CN115808483A - Method for determining related substances of liposome injection phospholipid of irinotecan hydrochloride - Google Patents
Method for determining related substances of liposome injection phospholipid of irinotecan hydrochloride Download PDFInfo
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- CN115808483A CN115808483A CN202211705183.8A CN202211705183A CN115808483A CN 115808483 A CN115808483 A CN 115808483A CN 202211705183 A CN202211705183 A CN 202211705183A CN 115808483 A CN115808483 A CN 115808483A
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- 238000002347 injection Methods 0.000 title claims abstract description 39
- 239000007924 injection Substances 0.000 title claims abstract description 39
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- 239000000126 substance Substances 0.000 title claims abstract description 29
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- GURKHSYORGJETM-WAQYZQTGSA-N irinotecan hydrochloride (anhydrous) Chemical compound Cl.C1=C2C(CC)=C3CN(C(C4=C([C@@](C(=O)OC4)(O)CC)C=4)=O)C=4C3=NC2=CC=C1OC(=O)N(CC1)CCC1N1CCCCC1 GURKHSYORGJETM-WAQYZQTGSA-N 0.000 title description 7
- 229960000779 irinotecan hydrochloride Drugs 0.000 title description 5
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Abstract
The invention relates to a method for determining related substances of phospholipid in irinotecan hydrochloride liposome injection, which comprises the steps of separating and detecting a pharmaceutical preparation sample to be detected by a high performance liquid chromatograph combined evaporation photodetector, wherein the related substances are Lysophosphatidylcholine (LPC) and Stearic Acid (SA); the method adopts a mobile phase A: 0.13% triethylamine-0.04 v/v% formic acid buffer, phase B: methanol; analysis was performed using a gradient elution procedure. Compared with the gas chromatography in the prior art, the method adopts the conventional liquid phase condition, and greatly improves the detection efficiency of related substances. And the method is inspected from the aspects of method specificity, detection limit, quantitative limit, linearity, accuracy, repeatability, durability and the like.
Description
Technical Field
The invention belongs to the technical field of pharmaceutical preparations, and relates to a method for determining relevant substances of liposome injection phospholipid of irinotecan hydrochloride.
Background
The liposome is used as an important mode of drug delivery, and the natural advantages of the biomembrane-like structure are utilized, so that the drug is encapsulated in the liposome and specifically reaches a focus position, thereby realizing the dual effects of targeting and low toxicity and improving the treatment effect. Phospholipids are an important component of liposomes and are the basis for achieving drug encapsulation. Compared with natural lecithin, many artificially synthesized phospholipids appear in the market at present, and specific functions and purposes of the phospholipids are improved and widened by a modification mode, such as neutral phospholipids, DPPC (dipalmitoylphosphatidylcholine), DMPC (dimyristoylphosphatidylcholine) and DSPC (distearoylphosphatidylcholine), and the like.
Lysophosphatidylcholine (LPC) is a part of phospholipid and is also an inherent metabolite of phospholipid products, and a small amount of the lysophosphatidylcholine exists in cell membranes and tissues, and if the content of the lysophosphatidylcholine is too high in vivo, the lysophosphatidylcholine can cause rupture of erythrocytes and the like, and further cause hemolysis or cell necrosis. Therefore, accurate determination of LPC content is one of the key indicators for measuring the quality stability and safety of liposome.
Irinotecan hydrochloride liposome injection contains irinotecan as a main component and phospholipid in a specific proportion, and the specific proportion is related to the long-term stability of the medicine. Stearic Acid (SA), a fatty acid widely found in nature, is another metabolite of phospholipids and also plays an indicator of the stability of liposomes.
