A method for rapidly detecting the content of index components in PLA / PLGA microsphere preparation

Abstract

This invention relates to a rapid method for detecting the content of indicative components in PLA / PLGA microsphere formulations. The method includes the following steps: (1) preparation of a reference solution; (2) preparation of a test solution; and (3) detection of the reference solution and the test solution using high-performance liquid chromatography (HPLC). The chromatographic detection results of this invention exhibit good peak shape, small deviation, and good reproducibility, and significantly shorten the detection cycle, reducing the detection time for a single injection from 20 minutes to 5 minutes.

Description

Technical Field

[0001] This invention relates to the field of detection technology, specifically to a method for rapidly detecting the content of indicative components in PLA / PLGA microsphere formulations, and more particularly to a method for rapidly detecting the content of indicative components in leuprorelin acetate microsphere formulations. Background Technology

[0002] Microspheres are tiny spherical entities formed by dispersing or adsorbing drugs within a polymer matrix, typically with a particle size between 1 and 250 μm. Due to their long-lasting or targeted effects, microsphere formulations significantly improve patient convenience and compliance, demonstrating considerable advantages in clinical practice and representing a highly promising dosage form. Furthermore, microsphere formulations offer substantial added value and a broad market prospect, making them a hot topic in drug development in recent years.

[0003] Research on microspheres as drug carriers began in the mid-1970s. With the development of new technologies, processes, and materials, long-acting biodegradable injectable microspheres have become an important research area for novel drug formulations. Especially in the last decade or so, novel biodegradable polymers have become important carriers for microsphere formulations, commonly including polylactic acid (PLA), polyglycolic acid (PGA), and lactoglycolic acid copolymer (PLGA). Microspheres prepared using these polymers, after administration, gradually degrade in the body, creating numerous micropores through which the drug dissolves and exerts its effect. PLA and PLGA, in particular, exhibit good safety, biocompatibility, and biodegradability.

[0004] The content of indicative components in pharmaceutical preparations is an important indicator for evaluating drug quality. Normally, the content is determined after the sample is prepared into a homogeneous solution. However, for microsphere samples containing poorly soluble polymeric excipients such as PLA / PLGA, the solubility of the indicative component differs significantly from that of the excipient. Using solvents such as ethanol or acetonitrile during sample preparation cannot simultaneously and adequately dissolve both the indicative component and the excipient. Therefore, only broad-spectrum solvents such as dimethyl sulfoxide (DMSO) can be used. Existing pharmaceutical standards for PLA / PLGA microsphere formulations all use DMSO as the solvent to dissolve the drug carrier PLA / PLGA and the drug components before determining the content of the indicative component.

[0005] However, using dimethyl sulfoxide (DMSO) as a solvent has significant drawbacks. Firstly, DMSO exhibits a strong absorption peak in the ultraviolet (UV) band, interfering with the detection of indicative components. To avoid this interference, the elution time of indicative components must be prolonged, leading to extended sample testing cycles and increased costs. Secondly, using DMSO as a solvent results in a significant solvation effect, causing severe peak leading of indicative components. This increases the error when calculating using peak area integration, leading to poor accuracy in content determination.

[0006] The PLA / PLGA carrier in PLA / PLGA microsphere formulations is a biodegradable material, and its degradation rate is significantly affected by pH value. The acetic acid content in the formulation significantly affects the stability during storage. PLA / PLGA degradation can accelerate drug release in vivo, increase blood drug concentration, shorten the drug release cycle, and worsen both drug safety and efficacy.

[0007] Therefore, it is of great significance to provide a rapid, accurate, and sensitive method for detecting the content of indicative components in PLA / PLGA microsphere formulations. Summary of the Invention

[0008] Through in-depth research, the inventors of this invention have successfully developed a rapid method for detecting the content of indicative components in PLA / PLGA microsphere formulations. This method includes a special sample preparation method and detection using high-performance liquid chromatography (HPLC). The method proposed in this invention solves the problems of solvent absorption peak interference and solvation effects when using dimethyl sulfoxide as a solvent. It has advantages such as simple operation, high detection efficiency, high sensitivity, good precision, high repeatability, and stability and reliability, which is beneficial for the quality control of PLA / PLGA microsphere formulations. This invention provides a rapid method for detecting the content of indicative components in PLA / PLGA microsphere formulations, comprising the following steps:

[0009] (1) Preparation of reference solution;

[0010] (2) Preparation of the test solution; and

[0011] (3) The reference solution and the test solution were detected by high performance liquid chromatography.

