An HPLC method for the detection of enantiomers in lemetmovir injection

Abstract

The application relates to the technical field of drug detection, in particular to an HPLC detection method for enantiomers in letimovir injection, which comprises the following steps: preparing letimovir sample solution; preparing control sample solution; and detecting by adopting a reversed-phase high performance liquid chromatography system, wherein the high performance liquid chromatography conditions are as follows: a chromatographic column filled with ovomucoid bonded silica gel; a mobile phase A: 0.01 mol / L phosphate solution with a pH value of 3.8-4.2; a mobile phase B: a mixed solution of methanol and acetonitrile, wherein the volume ratio of the methanol to the acetonitrile is 90:10; a flow rate of 0.35-0.45 ml / min; a column temperature of 32-38 DEG C; and a detection wavelength of 250-260 nm. The letimovir and isomers thereof can be effectively separated by the application, the separation capacity is high, the chromatographic medium performance is stable, and the operation process is simple.

Description

Technical Field

[0001] This invention relates to the field of drug detection technology, specifically to an HPLC method for the detection of enantiomers in lemetmovir injection. Background Technology

[0002] Letermovir is a novel anti-cytomegalovirus (CMV) drug, primarily used clinically to prevent CMV infection in hematopoietic stem cell transplant recipients. Its chemical name is (4... S )-2-[8-fluoro-2-[4-(3-methoxyphenyl)piperazin-1-yl]-3-[2-methoxy-5-(trifluoromethyl)phenyl]-3,4-dihydroquinazolin-4-yl]acetic acid, with the molecular formula C 29 H 28 F4N4O4 has a molecular weight of 572.56. Its molecular structure contains a chiral center and exhibits enantiomers (S-type and R-type). Its chemical structural formula is as follows:

[0003]

[0004] Because its enantiomers can vary significantly in terms of efficacy, toxicity, or metabolism, it is crucial to strictly control the enantiomer impurity content in lemetmovir active pharmaceutical ingredient and formulations.

[0005] Most existing methods for detecting enantiomers of letermovir in active pharmaceutical ingredients use normal-phase chromatography systems. However, in practice, normal-phase chromatography systems often require instrument replacement, which is cumbersome and the chromatographic reagents used are relatively expensive. Furthermore, normal-phase chromatographic columns cannot come into contact with water, and if used for the detection of letermovir injection, it will cause a rapid decline in column efficiency. Summary of the Invention

[0006] To address the shortcomings of existing technologies, this invention provides an HPLC method for the detection of enantiomers in lemetmovir injection. This invention effectively separates lemetmovir and its isomers, exhibiting strong separation capability, stable chromatographic media, and a simple operation process.

[0007] To achieve the above objectives, the present invention adopts the following technical solution:

[0008] This invention provides an HPLC method for the detection of enantiomers in lemetmovir injection, comprising the following steps:

[0009] Prepare the Letermovir test solution;

[0010] Prepare the reference solution;

[0011] The detection was performed using a reversed-phase high-performance liquid chromatography (RP-HPLC) system. The HPLC conditions were as follows: column: packed with ovomucoid-bonded silica gel; mobile phase: mobile phase A: 0.01 mol / L phosphate solution with a pH of 3.8–4.2, mobile phase B: a mixture of methanol and acetonitrile with a volume ratio of 90:10; flow rate: 0.35–0.45 mL / min; column temperature: 32–38 °C; detection wavelength: 250–260 nm.

[0012] Preferably, the test sample and reference solution are prepared using 50% methanol or 50% acetonitrile as solvent.

[0013] Preferably, the chromatographic column has the following specifications: inner diameter of 2.0 mm, length of 150 mm, and packing particle size of 5 μm.

[0014] Preferably, the pH adjuster for the phosphate solution is glacial acetic acid or phosphoric acid.

[0015] Preferably, the phosphate solution is selected from one or more of sodium dihydrogen phosphate solution, potassium dihydrogen phosphate solution, and ammonium dihydrogen phosphate solution.

[0016] For example, the pH value is any one of 3.8, 4.0, and 4.2, or a value between two of them. Preferably, the pH value is 4.0 to 4.2.

[0017] Preferably, the elution gradient of the mobile phase is:

[0018]

[0019] For example, the mobile phase flow rate is any one of 0.35 ml / min, 0.40 ml / min, and 0.45 ml / min, or a value between two of them. Preferably, the mobile phase flow rate is 0.40~0.45 ml / min.

[0020] For example, the column temperature is any one of 32°C, 35°C, and 38°C, or a value between two of them. Preferably, the column temperature is 35°C to 38°C.

