Detection method of L-alanine isopropyl ester in emtricitabine, propiophenol and tenofovir tablet

Abstract

The invention relates to the technical field of pharmaceutical analysis, and particularly discloses a high performance liquid chromatography-mass spectrometry tandem method (HPLC-MS / MS) for detecting a genotoxic impurity L-alanine isopropyl ester in an emtricitabine-propofol tenofovir tablet. By optimizing chromatographic column selection, a mobile phase gradient elution procedure and mass spectrometric detection parameters, the method realizes exclusive, sensitive and accurate detection of the L-isopropyl alanine. The methodological verification result shows that the detection limit is 1.00 ng / mL, the quantification limit is 2.00 ng / mL, the linear range is 2.00-100.00 ng / mL (the correlation coefficient r is equal to 0.9994), the recovery rate is stabilized between 92% and 98%, and the precision and repeatability are good. The detection method has the advantages of high sensitivity and strong selectivity, is suitable for quality control of emtricitabine propofol tenofovir tablets, meets the control requirements of ICH M7 guide on genotoxic impurities, and has good practicability and popularization value.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of pharmaceutical analysis, in particular to a high performance liquid chromatography-mass spectrometry (HPLC-MS / MS) detection method for detecting L-alanine isopropyl ester impurity in emtricitabine tenofovir alafenamide tablets. BACKGROUND

[0002] Emtricitabine tenofovir alafenamide is an important antiviral drug, which is suitable for use in combination with other antiretroviral drugs for the treatment of human immunodeficiency virus type I (HIV-I) infection in adults and adolescents (aged 12 years and above and weighing at least 35 kg).

[0003] Emtricitabine tenofovir alafenamide (trade name: Descovy) is developed by Gilead, which was approved by FDA in April 2016 for the treatment of HIV infection in combination with other antiretroviral drugs for patients weighing ≥35 kg. In December 2018, it was approved by NMPA for the treatment of HIV-I infection in adults and adolescents (aged 12 years and above and weighing at least 35 kg) in combination with other antiretroviral drugs, and the trade name is Descovy. In October 2019, the drug was also approved by FDA for HIV pre-exposure prophylaxis (PrEP).

[0004] Emtricitabine tenofovir alafenamide is a compound preparation, and the current 2025 edition of Chinese Pharmacopoeia only includes the related substance detection method of emtricitabine raw material and single preparation. Tenofovir alafenamide raw material and single preparation are not included in the Pharmacopoeia. Patent publication numbers CN115524408A and CN120233008A have both published the related substance detection method of emtricitabine tenofovir alafenamide compound preparation, but neither of them mentions the possible L-alanine isopropyl ester impurity. As a potential impurity, L-alanine isopropyl ester needs to be strictly controlled, and its accurate detection is of great significance to ensure the quality and safety of the drug.

[0005] The current detection methods for other impurities all use high performance liquid chromatography technology, and the conventional detection methods have the following technical problems:

[0006] 1. Insufficient detection sensitivity, difficult to detect low concentration of L-alanine isopropyl ester;

[0007] 2. Poor method specificity, easily interfered by other components;

[0008] 3. Narrow linear range, limited quantitative accuracy;

[0009] 4. Method stability and durability need to be improved.

[0010] Therefore, it is urgent to develop a L-alanine isopropyl ester detection method with high sensitivity, strong specificity, accuracy and reliability. SUMMARY

[0011] The present application aims to provide a high-sensitivity, high-specificity, accurate and reliable L-alanine isopropyl ester high-performance liquid chromatography-mass spectrometry detection method to solve the problems in the prior art.

[0012] In order to achieve the above-mentioned purposes of the application, the embodiments of the present application adopt the following technical solutions:

[0013] A detection method for L-alanine isopropyl ester in emtricitabine propyl tenofovir tablets, specifically comprising the following steps:

[0014] Preparation of solutions, including test solution and control solution;

[0015] Take the fine powder of the emtricitabine propyl tenofovir tablet to be tested, dissolve it with a solvent to prepare a test solution;

[0016] Preferably, the concentration of the test solution is 5 μg / ml to 15 μg / ml.