[0012] Preferably, in step (1), the concentration of the reference solution is 0.1 mg / mL. For example, when the PLA / PLGA microsphere formulation is leuprolide acetate microsphere formulation, the reference solution contains 0.1 mg of leuprolide acetate per 1 ml.

[0013] Preferably, in step (1), the reference solution uses acetonitrile as the solvent;

[0014] Preferably, in step (1), the preparation method of the reference solution is as follows: first, the reference standard is dissolved in acetonitrile, which accounts for 5% to 40% of the total integral, preferably 30%, and then diluted and made up to volume with a first aqueous solution;

[0015] Preferably, in step (1), the reference standard is leuprolide acetate;

[0016] Preferably, in step (2), the test solution is prepared as follows: first, the PLA / PLGA microsphere formulation is dissolved in acetonitrile, which accounts for 5% to 40% of the total integral, preferably 30%, and then diluted and made up to volume with a second aqueous solution;

[0017] Preferably, the first aqueous solution and the second aqueous solution include, but are not limited to, pure water and various buffer salt solutions, such as acetic acid solution, phosphate solution, acetate solution, trifluoroacetate solution, formate solution, hydrochloride solution, etc.

[0018] Preferably, in step (3), the detection conditions for detecting the reference solution and the test solution using high performance liquid chromatography are as follows:

[0019] The chromatographic column was packed with octadecylsilane-bonded silica gel, the flow rate was 1.0 ml / min, the detection wavelength was 220 nm, the mobile phase was a methanol-0.02 mol / L ammonium dihydrogen phosphate solution with a volume ratio of 60:40, the pH of the mobile phase was 7.0 ± 0.1, and isocratic elution was used.

[0020] Preferably, the PLA / PLGA microsphere formulation is a leuprolide acetate microsphere formulation, which uses polylactic acid (PLA) or lactoglycolic acid copolymer (PLGA) as a carrier, and the leuprolide acetate loading is 5%-15%, wherein the ratio of leuprolide acetate to PLA / PLGA is 1:19~5.7 (mass ratio).

[0021] Preferably, the formulation of the PLA / PLGA microspheres is: leuprolide acetate, PLA / PLGA, and mannitol.

[0022] Preferably, the indicative components are leuprolide acetate and acetic acid.

[0023] Preferably, the external standard method is used for detection.

[0024] In one specific implementation, a method for rapidly detecting the content of indicative components in PLA / PLGA microsphere formulations includes the following steps:

[0025] (1) Preparation of reference solution: Weigh about 20 mg of leuprolide acetate reference standard, accurately weigh it, place it in a 200 ml volumetric flask, add 5%~40% of the volumetric flask volume of acetonitrile, preferably 30% of the volumetric flask volume of acetonitrile, sonicate for 30s-60s, dilute with water and make up to volume, sonicate for 5 min, shake well to obtain the solution, and prepare two parallel solutions.

[0026] (2) Preparation of test sample solution: Accurately weigh an appropriate amount of sample (e.g., 125 mg), place it in a suitable volumetric flask (e.g., 100 ml), add 5%~40% of the volumetric flask volume of acetonitrile, preferably 30% of the volumetric flask volume of acetonitrile, sonicate for 30s-60s (if the water temperature is high, it needs to be restored to room temperature after sonication), dilute with water and make up to volume, sonicate for 5 min, shake well to obtain the solution, prepare two parallel portions, do not filter;

[0027] (3) Prepare the mobile phase: 0.02 mol / L ammonium dihydrogen phosphate buffer (weigh about 2.3 g of NH4H2PO4, add 1000 ml of water to dissolve, adjust the pH to 7.0±0.1 with ammonia water and then filter) - methanol = 40:60.