[0021] For example, the detection wavelength is any one of 250nm, 255nm, and 260nm, or a value between two of them. Preferably, the detection wavelength is 255~260nm.

[0022] Secondly, the present invention provides an application of the HPLC detection method for enantiomers in the above-mentioned lemetmovir injection in the quality control of lemetmovir injection.

[0023] The beneficial effects of this invention are:

[0024] This invention provides a method for detecting enantiomers in lemetmovir injection based on a reversed-phase chromatography system. Compared with traditional normal-phase chromatography systems, reversed-phase chromatography offers more stable media performance, stronger separation capabilities, and a simpler operation procedure. Secondly, this method allows for direct dilution of the injection solution before injection, eliminating the need for complex sample pretreatment steps and significantly improving detection efficiency. Most importantly, this method simultaneously achieves the dual functions of determining the content of the lemetmovir active ingredient and separating and detecting its enantiomers, demonstrating outstanding practical value.

[0025] The detection range of this invention covers 0.0202 μg / ml to 0.4035 μg / ml, with a detection limit as low as 0.0101% and a quantitation limit of 0.0202%, demonstrating excellent sensitivity and accuracy, and providing reliable assurance for drug quality control. Furthermore, this method uses conventional reagents to construct a reversed-phase chromatography system, which not only avoids the use of hazardous reagents but also significantly reduces detection costs, demonstrating good economic benefits and application prospects. This method is particularly suitable for the accurate detection of R-letermovir in injectable solutions, providing a new technical means for drug quality monitoring. Attached Figure Description

[0026] Figure 1 The chromatogram of the system test solution in Example 1;

[0027] Figure 2 Here is the limit of detection (LOD) chromatogram of lemetmovir and its enantiomers in Example 1;

[0028] Figure 3 Here is the limit of quantification (LOQ) chromatogram of lemetmovir and its enantiomers from Example 1;

[0029] Figure 4 The detection chromatogram is shown at a detection wavelength of 250 nm.

[0030] Figure 5 The detection chromatogram is shown at a detection wavelength of 255 nm.

[0031] Figure 6 The detection chromatogram is shown at a detection wavelength of 260 nm.

[0032] Figure 7 The chromatogram is the detection chromatogram at a column temperature of 32℃.

[0033] Figure 8 The chromatogram for detection at a column temperature of 35℃ is shown.

[0034] Figure 9 The chromatogram for detection at a column temperature of 38℃ is shown.

[0035] Figure 10 The chromatogram is for detection at a flow rate of 0.35 ml / min;

[0036] Figure 11 The chromatogram is for detection at a flow rate of 0.40 ml / min;

[0037] Figure 12 The chromatogram is for detection at a flow rate of 0.45 ml / min;

[0038] Figure 13 The chromatogram is for detection when the pH of mobile phase A is 4.2;

[0039] Figure 14 The chromatogram is for detection when the pH of mobile phase A is 4.0;

[0040] Figure 15 The chromatogram is for detection when the pH of mobile phase A is 3.8;

[0041] Figure 16 The detection chromatogram is shown when the organic phase of mobile phase B is pure methanol.

[0042] Figure 17 The detection chromatogram is shown when the organic phase of mobile phase B is methanol-acetonitrile (volume ratio 90:10);

[0043] Figure 18 This is the specific hybrid spectrum of Example 1;

[0044] Figure 19 The chromatogram for the detection in Comparative Example 1 is shown below.

[0045] Figure 20 This is the detection chromatogram for Comparative Example 2. Detailed Implementation

[0046] To enable those skilled in the art to better understand the technical solution of the invention, the invention will be further described in detail below with reference to specific embodiments.

[0047] Main instruments: High performance liquid chromatograph (model: LC-2050C, manufacturer: Shimadzu), electronic balance (model: XPR2, manufacturer: Mettler).

[0048] Main samples and reagents: Letermovir reference standard (Guangzhou Jiatu Technology Co., Ltd.), R-Letermovir reference standard (Guangzhou Jiatu Technology Co., Ltd.), Letermovir injection (self-made), chromatographic grade methanol (Beijing Mairuida Technology Co., Ltd.), chromatographic grade acetonitrile (Beijing Mairuida Technology Co., Ltd.), analytical grade anhydrous potassium dihydrogen phosphate (Chengdu Kelong Chemical Co., Ltd.), analytical grade glacial acetic acid (Chengdu Kelong Chemical Co., Ltd.).