[0017] Take the L-alanine isopropyl ester control product, dissolve it with a solvent to prepare a control solution;

[0018] Preferably, the concentration of the control solution is 40 ng / ml to 60 ng / ml.

[0019] Preferably, liquid chromatography tandem mass spectrometry is used to detect the test solution and the control solution.

[0020] Chromatographic conditions are set, including liquid chromatography and mass spectrometry conditions:

[0021] The chromatographic column is a reversed-phase column with octadecylsilane-bonded silica gel (C18) as the filler, the particle size of the filler is 1.8 μm, the length is 50 mm, and the diameter (inner diameter) is 4.6 mm;

[0022] Further, an optimization scheme for high-performance liquid chromatography-mass spectrometry tandem method (HPLC-MS / MS) for detecting genotoxic impurity L-alanine isopropyl ester in emtricitabine propyl tenofovir tablets is provided.

[0023] The method uses a gradient elution program to achieve efficient separation and sensitive detection of the target impurity by precisely controlling the composition and proportion of the mobile phase.

[0024] The mobile phase includes:

[0025] Mobile phase A: ammonium acetate buffer (take ammonium acetate 1.54 g, add formic acid 1 ml, and then dissolve and dilute with water to 1000 ml)

[0026] Mobile phase B: acetonitrile (HPLC grade)

[0027] The linear gradient elution was performed according to the following table: Time (min) Mobile phase A (%) Mobile phase B (%) 1.0 95 5 3.5 10 90 4.5 10 90 4.6 95 5 6.0 95 5

[0028] Preferably, the flow rate is 0.6 ml per minute;

[0029] Preferably, the column temperature of the chromatographic column is 35℃;

[0030] Preferably, the injector temperature is 10℃;

[0031] Preferably, the injection volume is 5 μl;

[0032] Preferably, the detection mode is MRM (multiple reaction monitoring); the scanning mode is positive ion; the ion source is ESI source, and the parent ion / daughter ion (m / z) is 132.30 / 89.9.

[0033] Compared with the prior art, the method for detecting L-alanine isopropyl ester in emtricitabine propofol tenofovir tablets provided by the application realizes qualitative and quantitative analysis of L-alanine isopropyl ester by adopting a C18 reverse-phase chromatographic column (Aglient Eclipse Plus C18 4.6×50mm 1.8μm) and optimizing mass spectrometry parameters and setting a characteristic ion pair (m / z, 132.30 / 89.9). The detection method has strong specificity, low detection limit and quantitative limit, good linear relationship, high recovery rate, good repeatability, strong stability, good durability, simple and fast operation, accurate and reliable results, and can provide effective data support for the quality of emtricitabine propofol tenofovir tablets.

[0034] Preferably, the test sample solution is prepared by dilution with ammonium acetate solution containing 0.1% formic acid.

[0035] Preferably, the control sample solution is prepared by dilution with ammonium acetate solution containing 0.1% formic acid.

[0036] Preferably, the specification of the chromatographic column is Aglient Eclipse Plus C18 4.6×50mm 1.8μm.

[0037] Preferably, the injector temperature is 5℃-15℃.

[0038] Preferably, the column oven temperature is 30℃-40℃.

[0039] Preferably, the injection volume is 5 μl-10 μl.

[0040] Preferably, the characteristic ion pair parent ion / daughter ion (m / z) is 132.30 / 89.9. BRIEF DESCRIPTION OF DRAWINGS

[0041] Figure 1 is a chromatogram of a blank solvent in an embodiment of the present application

[0042] Figure 2 is a chromatogram of a reference solution in an embodiment of the present application

[0043] Figure 3 is a chromatogram of a test solution in an embodiment of the present application

[0044] Figure 4 is a chromatogram of a spiked test solution in an embodiment of the present application DETAILED DESCRIPTION

[0045] The following detailed description is merely exemplary in nature and is not intended to limit the present application or the application and uses of the present application. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the application pertains. The terminology used herein is for describing particular embodiments only and is not intended to be limiting of the present application.

[0046] It is to be noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the present application. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the application pertains. The terminology used herein is for describing particular embodiments only and is not intended to be limiting of the present application.