[0028] (4) High performance liquid chromatography determination: Accurately pipette 10 μl of the reference solution and the test solution into the high performance liquid chromatograph, measure and record the chromatogram, and determine the content of the index component by external standard method.

[0029] In step (4), the measurement conditions for the high-performance liquid chromatograph are as follows:

[0030] The chromatographic column was packed with octadecylsilane-bonded silica gel, the mobile phase was a methanol-0.02 mol / L ammonium dihydrogen phosphate solution with a volume ratio of 60:40, the mobile phase pH was 7.0, the flow rate was 1.0 ml / min, the detection wavelength was 220 nm, and isocratic elution was used.

[0031] The test results show that the chromatographic conditions used in this invention have good linearity, precision, repeatability, stability and recovery rate, and all comply with the requirements of the 2020 edition of the Chinese Pharmacopoeia.

[0032] Compared with the prior art, the present invention has the following beneficial effects:

[0033] This invention proposes a rapid method for detecting the content of indicative components in PLA / PLGA microsphere formulations, solving the problem of the lack of suitable solvents for preparing test solutions when determining the content of PLA / PLGA microspheres. This invention employs a non-pure organic phase, adding a certain proportion of aqueous phase to the organic phase. This satisfies the solubility requirements of both water-soluble indicative components and poorly soluble excipients in the microsphere formulations, while reducing solvent peak interference and significant solvation effects caused by the pure organic phase as a solvent. This results in chromatographic detection results with good peak shape, small deviation, and good reproducibility, and significantly shortens the detection cycle, reducing the detection time per injection from 20 minutes to 5 minutes.

[0034] For quantitative liquid chromatography (LC) methods, samples are typically prepared by directly adjusting the volume using a solvent capable of dissolving the target substance. After sample preparation, there are usually two scenarios: 1. The sample is prepared as a homogeneous solution and directly injected for detection; 2. The sample is prepared as a suspension, where some excipients remain undissolved but the target substance is completely dissolved, and then filtered before detection. The sample preparation method in this invention differs from conventional methods: If water is used directly as the solvent, the sample will be a suspension, but this method is unacceptable because the target substance is encapsulated in PLA / PLGA and difficult to extract. If acetonitrile is used as the solvent, the sample will also be a suspension, but this method is also unacceptable because the target substance is insoluble in acetonitrile. If a 5-40% acetonitrile aqueous solution is used directly for sample preparation, it is found that the PLA / PLGA microspheres cannot dissolve, still failing to meet the detection requirements. This invention utilizes a method where a small amount of acetonitrile is first used to dissolve the PLA / PLGA packaging material, followed by dilution with an aqueous solution to form an O / W microemulsion. The water-insoluble PLA / PLGA dissolves in the microemulsion droplets, while the target analyte dissolves in water, allowing for direct injection and detection. Therefore, compared to conventional sample preparation methods, this method requires first dissolving the PLA / PLGA microspheres in 5%–40% of the total volume of acetonitrile to fully expose the indicative component, then diluting with the remaining 95%–60% of the volume of an aqueous solution. At this point, the indicative component is dissolved throughout the system, while the excipient PLA / PLGA is uniformly distributed in the form of microemulsion droplets. A macroscopically homogeneous and stable solution is essential for accurate detection of the indicative component. The diluent is an aqueous solution, including but not limited to pure water and various buffer salt solutions.

[0035] The detection method of this invention has the advantages of simple operation, high sensitivity, good precision, high repeatability, stability and reliability, and high work efficiency.

[0036] The detection method of the present invention is particularly applicable to leuprolide acetate PLA / PLGA microsphere formulations, wherein leuprolide acetate: PLA / PLGA = 1:19~5.7 (mass ratio), for example, the microsphere formulation composition is: about 11.25 mg of leuprolide acetate, about 99.3 mg of PLA / PLGA, and about 29.45 mg of mannitol.

