A method for simultaneously detecting the content of three impurities in caspofungin acetate

Abstract

The application discloses a method for simultaneously detecting the content of three impurities in caspofungin acetate. The method adopts LC-MS method for detection, and liquid chromatography conditions comprise the following steps: a C18+ chromatographic column is used, a mobile phase gradient elution method is used for separating a sample solution to be detected, a mobile phase A is formic acid-water solution, and a mobile phase B is formic acid-acetonitrile solution; mass spectrometry conditions comprise the following steps: an ion source is an ESI source, mass spectrometry is single quadrupole mass spectrometry, a selected ion detection mode is used, impurity A and impurity C are in a positive ion mode, and impurity D is in a negative ion mode. The method for simultaneously detecting the content of three impurities in caspofungin acetate has the advantages of simple operation, high sensitivity and strong applicability, and can quickly and accurately complete quantitative detection of impurities A, C and D in caspofungin acetate simultaneously.

Description

Technical Field

[0001] This invention relates to the field of compound detection technology, specifically to a method for simultaneously detecting the content of three impurities in caspofungin acetate. Background Technology

[0002] Caspofungin acetate is an antifungal drug used to treat fungal infections in patients with febrile neutropenia, as well as invasive aspergillosis that is unresponsive to or poorly tolerated by other treatments. It is a first-line drug recommended by the 2009 Infectious Diseases Society of America guidelines for empirical or initial treatment of invasive candidiasis. Caspofungin acetate is a cyclic hexapeptide compound obtained through semi-synthesis from fungal fermentation products. The starting material for synthesis is Pneumocandin B0 produced by the fermentation of the filamentous fungus Glarea lozoyensis. Impurities are generated during the synthesis of caspofungin acetate. Furthermore, its structural stability is poor, and it is easily degraded by environmental factors, producing impurities such as impurities A and C generated during the synthesis process, and impurity D generated during degradation.

[0003]

[0004]

[0005] The level of impurities in caspofungin acetate directly affects the quality of the drug and whether it meets relevant standards. While there are reports on the analysis of caspofungin acetate in plasma samples using LC-MS, reports on methods for determining the content of impurities in caspofungin acetate are scarce. Furthermore, the HPLC methods reported in the literature for analyzing related impurities in caspofungin acetate contain sodium chloride and perchloric acid solutions in the mobile phase. Considering the strong corrosiveness and oxidizing properties of perchloric acid, it is necessary to improve the analytical methods for related impurities in caspofungin acetate. Summary of the Invention

[0006] To address the aforementioned shortcomings of existing technologies, the present invention aims to provide a method for simultaneously detecting the content of three impurities in caspofungin acetate, thereby resolving the problem of poor applicability of existing methods for determining the content of impurities in caspofungin acetate.

[0007] The technical solution of the present invention to solve the above-mentioned technical problems is as follows:

[0008] A method for simultaneously detecting the content of three impurities in caspofungin acetate includes the following steps:

[0009] Prepare a caspofungin acetate sample solution, and then perform LC-MS detection on the sample solution to analyze impurities A, C, and D in the sample solution.

[0010] Furthermore, the LC used a C18+ column, and the LC employed a gradient elution method to separate the sample solution. The column dimensions were 2.7 μm and 4.6 × (50–150) mm.

[0011] Furthermore, the mobile phase gradient elution method is as follows: at 0 min, mobile phase A is 85-65% and mobile phase B is 15-35%; at 8.0 min, mobile phase A is 60-40% and mobile phase B is 40-60%; at 10.0 min, mobile phase A is 40-0% and mobile phase B is 60-100%; at 12.0 min, mobile phase A is 40-0% and mobile phase B is 60-100%; at 13.0 min, mobile phase A is 0% and mobile phase B is 100%; at 20.0 min, mobile phase A is 0% and mobile phase B is 100%; both mobile phase A and mobile phase B are expressed as volume percentages.

[0012] Furthermore, the chromatographic column specifications are 2.7μm, 4.6×50mm, 2.7μm, 4.6×100mm, or 2.7μm, 4.6×150mm.

[0013] Preferably, the chromatographic column has a specification of 2.7 μm and a diameter of 4.6 × 150 mm. The preferred gradient elution method is as follows: at 0 min, mobile phase A is 75% and mobile phase B is 25%; at 8.0 min, mobile phase A is 50% and mobile phase B is 50%; at 10.0 min, mobile phase A is 40% and mobile phase B is 60%; at 12.0 min, mobile phase A is 40% and mobile phase B is 60%; at 13.0 min, mobile phase A is 0% and mobile phase B is 100%; at 20.0 min, mobile phase A is 0% and mobile phase B is 100%. Both mobile phase A and mobile phase B are expressed as volume percentages.

