A method for detecting related substances in palonosetron-containing fat emulsion injection

Abstract

The application belongs to the technical field of medicine extraction and medicine detection, and discloses a detection method of related substances in palonosetron-containing fat emulsion injection. Methanol is used as a main demulsifier, and a sodium dihydrogen phosphate buffer salt solution with a suitable ionic strength is used for assistance, so that full demulsification is realized under the premise of not introducing a third organic phase; a water bath evaporation and redissolution method is used to enrich target components in the solution after demulsification, so that good chromatographic behavior and complete recovery of the target components are ensured, and the detection sensitivity is effectively improved. The palonosetron and related impurities have good separation degree, the method is simple to operate, accurate to measure, and strong in specificity, and is suitable for the determination of palonosetron related substances in rolapitant palonosetron fat emulsion.

Description

Technical Field

[0001] This invention relates to the field of drug extraction and drug detection technology, and in particular to a method for detecting related substances in a fat emulsion injection containing palonosetron. Background Technology

[0002] Palonosetron hydrochloride is a highly selective, high-affinity second-generation 5-HT3 receptor antagonist developed by Helsinn Healthcare SA in Switzerland. It was approved by the U.S. Food and Drug Administration (FDA) in July 2003 under the brand name Aloxi®. It is primarily used for the prevention and treatment of chemotherapy-induced nausea and vomiting (CINV). Clinical studies have shown that palonosetron has good preventative effects against both acute and delayed CINV induced by moderate to highly emetogenic chemotherapy, and it is the only 5-HT3 receptor antagonist approved for delayed CINV.

[0003] The chemical name of palonosetron hydrochloride is 2-[1-azabicyclo(2.2.2)oct-3S-yl]-2,3,3aS,4,5,6-hexahydro-1H-benzo[de]isoquinoline-1-one hydrochloride, and its molecular formula is C2. 19 H 24 N₂O·HCl, with a molecular weight of 332.87, has the following structural formula:

[0004] Palonosetron hydrochloride contains a lactam ring and a tertiary amine group in its structure, making it susceptible to hydrolysis or oxidative degradation. The main degradation impurity is palonosetron impurity A (also known as palonosetron nitrides), and its degradation pathway is as follows:

[0005] Lorapitane-palonosetron fat emulsion injection is a new generation of combination therapy targeting NK-1 and 5-HT3 receptors for the treatment of post-chemotherapy nausea and vomiting, offering significant clinical advantages compared to existing antiemetics. Palonosetron hydrochloride is a key active ingredient in this injection, and its quality stability directly affects the safety and efficacy of the formulation. Therefore, it is necessary to establish accurate and sensitive analytical methods to detect and control its related substances. While there are existing reports on the use of high-performance liquid chromatography (HPLC) to determine the content of palonosetron hydrochloride and related substances, rorapitane-palonosetron fat emulsion, being an emulsion, typically requires demulsification to release the drug before detection. Meanwhile, the concentration of palonosetron hydrochloride in the formulation is extremely low (approximately 8~16 μg / mL), and conventional pretreatment methods cannot meet the sensitivity requirements for impurity detection. Furthermore, for the rorapitan-palonosetron fat emulsion compound injection system, the presence of rorapitan and its related substances may interfere with the detection of palonosetron-related substances, making existing single-component impurity detection methods difficult to apply directly.

[0006] Therefore, there is an urgent need to develop a simple, accurate, and stable analytical method to detect related substances of palonosetron in rorapitan-palonosetron fat emulsion, so as to ensure the quality controllability of this compound preparation. Summary of the Invention

[0007] The purpose of this invention is to provide a method for detecting related substances in fat emulsion injections containing palonosetron, thereby solving the aforementioned problems existing in the prior art.