[0037] The detection method of this invention also has the following advantages: First, it can not only detect leuprolide acetate in PLA / PLGA microspheres more quickly and accurately, reducing the single-needle detection time from 20 minutes to 5 minutes; it can also detect other indicator components in leuprolide acetate microspheres, such as acetic acid. Existing methods can only detect water-soluble acetic acid, but cannot detect the acetic acid content in the microspheres. Second, the sample preparation method is also applicable to other PLA / PLGA microsphere formulations. By adjusting the liquid chromatography detection conditions, the content of different target components in PLA / PLGA microsphere formulations can be detected. Attached Figure Description

[0038] Figure 1 The graphs show the ratio of organic phase to buffer salt solution. The lower graph shows the ratio of organic phase to ammonium dihydrogen phosphate buffer at 60:40, and the upper graph shows the ratio of organic phase to ammonium dihydrogen phosphate buffer at 26:74.

[0039] Figure 2 Screening results obtained for mobile phases with different pH values;

[0040] Figure 3 Screening results of different types of diluents prepared for the test sample;

[0041] Figure 4 Optimal results for different proportions of dilution solvents prepared for the test sample;

[0042] Figure 5 The linear correlation results of the method for detecting leuprolide acetate content in PLA microspheres;

[0043] Figure 6 Linear correlation results for the determination method of leuprolide acetate content in PLGA microspheres;

[0044] Figure 7 This is a chromatogram for the detection of acetic acid;

[0045] Figure 8 The linear correlation results are for the method of detecting acetic acid content in microspheres. Detailed Implementation Plan

[0046] The present invention will be further described in detail below with reference to specific embodiments. The embodiments given are only for illustrating the present invention and are not intended to limit the scope of the present invention.

[0047] Unless otherwise specified, the experimental methods in the following examples are conventional methods, and the raw materials, reagents and other materials used in the following examples are commercially available products.

[0048] Example 1: Selection of mobile phase for the detection of leuprolide acetate content in PLA microspheres

[0049] 1. Testing conditions:

[0050] (1) Instrument: HPLC;

[0051] (2) Chromatographic column: Octadecylsilane-bonded silica gel column (RD-C18, 4.6×100mm, 4μm)

[0052] (or a chromatographic column with equivalent performance);

[0053] (3) Mobile phase: See point 3;

[0054] (4) Detection wavelength: 220nm;

[0055] (5) Flow rate: 1.0 ml / min;

[0056] (6) Column temperature: 30℃;

[0057] (7) Injection volume: 10 μl;

[0058] (8) Running time: greater than or equal to 5 minutes;

[0059] 2. Microsphere formulation:

[0060] Leuprolide acetate approximately 11.21 mg, PLA approximately 64.13 mg, and mannitol approximately 42.64 mg.

[0061] 3. Preparation methods of organic phase and buffer solution in the mobile phase

[0062]

[0063] 4. Selection scheme for organic phase and buffer solution in mobile phase

[0064] The volume ratio of the organic phase to the buffer salt solution in the mobile phase is 60:40.

[0065]

[0066] 5. Results of the selection of organic phase and buffer solution in the mobile phase

[0067]

[0068] 6. Summary

[0069] The results show that when the ratio of organic phase to buffer salt solution (buffer solution) in the mobile phase is the same, the asymmetry is closer to 1 when sodium dihydrogen phosphate and ammonium dihydrogen phosphate are used. Considering that ammonia is more convenient when pH needs to be adjusted later, ammonium dihydrogen phosphate is chosen as the buffer salt. When the ratio of organic phase to buffer salt solution and the selected buffer salt are the same, the asymmetry increases when the organic phase is changed from methanol to acetonitrile. Therefore, methanol is the better choice for the organic phase.

[0070] Example 2: Selection of mobile phase ratio for the detection of leuprolide acetate content in PLA microspheres

[0071] 1. Testing conditions:

[0072] (1) Instrument: HPLC;

[0073] (2) Chromatographic column: Octadecylsilane-bonded silica gel column (RD-C18, 4.6×100mm, 4μm)

[0074] (or a chromatographic column with equivalent performance);

[0075] (3) Mobile phase: methanol-ammonium dihydrogen phosphate buffer;

[0076] (4) Detection wavelength: 220nm;

[0077] (5) Flow rate: 1.0 ml / min;

[0078] (6) Column temperature: 30℃;

[0079] (7) Injection volume: 10 μl;

[0080] 2. Microsphere formulation

[0081] Leuprolide acetate approximately 10.23 mg, PLA approximately 112.5 mg, and mannitol approximately 15.13 mg.