[0047] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the application pertains. The terminology used herein is for describing particular embodiments only and is not intended to be limiting of the present application.

[0048] In addition, unless otherwise specified, the methods not specifically described or steps are routine methods. The reagents and materials used are commercially available.

[0049] In order to enable persons skilled in the art to more clearly understand the technical solutions of the present application, the present application will be further described in detail below with specific embodiments.

[0050] Example 1

[0051] A detection method of L-alanine isopropyl ester in an emtricitabine propyl phosphono tenofovir tablet, specifically comprising the following steps:

[0052] (1) Solution preparation

[0053] Solvent: ammonium acetate solution (take ammonium acetate 1.54 g, add formic acid 1 ml, then dissolve and dilute to 1000 ml with water, and then obtain)

[0054] Test solution: Take the emtricitabine tenofovir alafenamide tablets and grind them into fine powder, and accurately weigh 144 mg of the fine powder (about equivalent to 10 mg of tenofovir alafenamide), and then place the fine powder into a 100 ml volumetric flask. Add a suitable amount of solvent to dissolve the fine powder, and then dilute to the mark with the same solvent, and shake well. Filter the solution, and discard the initial filtrate. Collect 1 ml of the subsequent filtrate, and place the filtrate into a 10 ml volumetric flask. Dilute to the mark with the same solvent, and shake well to obtain the test solution.

[0055] Reference solution: Accurately weigh a suitable amount of L-alanine isopropyl ester reference substance, and dissolve and dilute the reference substance to obtain a solution containing about 0.25 μg per 1 ml, and then shake well to obtain the reference solution.

[0056] Reference solution: Accurately weigh 2 ml of the reference solution, and place the solution into a 10 ml volumetric flask. Dilute to the mark with the solvent, and shake well to obtain the final reference solution.

[0057] Preferably, all glassware is subjected to A-level capacity certification, and the operation environment is controlled in a clean laboratory at a temperature of 15-30 °C and a relative humidity of 20%-80%. The solution preparation process avoids direct sunlight, and the solution is prepared immediately for use or is sealed and stored at 2-8 °C for not more than 24 hours to ensure chemical stability.

[0058] Preferably, all glassware is subjected to A-level capacity certification, and the operation environment is controlled in a clean laboratory at a temperature of 15-30 °C and a relative humidity of 20%-80%. The solution preparation process avoids direct sunlight, and the solution is prepared immediately for use or is sealed and stored at 2-8 °C for not more than 24 hours to ensure chemical stability.

[0059] Test solution: Take the emtricitabine tenofovir alafenamide tablets and grind them into fine powder, and accurately weigh 144 mg of the fine powder (about equivalent to 10 mg of tenofovir alafenamide), and then place the fine powder into a 100 ml volumetric flask. Add a suitable amount of solvent to dissolve the fine powder, and then dilute to the mark with the same solvent, and shake well. Filter the solution, and discard the initial filtrate. Collect 1 ml of the subsequent filtrate, and place the filtrate into a 10 ml volumetric flask. Dilute to the mark with the same solvent, and shake well to obtain the test solution.

[0060] Accurately weigh 2 ml of the reference solution, and place the solution into a 10 ml volumetric flask. Dilute to the mark with the solvent, and shake well to obtain the final reference solution.

[0061] Further, the applicability of the reference solution in different matrices is verified by a standard addition recovery test. The results show that the reference solution meets the requirements of the ICH Q2 (R1) analytical method validation guideline in terms of linearity, precision, accuracy, and solution stability.

[0062] (2) L-alanine isopropyl ester in emtricitabine tenofovir alafenamide tablets is determined by high performance liquid chromatography (general rule 0512) and mass spectrometry (general rule 0431).

[0063] Preferably, the chromatographic column is a reversed-phase column with octadecylsilane-bonded silica (C18) as the packing material, the packing material has a particle size of 1.8 μm, a length of 50 mm, and a diameter (inner diameter) of 4.6 mm.

[0064] Preferably, the flow rate is set to 0.6 ml / min.

[0065] Preferably, the injector temperature is 10°C.

[0066] Preferably, the injection volume is 5 μl.