[0008] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides a method for detecting related substances in a fat emulsion injection containing palonosetron, comprising the following steps: (1) Mix the fat emulsion injection containing palonosetron with sodium dihydrogen phosphate buffer solution, and make up to volume with methanol as demulsifier. Vortex the resulting mixture to obtain a demulsified solution. Centrifuge the demulsified solution for the first time, and take the supernatant after centrifugation and place it in an evaporating dish to evaporate to dryness in a water bath. Add diluent to the residue after evaporation in the water bath and vortex to dissolve it. Then centrifuge for the second time and take the supernatant after centrifugation as the test solution. (2) The test solution was examined by high performance liquid chromatography to confirm the content of palonosetron impurities in the fat emulsion injection containing palonosetron.

[0009] Preferably, the volume ratio of the palonosetron-containing fat emulsion injection to the sodium dihydrogen phosphate buffer solution is 1~3:0.1~0.5.

[0010] Preferably, the conditions for the first centrifugation include: a rotation speed of 8000~10000 r / min and a time of 10~20 min.

[0011] Preferably, the conditions for the second centrifugation include: a rotation speed of 10,000 to 12,000 r / min and a time of 10 to 20 min.

[0012] Preferably, the temperature for the water bath evaporation is 40~50℃.

[0013] Preferably, the diluent is a mixture of methanol and sodium dihydrogen phosphate solution in a volume ratio of 30:70; the concentration of the sodium dihydrogen phosphate solution is 0.01~0.05 mol / L, and the pH is 7.

[0014] Preferably, the chromatographic conditions for the high-performance liquid chromatography include: The chromatographic column was a Phenomenex Luna C8 column; the column temperature was 35℃; the flow rate was 1 mL / min; the detection wavelength was 210 nm; the injection volume was 100 μL; mobile phase A was 0.02 mol / L sodium dihydrogen phosphate solution at pH 7, mobile phase B was methanol, and mobile phase C was acetonitrile; the elution method was gradient elution. The gradient elution is performed according to the following table:

[0015] Where % represents the volume fraction.

[0016] As can be seen from the above technical solution, compared with the prior art, the present invention has the following beneficial effects: This invention uses methanol as the main demulsifier, supplemented by a sodium dihydrogen phosphate buffer solution of suitable ionic strength, to achieve complete demulsification without introducing a third organic phase. The target component in the demulsified solution is enriched by water bath evaporation and reconstitution. The method is simple to operate and ensures good chromatographic behavior and complete recovery of the target component. The average recovery rate of palonosetron impurity A is 103.1%, effectively improving detection sensitivity. The detection limit and quantitation limit concentrations of palonosetron in this invention are 0.0032 μg / mL and 0.0064 μg / mL (relative concentrations of 0.05% and 0.11%, respectively), and the detection limit and quantitation limit concentrations of palonosetron impurity A are 0.0027 μg / mL and 0.0053 μg / mL (relative concentrations of 0.04% and 0.09%, respectively). Palonosetron and related impurities show good separation. The method is simple to operate, accurate, and highly specific, suitable for the determination of palonosetron-related substances in rorapitane-palonosetron fat emulsions. Attached Figure Description

[0017] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below.

[0018] Figure 1 This is the high-performance liquid chromatogram of the blank solution; Figure 2 High-performance liquid chromatography (HPLC) chromatogram of the solution for system suitability; Figure 3 This is a high-performance liquid chromatogram of a single blank fat emulsion. Figure 4 The high-performance liquid chromatogram of the test solution; Figure 5 High-performance liquid chromatography (HPLC) chromatogram of palonosetron solution at its detection limit; Figure 6 High-performance liquid chromatography (HPLC) chromatogram of palonosetron solution at the limit of quantitation; Figure 7 High-performance liquid chromatography (HPLC) chromatogram of palonosetron impurity A solution at the detection limit; Figure 8 High-performance liquid chromatography (HPLC) chromatogram of palonosetron impurity A solution at the limit of quantitation; Figure 9 For the linear operating curve of palonosetron; Figure 10 The linear operating curve for palonosetron impurity A is shown. Detailed Implementation

[0019] This invention provides a method for detecting related substances in a fat emulsion injection containing palonosetron, comprising the following steps: (1) Mix the fat emulsion injection containing palonosetron with sodium dihydrogen phosphate buffer solution, and make up to volume with methanol as demulsifier. Vortex the resulting mixture to obtain a demulsified solution. Centrifuge the demulsified solution for the first time, and take the supernatant after centrifugation and place it in an evaporating dish to evaporate to dryness in a water bath. Add diluent to the residue after evaporation in the water bath and vortex to dissolve it. Then centrifuge for the second time and take the supernatant after centrifugation as the test solution. (2) The test solution was examined by high performance liquid chromatography to confirm the content of palonosetron impurities in the fat emulsion injection containing palonosetron.