The ELSD detector, a general purpose detector, has been widely used in the field of drug detection. The use of the ELSD method for the determination of lysophosphatidylcholine has been studied. Chenhua et al (journal of drug analysis, 20)14, 34, 3 rd, 442-446) published the HPLLC-ELSD method for simultaneously determining the content of lysophosphatidylcholine and lysophosphatidylethanolamine in propofol injection, but lysophosphatidylcholine shows a bilateral peak, which is not favorable for accurate quantification. An improvement of a method for determining lysophosphatidylcholine in alprostadil injection by an HPLC-ELSD method is published by Wangquan (journal of drug analysis, 34, volume 7, 2014, 1291-1295). The method eliminates the situation that lysophosphatidylcholine presents a double shoulder peak by improving a pharmacopoeia method, but the chromatographic condition adopts a normal phase chromatographic condition, needs a special chromatographic column, a reagent and the like, and is not friendly to a laboratory with limited resources. Liu Wan Jun et al (J. Chinese pharmacy, vol. 13, no. 1 in 2015) published the HPLC-ELSD method for the content of polyene phosphatidyl choline capsules and the detection of related substances. Chenhua et al (J.Biochemical pharmaceuticals, 2016, vol.36, 10, pages 15-18) published HPLC-ELSD method for simultaneous determination of phosphatidylcholine and lysophosphatidylcholine in etomidate emulsion injection. The same gradient elution method is adopted by the two methods, the preparation of a mobile phase system is complex, n-hexane-isopropanol-glacial acetic acid-triethylamine (814: 170: 15: 0.8) is used as a mobile phase A, and isopropanol-water-glacial acetic acid-triethylamine (844: 140: 15: 0.8) is used as a mobile phase B, and the problems of long instrument balance time, increased flow rate and increased system pressure exist. Jing et al (chemical Engineer, 2 nd 2019) published HPLC-ELSD method for determining the content of lysophosphatidylcholine in ginkgolide B lipid microspheres. The mobile phase is methanol-glacial acetic acid (500: 10, triethylamine is adopted to adjust the pH value to 6.0) and is between 38.4 and 480 mu g/ml -1 The time linearity is good. Strictly speaking, pH represents the pH value of an aqueous solution, the mobile phase is an organic phase system, and the measuring method is not good enough in rigor. Butyl liyan et al (Shanghai medicine, vol. 41, 10, 2020) published a method for determining lysophosphatidylcholine and lysophosphatidylethanolamine in propofol medium/long chain fat emulsion injection. The linear verification range of the related LPC is high (20.56-411.12 mg/ml), and the method is only suitable for detecting the content of high-concentration LPC in propofol. Similarly, HPLC-ELSD method was published by using flurbiprofen axetil injection as the detection target and Cheng Rong et al (Chinese pharmacist, 23 rd vol. 1 of 2020)And simultaneously determining the content of lysophosphatidylcholine and lysophosphatidylethanolamine in the flurbiprofen axetil injection. The article basically completes each item of method verification, but the durability examination department does not mention the durability of chromatographic conditions except the parameter indexes of the ELSD detector, and the examination is not comprehensive. CN114324622A discloses an improved method for detecting lysophosphatidylcholine in pharmaceutical preparations, which uses forward chromatographic conditions to detect lysophosphatidylcholine. In both methods, two types of solvents were used in the control solutions: chloroform, which has serious potential safety hazard to human body, has been definitely forbidden by roche. CN107957467B discloses a method for separating and measuring lysophosphatidylcholine in pharmaceutical preparations. The method separates and detects a drug preparation sample to be detected by combining a high performance liquid chromatograph and an evaporation photodetector, wherein the adopted mobile phase is absolute methanol-absolute ethanol-glacial acetic acid, and the volume ratio of the absolute methanol-absolute ethanol-glacial acetic acid to the absolute methanol-absolute ethanol-glacial acetic acid is (300-800) to (15-25). However, this method requires too high a reagent grade, requires the use of absolute methanol and absolute ethanol, and even then, the service life of the column is maintained for only 6 months, which obviously increases the detection cost.
