Method for detecting low-content DSPC in medicine

Abstract

The invention discloses a method for detecting low-content DSPC in a medicine, and relates to a chemical analysis technology, and the method comprises the following steps: accurately weighing a medicine sample to be detected, adding the weighed sample into methanol, and ultrasonically dissolving the sample to prepare a solution to be detected with the concentration of 0.05 mg / m < 1 >; 1 ml, 2 ml, 2.5 ml, 4 ml and 5 ml of 1 mg / mL DSPC standard substance solutions are taken respectively and put into 50 ml measuring flasks respectively, the solutions are diluted to the scale through methyl alcohol, and linear solutions are prepared; a to-be-detected solution and a linear solution are detected through high performance liquid chromatography, the linear solution is used for establishing a standard curve, and the linear correlation coefficient is not smaller than 0.99; according to the method for detecting the low-content DSPC in the medicine, the low-content DSPC can be accurately detected by using an electrospray detector CAD, and the sensitivity is remarkably improved and the reproducibility is remarkably enhanced, so that the method is very important for ensuring the safety and the effectiveness of the medicine.

Description

Technical Field

[0001] The invention relates to the technical field of chemical analysis, and in particular to a method for detecting low-content DSPC in a drug. Background Art

[0002] Distearoylphosphatidylcholine (DSPC) is a commonly used pharmaceutical excipient used in pharmaceutical preparations to improve drug stability and bioavailability. Accurate determination of DSPC content in drugs is crucial to ensure drug safety and efficacy.

[0003] At present, the detection of DSPC content in drugs mainly relies on evaporative light detector (ELSD) technology. ELSD technology detects samples by measuring the light scattering intensity of particles after sample evaporation, but it has the following limitations: the detection limit of ELSD is usually around 10ng / μl, which is not sensitive enough for the detection of low-content DSPC; the reproducibility of ELSD is poor, resulting in low consistency of detection results; ELSD is easily contaminated during use, requiring frequent maintenance and high maintenance costs; ELSD technology is not suitable for rapid detection of large-scale samples.

[0004] Due to the limitations of ELSD technology, the existing DSPC content detection method cannot meet the pharmaceutical industry's detection needs for high sensitivity and high reproducibility. Summary of the invention

[0005] The purpose of the present invention is to provide a method for detecting low-content DSPC in drugs, so as to solve the above-mentioned problem in the prior art that the pharmaceutical industry cannot meet the detection requirements for high sensitivity and high reproducibility.

[0006] In order to achieve the above object, the present invention provides the following technical solution: a method for detecting low-content DSPC in a drug, comprising the following steps:

[0007] S1. Accurately weigh the drug sample to be tested, add the weighed sample into an organic solvent, and ultrasonically dissolve the sample to prepare a test solution with a concentration of 0.05 mg / ml;

[0008] S2. Take 1, 2, 2.5, 4, and 5 ml of 1 mg / mL DSPC standard solution, respectively, and place them in 50 ml volumetric flasks, and dilute to the scale with methanol to prepare 20 μg / mL, 40 μg / mL, 50 μg / mL, 80 μg / mL, and 100 μg / mL linear solutions;

[0009] S3. Detect DSPC in the test solution and the linear solution using high performance liquid chromatography.

[0010] Furthermore, the high performance liquid chromatography method described in S3 uses a chromatographic column with octadecylsilane bonded silica gel as the stationary phase, and the detector is an electrospray ionization detector CAD;

[0011] The specific parameters of the electrospray detector are that the data acquisition frequency is 10 Hz, the filtration constant is 5.0, and the evaporation tube temperature is 60°C.

[0012] Furthermore, the mobile phase of the high performance liquid chromatography described in S3 is methanol, and the flow rate is 0.8 ml / min to 1.2 mL / min.

[0013] Furthermore, the elution procedure of the high performance liquid chromatography described in S3 is: isocratic elution.

[0014] Furthermore, the parameters of the chromatographic column with octadecylsilane bonded silica gel as the stationary phase are 4.6 mm×150 mm, 5 μm.

[0015] Furthermore, the temperature of the chromatographic column is 25°C to 55°C.

[0016] Furthermore, the injection volume of the high performance liquid chromatography is 1 μl to 100 μl.

[0017] Furthermore, in the high performance liquid chromatography method described in S3, the temperature of the evaporation tube is 35°C to 80°C.

[0018] Furthermore, the running time of the high performance liquid chromatography method described in S3 is 15 minutes.

[0019] Furthermore, the linear solution described in S2 is used to establish a standard curve, and a linear curve is drawn with the logarithm of the peak area of ​​the linear solution and the logarithm of the concentration, and the linear correlation coefficient is not less than 0.99; the organic solvent described in S1 is methanol, ethanol or isopropanol.