[0020] In this invention, the palonosetron-containing fat emulsion injection is the rorapitan-palonosetron injection formulation described in patent application 202610966934.3, comprising the following components by mass percentage: rorapitan hydrochloride monohydrate 0.45%~1.1%; palonosetron hydrochloride 0.0006%~0.0016%; inorganic salt 0.03%~0.5%; metal chelating agent 0.004%~0.006%; injection oil 5%~10%; emulsifier 14%~15%; osmotic pressure regulator 5%~6%; first co-emulsifier 2.5%~3.2%; second co-emulsifier 0.5%~0.6%; antioxidant 0.02%~0.1%; balance water; the pH of the injection formulation is 8~9.

[0021] In this invention, the volume ratio of the palonosetron-containing fat emulsion injection to the sodium dihydrogen phosphate buffer solution is preferably 1~3:0.1~0.5, more preferably 3:0.35; the concentration of the sodium dihydrogen phosphate buffer solution is preferably 1 mol / L.

[0022] In this invention, the volume of the final volume is preferably 10 mL.

[0023] In this invention, the conditions for the first centrifugation include: a rotation speed preferably of 8000~10000 r / min, more preferably 10000 r / min, and a time preferably of 10~20 min, more preferably 10 min.

[0024] In this invention, the conditions for the second centrifugation include: a rotation speed preferably of 10,000 to 12,000 r / min, more preferably 12,000 r / min, and a time preferably of 10 to 20 min, more preferably 10 min.

[0025] In this invention, the water bath evaporation temperature is preferably 40~50℃, and more preferably 40℃.

[0026] In this invention, the diluent is preferably a mixture of methanol and sodium dihydrogen phosphate solution in a volume ratio of 30:70; the concentration of the sodium dihydrogen phosphate solution is preferably 0.01~0.05mol / L, more preferably 0.02mol / L, and the pH is preferably 7.

[0027] In this invention, the chromatographic conditions for the high-performance liquid chromatography include: The preferred chromatographic column is a Phenomenex Luna C8 column; the preferred column temperature is 35℃; the preferred flow rate is 1 mL / min; the preferred detection wavelength is 210 nm; the preferred injection volume is 100 μL; the preferred mobile phase A is a 0.02 mol / L sodium dihydrogen phosphate solution with pH 7; the preferred mobile phase B is methanol; and the preferred mobile phase C is acetonitrile; the preferred elution method is gradient elution. The gradient elution is performed according to the following table:

[0028] Where % represents the volume fraction.

[0029] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0030] The rorapitan-palonosetron fat emulsion used in the following embodiments and comparative examples of the present invention were all from the same batch, and the high-performance liquid chromatography analysis conditions were all the same.

[0031] Comparative Example 1

[0032] This comparative example provides a method for demulsifying a fat emulsion test solution, comprising the following steps: Take 500 μL of rorapitan-palonosetron fat emulsion, add 1 mL of methanol, vortex for 1 min, centrifuge at 13000 r / min for 20 min to demulsify, and take the supernatant.

[0033] The results showed that under these conditions, the upper layer was turbid and a clear test solution could not be obtained, indicating that the methanol ratio was not ideal for demulsification and could not be directly injected for analysis.

[0034] Comparative Example 2

[0035] This comparative example provides a method for demulsifying a fat emulsion test solution, comprising the following steps: Take 500 μL of rorapitan-palonosetron fat emulsion, add 900 μL of methanol and 100 μL of 0.5 mol / L sodium dihydrogen phosphate buffer solution, mix well, and centrifuge at 13000 r / min for 20 min and 15000 r / min for 5 min respectively to demulsify, and take the supernatant.