In the current Chinese pharmacopoeia, stearic acid is used as a medicinal auxiliary material, a gas phase detection method is adopted, and sample treatment and complexity are reduced. Compared with the prior art, the method adopts the conventional liquid phase condition, and greatly improves the detection efficiency of stearic acid.
Therefore, the method in the prior art cannot simultaneously detect a plurality of phospholipid-related substances in the irinotecan hydrochloride liposome injection, and therefore, a detection method which can simultaneously detect a plurality of phospholipid-related substances, has a good separation effect and realizes the effect of monitoring the quality of the irinotecan hydrochloride liposome injection is needed in the field.
Disclosure of Invention
In order to solve the technical problems, the invention provides the following technical scheme:
the invention provides a method for determining phospholipid related substances in irinotecan hydrochloride liposome injection, which is a high performance liquid chromatography;
furthermore, the related substances detected by the method are lysophosphatidylcholine and stearic acid;
further, the mobile phase adopted by the method is phase A: 0.13v/v% triethylamine-0.04 v/v% formic acid buffer, phase B: methanol;
furthermore, the pH adjusting range of the mobile phase A is 4-5; more preferably, the pH of the mobile phase A is adjusted within a range of 4.1 to 4.5, and still more preferably, the pH of the mobile phase A is 4.3;
further, the elution procedure adopted by the method is a gradient elution procedure, and the elution time is 35min; further, the gradient elution procedure is:
further, the method adopts a chromatographic column C 8 A column; furthermore, the method adopts a chromatographic column with the specification of 250 multiplied by 4.6mm and the inner diameter of 5 mu m;
further, the flow rate of the mobile phase adopted by the method is 0.5-1.5 ml/min; preferably 1.0ml/min
Further, the column temperature adopted by the method is 20-40 ℃; further preferably, the column temperature is 25 ℃; further, the temperature of a sample plate adopted by the method is 20-30 ℃; preferably 25 ℃;
furthermore, the sample injection volume adopted by the method is 10-50 mu l; preferably 20. Mu.l;
furthermore, the detector adopted by the method is an ELSD detector; further, the detector model is Waters2424 or AllChrom 6100;
further, the preparation method of the reference substance storage solution comprises the following steps: precisely weighing a lysophosphatidylcholine reference substance and a stearic acid reference substance respectively, placing the reference substances into a volumetric flask with the same proper volume, fixing the volume by adopting a diluent, and shaking up to obtain a mixed reference substance storage solution, wherein the concentration of the lysophosphatidylcholine is 0.5-2 mg/ml, and the concentration of the stearic acid is 0.25-1 mg/ml; further, the mixed control stock solution had a lysophosphatidylcholine concentration of 1mg/ml and a stearic acid concentration of 0.5mg/ml;
further, the preparation method of the test solution comprises the following steps: precisely measuring 0.5-2 ml of irinotecan hydrochloride liposome injection, placing the irinotecan hydrochloride liposome injection into a volumetric flask with proper volume, diluting the solution to a constant volume by 5-10 times by using a diluent, shaking up the solution, centrifuging the solution at 5000-15000 rpm for 3-10 min, and taking supernatant to obtain a test solution; further, the centrifugal rotating speed is 10000rpm; further, the centrifugation time is 5min;
further, the diluent is methanol;
further, the retention time t of the lysophosphatidylcholine and stearic acid R 14.7 and 18.0min, respectively.
Drawings
FIG. 1 is an HPLC chromatogram for simultaneously separating and detecting lysophosphatidylcholine and stearic acid in example 1
Advantageous effects
The HPLC-ELSD detection method is adopted, so that the simultaneous detection of lysophosphatidylcholine and stearic acid in the irinotecan hydrochloride liposome injection is realized, the method is strong in specificity, simple and convenient to operate, good in separation degree and high in sensitivity, and a reference value is provided for the detection of degradation products of other related phospholipids in the liposome injection.
Detailed Description
In one specific embodiment, the ELSD detector used is Waters2424, the operating gas pressure is 30-50 psi, the gain value is 40-60, and the drift tube temperature is 50-80 ℃;
the invention is described in detail below with reference to the figures and specific embodiments.