[0020] Compared with the prior art, the present invention provides a method for detecting low-content DSPC in drugs, which is used to detect low-content DSPC (distearoylphosphatidylcholine) in drugs. Compared with the traditional evaporative light detector technology, it has significantly improved sensitivity and enhanced reproducibility. By using an electrospray ionization detector CAD, low-content DSPC can be accurately detected, which is very important for ensuring the safety and effectiveness of drugs.

[0021] By optimizing the chromatographic conditions, interference in the detection process was reduced and the accuracy and reliability of the detection results were improved. This method has wide applicability and flexibility, enabling it to meet the detection needs of DSPC content in different drug preparations and different concentration ranges, which helps to improve the quality of corporate products, enhance market competitiveness, and accelerate the drug research and development process. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments recorded in the present invention. For ordinary technicians in this field, other drawings can also be obtained based on these drawings.

[0023] Figure 1 A schematic diagram of the overall detection method flow provided by an embodiment of the present invention;

[0024] Figure 2 A quantitative limit detection result diagram of the detection method provided in an embodiment of the present invention;

[0025] Figure 3 A graph showing the detection limit detection results of the detection method provided by an embodiment of the present invention;

[0026] Figure 4 A linear curve graph provided for an embodiment of the present invention. DETAILED DESCRIPTION

[0027] In order to enable those skilled in the art to better understand the technical solution of the present invention, the present invention will be further described in detail below with reference to the accompanying drawings.

[0028] Embodiment 1:

[0029] See also Figure 1 and Figure 4 , a method for detecting low-content DSPC in a drug, comprising the following steps:

[0030] S1. Accurately weigh the drug sample to be tested, add the weighed sample to an organic solvent, and ultrasonically dissolve the sample to prepare a test solution with a concentration of 0.05 mg / ml; the organic solvent is methanol, ethanol or isopropanol;

[0031] S2. Take 1, 2, 2.5, 4, and 5 ml of 1 mg / mL DSPC standard solution, respectively, and place them in 50 ml volumetric flasks, dilute to the scale with methanol, and prepare 20 μg / mL, 40 μg / mL, 50 μg / mL, 80 μg / mL, and 100 μg / mL linear solutions; the linear solution is used to establish a standard curve, and a linear curve is drawn with the logarithm of the peak area and concentration of the linear solution, and the linear correlation coefficient is not less than 0.99;

[0032] S3, using high performance liquid chromatography to detect DSPC in the test solution and the linear solution;

[0033] The high performance liquid chromatography method uses a chromatographic column with octadecylsilane bonded silica gel as the stationary phase, and the parameters of the chromatographic column are 4.6mm×150mm, 5μm; the chromatographic column temperature is 25℃~55℃; the detector is an electrospray detector; the injection volume is 1μl~100μl; the evaporation tube temperature is 35℃~80℃; the running time is 15 minutes; the elution procedure of the high performance liquid chromatography method is: isocratic elution;

[0034] The detector is an electrospray detector CAD;

[0035] The specific parameters of the electrospray detector are data acquisition frequency of 10 Hz, filter constant of 5.0, and evaporation tube temperature of 60 °C.

[0036] The specific implementation method is as follows: S1. Accurately weigh 12.5 mg of the drug sample to be tested, place it in a 20 ml volumetric flask, add methanol ultrasonic solution and dilute to the scale, shake well, accurately measure 2 ml, place it in a 25 ml volumetric flask, dilute it with methanol to make a 0.05 mg / ml solution, and obtain the solution to be tested;

[0037] In the sample preparation stage, accurate weighing is crucial for the repeatability and accuracy of the experiment. Choosing to weigh 0.5 mg of the drug sample to be tested can not only ensure the accuracy of the analysis, but also maximize the use of limited sample resources; methanol is used as a solvent because methanol is a polar solvent that can effectively dissolve polar lipids such as DSPC (distearoylphosphatidylcholine). Methanol is also less volatile and suitable for HPLC analysis. The solubility of the sample directly affects the peak shape and separation effect of the chromatogram. If the sample is not completely dissolved, abnormal peaks may appear in the chromatogram, affecting the accuracy of the analysis results. Therefore, ensuring that the sample is completely dissolved is a prerequisite for obtaining reliable analysis results.

[0038] Ultrasonic treatment accelerates the dissolution process of the drug sample to be tested in methanol. Ultrasonic waves can produce mechanical vibration and cavitation effects, thereby destroying aggregates in the sample, promoting the contact between sample molecules and solvent molecules, and accelerating the dissolution process. Ultrasonic treatment can not only shorten the dissolution time of the sample, but also improve the dissolution efficiency and ensure the uniformity and consistency of the sample, which is crucial for subsequent chromatographic analysis because the uniformity of the sample directly affects the peak shape and separation effect of the chromatogram.