[0082] 3. Comparison of the ratio of organic phase to buffer salt solution

[0083]

[0084] For a detailed comparison chart of the ratios of organic phase to buffer salt solution, please see the appendix. Figure 1 .

[0085] 4. Summary

[0086] As shown in the table, when the volume ratio of methanol to ammonium dihydrogen phosphate buffer in the mobile phase is 60:40, the retention time of the chromatographic peak is shortened by about 9 min compared with the volume ratio of 26:74, and the detection time is shortened by 15 min. Considering the time cost, a volume ratio of methanol to ammonium dihydrogen phosphate buffer of 60:40 is selected as the final ratio of the mobile phase.

[0087] Example 3: pH selection of the mobile phase for detecting leuprolide acetate content in PLA microspheres

[0088] 1. Testing conditions:

[0089] (1) Instrument: HPLC;

[0090] (2) Chromatographic column: Octadecylsilane bonded silica gel column (RD-C18, 4.6×100mm, 4μm or equivalent column).

[0091] (3) Mobile phase: The volume ratio of methanol to buffer salt solution is 60:40. The preparation method is described in point 3.

[0092] (4) Detection wavelength: 220nm;

[0093] (5) Flow rate: 1.0 ml / min;

[0094] (6) Column temperature: 30℃;

[0095] (7) Injection volume: 10 μl;

[0096] (8) Running time: greater than or equal to 5 minutes;

[0097] 2. Microsphere formulation:

[0098] Leuprolide acetate approximately 10.15 mg, PLA approximately 99.3 mg, mannitol approximately 19.45 mg

[0099] 3. Comparison of pH values ​​of different mobile phases

[0100]

[0101] High-performance liquid chromatography (HPLC) was performed using mobile phases with different pH values, and the resulting chromatograms are shown in the attached figure. Figure 2 As shown.

[0102] 4. Summary

[0103] As attached Figure 2 As shown, sodium dihydrogen phosphate was selected as the buffer salt: when the pH of the mobile phase was adjusted to 3.2, the peak shape was poor; when the pH of the mobile phase was 4.6, the preparation was simple, but the peak shape had a slight tailing; when the pH of the mobile phase was 7.0, the tailing phenomenon was significantly improved, so the pH of the mobile phase was chosen to be 7.0. Considering that using ammonia to adjust the pH of ammonium dihydrogen phosphate buffer is more convenient and safer than using sodium hydroxide for sodium dihydrogen phosphate buffer, ammonium dihydrogen phosphate was ultimately chosen as the buffer salt.

[0104] Example 4: Selection of dilution solvent for the determination of leuprolide acetate content in PLA microspheres

[0105] 1. Testing conditions:

[0106] (1) Instrument: HPLC;

[0107] (2) Chromatographic column: Octadecylsilane bonded silica gel column (RD-C18, 4.6×100mm, 4μm or equivalent column).

[0108] (3) Mobile phase: methanol-ammonium dihydrogen phosphate buffer, volume ratio 60:40, pH 7.0.

[0109] The preparation method is described in point 3 of Example 3;

[0110] (4) Detection wavelength: 220nm;

[0111] (5) Flow rate: 1.0 ml / min;

[0112] (6) Column temperature: 30℃;

[0113] (7) Injection volume: 10 μl;

[0114] (8) Running time: greater than or equal to 5 minutes;

[0115] 2. Microsphere formulation

[0116] Leuprolide acetate approximately 7.67 mg, PLA approximately 145.69 mg, and mannitol approximately 18.57 mg.

[0117] 3. Comparison of Diluent Types

[0118]

[0119] The test solution was prepared using the above-mentioned diluent, and the chromatogram results were obtained by high-performance liquid chromatography (HPLC) as shown in the attached figure. Figure 3 As shown.

[0120] 4. Summary 1

[0121] As shown in the chart, using pure solvents to prepare the test solution cannot meet the requirements of dissolving both API and PLA while maintaining good peak shape. Using pure acetonitrile also results in API insolubility, leading to the absence of characteristic API peaks in the chromatogram. However, using acetonitrile to dissolve the carrier before diluting with water not only improves API solubility but also eliminates impurities before the main peak, resulting in good peak shape and facilitating quantitative analysis.