[0067] Preferably, the detection mode is MRM.

[0068] Preferably, the scan mode is positive ion, the ion source is ESI source, and the characteristic ion pair parent ion / daughter ion (m / z) is 132.30 / 89.9.

[0069] Example 2: Investigation of the influence of the mobile phase in the chromatographic mass spectrometry conditions described in the present application

[0070] (1) In a preferred embodiment of the present application, a mobile phase system and a gradient elution method suitable for detecting L-alanine isopropyl ester impurities by high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS / MS) are provided. The mobile phase system consists of mobile phase A and mobile phase B;

[0071] Mobile phase A: ammonium acetate buffer

[0072] The ammonium acetate buffer comprises taking ammonium acetate 1.54 g, adding 1 ml of formic acid, dissolving and diluting to 1000 ml with water, and mixing uniformly.

[0073] Mobile phase B: acetonitrile

[0074] The above mobile phase A and mobile phase B are linearly gradient eluted according to the time-proportion relationship shown in the following table:

[0075] Time (min) Mobile phase A (%) Mobile phase B (%) 1.0 95 5 3.5 10 90 4.5 10 90 4.6 95 5 6.0 95 5

[0076] Among them, the proportion of mobile phase is rapidly switched from 10% A / 90% B to 95% A / 5% B between 4.5 min and 6.6 min to complete the column rebalancing process, ensuring system stability and reproducibility of continuous injection.

[0077] Preferably, the chromatographic system is fully degassed before use, and all mobile phases are filtered through a 0.22 μm filter; the gradient elution program is accurately controlled by a binary pump, the system pressure fluctuation is less than ±5%, the retention time RSD is less than 1.0% (n=6), which meets the high-precision detection requirements.

[0078] Furthermore, the samples containing L-alanine isopropyl ester were analyzed using this gradient elution condition. The results showed that the target impurity peaks were symmetrical and well separated (separation from adjacent peaks > 1.5), and still had an excellent signal-to-noise ratio (S / N ≥ 10) at levels as low as 1.0 ng, fully meeting the requirements for trace detection of genotoxic impurities.

[0079] (2) As a comparative experiment, mobile phase A was replaced with 0.1% formic acid aqueous solution (containing 20 mmol / L ammonium acetate), while keeping other conditions consistent. Experimental results showed that the chromatogram of the test solution obtained under these conditions did not change significantly; the retention time, peak shape, and response intensity of the target peak remained stable, indicating that the detection method described in this invention has a certain tolerance to ammonium acetate concentration and good method robustness. In summary, the mobile phase system and gradient elution program provided in this embodiment can achieve efficient, sensitive, and stable detection of potential impurities in L-alanine isopropyl ester, and are suitable for trace impurity analysis in pharmaceutical quality control.

[0080] Example 3

[0081] Methodological investigation of the detection method of L-alanine isopropyl ester in emtricitabine acetaminophen tenofovir tablets in Example 1

[0082] (1) Specificity examination

[0083] To verify the good specificity of the liquid chromatography-tandem mass spectrometry (HPLC-MS / MS) method for detecting L-alanine isopropyl ester provided in this invention, and to eliminate matrix interference and diluent effects, specificity tests were conducted.

[0084] The specific steps are as follows:

[0085] The solvent, test solution, reference solution, and spiked test solution prepared in Example 1 were used for detection under the liquid chromatography-mass spectrometry conditions described in Example 2. The detection results are shown in Table 1, and the chromatograms are shown in Appendix 1. Figures 1-4 ;

[0086] Appendix Figure 1 The MRM chromatogram for blank solvent shows no significant interfering peaks;

[0087] Appendix Figure 2 The MRM chromatogram of the reference solution shows that the target component has a clear response at 3.10 min;

[0088] Appendix Figure 3 The MRM chromatogram of the test solution shows that the target component responds at 3.10 min, and the retention time is exactly the same as that of the reference standard.

[0089] Appendix Figure 4The MRM chromatogram of the spiked sample solution showed obvious spiked response peaks, and the retention time was completely consistent with that of the reference substance.