[0014] Furthermore, the detection conditions for LC were: flow rate 0.3-0.7 mL / min, column temperature 30-45℃.

[0015] Furthermore, mobile phase A is a formic acid-water solution with a formic acid volume concentration of 0.05-0.20%; mobile phase B is a formic acid-acetonitrile solution with a formic acid volume concentration of 0.05-0.20%.

[0016] Furthermore, the MS detection conditions were as follows: ESI source, single quadrupole mass spectrometer, selected ion detection mode, impurity A (m / z 540) and impurity C (m / z 555) in positive ion mode, impurity D in negative ion mode (m / z 1049), drying gas temperature 300℃, drying gas flow rate 9L / min, nebulizer gas pressure 25psi, capillary voltage 3500V, and fragmentation voltage 135V.

[0017] The present invention has the following beneficial effects:

[0018] 1) This invention uses LC-MS and tests the column temperature, mobile phase A, and mobile phase B selected in this invention. The separation degree between caspofungin acetate and impurities A, C, and D is higher, and the baseline drift of the chromatogram is also significantly improved. Under the optimal liquid chromatography conditions, impurities C and D can be completely separated from caspofungin acetate, while impurity A cannot be completely separated from the baseline of caspofungin acetate. However, since the molecular weight of impurity A is different from that of caspofungin acetate, caspofungin acetate will not interfere with the quantitative detection of impurity A during mass spectrometry.

[0019] 2) The MS detection of this invention adopts a selected ion detection method. Impurities A and C are in positive ion mode, and impurity D is in negative ion mode. The dry gas temperature, dry gas flow rate, nebulizing gas pressure, capillary voltage and other ion source parameters and fragmentation voltage of this invention are used for testing, which has higher detection sensitivity for caspofungin acetate.

[0020] 3) The LC-MS method disclosed in this invention can simultaneously determine the content of impurities A, C and D in caspofungin acetate. This method is simple to operate, highly sensitive and widely applicable, and can provide a method reference for the quantitative analysis of other impurities in caspofungin acetate. Attached Figure Description

[0021] Figure 1 This is the LC-MS extractive ion chromatogram of Example 1, where A: blank solvent, B: mixed standard solution, C: test solution, IPA: impurity A, IPC: impurity C, IPD: impurity D;

[0022] Figure 2 This is a graph showing the peak area changes of the mixed standard and test sample solutions in Example 6 after being placed at room temperature for different times. A: Low-concentration mixed standard solution (concentrations of impurities A, C, and D are 0.260, 0.0400, and 0.200 μg·mL, respectively). -1 B: Medium-concentration mixed standard solution (impurities A, C, and D have concentrations of 1.30, 0.200, and 0.999 μg·mL, respectively). -1 IPA: impurity A, IPC: impurity C, IPD: impurity D;

[0023] Figure 3 This is a graph showing the peak area changes of the mixed standard and test sample solutions in Example 6 after being placed at room temperature for different times; where C: high-concentration mixed standard solution (impurities A, C, and D concentrations are 6.49, 1.00, and 5.00 μg·mL, respectively). -1 D: test solution, IPA: impurity A, IPC: impurity C, IPD: impurity D. Detailed Implementation

[0024] The examples given below are for illustrative purposes only and are not intended to limit the scope of the invention. Unless otherwise specified, conditions in the examples are performed under standard conditions or as recommended by the manufacturer. Reagents or instruments whose manufacturers are not specified are all commercially available products.

[0025] Example 1:

[0026] Specificity test:

[0027] 1) Preparation of reference stock solutions: Accurately weigh appropriate amounts of impurity A, C, and D reference standards, and dilute them with a diluent (an aqueous solution containing 0.2% acetic acid and 30% acetonitrile) to prepare solutions with concentrations of 1.10, 1.01, and 1.03 mg / mL, respectively. -1 The reference stock solution.

[0028] 2) Preparation of mixed standard solutions: Accurately measure appropriate amounts of stock solutions of impurities A, C, and D, and dilute them with a diluent (an aqueous solution containing 0.2% acetic acid and 30% acetonitrile) to prepare concentrations of impurities A, C, and D of 130, 20.0, and 99.9 μg·mL, respectively. -1 The mixed standard stock solution was prepared. An appropriate amount of the mixed standard stock solution was accurately measured and diluted with a diluent (an aqueous solution containing 0.2% acetic acid and 30% acetonitrile) to prepare impurities A, C, and D with concentrations of 5.19, 0.800, and 4.00 μg·mL, respectively. -1 A mixed standard solution.