Example 1
A method for determining phospholipid related substances lysophosphatidylcholine and stearic acid in irinotecan hydrochloride liposome injection is high performance liquid chromatography, and comprises the following specific steps:
1.1 chromatographic conditions
(1) And (3) chromatographic column: the method adopts a chromatographic column C 8 Column (Phenomenex)
C 8 ) The specification is 250 multiplied by 4.6mm, and the inner diameter is 5 mu m; the column temperature was 25 ℃;
(2) Mobile phase: phase A: 0.13v/v% triethylamine-0.04 v/v% formic acid buffer, the mobile phase A pH is 4.3; phase B: methanol;
(3) Gradient elution procedure: the elution time is 35min; the specific gradient elution procedure was:
(4) A detector: using an ELSD detector Waters2424, gas pressure: 40psi, drift tube temperature: 65 ℃, gain value: 50 (Waters 2424);
(5) The flow rate of the mobile phase is 1.0ml/min; the injection volume is 20 mul;
1.2 preparation of the solution
(1) The method of preparing the control stock solution comprises: accurately weighing 20mg of lysophosphatidylcholine reference substance and 10mg of stearic acid reference substance respectively, simultaneously placing the reference substances in a 20mg volumetric flask, fixing the volume by adopting methanol, and shaking up to obtain a reference substance storage solution, wherein the concentration of the lysophosphatidylcholine is 1mg/ml and the concentration of the stearic acid is 0.5mg/ml;
(2) The preparation method of the test solution comprises the following steps: precisely measuring 1ml of irinotecan hydrochloride liposome injection, placing in a 10ml volumetric flask, diluting to constant volume with methanol, shaking up, centrifuging the solution at 10000rpm for 5min, and taking supernatant to obtain a test solution;
1.3 assay method
The sample solution of the reference substance and the sample solution are injected with 20 μ L of sample solution respectively, the result is shown in figure 1, the separation degree between the reference substance and the sample solution is 15.3, and the specificity of the method is strong. Retention time t of lysophosphatidylcholine and stearic acid R 14.7 and 18.0min, respectively.
(II) methodological investigation
2.1 specialization investigation:
2.1.1 chromatographic conditions are shown in Table 1 below:
TABLE 1 chromatographic conditions
2.1.2 solution preparation (the diluent is methanol):
(1) Reference stock solution: accurately weighing lysophosphatidylcholine reference substance about 20mg and stearic acid reference substance about 10mg, placing into a 20ml measuring flask, dissolving with diluent to constant volume, and shaking to obtain reference substance stock solution;
(2) Test solution: precisely measuring 1ml of irinotecan hydrochloride liposome injection, placing into a 10ml measuring flask, diluting to a constant volume by using a diluent, and shaking uniformly; centrifuging the above solution at 10000rpm for 5min, and collecting supernatant to obtain sample solution;
(3) Adding a standard test solution: precisely measuring 1ml of irinotecan hydrochloride liposome injection and 1ml of reference substance stock solution, putting into the same 10ml measuring flask, diluting to a constant volume by using a diluent, and shaking uniformly; centrifuging the solution at 10000rpm for 5min, and collecting supernatant;
(4) System applicability solution: precisely measuring 1ml of the reference substance storage solution, placing the reference substance storage solution in a 20ml measuring flask, fixing the volume by using a diluent, and shaking up;
(5) Preparing positioning components:
LPC: weighing Lysolecithin (LPC) control sample about 1mg, placing into a 20ml measuring flask, dissolving with diluent to constant volume, and shaking up.
And b, SA: weighing Stearic Acid (SA) reference substance about 1mg, placing into a 20ml measuring flask, dissolving with diluent to constant volume, and shaking up.