[0122] 5. Selection of acetonitrile solvent addition method:

[0123]

[0124] 6. Summary 2

[0125] As shown in the table above, only after adding acetonitrile to dissolve PLA / PLGA, and then adding water to dissolve mannitol and...

[0126] Only leuprolide acetate can completely dissolve the sample. The final sample preparation method is to add acetonitrile first and then water.

[0127] 7. Comparison of acetonitrile solvent ratios

[0128]

[0129] The test solution was prepared using an acetonitrile aqueous solution in the above proportions as the solvent, and detected by high-performance liquid chromatography (HPLC). The chromatogram results are shown in the attached figure. Figure 4 As shown.

[0130] 8. Summary 2

[0131] As shown in the chart, dissolving PLA / PLGA microspheres in acetonitrile (preferably 30% of the total integral) and then diluting the solution with the remaining 95%–60% (preferably 70% of the total integral) of an aqueous solution to prepare the test solution before loading and detecting results in good peak shapes for the target components. This method is superior to using dimethyl sulfoxide as a solvent for sample preparation, as it eliminates solvent peak tailing interference, significantly shortening the detection time, and avoids solvation effects, resulting in better peak shapes for the analytes and improved detection accuracy and reproducibility.

[0132] Example 5: Determination of leuprolide acetate content in PLA microspheres

[0133] 1. Testing conditions:

[0134] (1) Instrument: HPLC;

[0135] (2) Chromatographic column: Octadecylsilane bonded silica gel column (RD-C18, 4.6×100mm, 4μm or equivalent column).

[0136] (3) Mobile phase: ammonium dihydrogen phosphate buffer (weigh about 2.3g of ammonium dihydrogen phosphate, add 1000ml of water to dissolve, adjust the pH to 7.0±0.1 with ammonia water and then filter) - methanol = 40:60;

[0137] (4) Detection wavelength: 220nm;

[0138] (5) Flow rate: 1.0 ml / min;

[0139] (6) Column temperature: 30℃;

[0140] (7) Injection volume: 10 μl;

[0141] (8) Running time: greater than or equal to 5 minutes;

[0142] Note: During analysis, the tailing factor (asymmetry) of the main peak should not exceed 2.0, the peak time of leuprolide acetate is about 3 min, and the linear range of the sample is 37.5 μg / ml to 112.5 μg / ml.

[0143] 2. Microsphere formulation:

[0144] Leuprolide acetate approximately 11.25 mg, PLA approximately 99.3 mg, and mannitol approximately 29.45 mg.

[0145] 3. Linearity test

[0146] 3.1 Preparation of reference solution

[0147] Accurately weigh leuprolide acetate, dilute to a final volume, and prepare a reference solution containing 0.1 mg of leuprolide acetate per ml. Dilute the reference solution to different concentrations, and accurately pipette 10 μl of each concentration for analysis. The results are shown in the table below.

[0148]

[0149] 3.2 Linear detection results are as follows Figure 5 As shown.

[0150] 3.3 Summary: The results show that under the chromatographic conditions, when the concentration of leuprolide acetate is in the range of 0.91 μg / ml-136.7 μg / ml (9.1 ng-1367 ng), the obtained concentration-peak area curve has a good linear relationship.

[0151] 4. Accuracy Test

[0152] Accurately weigh approximately 10 mg of PLA and approximately 2 mg of mannitol (9 portions in total), and place them separately into 10 mL volumetric flasks. Add 3 mL of acetonitrile to each flask and sonicate for 30 seconds. Then, transfer an appropriate amount of the reference standard stock solution to the same volumetric flask, add water to the mark, shake well, and sonicate for 5 minutes. Prepare three concentrations (high, medium, and low) based on the amount of reference standard added, and prepare three parallel aliquots for each concentration. Accurately pipette 10 μL of the filtrate and determine the content according to the assay method. Calculate the recovery rate. The results are shown in the table below:

[0153]

[0154] Example 6: Determination of Leuprolide Acetate Content in Microspheres (PLGA Microspheres)

[0155] 1. Testing conditions:

[0156] (1) Instrument: HPLC;

[0157] (2) Chromatographic column: Octadecylsilane bonded silica gel column (RD-C18, 4.6×100mm, 4μm or equivalent column).