[0029] 3) Preparation of test solution: Accurately weigh an appropriate amount of caspofungin acetate and dilute it with a diluent (an aqueous solution containing 0.2% acetic acid and 30% acetonitrile) to prepare a solution with a concentration of 1.003 mg / mL. -1 Prepare the test sample stock solution; accurately measure an appropriate amount of the test sample stock solution and dilute it to 501.5 μg·mL with a diluent (an aqueous solution containing 0.2% acetic acid and 30% acetonitrile). -1 That is, you get it.

[0030] 4) Chromatographic conditions: The LC column used was a Waters Cortecs. C18+ (2.7 μm, 4.6 × 150 mm); Mobile phase A was formic acid-water solution with a formic acid volume concentration of 0.10%; Mobile phase B was formic acid-acetonitrile solution with a formic acid volume concentration of 0.10%; The sample solution was separated using a mobile phase gradient elution method; The specific mobile phase gradient elution method was as follows: at 0 min, mobile phase A was 75% and mobile phase B was 25%; at 8.0 min, mobile phase A was 50% and mobile phase B was 50%; at 10.0 min, mobile phase A was 40% and mobile phase B was 60%; at 12.0 min, mobile phase A was 40% and mobile phase B was 60%; at 13.0 min, mobile phase A was 0% and mobile phase B was 100%; at 20.0 min, mobile phase A was 0% and mobile phase B was 100%; Mobile phase A and mobile phase B are both expressed as volume percentages; The mobile phase flow rate was 0.5 mL / min; The column temperature was 40 °C.

[0031] 5) Mass spectrometry conditions: The ion source is an ESI source, a single quadrupole mass spectrometer, and the selected ion detection mode is positive ion mode for impurity A (m / z 540) and impurity C (m / z 555), and negative ion mode (m / z 1049) for impurity D. The drying gas temperature is 300℃, the drying gas flow rate is 9L / min, the nebulizer gas pressure is 25psi, the capillary voltage is 3500V, the fragmentation voltage is 135V, and the mass spectrometry acquisition time is 3-20min, of which 3-9min is positive ion acquisition mode and 9-20min is negative ion acquisition mode.

[0032] 6) Take the blank solvent (i.e. the above diluent), mixed standard solution and test solution and perform LC-MS analysis according to the chromatographic and mass spectrometric conditions of steps 4) and 5) above to obtain chromatograms.

[0033] Results of specificity test:

[0034] The test results of the specificity test of this invention are as follows: Figure 1 As shown, no interfering peaks were found in the blank solvent, indicating that the blank solvent does not interfere with the detection of impurities A, C, and D. The coexisting chromatographic peaks in each EIC chromatogram of the test solution can be separated from impurities A, C, and D at the baseline, indicating that the method has good specificity.

[0035] Example 2:

[0036] Sample injection precision test:

[0037] 1) Preparation of mixed standard stock solutions: Accurately measure appropriate amounts of impurity A, C, and D reference standard stock solutions, and dilute them with a diluent (an aqueous solution containing 0.2% acetic acid and 30% acetonitrile) to prepare concentrations of impurities A, C, and D of 130, 20.0, and 99.9 μg·mL, respectively. -1 Prepare a mixed standard stock solution. Accurately measure an appropriate amount of the mixed standard stock solution to prepare impurity A, C, and D concentrations of 0.260, 0.0400, and 0.200 μg·mL, respectively. -1 The mixed standard solution was injected six times consecutively.

[0038] 2) Chromatographic conditions: The LC column used was a Waters CORTECS column. C18+ (2.7 μm, 4.6 × 150 mm); Mobile phase A was formic acid-water solution with a formic acid volume concentration of 0.10%; Mobile phase B was formic acid-acetonitrile solution with a formic acid volume concentration of 0.10%; The sample solution was separated using a mobile phase gradient elution method; The specific mobile phase gradient elution method was as follows: at 0 min, mobile phase A was 75% and mobile phase B was 25%; at 8.0 min, mobile phase A was 50% and mobile phase B was 50%; at 10.0 min, mobile phase A was 40% and mobile phase B was 60%; at 12.0 min, mobile phase A was 40% and mobile phase B was 60%; at 13.0 min, mobile phase A was 0% and mobile phase B was 100%; at 20.0 min, mobile phase A was 0% and mobile phase B was 100%; Mobile phase A and mobile phase B are both expressed as volume percentages; The mobile phase flow rate was 0.5 mL / min; The column temperature was 40 °C.