[0036] The results showed that under both centrifugation conditions, the supernatant was turbid and a clear test solution could not be obtained, indicating that the above-mentioned methanol-buffered salt system had an unsatisfactory demulsification effect and could not be directly injected for analysis.

[0037] Comparative Example 3

[0038] This comparative example provides a method for demulsifying a fat emulsion test solution, comprising the following steps: Take 500 μL of rorapitan-palonosetron fat emulsion, add 800 μL of methanol, 100 μL of ethanol and 100 μL of 0.5 mol / L sodium dihydrogen phosphate buffer solution, mix well, centrifuge at 13000 r / min for 10 min, obtain the clear supernatant, and perform high performance liquid chromatography analysis.

[0039] The results showed that the supernatant was clear and plentiful after centrifugation, indicating good demulsification. However, the chromatographic analysis showed that the main peak shape was slightly poor, and the separation performance of the chromatographic column was affected, which was not conducive to the accurate determination of related substances.

[0040] Comparative Example 4

[0041] This comparative example provides a method for demulsifying a fat emulsion test solution, comprising the following steps: Take 500 μL of rorapitan-palonosetron fat emulsion, add 450 μL of methanol, 450 μL of ethanol and 100 μL of 0.5 mol / L sodium dihydrogen phosphate buffer solution, mix well, centrifuge at 13000 r / min for 10 min, obtain the clear supernatant, and perform high performance liquid chromatography analysis.

[0042] The results showed that the supernatant was clear and plentiful after centrifugation, indicating good demulsification. However, chromatographic analysis showed that the main peak was split, indicating that the organic phase composition at this ratio caused abnormal chromatographic behavior and could not meet the requirements for the detection of related substances.

[0043] Comparative Example 5

[0044] This comparative example provides a method for demulsifying a fat emulsion test solution, comprising the following steps: Take 500 μL of rorapitan-palonosetron fat emulsion, add 850 μL of methanol, 50 μL of 1% trifluoroacetic acid aqueous solution and 100 μL of 0.5 mol / L sodium dihydrogen phosphate buffer solution, mix well, centrifuge at 13000 r / min for 15 min, and obtain the clear supernatant for high performance liquid chromatography analysis.

[0045] The results showed that the supernatant after centrifugation was opalescent and scarce, indicating an unsatisfactory demulsification effect. Meanwhile, chromatographic analysis showed that the peak area of ​​the main peak was only 0.5 times the normal value of other samples, indicating that the addition of trifluoroacetic acid caused degradation or loss of the sample, making it unsuitable for the detection of related substances in this fat emulsion formulation.

[0046] Comparative Example 6

[0047] This comparative example provides a method for demulsifying a fat emulsion test solution, comprising the following steps: Take 500 μL of rorapitan-palonosetron fat emulsion, add 850 μL of methanol, 50 μL of isopropanol and 100 μL of 0.5 mol / L sodium dihydrogen phosphate buffer solution, mix well, centrifuge at 15000 r / min for 5 min, and obtain the clear supernatant for high performance liquid chromatography analysis.

[0048] The results showed that the supernatant was clear but in small quantity after centrifugation, and the main peak had a good shape and normal peak area. However, the introduction of isopropanol increased the complexity of the organic phase composition, and the amount of supernatant recovered after centrifugation was small, which was not conducive to the general applicability of the method.

[0049] Comparative Example 7

[0050] This comparative example provides a method for demulsifying a fat emulsion test solution, comprising the following steps: Take 500 μL of rorapitan-palonosetron fat emulsion, add 950 μL of methanol and 50 μL of 1.0 mol / L sodium dihydrogen phosphate buffer solution, mix well, centrifuge at 14000 r / min for 1 min, and obtain the clear supernatant for high performance liquid chromatography analysis.

[0051] The results showed that the supernatant was clear and plentiful after centrifugation, indicating good demulsification. The main peak shape and peak area were good in chromatographic analysis, but still could not meet the sensitivity requirements for the detection of related substances, and the solution needed to be concentrated after demulsification.