DSPC: weighing about 2mg of distearoyl phosphatidylcholine (DSPC), placing in a 10ml measuring flask, dissolving with diluent to constant volume, and shaking;
chol: weighing about 1mg of Chol, placing the Chol in a 20ml measuring flask, dissolving the Chol with a diluent to a constant volume, and shaking up;
e. irinotecan glucose solution: weighing irinotecan hydrochloride of about 5mg, placing the irinotecan hydrochloride into a 10ml measuring flask, dissolving the irinotecan hydrochloride with 9 percent (g/100 ml) glucose solution to constant volume, and shaking up;
f. cleaning solution: weighing about 5mg of NaCl and 4- (2-hydroxyethyl) -1-piperazine ethanesulfonic acid (HEPES) respectively, placing in a 10ml measuring flask, adjusting pH to neutrality, dissolving with pure water to constant volume, and shaking;
SOS-TEA: weighing about 5mg of SOS-TEA, putting the SOS-TEA into a 10ml measuring flask, dissolving the SOS-TEA with pure water to a constant volume, and shaking up;
MPEG2000-DSPE: about 2mg of MPEG2000-DSPE is weighed and placed in a 10ml measuring flask, dissolved by diluent to a constant volume and shaken up.
As shown in Table 2, the other components do not interfere with the detection of LPC and SA, the separation degree between LPC and SA is 15.3, and the specificity of the method is high.
TABLE 2 Special attribute results Table
2.2 Linear investigation
2.2.1 overview
The linear response of this method was examined between relative concentrations within about 25% to 200% of the LPC limit (1 mg/ml), SA limit (0.5 mg/ml), and the results showed that the log of the response (peak area) was directly proportional to the log of the analyte concentration in the sample within the given range.
The chromatographic conditions were the same as in example 1.
2.2.2 solution preparation:
preparation of a reference stock solution: accurately weighing about 20mg of Lysolecithin (LPC) reference substance and about 10mg of Stearic Acid (SA) reference substance, placing the materials into a 20ml measuring flask, dissolving the materials by using methanol to a constant volume, and shaking up.
Each linear solution was formulated as in table 3 below:
TABLE 3 Linear solution formulation
The test results are shown in table 4:
TABLE 4 Linear results Table
| Name (R) | LPC | SA |
| Linear equation of equations | Y＝1.8230X+8.4808 | Y＝1.7335X+8.4392 |
| Correlation coefficient | 0.999 | 0.998 |
The result shows that the linear relationship of LPC is good within the range of 25.6-204.9% (0.0256-0.2049 mg/ml) of limit, and the linear relationship of SA is good within the range of 25.8-206.4% (0.0129-0.1032 mg/ml) of limit, thus meeting the acceptance standard.
2.3 limit of detection (LOD) and limit of quantitation (LOQ) studies.
The chromatographic conditions were the same as in example 1.
2.3.1 preparation of solutions
Detection limiting solution: the linear series solution Ll (concentration level 25%) in example 2 was precisely measured at 5ml, placed in a 10ml measuring flask, fixed in volume with methanol, and shaken up.
Quantitative limiting solution: a linear series of solutions L1 (concentration level 25%) was taken.
The test results are shown in tables 5 and 6:
TABLE 5 Table of detection limit results
TABLE 6 quantitative limit results table
The continuous 2-pin signal-to-noise ratios of the LPC detection limit are 6 and 7 respectively; the signal-to-noise ratio for the consecutive 2 pins at the SA detection limit was 7 and 9, respectively. The detection limit meets the acceptance standard that the signal-to-noise ratio is more than or equal to 3. The continuous 6 pin snr for LPC quantitation limit is: 35 38, 40, 50, 42 and 37; the LPC quantitative limit peak area RSD is 4.3%. The signal-to-noise ratios for 6 consecutive pins of the SA quantitation limit were: 31 30, 30, 38, 29 and 25; the SA quantitative limit peak area RSD is 4.1%. The quantitative limit meets the acceptance standard that the signal-to-noise ratio is more than or equal to 10 and the peak area RSD is less than or equal to 10.0 percent.