[0039] 3) Mass spectrometry conditions: The ion source is an ESI source, a single quadrupole mass spectrometer, and the selected ion detection mode is positive ion mode for impurity A (m / z 540) and impurity C (m / z 555), and negative ion mode (m / z 1049) for impurity D. The drying gas temperature is 300℃, the drying gas flow rate is 9L / min, the nebulizer gas pressure is 25psi, the capillary voltage is 3500V, the fragmentation voltage is 135V, and the mass spectrometry acquisition time is 3-20min, of which 3-9min is positive ion acquisition mode and 9-20min is negative ion acquisition mode.

[0040] Results of the injection precision test:

[0041] The relative standard deviations of the peak areas of impurities A, C, and D are shown in Table 1.

[0042] Table 1. Relative standard deviation of peak areas for impurities A, C, and D.

[0043] Substance Name Impurity A Impurity C Impurity D Peak area relative standard deviation 2.3％ 1.9％ 2.7％

[0044] The data in the table show that the relative standard deviation of the peak areas of impurities A, C, and D is less than 3%, indicating that the detection method has good precision.

[0045] Example 3:

[0046] Linearity, limit of quantitation, and limit of detection tests:

[0047] 1) Preparation of mixed standard stock solutions: Accurately measure appropriate amounts of impurity A, C, and D reference standard stock solutions, and dilute them with a diluent (an aqueous solution containing 0.2% acetic acid and 30% acetonitrile) to prepare concentrations of impurities A, C, and D of 130, 20.0, and 99.9 μg·mL, respectively. -1 The mixed standard stock solution. Linear solution preparation: Accurately measure an appropriate amount of the mixed standard stock solution and dilute it with a diluent (an aqueous solution containing 0.2% acetic acid and 30% acetonitrile) to a concentration of impurity A of 0.0649 μg·mL⁻¹. -1 ~7.79 μg·mL -1 The concentration of impurity C is 0.0100 μg·mL. -1 ~1.20 μg·mL -1 The concentration of impurity D is 0.0500 μg·mL. -1 ~6.00 μg·mL -1 A series of mixed standard solutions.

[0048] 2) Chromatographic conditions: The LC column used was a Waters CORTECS column. C18+ (2.7 μm, 4.6 × 150 mm); Mobile phase A was formic acid-water solution with a formic acid volume concentration of 0.10%; Mobile phase B was formic acid-acetonitrile solution with a formic acid volume concentration of 0.10%; The sample solution was separated using a mobile phase gradient elution method; The specific mobile phase gradient elution method was as follows: at 0 min, mobile phase A was 75% and mobile phase B was 25%; at 8.0 min, mobile phase A was 50% and mobile phase B was 50%; at 10.0 min, mobile phase A was 40% and mobile phase B was 60%; at 12.0 min, mobile phase A was 40% and mobile phase B was 60%; at 13.0 min, mobile phase A was 0% and mobile phase B was 100%; at 20.0 min, mobile phase A was 0% and mobile phase B was 100%; Mobile phase A and mobile phase B are both expressed as volume percentages; The mobile phase flow rate was 0.5 mL / min; The column temperature was 40 °C.

[0049] 3) Mass spectrometry conditions: The ion source is an ESI source, a single quadrupole mass spectrometer, and the selected ion detection mode is positive ion mode for impurity A (m / z 540) and impurity C (m / z 555), and negative ion mode (m / z 1049) for impurity D. The drying gas temperature is 300℃, the drying gas flow rate is 9L / min, the nebulizer gas pressure is 25psi, the capillary voltage is 3500V, the fragmentation voltage is 135V, and the mass spectrometry acquisition time is 3-20min, of which 3-9min is positive ion acquisition mode and 9-20min is negative ion acquisition mode.

[0050] 4) Take the linear solution prepared in step 2) and analyze it under the above chromatographic and mass spectrometric conditions. Analyze the concentrations (X, μg·mL⁻¹) of impurities A, C, and D using their peak areas (Y). -1 Linear regression analysis was performed.

[0051] Linearity, limit of quantitation, and limit of detection test results:

[0052] The relevant information on linear regression, as well as the limits of quantitation (S / N=10) and detection (S / N=3), are shown in Table 2.

[0053] Table 2. Linear relationships, limits of quantitation, and limits of detection for impurities A, C, and D.

