for gradient elution, the column temperature was 30~40°C, the detection wavelength was 200~270nm, and the flow rate was 0.8~1.5ml / min; The related substances include: cyclopentylphenylacetaldehyde, impurity A, impurity B, impurity C, impurity R and impurity S; impurity A is 3[2-cyclopentyl-2-phenyl-2(2-cyclopentyl-2-hydroxy-2-phenylethoxy)ethoxy]quinuclidine hydrochloride, impurity B is 3-(2-hydroxy-2,2-diphenylethoxy)quinuclidine hydrochloride, impurity C is phenylcyclopentyl ketone, impurity R is 1-cyclopentyl-1-phenyl-1,2-ethylene glycol, and impurity S is 3-(2-cyclopentyl-2-hydroxy-2-phenylethoxy)quinuclidine-1-oxide; The gradient elution program is as follows: 0-5 min, mobile phase A is 90%, mobile phase B is 10%; 5-25 min, mobile phase A is uniformly reduced from 90% to 70%, mobile phase B is increased from 10% to 30%; 25-55 min, mobile phase A is uniformly reduced from 70% to 0%, mobile phase B is increased from 30% to 100%; 55-70 min, mobile phase A is 0%, mobile phase B is 100%; 70-75 min, mobile phase A is increased from 0% to 90%, mobile phase B is reduced from 100% to 10%; and then balanced for 5 min with mobile phase A at 90% and mobile phase B at 10%.

2. The method for detecting related substances of penehyclidine hydrochloride injection by high performance liquid chromatography according to claim 1, characterized in that: The reverse phase C18 chromatographic column has a length of 250-300 mm, the filler type is octadecylsilane bonded silica gel, and the filler particle size is 3-5 μm; the chromatographic column is selected from SHIMADZEU Inertsil series, Waters Xbridge series or Agilent ZORBAX series.

3. The method for detecting related substances of penehyclidine hydrochloride injection by high performance liquid chromatography according to claim 1, characterized in that: The reverse phase C18 chromatographic column is SHIMADZU Inertsil ODS-3V, with an inner diameter of 4.6 mm×250 mm and a filler particle size of 5 μm.

4. The method for detecting related substances of penehyclidine hydrochloride injection by high performance liquid chromatography according to claim 1, characterized in that: The detector of the high performance liquid chromatograph is a diode array or an ultraviolet absorption detector, the temperature of the chromatographic column is 35° C., the detection wavelength is 206 nm, and the flow rate is 1.0 ml / min.

5. The method for detecting related substances of penehyclidine hydrochloride injection by high performance liquid chromatography according to claim 1, characterized in that: In the dipotassium hydrogen phosphate solution, the concentration range of dipotassium hydrogen phosphate is 0.015 mol / L to 0.035 mol / L; in the mobile phase B, the volume ratio of water to acetonitrile is 20:

80.

6. The method for detecting related substances of penehyclidine hydrochloride injection by high performance liquid chromatography according to claim 1, characterized in that: The test solution is penehyclidine hydrochloride injection; or the test solution is penehyclidine hydrochloride solution, including penehyclidine hydrochloride raw material or its preparation, wherein the concentration of penehyclidine hydrochloride is not less than 1 mg / ml.

7. The method for detecting related substances of penehyclidine hydrochloride injection by high performance liquid chromatography according to any one of claims 1 to 6, characterized in that: The sample injection plate temperature of the high performance liquid chromatograph is 4-8°C.

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