2.4 accuracy survey
2.4.1 overview
The accuracy is to determine the difference between the measurement result of the method in the measurement of the phospholipid-related substances LPC and SA and the true value, thereby confirming whether the method can obtain an accurate test result.
The chromatographic conditions were the same as in example 1.
2.4.2 solution preparation:
test solution: precisely measuring 1.0ml of a test sample, putting the test sample into a 10ml measuring flask, metering volume by using methanol, and shaking up; centrifuging the above solution at 10000rpm for 5min, and collecting supernatant.
Accuracy solution 1 (25%, relative to L3): precisely measuring 1.0ml of a test sample and 5.0ml of L2 linear solution, placing the test sample and the L2 linear solution in the same 10ml measuring flask, metering volume by using methanol, and shaking up. 3 parts are prepared in parallel.
Accuracy solution 2 (50%, relative to L3): precisely measuring 1.0ml of the sample and 0.5ml of the reference stock solution, placing in the same 10ml measuring flask, adding methanol to desired volume, and shaking. 3 parts are prepared in parallel.
Accuracy solution 3 (100%, relative to L3): precisely measuring 1.0ml of the sample and 1.0ml of the reference stock solution, placing in the same 10ml measuring flask, adding methanol to desired volume, and shaking. 3 parts are prepared in parallel.
Accuracy solution 4 (200%, relative to L3): precisely measuring 1.0ml of the sample and 2.0ml of the reference stock solution, placing in a 10ml measuring flask, adding methanol to desired volume, and shaking. 3 parts are prepared in parallel.
The test results are shown in tables 7 and 8
TABLE 7LPC accuracy results Table
TABLE 8SA accuracy results table
LPC: the 25% level recovery was: 101.1 percent, 95.7 percent and 101.5 percent, and the average recovery rate is 99.4 percent; the recovery at the 50% level was: 101.7%,103.5%,104.4%, average recovery rate of 103.2%; the recovery at 100% level was: 101.4%,103.6%,100.4%, average recovery rate of 101.8%; the recovery at the 200% level was: 99.1 percent, 99.0 percent and 97.2 percent, and the average recovery rate is 98.4 percent; the overall average recovery of the 12 samples was 100.7% with an RSD of 2.6%. The accuracy meets the acceptance standard that the recovery rate is between 80.0 and 120.0 percent and the RSD is less than or equal to 10.0 percent.
And SA: the 25% level recovery was: 88.0 percent, 84.9 percent and 90.3 percent, and the average recovery rate is 87.7 percent; the recovery at the 50% level was: 82.0 percent, 85.1 percent and 85.5 percent, and the average recovery rate is 84.2 percent; the recovery at 100% level was: 90.0 percent, 89.6 percent and 88.9 percent, and the average recovery rate is 89.5 percent; the recovery at the 200% level was: 98.2%,92.3%,98.3%, the average recovery rate is 96.3%; the overall average recovery of the 12 samples was 89.4% with an RSD of 5.6%. The accuracy meets the acceptance standard that the recovery rate is between 80.0 and 120.0 percent and the RSD is less than or equal to 10.0 percent.
2.5 precision investigation
2.5.1 overview: precision refers to the proximity between the results obtained from multiple measurements of the same homogeneous test sample under specified test conditions. The closeness between the results measured by the same analyst under the same conditions is called the reproducibility, and 6 test sample solutions of the same concentration are measured in parallel, and the RSD value of the content is calculated to obtain the reproducibility.
The chromatographic conditions were the same as in example 1.
2.5.2 solution preparation
Test solution: precisely measuring 1.0ml of the sample and 1.0ml of the reference stock solution, placing in the same 10ml measuring flask, adding methanol to desired volume, and shaking. 6 parts are prepared in parallel.