[0054]

[0055] Example 4:

[0056] Recovery rate test:

[0057] 1) Preparation of mixed standard stock solutions: Accurately measure appropriate amounts of impurity A, C, and D reference standard stock solutions, and dilute them with a diluent (an aqueous solution containing 0.2% acetic acid and 30% acetonitrile) to prepare concentrations of impurities A, C, and D of 130, 20.0, and 99.9 μg·mL, respectively. -1 The mixed standard stock solution. Preparation of the test sample stock solution: Accurately weigh an appropriate amount of caspofungin acetate and dilute it with a diluent (an aqueous solution containing 0.2% acetic acid and 30% acetonitrile) to prepare a solution with a concentration of 1.003 mg / mL. -1 The test sample stock solution.

[0058] 2) Chromatographic conditions: The LC column used was a Waters CORTECS column. C18+ (2.7 μm, 4.6 × 150 mm); Mobile phase A was formic acid-water solution with a formic acid volume concentration of 0.10%; Mobile phase B was formic acid-acetonitrile solution with a formic acid volume concentration of 0.10%; The sample solution was separated using a mobile phase gradient elution method; The specific mobile phase gradient elution method was as follows: at 0 min, mobile phase A was 75% and mobile phase B was 25%; at 8.0 min, mobile phase A was 50% and mobile phase B was 50%; at 10.0 min, mobile phase A was 40% and mobile phase B was 60%; at 12.0 min, mobile phase A was 40% and mobile phase B was 60%; at 13.0 min, mobile phase A was 0% and mobile phase B was 100%; at 20.0 min, mobile phase A was 0% and mobile phase B was 100%; Mobile phase A and mobile phase B are both expressed as volume percentages; The mobile phase flow rate was 0.5 mL / min; The column temperature was 40 °C.

[0059] 3) Mass spectrometry conditions: The ion source is an ESI source, a single quadrupole mass spectrometer, and the selected ion detection mode is positive ion mode for impurity A (m / z 540) and impurity C (m / z 555), and negative ion mode (m / z 1049) for impurity D. The drying gas temperature is 300℃, the drying gas flow rate is 9L / min, the nebulizer gas pressure is 25psi, the capillary voltage is 3500V, the fragmentation voltage is 135V, and the mass spectrometry acquisition time is 3-20min, of which 3-9min is positive ion acquisition mode and 9-20min is negative ion acquisition mode.

[0060] 4) Accurately measure appropriate amounts of the test sample stock solution and the reference stock solution, and dilute with diluent to prepare a caspofen acetate net concentration of 50.15 μg·mL. -1 The spiking concentration of impurity A was 0.649 μg·mL. -1 Prepare a separate spiking solution containing caspofungin acetate at a concentration of 501.5 μg / mL. -1 The spiked concentrations of impurities C and D were 1.00 μg·mL, respectively. -1 and 5.00 μg·mL -1 Six parallel aliquots of the spiked solution of the test sample were prepared. The samples were analyzed under the chromatographic and mass spectrometric conditions described above, and the recoveries of impurities A, C, and D were calculated.

[0061] Recovery rate test results:

[0062] The samples were analyzed and tested, and the recovery rates were calculated (see Table 3).

[0063] Table 3 Recovery results of impurities A, C, and D

[0064]

[0065] The data in the table show that the average recoveries of impurities A, C, and D are 100.5%, 104.1%, and 105.2%, respectively, with RSDs of 2.5%, 3.6%, and 2.2%, indicating that the method has good accuracy.

[0066] Example 5:

[0067] Intraday precision and interday precision testing:

[0068] 1) Preparation of mixed standard stock solutions: Accurately measure appropriate amounts of impurity A, C, and D reference standard stock solutions, and dilute them with a diluent (an aqueous solution containing 0.2% acetic acid and 30% acetonitrile) to prepare concentrations of impurities A, C, and D of 130, 20.0, and 99.9 μg·mL, respectively. -1 Mixed standard stock solution.

[0069] 2) Preparation of the test sample stock solution: Accurately weigh an appropriate amount of caspofungin acetate and dilute it with a diluent (an aqueous solution containing 0.2% acetic acid and 30% acetonitrile) to prepare a solution with a concentration of 1.003 mg·mL⁻¹. -1 The test sample stock solution.

[0070] 3) Chromatographic conditions: The LC column used was a Waters Cortecs. C18+ (2.7 μm, 4.6 × 150 mm); Mobile phase A was formic acid-water solution with a formic acid volume concentration of 0.10%; Mobile phase B was formic acid-acetonitrile solution with a formic acid volume concentration of 0.10%; The sample solution was separated using a mobile phase gradient elution method; The specific mobile phase gradient elution method was as follows: at 0 min, mobile phase A was 75% and mobile phase B was 25%; at 8.0 min, mobile phase A was 50% and mobile phase B was 50%; at 10.0 min, mobile phase A was 40% and mobile phase B was 60%; at 12.0 min, mobile phase A was 40% and mobile phase B was 60%; at 13.0 min, mobile phase A was 0% and mobile phase B was 100%; at 20.0 min, mobile phase A was 0% and mobile phase B was 100%; Mobile phase A and mobile phase B are both expressed as volume percentages; The mobile phase flow rate was 0.5 mL / min; The column temperature was 40 °C.