[0049] Example 1

[0050] This embodiment provides an HPLC method for the detection of enantiomers in lemetmovir injection, including the following steps:

[0051] 1. Prepare samples

[0052] System test solution: Take appropriate amounts of lemetmovir reference standard and R-lemetmovir reference standard, dissolve and dilute with 50% methanol to obtain system test solution of lemetmovir 0.1 mg / ml and R-lemetmovir 0.2 μg / ml;

[0053] Test solution: 0.1 mg / ml (calculated as Letermovir);

[0054] Reference solution: Accurately weigh an appropriate amount of R-letermovir reference standard, dissolve and dilute it with 50% methanol to prepare a solution containing approximately 0.2 μg per 1 ml.

[0055] 2. Detection

[0056] The detection was performed using a reversed-phase high-performance liquid chromatography system. Detection conditions:

[0057] Instrument: Shimadzu LC-2050C high performance liquid chromatograph;

[0058] Chromatographic column: Column model: Ultron ES-OVM; Column packing material: ovomucoid-bonded silica gel; Column specifications: inner diameter 2.0 mm, length 150 mm, packing particle size 5 μm;

[0059] Mobile phase: Mobile phase A: 0.01 mol / L potassium dihydrogen phosphate (adjusted to pH 4.0 with glacial acetic acid); Mobile phase B: methanol-acetonitrile (90:10);

[0060] Elution gradient:

[0061]

[0062] Detection wavelength: 255nm;

[0063] Column temperature: 35℃;

[0064] Flow rate: 0.4 ml / min;

[0065] Injection volume: 10 μl.

[0066] 3. Test Results:

[0067] Figure 1 The chromatogram of the system test solution is shown. Figure 2 Chromatograms showing the limits of detection (LOD) for lemetmovir and its enantiomers. Figure 3 This is a chromatogram showing the limit of quantitation (LOQ) of lemetmovir and its enantiomers. Figure 1-3As shown, letermovir and R-letermovir were separated at the baseline. Compared with Comparative Example 2, the main peak shape was better and the method sensitivity was better after adding an appropriate amount of acetonitrile because acetonitrile has a stronger elution ability than methanol.

[0068] 4. Investigation of chromatographic conditions

[0069] 4.1 Wavelength Selection

[0070] Keeping other chromatographic parameters constant, the detection wavelengths were set to 250 nm, 255 nm, and 260 nm, respectively, for analysis and investigation. The results are shown below. Figures 4-6 .

[0071] Depend on Figures 4-6 It can be seen that there is no significant difference in the chromatograms when the detection wavelengths are 250nm, 255nm and 260nm respectively. The detection wavelengths of 250nm to 260nm can all achieve the purpose of this invention, and the maximum absorption wavelength is preferably 255nm.

[0072] 4.2 Column Temperature Investigation

[0073] Keeping other chromatographic parameters constant, the column temperature was set to 32℃, 35℃, and 38℃ respectively for analysis and investigation. The results are shown in the figure. Figures 7-9 .

[0074] Depend on Figures 7-9 It can be seen that there are no significant differences in the chromatograms when the column temperature is 32℃, 35℃, and 38℃, and all of them can achieve the purpose of this invention.

[0075] 4.3 Flow velocity investigation

[0076] Keeping other chromatographic parameters constant, the flow rates were set to 0.35 ml / min, 0.40 ml / min, and 0.45 ml / min, respectively, for analysis and investigation. The results are shown in [Figure number missing]. Figures 10-12 .

[0077] Depend on Figures 10-12 As can be seen, there is no significant difference in the chromatograms when the flow rates are 0.35 ml / min, 0.40 ml / min, and 0.45 ml / min, and all of them can achieve the purpose of this invention.

[0078] 4.4 Investigation of the pH value of mobile phase A

[0079] Keeping other chromatographic parameters constant, the pH of mobile phase A was adjusted to 4.2, 4.0, and 3.8 with glacial acetic acid, and the results were analyzed and investigated. (See attached table). Figures 13-15 .

[0080] Depend on Figures 13-15 It can be seen that the chromatograms show no significant differences when the pH value of the dynamic phase A is 4.2, 4.0, and 3.8, and all of them can achieve the purpose of this invention.

[0081] 4.5 Investigation of the types of organic phases in mobile phase B

[0082] Keeping other chromatographic parameters constant, the type of mobile phase B was changed to pure methanol and methanol-acetonitrile (volume ratio 90:10), and the analysis was carried out. The results are shown in the figure. Figures 16-17 .