[0158] (3) Mobile phase: ammonium dihydrogen phosphate buffer (weigh about 2.3g of ammonium dihydrogen phosphate, add 1000ml of water to dissolve, adjust the pH to 7.0±0.1 with ammonia water and then filter) - methanol = 40:60;

[0159] (4) Detection wavelength: 220nm;

[0160] (5) Flow rate: 1.0 ml / min;

[0161] (6) Column temperature: 30℃;

[0162] (7) Injection volume: 10 μl;

[0163] (8) Running time: greater than or equal to 5 minutes;

[0164] Note: During analysis, the tailing factor (asymmetry) of the main peak should not exceed 2.0, the peak time of leuprolide acetate is about 3 min, and the linear range of the sample is 37.5 μg / ml to 112.5 μg / ml.

[0165] 2. Microsphere formulation:

[0166] Leuprolide acetate approximately 3.46 mg, PLGA approximately 41.42 mg, mannitol approximately 3.88 mg

[0167] 3. Linearity Validation

[0168] 3.1 Preparation of acetic acid aqueous solution: Dissolve 0.5 ml of glacial acetic acid in 1000 ml of water to obtain the solution;

[0169] 3.2 Solvent preparation (30% acetonitrile-acetic acid aqueous solution): Acetonitrile:acetic acid aqueous solution = 30:70;

[0170] 3.3 Preparation of API stock solution: Take about 38 mg of API and place it in a 100 ml volumetric flask. Dissolve it in solvent and dilute to the mark. Shake well to prepare the reference stock solution.

[0171] 3.4 Preparation of reference standard: 100% solution of the same linearity.

[0172] 3.5 The results of the linearity study are shown in the table below. Figure 6 As shown:

[0173]

[0174] 3.6 Summary: From the table and graph, we can see that the correlation coefficient R of the regression line is... 2 The linearity of the content analysis method is 0.9999, ≥0.998; the linear range is 0.00075 mg / ml to 0.15 mg / ml.

[0175] 4. Content recovery rate verification

[0176] 4.1 The reference standard is a 100% linear solution.

[0177] 4.2 Solution preparation: Take about 9 mg of PLGA and about 3 mg of mannitol and place them in a 10 ml volumetric flask. Add 3 ml of acetonitrile to dissolve them and sonicate for 30 s. Add an appropriate amount of reference stock solution and dilute to volume with acetic acid aqueous solution. Sonicate for 5 min to obtain the solution. Prepare 50%, 100%, and 150% recovery solutions. Prepare 3 parallel aliquots for each concentration.

[0178] 4.3 The results of the content recovery rate verification are shown in the table below:

[0179]

[0180] 4.4 Summary: As shown in the table, the recovery rates were all between 95% and 102%, with an average recovery rate of 99.00%. The RSD (n=9) was 1.33% < 3%.

[0181] Example 7: Detection of Acetic Acid Content in Microspheres

[0182] Chromatogram of acetic acid detection is shown below Figure 7 As shown.

[0183] 1. Testing conditions

[0184] (1) Instrument: Waters H-plus or other equivalent equipment;

[0185] (2) Chromatographic column: Octadecylsilane bonded silica gel column (RD-C18, 4.6×100mm, 4μm or equivalent column).

[0186] (3) Mobile phase: Mobile phase A is 0.02 mol / L sodium dihydrogen phosphate buffer (weigh about 3.12 g of sodium dihydrogen phosphate, add 1000 ml of water to dissolve, adjust the pH to 2.0 with phosphoric acid and then filter); Mobile phase B is acetonitrile; elute according to the gradient in the table below.

[0187]

[0188] (4) Detection wavelength: 210nm;

[0189] (5) Flow rate: 1.0 ml / min;

[0190] (6) Column temperature: 30℃;

[0191] (7) Injection volume: 50 μl;

[0192] (8) Running time: 10 min;

[0193] Note: During the analysis, the peak time of acetic acid was approximately 1.7 min.