The test results are shown in table 9:
TABLE 9 repeatability results table
| Name(s) | LPC content (mg/ml) | SA content (mg/ml) |
| 1 | 0.1112 | 0.0560 |
| 2 | 0.1105 | 0.0566 |
| 3 | 0.1132 | 0.0555 |
| 4 | 0.1087 | 0.0569 |
| 5 | 0.1085 | 0.0545 |
| 6 | 0.1096 | 0.0566 |
| Mean value of | 0.1103 | 0.0560 |
| RSD％ | 1.6％ | 1.6％ |
As a result: in 6 test sample solutions, the RSD of LPC content is 1.6%, the RSD of SA content is 1.6%, and the repeatability meets the acceptance standard that the RSD is less than or equal to 5.0%.
2.6 durability examination
2.6.1 overview: durability is the ability to test small changes in process parameters (such as sample residence time, flow rate, column temperature, changes in carrier gas pressure, etc.) while remaining unaffected, and studies were conducted to demonstrate process reliability.
2.6.2 chromatographic conditions are shown in Table 10 below:
TABLE 10 chromatographic conditions
2.6.3 solution preparation:
test solution: precisely measuring 2.0ml of the sample and 2.0ml of the reference stock solution, placing in a 20ml measuring flask, adding methanol to desired volume, and shaking.
2.6.4 test results: as shown in tables 11-14 below.
Table 11 flow rate results table
Table 12 results table for varying column temperatures
Table 13 results table for carrier gas pressure change
TABLE 14 solution stability results Table
And (4) testing results:
under the condition of room temperature, the RSD of the LPC content of the test solution is 1.0 percent, the RSD of the SA content of the test solution is 1.4 percent, and the test solution is proved to be stable within 24 hours. The flow rate was varied: RSD of LPC content is 3.0%, RSD of SA content is 0.9%; changing the column temperature: RSD of LPC content is 1.8%, RSD of SA content is 0.9%; changing the carrier gas pressure: RSD for LPC content was 4.8% and RSD for SA content was 4.5%. In the above experiments, the RSD meets the acceptance standard of less than or equal to 5.0%, which indicates that the small changes of the flow rate, the column temperature and the carrier gas pressure have no influence on the content detection of LPC and SA. LPC and SA are adjacent peaks, the separation degree between LPC and SA is not less than 13, and the result meets the acceptance standard that the separation degree of different components is not less than 1.5.
And (4) conclusion:
(1) The blank solution and other component substances in the product do not interfere the detection of LPC and SA, the separation degree between the LPC and SA is as high as 15, and strong specificity is shown;
(2) The limit concentration of detection of LPC and SA can be as low as about 13ug/ml and 7ug/ml, and the limit concentration of quantification can reach about 26 mug/ml and 13 mug/ml respectively;
(3) LPC linear equation: y =1.8230X +8.4808, and the correlation coefficient r is 0.999; SA Linear equation: y =1.7335X +8.4392, and the correlation coefficient r is 0.998;
(4) The recovery rate of LPC and SA accuracy is between 80.0 and 120.0 percent;
(5) The repeatability of the LPC and SA is good, and the RSD of the LPC and SA content is 1.6%;
(6) Through slight changes of parameters such as flow velocity, column temperature, carrier gas pressure and the like, the durability is investigated, and the result shows that the contents RSD of LPC and SA are both less than 5.0 percent, and the separation degrees of the LPC and SA are both good and are both more than 15; under the condition of room temperature, the RSD of the LPC content of the test solution is 1.0 percent, the RSD of the SA content of the test solution is 1.4 percent, and the test solution is proved to be stable within 24 hours.
The method provides a good reference value for detection of degradation products of the phospholipid, and realizes the quality monitoring effect on the irinotecan hydrochloride liposome injection.
It will be readily apparent to those skilled in the art that various modifications to these embodiments may be made, and the generic principles described herein may be applied to other embodiments without the use of the inventive faculty. Therefore, the present invention is not limited to the above embodiments, and those skilled in the art should make improvements and modifications within the scope of the present invention based on the disclosure of the present invention.