[0052] Comparative Example 8

[0053] This comparative example provides a method for concentrating a fat emulsion test solution, comprising the following steps: Accurately measure 3 mL of rorapitan-palonosetron fat emulsion and place it in a 10 mL volumetric flask. Add 350 μL of 1.0 mol / L potassium dihydrogen phosphate buffer solution, dilute to the mark with methanol, mix well, centrifuge, and decant the supernatant. Rotary evaporate the solution at 35 °C until it is less than 2 mL. Dilute to 5 mL with methanol and methanol-mobile phase A (50:50) respectively, mix well, centrifuge, and take the supernatant as the test solution for high performance liquid chromatography analysis.

[0054] The results showed that when the diluent for the test solution was methanol, the main peak exhibited peak splitting, while the main peak shape was good when the diluent was methanol-mobile phase A (50:50). Rotary evaporation was prone to bumping, making complete solvent removal impossible, and the resolution of residues required a large amount of solvent. The theoretical concentration of palonosetron in the resulting test solution was 4.8 μg / mL, and the main peak area was low, failing to meet the sensitivity requirements for related substance detection.

[0055] Example 1

[0056] Accurately measure 3 mL of rorapitan-palonosetron fat emulsion and place it in a 10 mL volumetric flask. Add 350 μL of 1 mol / L sodium dihydrogen phosphate buffer solution, shake well, and dilute to the mark with methanol. Vortex to break the emulsion. Centrifuge the solution at 10000 r / min for 10 min, decant the supernatant, accurately measure 5 mL, place it in an evaporating dish, and evaporate to dryness in a 40℃ water bath. Accurately add 2 mL of diluent (methanol-mobile phase A (30:70)) to the residue and vortex to dissolve. Centrifuge the solution at 12000 r / min for 10 min, and take the supernatant as the test solution for high performance liquid chromatography analysis.

[0057] The results showed that the method of the present invention for treating rorapitan-palonosetron fat emulsion resulted in complete demulsification, clear supernatant after centrifugation, and sufficient recovery. Through water bath evaporation and concentration, the theoretical concentration of palonosetron could reach 6 μg / mL, with a good main peak shape, meeting the sensitivity requirements for related substance detection. Furthermore, the main component and impurities were well separated, making the method suitable for the detection of palonosetron-related substances in rorapitan-palonosetron fat emulsion.

[0058] Comparison and summary

[0059] The comparative examples 1-7 above demonstrate that the selection and dosage of demulsifiers have a significant impact on the demulsification effect and chromatographic behavior of fat emulsions. Simply increasing the methanol ratio or adding ethanol can yield a clear supernatant, but it easily leads to peak deterioration or even peak splitting. Adding trifluoroacetic acid, while aiding demulsification, causes degradation and loss of the main component. While using isopropanol instead of ethanol can achieve a good peak shape, the amount of supernatant recovered is low. The method of this invention, by optimizing the methanol dosage and using a sodium dihydrogen phosphate buffer solution of suitable ionic strength (1 mol / L), achieves thorough demulsification without introducing a third organic phase, ensuring good chromatographic behavior and complete recovery of the target component.

[0060] Comparative Example 8 shows that when the sample is concentrated using rotary evaporation, the theoretical concentration of palonosetron is only 4.8 μg / mL, and the peak area is too low to meet the sensitivity requirements for related substance detection. The method of this invention uses a water bath to evaporate the sample to dryness, fully enriching the target component in 5 mL of supernatant before redissolving it with 2 mL of diluent. This achieves a theoretical concentration of palonosetron of up to 6 μg / mL, effectively improving detection sensitivity. Furthermore, the method is simple to operate and avoids peak shape abnormalities that may occur during rotary evaporation due to boiling over or transfer losses.

[0061] Example 2

[0062] 1. Instruments and reagents

[0063] Agilent 1260 liquid chromatograph (equipped with a G7117C DAD detector). Acetonitrile (chromatographic grade), methanol (chromatographic grade), sodium dihydrogen phosphate (analytical grade), sodium hydroxide (analytical grade), purified water.