[0194] 3. Microsphere formulation

[0195] Leuprolide acetate approximately 11.38 mg, PLA approximately 102.37 mg, mannitol approximately 16.44 mg

[0196] 2. Linearity Validation

[0197] 2.1 Solvent: Initial mobile phase;

[0198] 2.2 Linearity Test Table:

[0199]

[0200] 2.3 Linear detection results are as follows Figure 8 As shown.

[0201] 2.4 Summary: From the table and graph, the linear regression equation of acetic acid concentration-peak area is y=361.92x+0.2708, the correlation coefficient R2 of the regression line is 0.9989, which is greater than or equal to 0.998; the Y-intercept is 0.2708. The linearity verification parameters of the content analysis method meet the requirements, and the linear range is 0.0625μg~0.5μg.

[0202] 3. Accuracy

[0203] 3.1 Solution preparation:

[0204] 3.1.1 Acetic acid stock solution: Same as 2.2 Stock Solution B;

[0205] 3.1.2 Preparation of recovery solution: Weigh 100mg PLA and 30mg mannitol (9 portions in total), accurately weigh them, and place them in a 20ml volumetric flask. First, add 1mL acetonitrile and sonicate for 30s. Then, accurately add 3mL, 2mL, and 1mL of acetic acid stock solution B (three portions of each concentration). Finally, dilute to the mark with buffer solution and shake well to obtain the test solution.

[0206] 3.2 Summary table of recovery rate results:

[0207]

[0208] 3.3 Summary: As shown in the table, the recovery rate of the 50% solution is 105%, the recovery rate of the 100% solution is 98%, and the recovery rate of the 150% solution is 97%, all of which are within the range of 92%-105% and meet the requirements.

Claims

1. A method for rapid detection of leuprolide acetate and acetic acid in polylactic acid or lactoglycolic acid copolymer microsphere formulations, the method comprising the following steps: (1) Preparation of reference solution: First, dissolve the reference standard leuprolide acetate in acetonitrile, which accounts for 5% to 40% of the total volume, and then dilute with water to make up to a concentration of 0.1 mg / mL. (2) Preparation of the test solution: First, dissolve the polylactic acid or lactic acid-glycolic acid copolymer microsphere formulation in acetonitrile, accounting for 5% to 40% of the total volume, and then dilute with water to a fixed volume; and (3) The reference solution and the test solution were detected by high performance liquid chromatography. The determination conditions for leuprolide acetate by high performance liquid chromatography were as follows: the column was packed with octadecylsilane bonded silica gel, the flow rate was 1.0 ml / min, the detection wavelength was 220 nm, the mobile phase was methanol-0.02 mol / L ammonium dihydrogen phosphate solution with a volume ratio of 60:40, the pH of the mobile phase was 7.0±0.1, and isocratic elution was used. The determination conditions for acetic acid using high performance liquid chromatography are as follows: Chromatographic column: Octadecylsilane-bonded silica gel column; Mobile phase: Mobile phase A is 0.02 mol / L sodium dihydrogen phosphate buffer; Mobile phase B is acetonitrile; Elution is performed according to the gradient in the table below: Detection wavelength: 210nm; Flow rate: 1.0 ml / min; Column temperature: 30℃; The polylactic acid or lactoglycolic acid copolymer microsphere formulation is a leuprolide acetate microsphere formulation, which uses polylactic acid or lactoglycolic acid copolymer as a carrier, with a leuprolide acetate loading of 5%-15%, wherein the mass ratio between leuprolide acetate and polylactic acid or lactoglycolic acid copolymer is 1:19~5.7, and the polylactic acid or lactoglycolic acid copolymer microsphere formulation also includes mannitol.

2. The method according to claim 1, wherein, In step (1), the reference standard leuprolide acetate is first dissolved in acetonitrile, which accounts for 30% of the total integral, and then diluted with water.

3. The method according to claim 1 or 2, wherein, In step (2), the polylactic acid or lactic acid-glycolic acid copolymer microsphere formulation is first dissolved in acetonitrile, which accounts for 30% of the total integral, and then diluted with water.

4. The method according to claim 1 or 2, wherein, The external standard method was used for testing.

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