[0090] Table 1 results of specificity test Solution name Characteristic chromatographic peak of ion pair Blank diluent Not detected Reference solution Characteristic chromatographic peak of ion pair detected at 3.1 min Test solution Characteristic chromatographic peak of ion pair detected at 3.1 min Spiked test solution Characteristic chromatographic peak of ion pair detected at 3.1 min

[0091] From the above experimental results, in the presence of the test sample matrix and diluent, the target impurity L-alanine isopropyl ester can be specifically detected at 3.10 min, and there is no related signal response in the blank sample, which proves that the analysis method described in the present application can effectively distinguish the target from the background interference, and has excellent method specificity.

[0092] Further, the present application successfully establishes a specific detection method for the genotoxic impurity L-alanine isopropyl ester by designing a reasonable sample pretreatment process, optimizing the composition and gradient program of the mobile phase, and combining with high selectivity mass spectrometry detection parameters. The method is not interfered by the diluent and sample matrix, and still has high selectivity at low concentration level, meets the requirements of ICH M7 and Q2(R1) guidelines for the specificity of impurity analysis method, and is suitable for the quality control and safety evaluation of tenofovir alafenamide propyl.

[0093] (2) Detection limit and quantification limit investigation

[0094] In order to evaluate the sensitivity of the liquid chromatography-mass spectrometry method (HPLC-MS / MS) provided in the present application to the genotoxic impurity L-alanine isopropyl ester, the L-alanine isopropyl ester reference substance stock solution was prepared according to the method described in Example 1, and a series of low concentration solutions were prepared by gradually diluting the solvent, which were used to determine the detection limit (Limit of Detection, LOD) and quantification limit (Limit of Quantitation, LOQ) of the method.

[0095] The specific operation is as follows:

[0096] An appropriate amount of L-alanine isopropyl ester reference substance was accurately weighed and prepared into a reference substance stock solution containing about 10 μg per 1 ml according to the method of Example 1. Then, the same diluent was further diluted step by step to prepare low concentration test solutions close to the expected detection limit and quantification limit.

[0097] The diluted solutions were injected into the liquid chromatography-tandem mass spectrometry system, and detected under the chromatographic and mass spectrometric conditions described in Example 1, and the signal-to-noise ratio (Signal-to-Noise Ratio, S / N) of the main peak of the target compound at the retention time of about 3.1 min was recorded.

[0098] According to the internationally recognized standard:

[0099] When the signal-to-noise ratio S / N is greater than or equal to 3, the corresponding concentration is defined as the detection limit (LOD);

[0100] When the signal-to-noise ratio S / N is greater than or equal to 10, the corresponding concentration is defined as the quantification limit (LOQ).

[0101] At the same time, the quantification limit concentration solution is continuously sampled for 6 times (n = 6), the retention time (TR) is recorded, and the relative standard deviation (RSD) of the relative retention time is calculated to evaluate the repeatability of the method at a low concentration level.

[0102] The experimental test results are shown in Table 2:

[0103] Table 2 Test results of detection limit and quantification limit Parameter Concentration (ng / ml) Percentage equivalent to test solution concentration (%) Signal to noise RSD (%) (TR, n = 6) Limit of quantification 2.00 0.02 124.8、88.4、172.0、106.0、103.7、199.8 0.17 Limit of detection 1.00 0.01 107.2、77.9 /

[0104] From Table 2, it can be seen that:

[0105] The quantification limit of L-alanine isopropyl ester is 2.0 ng / ml, which is equivalent to 0.02% of the concentration of the test sample;

[0106] The detection limit is 1.0 ng / ml, which is equivalent to 0.01% of the concentration of the test sample;

[0107] At the quantification limit concentration, 6 quantification limit solutions are repeatedly measured for 6 times, and the relative retention time RSD of the target peak is 0.17%, which is much smaller than the limit of 5% specified by ICH Q2(R1), indicating that the method still has excellent retention time reproducibility and system stability at very low concentration;

[0108] Further, the detection method provided by the present application has very high sensitivity, and can accurately identify and quantify trace amounts of L-alanine isopropyl ester impurities. The low detection limit and quantification limit benefit from the optimized mobile phase system, efficient C18 chromatographic separation, and high selectivity of the multiple reaction monitoring (MRM) mode, effectively improving the ionization efficiency and background noise suppression capability. In addition, the good repeatability at the quantification limit level further verifies the reliability and practicality of the method in actual quality control.