[0064] 2. Chromatographic conditions

[0065] Chromatographic column: Phenomenex Luna C8 column (4.6 mm × 250 mm, 5 μm)

[0066] Detection wavelength: 210nm

[0067] Column temperature: 35℃

[0068] Flow rate: 1.0 mL / min

[0069] Injection volume: 100 μL

[0070] Diluent: Methanol-Mobile Phase A (30:70)

[0071] Mobile phase: Mobile phase A: 0.02M sodium dihydrogen phosphate solution (adjusted to pH=7.0 with sodium hydroxide), mobile phase B: methanol, mobile phase C: acetonitrile, gradient elution is performed according to the table below.

[0072]

[0073] 3. Solution preparation

[0074] (1) Blank solution / diluent: methanol-mobile phase A (30:70).

[0075] (2) Blank fat emulsion solution: Measure 3 mL of blank fat emulsion (without palonosetron hydrochloride) into a 10 mL volumetric flask, add 350 μL of 1 mol / L sodium dihydrogen phosphate buffer solution, shake well, dilute to the mark with methanol, and vortex to break the emulsion. Centrifuge the solution at 10000 r / min for 10 min, pour off the supernatant, accurately measure 5 mL, place it in an evaporating dish, evaporate to dryness in a 40℃ water bath, accurately add 2 mL of diluent to the residue, vortex to dissolve, centrifuge the solution at 12000 r / min for 10 min, and take the supernatant.

[0076] (3) System suitability solution: Take appropriate amounts of palonosetron and palonosetron impurity A, dissolve them in diluent and dilute quantitatively to prepare solutions containing 5 μg and 0.25 μg per mL, respectively.

[0077] (4) Test solution: Measure 3 mL of rorapitan-palonosetron fat emulsion into a 10 mL volumetric flask, add 350 μL of 1 mol / L sodium dihydrogen phosphate buffer solution, shake well, dilute to the mark with methanol, and vortex to break the emulsion. Centrifuge the solution at 10000 r / min for 10 min, pour off the supernatant, accurately measure 5 mL, place it in an evaporating dish, evaporate to dryness in a 40℃ water bath, accurately add 2 mL of diluent to the residue, vortex to dissolve, centrifuge the solution at 12000 r / min for 10 min, and take the supernatant.

[0078] (5) Palonosetron Impurity A Reference Solution: Take an appropriate amount of palonosetron impurity A reference standard, dissolve it in methanol and dilute quantitatively to prepare a solution containing 5 μg per mL, which is used as the palonosetron impurity A reference standard stock solution. Accurately measure 1 mL of the stock solution, place it in a 20 mL volumetric flask, dilute to the mark with methanol, and shake well to obtain the solution (concentration of 0.25 μg / mL).

[0079] (6) Spike A solution: Accurately measure 1 mL of palonosetron impurity A reference standard stock solution, place it in a 20 mL volumetric flask, dilute with methanol to the mark, and shake well to obtain the solution.

[0080] (7) Impurity Recovery Solution: Measure 3 mL of rorapitan-palonosetron fat emulsion into a 10 mL volumetric flask, add 350 μL of 1 mol / L sodium dihydrogen phosphate buffer solution, and then add a certain amount of spiking impurity A solution (0.1 mL - 10% (LOQ), 1 mL - 100%, 1.5 mL - 150%), shake well, dilute to the mark with methanol, and vortex to break the emulsion. Centrifuge the solution at 10000 r / min for 10 min, decant the supernatant, accurately measure 5 mL, place it in an evaporating dish, evaporate to dryness in a 40℃ water bath, accurately add 2 mL of diluent to the residue, vortex to dissolve, centrifuge the solution at 12000 r / min for 10 min, and take the supernatant. Prepare 3 parallel aliquots for each concentration level.