[0083] Depend on Figures 16-17 It can be seen that when the mobile phase B is pure methanol, the tailing factor is 3.208 and the theoretical plate number is 671. When the mobile phase B is methanol-acetonitrile (90:10), the tailing factor is 2.542 and the theoretical plate number is 2121. The preferred mobile phase B is methanol-acetonitrile (90:10).

[0084] 5. Methodological Examination

[0085] 5.1 Specificity and System Applicability

[0086] See specific hybrid maps Figure 18 The blank solvent did not affect the detection of isomers. The resolution between lemetmovir and R-lemetmovir was 4.934, greater than 1.5; the tailing factor of the main peak was 2.521, less than 3.0; and the theoretical plate number was 8670, greater than 3000. In the specificity mixed solution, the purity similarity of the minimum peak of the main peak was 1.000000, greater than 0.990. The method has strong specificity. In the system suitability solution, the resolution between the main peak and R-lemetmovir was 5.030, greater than 1.5; the theoretical plate number of the main peak was 9006, greater than 3000; and the tailing factor was 2.466, less than 3.0.

[0087] 5.2 Limit of Quantitation and Limit of Detection

[0088] Chromatogram (see) Figure 2 and Figure 3 The detection limit (LOD) of R-letermovir was 0.0101 μg / ml, accounting for 0.0101% of the sample detection concentration; the quantitation limit (LOQ) of R-letermovir was 0.0202 μg / ml, accounting for 0.0202% of the sample detection concentration, which is much lower than the limit concentration of R-letermovir (0.2 μg / ml) and the quantitation limit (LOQ) of letermovir was 0.0202 μg / ml, accounting for 0.0202% of the sample detection concentration. This method exhibits high detection sensitivity.

[0089] 5.3 Linearity and Range

[0090] The isomer limit for letermovir injection is no more than 0.2% for R-letermovir; therefore, the linear range for R-letermovir is LOQ ~ 0.4% (0.4 μg / ml). Linear solutions of R-letermovir with concentrations of 0.0202 μg / ml, 0.0403 μg / ml, 0.1009 μg / ml, 0.2017 μg / ml, 0.3026 μg / ml, and 0.4035 μg / ml were prepared. 10 μl of each linear solution was injected, and the chromatograms were recorded. The peak area of ​​the main peak was recorded, and a linear regression equation was performed using the peak area against each concentration. The isomer R-letmovir showed a linear equation of y=56694.3343x-328.9524 in the concentration range of 0.0202μg / ml to 0.4035μg / ml, with a linear correlation coefficient r=0.9999, which is greater than 0.999. The y-intercept deviation was 2.99%, which is less than 25%, indicating that the peak area and concentration showed a good linear relationship.

[0091] 5.4 Precision

[0092] The test solutions prepared from the same batch of samples were tested by different analysts using different instruments. The RSDs of the test results for 6 test samples were 1.6% and 1.2%, respectively; the RSD of the test results for 12 test samples was 2.1%. The results indicate that the method has good precision.

[0093] 5.5 Accuracy

[0094] Recovery was determined by spiking the test solution, with three concentration levels of 50%, 100%, and 150% selected for testing. The recoveries of R-letermovir ranged from 98.35% to 103.91%, with an average recovery of 101.3%, between 90.0% and 108.0%, and an RSD of 1.8%, less than 3.0%. This indicates that the method for detecting R-letermovir is accurate.

[0095] 5.6 Durability

[0096] Under normal test conditions, the flow rate, column temperature, wavelength, and mobile phase pH were finely adjusted to test the method's durability.

[0097] Chromatographic durability conditions

[0098]

[0099] Compared to normal conditions, this method meets the robustness verification requirements at flow rates of 0.35–0.45 ml / min, column temperatures of 32–38 °C, wavelengths of 250–260 nm, and mobile phase pH of 3.8–4.2. The minimum resolution between lemetribone and isomer impurities in the system suitability solution is 4.879, greater than 1.5. The maximum absolute deviation of R-lemetribone content compared to normal conditions is 3.7%, less than 5%. The method is robust under the above conditions.

[0100] After detailed analytical methodology validation, this method demonstrates strong specificity, high sensitivity, good linearity and precision, and high accuracy, meeting the requirements for the detection of isomers in lemetmovir injection.

[0101] 6. Summary of Methodological Investigation

[0102]

[0103] This method is highly specific, sensitive, linear, precise, and accurate, meeting the requirements for the detection of enantiomers in lemetmovir injection.

[0104] Comparative Example 1

[0105] 1. Prepare samples

[0106] System test solution: Take appropriate amounts of lemetmovir reference standard and R-lemetmovir reference standard, dissolve and dilute with 50% acetonitrile to obtain system test solutions of 0.1 mg / ml lemetmovir and 0.1 mg / ml R-lemetmovir.