[0109] The method fully meets the strict requirements of ICH M7 on the control of genotoxic impurities, and is particularly suitable for quantitative analysis and safety evaluation of potential nitrosamine impurities in propylthiophene tenofovir raw materials and preparations.

[0110] (3) Linear relationship investigation

[0111] In order to verify that the liquid chromatography-tandem mass spectrometry (HPLC-MS / MS) provided by the present application has good concentration-response linear relationship when determining L-alanine isopropyl ester impurities, linear experiments are specially carried out.

[0112] The specific steps are as follows:

[0113] An appropriate amount of L-alanine isopropyl ester control sample was precisely weighed and prepared into a control sample solution according to the method described in Example 1, and on this basis, six sample solutions with different concentrations were prepared by stepwise dilution, and the target compound concentrations were 2 ng / ml, 5 ng / ml, 10 ng / ml, 20 ng / ml, 50 ng / ml and 100 ng / ml, respectively;

[0114] The sample solutions with the above concentrations were injected and analyzed according to the liquid chromatography-mass spectrometry conditions described in Example 1, and the peak area of the target compound at a retention time of about 3.10 min was recorded;

[0115] Further, the concentration of L-alanine isopropyl ester in each sample solution (ng / ml) was taken as the abscissa (x), and the corresponding mass spectrometry response peak area was taken as the ordinate (y), linear regression was performed by the least square method, a standard working curve was drawn, and a linear equation was established;

[0116] The experimental results are shown in Table 3:

[0117] Table 3 Linear relationship test results of L-alanine isopropyl ester Serial number 1 2 3 4 5 6 Concentration (ng / ml) 2.00 5.00 10.00 20.00 50.00 100.00 Peak area 19600 51740 101100 194600 459700 861000

[0118] Regression equation: y = 8566.3238x + 14306.2414 correlation coefficient r = 0.9994

[0119] From the above data, it can be seen that within the concentration range of 2.0-100.0 ng / ml, the concentration of L-alanine isopropyl ester and the peak area of the instrument response show a good linear relationship, the correlation coefficient is close to 1, which meets the requirements of the ICH Q2(R1) guidance principle for linear verification of the analysis method (usually r ≥ 0.99). The data points are uniformly distributed without significant deviation trend, indicating that the method has stable response and good reproducibility in the entire determination range.

[0120] Further, the use of a mobile phase system containing ammonium acetate buffer salt effectively improves the desolvation efficiency and protonation level of the target compound in the electrospray ion source, enhances the signal intensity and stability at low concentrations, and is one of the key factors to achieve a wide linear range and high sensitivity.

[0121] In summary, the HPLC-MS / MS detection method provided by the present application has excellent quantitative ability in the linear range, and can be accurately and reliably used for the determination of trace L-alanine isopropyl ester impurities in bulk drugs and preparations, especially for the accurate quantitative control of low-level impurities, which meets the strict requirements of genotoxicity impurity detection on methodological performance.

[0122] This property ensures that the present method can be used for accurate content determination of trace genotoxic impurities in drug substances and formulations, especially for accurate quantitative control of low level impurities.

[0123] (4) Recovery rate investigation

[0124] To verify the accuracy and reliability of the liquid chromatography-tandem mass spectrometry (HPLC-MS / MS) provided in the present application in determining the genotoxic impurity L-alanine isopropyl ester in propyl tenofovir drug substance or preparation, a recovery rate test was carried out.

[0125] The specific steps are as follows:

[0126] An appropriate amount of L-alanine isopropyl ester reference substance was precisely weighed and prepared into a reference substance stock solution containing about 10 μg per 1 mL according to the method described in Example 1;

[0127] Another 144 mg of finely ground eniltegravir propyl tenofovir test substance (equivalent to 10 mg of propyl tenofovir) was placed in a 100 ml volumetric flask, an appropriate amount of solvent was added to dissolve and dilute to the mark, shaken well, filtered, and 1 ml of the filtrate was precisely measured and placed in a 10 ml volumetric flask to prepare 9 blank test solution samples in parallel.