[0081] (8) Palonosetron Limit of Quantification Solution: Take an appropriate amount of palonosetron hydrochloride reference standard, dissolve it in diluent and dilute quantitatively to prepare a solution containing 1 μg per mL, which is used as the palonosetron reference standard stock solution. Accurately measure 0.6 mL of the stock solution, place it in a 100 mL volumetric flask, dilute to the mark with diluent, and shake well to obtain the solution.

[0082] (9) Palonosetron detection limit solution: Accurately measure 5 mL of palonosetron quantitation limit solution, place it in a 10 mL volumetric flask, add diluent to dilute to the mark, and shake well to obtain the solution.

[0083] (10) Palonosetron impurity A limit of quantitation solution: Accurately measure 1 mL of palonosetron impurity A reference solution, place it in a 50 mL volumetric flask, add diluent to dilute to the mark, shake well, and the solution is ready.

[0084] (11) Palonosetron impurity A detection limit solution: Accurately measure 5 mL of palonosetron impurity A quantitation limit solution, place it in a 10 mL volumetric flask, add diluent to dilute to the mark, shake well, and it is ready.

[0085] (12) Linear solutions: Accurately measure appropriate amounts of palonosetron reference stock solution and impurity A reference stock solution, and dilute them with diluent to different concentrations, which are equivalent to 0.1% (LOQ), 0.2%, 0.5%, 1.0%, 1.5% and 2.0% of the concentration of the main component of the test solution, respectively, as linear solutions.

[0086] (13) Reproducible solution: Measure 3 mL of rorapitan-palonosetron fat emulsion into a 10 mL volumetric flask, add 350 μL of 1 mol / L sodium dihydrogen phosphate buffer solution, then add 1 mL of spiking impurity A solution, shake well, dilute to the mark with methanol, and vortex to break the emulsion. Centrifuge the solution at 10000 r / min for 10 min, decant the supernatant, accurately measure 5 mL, place it in an evaporating dish, evaporate to dryness in a 40℃ water bath, accurately add 2 mL of diluent to the residue, vortex to dissolve, centrifuge the solution at 12000 r / min for 10 min, and collect the supernatant. Prepare 6 parallel aliquots.

[0087] 4. Method Validation

[0088] 4.1 Specificity

[0089] Accurately measure 100 μL of each of the above solutions (1) to (4), inject them into the liquid chromatograph, and record the chromatograms. See the chromatograms below. Figures 1-4 The results showed that neither the blank solution nor the single-component blank fat emulsion solution interfered with palonosetron at the retention time of impurity A; in the system suitability solution chromatogram, the resolution between palonosetron and impurity A was 8.6, which was good, indicating that the method has good specificity.

[0090] 4.2 Limit of Detection and Limit of Quantification

[0091] Accurately measure 100 μL of each of the above solutions (8) to (11), inject them into the liquid chromatograph, and record the chromatograms. The results of the limits of detection and quantitation are shown in Table 1, and the chromatograms are shown in [Table 1]. Figures 5-8 .

[0092] Table 1 Results of Limit of Detection and Limit of Quantitation Tests

[0093] 4.3 Linear

[0094] Accurately measure 100 μL of the above solution (12) and inject it into the liquid chromatograph, and record the chromatogram. Linear regression was performed with the concentrations of palonosetron and palonosetron impurity A as the abscissa and the peak area as the ordinate to obtain the linear equation. The linear equation for palonosetron was y = 8.8080x + 0.0467, with a correlation coefficient of 0.9992, greater than 0.999; the linear equation for palonosetron impurity A was y = 9.1714x + 0.0045, with a correlation coefficient of 0.9999, greater than 0.999. The results showed that within the concentration range of 0.006~0.12 μg / mL, the linear relationship between palonosetron and palonosetron impurity A was good. The correction factor for palonosetron impurity A relative to palonosetron was calculated to be 0.97 based on the ratio of their linear slopes. The determination results are shown in Table 2, and the linear working curve is shown in Table 3. Figures 9-10 .

[0095] Table 2 Results of linear experiments

[0096] 4.4 Repeatability

[0097] Accurately measure 100 μL of the above solution (13) and inject it into the liquid chromatograph, and record the chromatogram. The results show that the RSD of palonosetron impurity A and total impurity content in the 6 solutions is 2.8%, and other individual impurities are not detected, indicating that the method has good repeatability. The determination results are shown in Table 3.