[0039] S2. Take 1, 2, 2.5, 4, and 5 ml of 1 mg / mL DSPC standard solution, respectively, and place them in 50 ml volumetric flasks, dilute to the scale with methanol to prepare 20μg / mL, 40μg / mL, 50μg / mL, 80μg / mL, and 100μg / mL linear solutions; the linear solutions are used to establish the standard curve, and the linear curve is drawn with the logarithm of the peak area and concentration of the linear solution, and the linear correlation coefficient is not less than 0.99.

[0040] In chromatographic analysis, the preparation of standard solution is a key step in establishing a quantitative analysis method. A series of linear solutions with different concentrations were prepared by taking different volumes of DSPC standard solution.

[0041] The accurate preparation of standard solution is the basis for establishing an accurate standard curve. In chromatographic analysis, the standard curve is used to convert the response of the detector (such as peak area) to the concentration of the analyte in the sample. If the concentration of the standard solution is inaccurate, the slope and intercept of the standard curve will be inaccurate, which will affect the quantitative results of all samples. Therefore, the accurate preparation of standard solution is the key to ensure the accuracy and reliability of quantitative analysis. Linear solutions of different concentrations (20μg / mL, 40μg / mL, 50μg / mL, 80μg / mL, 100μg / mL) are prepared to ensure that the standard curve can cover the concentration range of DSPC in the expected sample. This ensures that when analyzing the sample, the chromatographic signal falls within the linear range of the standard curve, thereby ensuring the accuracy of the quantitative results. If the concentration of DSPC in the sample exceeds the range of the standard curve, accurate quantitative results will not be obtained.

[0042] The establishment of standard curve: by calculating the linear correlation coefficient (R 2 ) to verify the linearity of the standard curve, R 2 The closer the value is to 1, the better the linear relationship of the standard curve is, and the higher the accuracy of the quantitative analysis is; ensure that the standard curve covers the expected concentration range of DSPC in the sample and has a good linear relationship within this range, providing a solid foundation for subsequent sample quantitative analysis. The precise execution of these steps is crucial to obtaining accurate and reliable analysis results.

[0043] S3, using high performance liquid chromatography to detect DSPC in the test solution and the linear solution;

[0044] The high performance liquid chromatography method uses a chromatographic column with octadecylsilane bonded silica gel as the stationary phase, and the parameters of the chromatographic column are 4.6mm×150mm, 5μm; the chromatographic column temperature is 40℃; the detector is an electrospray detector; the injection volume is 50μl; the evaporation tube temperature is 60℃; the running time is 15 minutes;

[0045] In HPLC detection, the selection of chromatographic columns is crucial to the separation effect of samples. Octadecylsilane bonded silica gel chromatographic columns have good stability and high affinity for lipid substances such as DSPC; the parameters of the chromatographic column are 4.6mm×150mm, 5μm, which determine the separation efficiency and column pressure of the chromatographic column. The column temperature of 40°C is conducive to the separation and peak shape optimization of DSPC. This temperature can reduce the interaction between the analyte and the stationary phase, thereby obtaining a better separation effect. The electrospray detector (CAD) has high sensitivity and a wide range of applications. The CAD detector detects samples by measuring the charge of the sample's charged aerosol and is suitable for the detection of a variety of compounds. This detector is very sensitive to changes in sample concentration and can provide accurate quantitative analysis. The injection volume of 50 μl ensures the consistency and repeatability of the sample, which is critical for obtaining reliable and repeatable analytical results. The evaporation tube temperature has an important influence on the performance of the CAD detector. The evaporation tube temperature of 60°C contributes to the stable operation of the CAD detector and the enhancement of the signal. This temperature can ensure that the sample particles are effectively evaporated in the evaporation tube, thereby improving the detection sensitivity. The 15-minute running time is set based on the needs of chromatographic separation and the complexity of the sample. This time can ensure the complete separation and detection of DSPC while maintaining high efficiency of the analysis.

[0046] The detector is an electrospray detector CAD;

[0047] The specific parameters of the electrospray detector are data acquisition frequency of 10 Hz, filter constant of 5.0, and evaporation tube temperature of 60 °C.

[0048] Electrospray ionization detector (CAD) is used as a detector to detect low-content DSPC (distearoylphosphatidylcholine) in drugs. CAD has high sensitivity to lipid substances such as DSPC. CAD provides quality-based detection while ensuring the accuracy and reliability of the results; through the above detailed chromatographic condition settings and detector parameter optimization, the accuracy, repeatability and sensitivity of the detection method can be ensured, providing reliable data support for drug quality control.

[0049] Embodiment 2:

[0050] See also Figure 2-Figure 3 This embodiment provides a technical solution based on the first embodiment: the detection limit, sensitivity and reproducibility of the detection method are tested.