[0071] 4) Mass spectrometry conditions: The ion source is an ESI source, a single quadrupole mass spectrometer, and the selected ion detection mode is positive ion mode for impurity A (m / z 540) and impurity C (m / z 555), and negative ion mode (m / z 1049) for impurity D. The drying gas temperature is 300℃, the drying gas flow rate is 9L / min, the nebulizer gas pressure is 25psi, the capillary voltage is 3500V, the fragmentation voltage is 135V, and the mass spectrometry acquisition time is 3-20min, of which 3-9min is positive ion acquisition mode and 9-20min is negative ion acquisition mode.

[0072] 5) Intra-day precision test: Accurately measure appropriate amounts of the test sample stock solution and the reference stock solution, and dilute with diluent to prepare a solution containing a net concentration of caspofungin acetate of 50.15 μg·mL. -1 The concentration of impurity A was 0.649 μg·mL. -1 Prepare a separate spiking solution containing caspofungin acetate at a concentration of 501.5 μg / mL. -1 The spiked concentrations of impurities C and D were 1.00 μg·mL, respectively. -1 and 5.00 μg·mL -1 The spiked solution of the test sample was prepared in 6 parallel samples and analyzed under the above chromatographic and mass spectrometric conditions. The concentrations of impurities A, C and D were calculated.

[0073] 6) Daytime precision test: Accurately measure appropriate amounts of the test sample stock solution and the reference stock solution, and dilute with diluent to prepare a solution containing a net concentration of caspofungin acetate of 50.15 μg·mL. -1 The concentration of impurity A was 0.649 μg·mL. -1 Prepare a separate spiking solution containing caspofungin acetate at a concentration of 501.5 μg / mL. -1 The spiked concentrations of impurities C and D were 1.00 μg·mL, respectively. -1 and 5.00 μg·mL -1 The spiked solution of the test sample was injected and analyzed for 6 consecutive days under the above chromatographic and mass spectrometric conditions, and the concentrations of impurities A, C and D were calculated.

[0074] Results of intraday precision test:

[0075] The relative standard deviations of the concentrations of impurities A, C, and D are shown in Table 4.

[0076] Table 4. Relative standard deviations of impurity A, C, and D concentrations

[0077] Tests and trials Substance Name Impurity A Impurity C Impurity D Intraday precision relative standard deviation of concentration 1.7％ 3.5％ 1.9％ Daytime precision relative standard deviation of concentration 2.5％ 3.2％ 3.3％

[0078] The data in the table show that the relative standard deviations of the concentrations of impurities A, C, and D are all less than 4%, indicating that this detection method has good intra-day and inter-day precision.

[0079] Example 6:

[0080] Room temperature stability test:

[0081] 1) Preparation of mixed standard solutions: Accurately measure appropriate amounts of stock solutions of impurities A, C, and D, and dilute them with a diluent (an aqueous solution containing 0.2% acetic acid and 30% acetonitrile) to prepare concentrations of impurities A, C, and D of 130, 20.0, and 99.9 μg·mL, respectively. -1 Prepare a mixed standard stock solution; accurately measure an appropriate amount of the mixed standard stock solution and dilute it with a diluent (an aqueous solution containing 0.2% acetic acid and 30% acetonitrile) to prepare low concentrations (impurities A, C, and D concentrations of 0.260, 0.0400, and 0.200 μg·mL, respectively). -1 Medium concentrations (impurities A, C, and D concentrations were 1.30, 0.200, and 0.999 μg·mL, respectively). -1 ) and high concentrations (impurities A, C, and D concentrations were 6.49, 1.00, and 5.00 μg·mL, respectively). -1 ) mixed standard solution.

[0082] 2) Preparation of test solution: Accurately weigh an appropriate amount of caspofungin acetate and prepare a solution with a concentration of 2.644 mg·mL using a diluent (an aqueous solution containing 0.2% acetic acid and 30% acetonitrile). -1 Prepare the test sample stock solution; accurately measure an appropriate amount of the test sample stock solution and dilute it to 499.7 μg·mL with a diluent (an aqueous solution containing 0.2% acetic acid and 30% acetonitrile by volume). -1 The test solution was prepared.