[0098] Table 3 Repeatability Test Results

[0099] Note: The " / " symbol is not applicable.

[0100] 4.5 accuracy

[0101] Accurately measure 100 μL of each of the above solutions (5) and (7), inject them into the liquid chromatograph, and record the chromatograms. The results show that the recovery rates of palonosetron impurity A at the three concentration levels of 10% (LOQ), 100%, and 150% are all between 95.0% and 105.0%, with an average recovery rate of 103.1% and an RSD of 1.5%, which is less than 2.0%, indicating that the method has good accuracy. The determination results are shown in Table 4.

[0102] Table 4 Accuracy Test Results

[0103] 4.6 Solution stability

[0104] Take the first portion of the above solution (13), and accurately measure 100 μL at 0 h, 5 h, 10 h, 15 h, 20 h, and 25 h respectively, inject it into the liquid chromatograph, and record the chromatogram. The results show that after 25 h at room temperature, compared with 0 h, the maximum change in the content of palonosetron impurity A and total impurities is 0.02%, both less than 0.1%, indicating that the solution has good stability after 25 h at room temperature. The determination results are shown in Table 5.

[0105] Table 5 Solution stability results

[0106] 4.7 Sample Testing

[0107] Accurately measure 100 μL of the above solution (4) and inject it into the liquid chromatograph, then record the chromatogram. See the chromatogram below. Figure 4 The results showed that the main component and impurity A were well separated from adjacent chromatographic peaks in the chromatogram of the test solution.

[0108] In summary, this method is simple to operate, accurate, and highly specific, and is suitable for the determination of related substances of palonosetron in rorapitane-palonosetron fat emulsion.

[0109] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for detecting related substances in a fat emulsion injection containing palonosetron, characterized in that, Includes the following steps: (1) Mix the fat emulsion injection containing palonosetron with sodium dihydrogen phosphate buffer solution, and make up to volume with methanol as demulsifier. Vortex the resulting mixture to obtain a demulsified solution. Centrifuge the demulsified solution for the first time, and take the supernatant after centrifugation and place it in an evaporating dish to evaporate to dryness in a water bath. Add diluent to the residue after evaporation in the water bath and vortex to dissolve it. Then centrifuge for the second time and take the supernatant after centrifugation as the test solution. (2) The test solution was examined by high performance liquid chromatography to confirm the content of palonosetron impurities in the fat emulsion injection containing palonosetron.

2. The detection method according to claim 1, characterized in that, The volume ratio of the palonosetron-containing fat emulsion injection to the sodium dihydrogen phosphate buffer solution is 1~3:0.1~0.

5.

3. The detection method according to claim 1, characterized in that, The conditions for the first centrifugation include: a rotation speed of 8000~10000 r / min and a time of 10~20 min.

4. The detection method according to claim 1, characterized in that, The conditions for the second centrifugation include: a rotation speed of 10,000 to 12,000 r / min and a time of 10 to 20 min.

5. The detection method according to claim 1, characterized in that, The temperature for the water bath evaporation is 40~50℃.

6. The detection method according to claim 1, characterized in that, The diluent is a mixture of methanol and sodium dihydrogen phosphate solution in a volume ratio of 30:70; the concentration of the sodium dihydrogen phosphate solution is 0.01~0.05 mol / L, and the pH is 7.

7. The detection method according to claim 1, characterized in that, The chromatographic conditions for the high-performance liquid chromatography include: The chromatographic column was a Phenomenex Luna C8 column; the column temperature was 35℃; the flow rate was 1 mL / min; the detection wavelength was 210 nm; the injection volume was 100 μL; mobile phase A was 0.02 mol / L sodium dihydrogen phosphate solution at pH 7, mobile phase B was methanol, and mobile phase C was acetonitrile; the elution method was gradient elution. The gradient elution is performed according to the following table: Where % represents the volume fraction.

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