[0128] Further, the aforementioned reference substance stock solution was precisely added to each of the above solutions to obtain three levels of L-alanine isopropyl ester, with 3 parallel samples for each concentration level. After the addition was completed, the volume was adjusted to 10 ml with diluent, shaken well, and the recovery rate test sample solution was obtained.

[0129] Preferably, the above recovery rate test sample solution was analyzed and determined according to the detection method and chromatographic conditions described in Example 1.

[0130] According to the measured peak area, the amount of L-alanine isopropyl ester in each sample was calculated by the standard curve method, and the background content (amount itself) of the sample was deducted, and the recovery rate at each concentration level was calculated according to the following formula.

[0131] Recovery rate (%) = [(measured amount - amount itself) / added amount] * 100%

[0132] The test results are shown in Table 4:

[0133] Table 4 Recovery rate test results

Claims

1. A method for detecting L-alanine isopropyl ester in emtricitabine acetonide and tenofovir disoproxil fumarate tablets, characterized in that, Specifically, the following steps are included: Take the test sample, grind it, dissolve it in a diluent, make up to volume, and filter it to prepare a test sample solution; Take L-alanine isopropyl ester reference standard, dissolve it in a diluent and dilute it quantitatively to prepare a reference standard solution; The test solution and the reference solution were detected by high performance liquid chromatography-mass spectrometry tandem method.

2. The method for detecting L-alanine isopropyl ester in emtricitabine acetonide / tenofovir disoproxil fumarate tablets as described in claim 1, characterized in that, The diluent is an ammonium acetate-formic acid aqueous solution, which is prepared by dissolving 1.54g of ammonium acetate and 1ml of formic acid in water and diluting to 1000ml.

3. The method for detecting L-alanine isopropyl ester in emtricitabine acetonide / tenofovir disoproxil fumarate tablets as described in claim 1, characterized in that, The chromatographic column is a reversed-phase column packed with octadecylsilane-bonded silica gel, with dimensions of 4.6 × 50 mm and 1.8 μm.

4. The method for detecting L-alanine isopropyl ester in emtricitabine acetonide / tenofovir disoproxil fumarate tablets as described in claim 1, characterized in that, Mobile phase A is an aqueous solution of ammonium acetate and formic acid, and mobile phase B is acetonitrile, eluted according to the following gradient: The flow rate was 0.6 ml per minute; The column temperature was 35℃. The injector temperature is 10℃; The injection volume was 5 μl.

5. The method for detecting L-alanine isopropyl ester in emtricitabine acetonide / tenofovir disoproxil fumarate tablets as described in claim 1, characterized in that: Mass spectrometry detection was performed using multiple reaction monitoring mode, with an ESI source as the ion source, a positive ion scanning mode, and the monitored ion pair from m / z 132.30 to 89.

9.

6. The method for detecting L-alanine isopropyl ester in emtricitabine acetonide / tenofovir disoproxil fumarate tablets as described in claim 1, characterized in that: The concentration of the test solution was approximately equivalent to 10 μg / ml of tenofovir alafenamide.

7. The method for detecting L-alanine isopropyl ester in emtricitabine acetonide / tenofovir disoproxil fumarate tablets as described in claim 1, characterized in that: The concentration of the reference solution was 0.05 μg / ml.

8. The method for detecting L-alanine isopropyl ester in emtricitabine acetonide / tenofovir disoproxil fumarate tablets as described in claim 1, characterized in that: The allowable column temperature range is 30℃~40℃.

9. The method for detecting L-alanine isopropyl ester in emtricitabine acetonide / tenofovir disoproxil fumarate tablets as described in claim 1, characterized in that: The allowable flow rate range is 0.55~0.65 ml / min.

10. The method for detecting L-alanine isopropyl ester in emtricitabine acetonide / tenofovir disoproxil fumarate tablets as described in claim 1, characterized in that: The chromatographic columns include the Agilent Eclipse Plus C18 or equivalent columns.

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