[0051] The method detection limit (MDL) refers to the lowest concentration or amount that can be detected by the method. This embodiment adopts the signal-to-noise ratio method, that is, by preparing a series of DSPC standard solutions of different concentrations, and measuring them according to the detection method of Example 1, when the signal-to-noise ratio is about 3, the concentration or the amount injected into the instrument is the lowest concentration or amount; when the signal-to-noise ratio is about 10, the concentration or the amount injected into the instrument is the quantitative limit.

[0052] Sensitivity refers to the degree of response of the method to changes in concentration, which is evaluated by the slope of the calibration curve; prepare a series of DSPC standard solutions of known concentrations and measure their response signals; draw a standard curve, that is, a graph of concentration versus response signal, and calculate the slope of the linear regression equation. The larger the slope, the higher the sensitivity.

[0053] Reproducibility refers to the consistency of the results obtained by repeating the experiment under the same conditions. Intra-day reproducibility is to measure the same concentration of DSPC test solution multiple times (at least 5 times) within one day, and calculate the relative standard deviation (RSD). The smaller the RSD, the better the reproducibility. Inter-day reproducibility is to measure the same concentration of DSPC test solution multiple times on different days, and calculate the RSD to evaluate the inter-day reproducibility.

[0054] The test solution was measured by experimenter A according to the detection method of Example 1; and the test solution was measured by experimenter B according to the detection method of Example 1. After the determination, the content of DSPC in the 6 test solutions determined by experimenter A and experimenter B was calculated, and the RSD of each and the RSD of the content of the 12 test samples of the two were calculated. The specific experimental results are as follows:

[0055]

[0056]

[0057] See also Figure 2-Figure 3 The detection limit of this method is 0.0076 ng / μl, and the quantification limit is 0.0759 ng / μl.

[0058] The above description is only by way of illustration of certain exemplary embodiments of the present invention. It is undoubted that those skilled in the art can modify the described embodiments in various ways without departing from the spirit and scope of the present invention. Therefore, the above drawings and descriptions are illustrative in nature and should not be construed as limiting the scope of protection of the claims of the present invention.

Claims

1. A method for detecting low-content DSPC in a drug, characterized in that: The following steps are involved: S1. Accurately weigh the drug sample to be tested, add the weighed sample into an organic solvent, and ultrasonically dissolve the sample to prepare a test solution with a concentration of 0.05 mg / ml; S2. Take 1, 2, 2.5, 4, and 5 ml of 1 mg / mL DSPC standard solution, respectively, place them in 50 ml volumetric flasks, and dilute to the scale with methanol to prepare 20 μg / mL, 40 μg / mL, 50 μg / mL, 80 μg / mL, and 100 μg / mL linear solutions; S3. Detect DSPC in the test solution and the linear solution using high performance liquid chromatography.

2. The method for detecting low-content DSPC in a drug according to claim 1, characterized in that: The high performance liquid chromatography method described in S3 uses a chromatographic column with octadecylsilane bonded silica gel as the stationary phase, and the detector is an electrospray ionization detector CAD; The specific parameters of the electrospray detector are that the data acquisition frequency is 10 Hz, the filter constant is 5.0, and the evaporation tube temperature is 60°C.

3. The method for detecting low-content DSPC in a drug according to claim 1, characterized in that: The mobile phase used in the high performance liquid chromatography method described in S3 is methanol, and the flow rate is 0.8 ml / min to 1.2 ml / min.

4. The method for detecting low-content DSPC in a drug according to claim 1, characterized in that: The elution procedure of the high performance liquid chromatography described in S3 is: isocratic elution.

5. The method for detecting low-content DSPC in a drug according to claim 2, characterized in that: The parameters of the chromatographic column with octadecylsilane bonded silica gel as the stationary phase are 4.6 mm×150 mm, 5 μm.

6. The method for detecting low-content DSPC in a drug according to claim 2, characterized in that: The temperature of the chromatographic column is 25°C to 55°C.

7. The method for detecting low-content DSPC in a drug according to claim 1, characterized in that: The injection volume of the high performance liquid chromatography method is 1 μl to 100 μl.

8. The method for detecting low-content DSPC in a drug according to claim 1, characterized in that: The temperature of the evaporating tube in the high performance liquid chromatography described in S3 is 35°C to 80°C.

9. The method for detecting low-content DSPC in a drug according to claim 1, characterized in that: The running time in the HPLC method described in S3 is 15 minutes.

10. The method for detecting low-content DSPC in a drug according to claim 1, characterized in that: The linear solution described in S2 is used to establish a standard curve, and a linear curve is drawn using the logarithm of the peak area and concentration of the linear solution, and the linear correlation coefficient is not less than 0.99; the organic solvent described in S1 is methanol, ethanol or isopropanol.

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