[0083] 3) Chromatographic conditions: The LC column used was a Waters Cortecs. C18+ (2.7 μm, 4.6 × 150 mm); Mobile phase A was formic acid-water solution with a formic acid volume concentration of 0.10%; Mobile phase B was formic acid-acetonitrile solution with a formic acid volume concentration of 0.10%; The sample solution was separated using a mobile phase gradient elution method; The specific mobile phase gradient elution method was as follows: at 0 min, mobile phase A was 75% and mobile phase B was 25%; at 8.0 min, mobile phase A was 50% and mobile phase B was 50%; at 10.0 min, mobile phase A was 40% and mobile phase B was 60%; at 12.0 min, mobile phase A was 40% and mobile phase B was 60%; at 13.0 min, mobile phase A was 0% and mobile phase B was 100%; at 20.0 min, mobile phase A was 0% and mobile phase B was 100%; Mobile phase A and mobile phase B are both expressed as volume percentages; The mobile phase flow rate was 0.5 mL / min; The column temperature was 40 °C.

[0084] 4) Mass spectrometry conditions: The ion source is an ESI source, a single quadrupole mass spectrometer, and the selected ion detection mode is positive ion mode for impurity A (m / z 540) and impurity C (m / z 555), and negative ion mode (m / z 1049) for impurity D. The drying gas temperature is 300℃, the drying gas flow rate is 9L / min, the nebulizer gas pressure is 25psi, the capillary voltage is 3500V, the fragmentation voltage is 135V, and the mass spectrometry acquisition time is 3-20min, of which 3-9min is positive ion acquisition mode and 9-20min is negative ion acquisition mode.

[0085] 5) Take low-concentration, medium-concentration, and high-concentration mixed standard solutions respectively, and perform LC-MS analysis every 0.5 h according to the above chromatographic and mass spectrometric conditions. Take the test sample solution and perform LC-MS analysis every 0.5 h according to the above chromatographic and mass spectrometric conditions to investigate the room temperature stability of mixed standard solutions and test sample solutions of different concentrations.

[0086] Results of room temperature stability test:

[0087] The trends of peak area changes of impurities A, C, and D at different times after being placed at room temperature are as follows: Figure 2 and Figure 3 As shown in the figure, the peak areas of impurities A and D in the mixed standard showed no significant fluctuation within 5 hours, with an RSD of less than 4%, indicating that impurities A and D were stable within 5 hours at room temperature. However, the peak area of ​​low-concentration impurity C decreased significantly after 1 hour at room temperature, with an RSD of 6.2%. The RSD of medium-concentration impurity C after 1.5 hours at room temperature was 8.0%, and the RSD of high-concentration impurity C after 3 hours at room temperature was 6.6%. In the test solution, the peak area of ​​impurity A showed no significant fluctuation within 1 hour, but the peak area of ​​impurity C decreased significantly, while the peak area of ​​impurity D increased significantly. The RSDs of the peak areas of impurities A, C, and D were 1.2%, 22.9%, and 40.6%, respectively. These results indicate that impurity C degrades more severely when placed at room temperature, while caspofungin acetate degrades to produce impurity D when placed at room temperature. Therefore, when analyzing impurities A, C, and D simultaneously, the time the sample solution is placed at room temperature should still be minimized.

[0088] Example 7:

[0089] Sample determination test:

[0090] 1) Preparation of test solution: Accurately weigh 10 mg of each of three batches of caspofungin acetate raw material samples, and prepare solutions with diluent (an aqueous solution containing 0.2% acetic acid and 30% acetonitrile) to concentrations of 1.012, 1.000, and 1.012 mg / mL, respectively. -1Prepare 10 mL of the test sample stock solution; accurately measure an appropriate amount of the test sample stock solution and prepare a solution with a labeled concentration of 500 μg / mL using a diluent (an aqueous solution containing 0.2% acetic acid and 30% acetonitrile). -1 The test solution and the labeled concentration are 50 μg·mL -1 Each test solution was prepared in triplicate.

[0091] 2) Chromatographic conditions: The LC column used was a Waters CORTECS column. C18+ (2.7 μm, 4.6 × 150 mm); Mobile phase A was formic acid-water solution with a formic acid volume concentration of 0.10%; Mobile phase B was formic acid-acetonitrile solution with a formic acid volume concentration of 0.10%; The sample solution was separated using a mobile phase gradient elution method; The specific mobile phase gradient elution method was as follows: at 0 min, mobile phase A was 75% and mobile phase B was 25%; at 8.0 min, mobile phase A was 50% and mobile phase B was 50%; at 10.0 min, mobile phase A was 40% and mobile phase B was 60%; at 12.0 min, mobile phase A was 40% and mobile phase B was 60%; at 13.0 min, mobile phase A was 0% and mobile phase B was 100%; at 20.0 min, mobile phase A was 0% and mobile phase B was 100%; Mobile phase A and mobile phase B are both expressed as volume percentages; The mobile phase flow rate was 0.5 mL / min; The column temperature was 40 °C.