Claims (13)
1. A method for determining phospholipid related substances in irinotecan hydrochloride liposome injection is characterized in that the method is high performance liquid chromatography; furthermore, the related substances detected by the method are lysophosphatidylcholine and stearic acid.
2. The method for determining substances related to phospholipids in irinotecan hydrochloride liposome injection according to claim 1, characterized in that the method adopts a mobile phase of phase A: 0.13v/v% triethylamine-0.04 v/v% formic acid buffer, phase B: methanol.
3. The method for measuring substances related to phospholipids in irinotecan hydrochloride liposome injection according to claim 1, characterized in that the mobile phase ApH regulation range is 4 to 5; more preferably, the pH of the mobile phase a is adjusted in a range of 4.1 to 4.5, and still more preferably, the pH of the mobile phase a is 4.3.
4. The method for determining substances related to phospholipids in irinotecan hydrochloride liposome injection according to claim 1, characterized in that the method adopts a gradient elution procedure with an elution time of 35min; further, the gradient elution procedure is:
5. the method for determining phospholipid-related substances in irinotecan hydrochloride liposome injection according to claim 1, wherein the chromatographic column C is adopted 8 A column; furthermore, the method adopts a chromatographic column with the specification of 250 multiplied by 4.6mm and the inner diameter of 5 μm.
6. The method for determining phospholipid-related substances in irinotecan hydrochloride liposome injection according to claim 1, wherein the flow rate of the mobile phase used in the method is 0.5 to 1.5ml/min; preferably 1.0ml/min.
7. The method for determining phospholipid-related substances in irinotecan hydrochloride liposome injection according to claim 1, wherein the method is carried out at a column temperature of 20-40 ℃; further preferably, the column temperature is 25 ℃; further, the temperature of a sample plate adopted by the method is 20-30 ℃; preferably 25 deg.c.
8. The method for determining substances related to phospholipids in irinotecan hydrochloride liposome injection according to claim 1, characterized in that the method adopts a sample injection volume of 10 to 50 μ l; preferably 20. Mu.l.
9. The method for determining phospholipid-related substances in irinotecan hydrochloride liposome injection according to claim 1, characterized in that the detector adopted in the method is an ELSD detector; further, the detector is model number Waters2424 or AllChrom 6100.
10. The method for measuring phospholipid-related substances in irinotecan hydrochloride liposome injection according to claim 1, characterized in that the preparation method of the control stock solution comprises: precisely weighing a lysophosphatidylcholine reference substance and a stearic acid reference substance respectively, placing the reference substances in a volumetric flask with the same proper volume, fixing the volume by adopting a diluent, and shaking up to obtain a reference substance storage solution, wherein the concentration of the lysophosphatidylcholine is 0.5-2 mg/ml, and the concentration of the stearic acid is 0.25-1 mg/ml; further, the control stock solution had a lysophosphatidylcholine concentration of 1mg/ml and a stearic acid concentration of 0.5mg/ml.
11. The method for determining substances related to phospholipids in irinotecan hydrochloride liposome injection according to claim 1, characterized in that the preparation method of the test solution comprises: precisely measuring 0.5-2 ml of irinotecan hydrochloride liposome injection, placing in a volumetric flask with proper volume, diluting with a diluent to a constant volume of 5-10 times, shaking up, centrifuging the solution at 5000-15000 rpm for 3-10 min, and taking supernatant to obtain a test solution; further, the centrifugal rotating speed is 10000rpm; further, the centrifugation time is 5min.
12. The method for measuring phospholipid-related substances in irinotecan hydrochloride liposome injection according to claim 10 or 11, wherein the diluent is methanol.
13. The method for measuring phospholipid-related substances in irinotecan hydrochloride liposome injection according to claim 1, wherein the retention time t of the lysophosphatidylcholine and the stearic acid is R 14.7 and 18.0min, respectively.
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