[0107] 2. Detection

[0108] The detection was performed using a reversed-phase high-performance liquid chromatography system. Detection conditions:

[0109] Chromatographic column: Column model: LuxTM i-Amylose-3; Column packing material: linear starch-tris(3-chloro-5-methylphenylcarbamate) covalent bond and silica gel; Column specifications: inner diameter 4.6 mm, length 250 mm, packing particle size 5 μm;

[0110] Mobile phase: Mobile phase A: 0.01 mol / L potassium dihydrogen phosphate; Mobile phase B: acetonitrile;

[0111] Elution gradient:

[0112]

[0113] Detection wavelength: 236nm;

[0114] Column temperature: 40℃;

[0115] Flow rate: 1.0 ml / min;

[0116] Injection volume: 10 μl.

[0117] 3. Test Results:

[0118] like Figure 19 As shown, letermovir and R-letermovir completely overlap, and this method cannot effectively detect the corresponding isomer of letermovir.

[0119] Comparative Example 2

[0120] 1. Prepare samples

[0121] System test solution: Take appropriate amounts of lemetmovir reference standard and R-lemetmovir reference standard, dissolve and dilute with 50% acetonitrile to obtain a system test solution of 0.1 mg / ml lemetmovir and 2 μg / ml R-lemetmovir.

[0122] 2. Detection

[0123] The detection was performed using a reversed-phase high-performance liquid chromatography system. Detection conditions:

[0124] Chromatographic column: Column model: Ultron ES-OVM; Column packing material: ovomucoid-bonded silica gel; Column specifications: inner diameter 2.0 mm, length 150 mm, packing particle size 5 μm;

[0125] Mobile phase: Mobile phase A: 0.01 mol / L potassium dihydrogen phosphate (adjusted to pH 4.0 with glacial acetic acid); Mobile phase B: methanol;

[0126] Elution gradient:

[0127]

[0128] Detection wavelength: 236nm;

[0129] Column temperature: 40℃;

[0130] Flow rate: 0.4 ml / min;

[0131] Injection volume: 10 μl.

[0132] 3. Test Results:

[0133] like Figure 20 As shown, although the baselines of lemetmovir and R-lemetmovir are separated, the main peak has severe tailing (tailing factor 3.1) and the theoretical plate number is low (below 3000).

[0134] The above are merely preferred embodiments of the present invention. It should be noted that the above preferred embodiments should not be considered as limitations on the present invention, and the scope of protection of the present invention should be determined by the scope defined in the claims. For those skilled in the art, several improvements and modifications can be made without departing from the spirit and scope of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for detecting enantiomers in an injection solution of letimovir, characterized by, The method comprises the following steps: Preparation of the test solution of letimovir; Preparation of the control solution; The detection is performed by using a reversed-phase high performance liquid chromatography system, and the high performance liquid chromatography is performed under the following conditions: a column filled with egg ovomucoid bonded silica gel; a mobile phase A: 0.01 mol / L phosphate solution with a pH value of 3.8-4.2, a mobile phase B: a mixture of methanol and acetonitrile with a volume ratio of 90:10; a flow rate of 0.35-0.45 ml / min; a column temperature of 32-38 ℃; and a detection wavelength of 250-260 nm; The elution gradient of the mobile phase is as follows: 。 2. The detection method according to claim 1, characterized in that, The test solution and the control solution are prepared by using 50% methanol or 50% acetonitrile as a solvent.

3. The detection method according to claim 1 or 2, characterized in that, The column has an inner diameter of 2.0 mm, a length of 150 mm, and a filler particle size of 5 μm.

4. The detection method according to claim 1 or 2, characterized in that, The pH value adjusting agent of the phosphate solution is glacial acetic acid or phosphoric acid.

5. The detection method according to claim 1 or 2, characterized by, The phosphate solution is selected from one or more of sodium dihydrogen phosphate solution, potassium dihydrogen phosphate solution, and ammonium dihydrogen phosphate solution.

6. The detection method according to claim 1 or 2, characterized by, The flow rate of the mobile phase is 0.40-0.45 ml / min.

7. The detection method according to claim 1 or 2, characterized by, The column temperature is 35 ℃-38 ℃.

8. The detection method according to claim 1 or 2, characterized by, The detection wavelength is 255-260 nm.

9. Application of the HPLC detection method of the enantiomer in the letimovir injection as defined in any one of claims 1-8 in the quality control of the letimovir injection.

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