[0092] 3) Mass spectrometry conditions: The ion source is an ESI source, a single quadrupole mass spectrometer, and the selected ion detection mode is positive ion mode for impurity A (m / z 540) and impurity C (m / z 555), and negative ion mode (m / z 1049) for impurity D. The drying gas temperature is 300℃, the drying gas flow rate is 9L / min, the nebulizer gas pressure is 25psi, the capillary voltage is 3500V, the fragmentation voltage is 135V, and the mass spectrometry acquisition time is 3-20min, of which 3-9min is positive ion acquisition mode and 9-20min is negative ion acquisition mode.

[0093] 4) Take the sample prepared in step 1) with a labeled concentration of 50 μg·mL -1 The test solution was analyzed by LC-MS under the chromatographic and mass spectrometric conditions described above to calculate the content of impurity A in the sample. Separately, a sample prepared in step 1) with a labeled concentration of 500 μg·mL⁻¹ was also analyzed. -1 The test solution was analyzed by LC-MS under the chromatographic and mass spectrometric conditions described above, and the contents of impurity C and impurity D in the sample were calculated.

[0094] The test results for the samples are shown in Table 5:

[0095] Table 5. Contents of impurities A, C, and D in three batches of caspofungin acetate raw material.

[0096]

[0097] The test results showed that the content of impurity A in all three batches of samples was <0.7%, the content of impurity C was <0.007%, and the content of impurity D was ≤0.14%. This indicates that all three batches of active pharmaceutical ingredients meet the impurity limits required by the quality standard.

[0098] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for simultaneously detecting the content of three impurities in caspofungin acetate, characterized in that, The detection method using LC-MS includes the following steps: Prepare a caspofungin acetate sample solution, and then perform LC-MS detection on the sample solution to analyze the content of impurities A, C and D in the sample solution. The LC used a C18+ column, and the LC employed a gradient elution method to separate the sample solution. The column dimensions were 2.7 μm and 4.6 × (50~150) mm. The gradient elution method was as follows: at 0 min, mobile phase A was 85-65% and mobile phase B was 15-35%. 8.0 min, mobile phase A is 60-40%, mobile phase B is 40-60%; At 10.0 min, mobile phase A was 40-0% and mobile phase B was 60-100%. At 12.0 min, mobile phase A was 40-0% and mobile phase B was 60-100%. At 13.0 min, mobile phase A was 0% and mobile phase B was 100%; at 20.0 min, mobile phase A was 0% and mobile phase B was 100%; both mobile phase A and mobile phase B are expressed as volume percentage. The mobile phase A is a formic acid-water solution with a formic acid volume concentration of 0.05-0.20%; the mobile phase B is a formic acid-acetonitrile solution with a formic acid volume concentration of 0.05-0.20%. The detection conditions for the MS were as follows: the ion source was an ESI source, the mass spectrometer was a single quadrupole mass spectrometer, selected ion detection was used, impurity A and impurity C were in positive ion mode, and impurity D was in negative ion mode; wherein... The positive ion of impurity A is m / z 540, the positive ion of the impurity C is m / z 555, the negative ion of impurity D is m / z 1049; The structural formula of impurity A is: ; The structural formula of the impurity C is: ; The structural formula of the impurity D is: .

2. The method for simultaneously detecting the content of three impurities in caspofungin acetate according to claim 1, characterized in that, The chromatographic column has a size of 2.7 μm and a diameter of 4.6 × 150 mm. The specific method for gradient elution of the mobile phase is as follows: at 0 min, mobile phase A is 75% and mobile phase B is 25%; at 8.0 min, mobile phase A is 50% and mobile phase B is 50%. At 10.0 min, mobile phase A was 40% and mobile phase B was 60%; at 12.0 min, mobile phase A was 40% and mobile phase B was 60%. At 13.0 min, mobile phase A was 0% and mobile phase B was 100%; at 20.0 min, mobile phase A was 0% and mobile phase B was 100%; both mobile phase A and mobile phase B are expressed as volume percentages.

3. The method for simultaneously detecting the content of three impurities in caspofungin acetate according to claim 1, characterized in that, The detection conditions for the LC were: flow rate 0.3-0.7 mL / min, column temperature 30-45 ℃.

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