LC-HRMS (liquid chromatography-high resolution mass spectrometry)-based detection method for Maillard reaction impurities in efemitide tablets

Abstract

The invention belongs to the technical field of detection, and particularly relates to a method for detecting Maillard reaction impurities in efemitide tablets based on LC-HRMS. The method comprises steps as follows: a sample is prepared, qualitative analysis and quantitative analysis are performed under LC-HRMS conditions, and the LC-HRMS conditions include that a chromatographic column is Waters Xselect HSS T3 C18, 3.5 mu m and 4.6 * 100 mm; a mobile phase A is 10 mmol / L ammonium acetate-aqueous solution; a mobile phase B is acetonitrile; the detection wavelength is 260 nm; the ion source is ESI; the mode is positive ions. The invention provides a high-specificity and high-sensitivity analysis method which can accurately identify and quantify four specific Maillard reaction impurities in efemitib tablets.

Description

Technical Field

[0001] This invention belongs to the field of detection technology, and in particular relates to a method for detecting meladard reaction impurities in efamiti tablets based on LC-HRMS. Background Technology

[0002] Efamiti tablets (I) are a combination anti-HIV medication containing efavirenz, lamivudine, and tenofovir disoproxil fumarate. Both lamivudine and tenofovir disoproxil fumarate contain primary amine groups in their molecular structures, while lactose is commonly used as a filler in the formulation. During the manufacturing or storage of the formulation, the primary amine readily undergoes a Maillard reaction with the reducing sugar (lactose), generating adduct impurities.

[0003] The existing technology has the following main defects: The structure of Maillard reaction impurities cannot be accurately identified: Conventional HPLC-UV relies solely on retention time for qualitative analysis, which is easily affected by excipients or degradation products and cannot confirm the chemical structure of impurities.

[0004] Insufficient sensitivity: For potential genotoxic or highly active impurities that need to be controlled within 0.05%, the limits of detection (LOD) and quantitation (LOQ) of conventional methods are difficult to meet the requirements of ICH Q3A / Q3B.

[0005] False positive interference exists: Efamiti tablets often contain PEG or polysorbate excipients, which can generate m / z signals similar to the target impurities in mass spectrometry (such as a series of peaks around m / z 612 and 728 that differ by 44 Da), leading to misjudgment.

[0006] Lack of simultaneous detection capability for multiple impurities: Existing methods are usually developed for single impurities, making it difficult to achieve simultaneous qualitative and quantitative analysis of multiple Maillard reaction products. Summary of the Invention

[0007] In view of the shortcomings of the prior art described above, the purpose of this invention is to provide a method for detecting melatonin reaction impurities in efamiti tablets based on LC-HRMS.

[0008] This invention is the first to clearly define the chemical structures of four Maillard reaction impurities in efemitiate tablets and their LC-HRMS characteristic parameters (retention time, precise m / z, fragment ions). A method for simultaneous detection of multiple impurities based on LC-HRMS is established, which can simultaneously qualitatively and quantitatively detect the above four impurities in a single run. A triple verification mechanism of retention time, precise mass number, and characteristic fragments is proposed to effectively eliminate the interference of excipients such as PEG / polysorbate in the m / z 612 / 728 region and ensure the accuracy of the detection results. The specificity, sensitivity, precision, accuracy, and stability of the validation method meet ICH requirements and are suitable for pharmaceutical quality control.

[0009] To achieve the above and other related objectives, the present invention provides the following technical solutions: A method for detecting melatonin reaction impurities in efamicin tablets based on LC-HRMS includes the following steps: Samples were prepared and qualitative and quantitative analyses were performed using LC-HRMS conditions, including: Chromatographic column: Waters Xselect HSS T3 C18, 3.5 μm, 4.6 × 100 mm; Mobile phase A: 10 mmol / L ammonium acetate aqueous solution; Mobile phase B: Acetonitrile; Detection wavelength: 260nm; Ion source: ESI; Mode: Positive ion.

[0010] Preferably, the LC-HRMS conditions also include: Injection volume: 5 μL; Flow rate: 0.7 mL / min; Column temperature: 35 °C; Capillary voltage: 3.8 kV; Sheath flow rate: 60 arbitrary units; Collision energy: 20 eV; Resolution: 70000; Capillary temperature: 320 °C.

[0011] Preferably, the LC-HRMS conditions also include: Elution gradient: 0 minutes: Phase B ratio 0%; 5 minutes: Phase B ratio 0%; 23 minutes: Phase B ratio 60%; 24 minutes: Phase B ratio 95%; 25 minutes: Phase B ratio 95%; 26 minutes: Phase B ratio 0%; 30 minutes: Phase B ratio 0%.

[0012] Preferably, sample preparation includes the following steps: Take efamiti tablets, shake with water to disintegrate them, add methanol, sonicate, add water to make up to volume, centrifuge, take the supernatant, dilute with methanol, and obtain the test solution as the sample.

[0013] Preferably, the qualitative analysis includes the following steps: Based on the theoretical monoisotopic molecular weight calculations of each impurity, [M+H] is calculated. + The theoretical m / z value was determined, and ion chromatographic peaks were extracted within a ±10 ppm window. The structure was then confirmed using a secondary fragment ion map.

[0014] Preferably, the quantitative analysis includes the following steps: The external standard method was used to quantify impurities 1, 2, and 3 using impurity 1 reference standard; and impurity 4 was quantified using impurity 4 reference standard.

[0015] Furthermore, impurity 1 is C 31 H 50 N5O 20 P, impurity 2 is C 21 H 34 N5O 14 P.

[0016] Furthermore, impurity 3 is C 26 H 42 N5O 17 P, impurity 4 is C 20 H 31 N3O 13 S.

[0017] Furthermore, interference is eliminated using LC-HRMS conditions, including the following steps: Interference peaks caused by excipients can be effectively distinguished by differences in retention time and fragment ion characteristics, thus avoiding misjudgment.

[0018] Furthermore, excipients include PEG.

[0019] The beneficial effects of this invention are: This invention provides a highly specific and sensitive analytical method that can accurately identify and quantify four specific Maillard reaction impurities in efamiti tablets (i.e., tenofovir disoproxil fumarate-lactose, tenofovir-lactose, tenofovir monoclonal ester-lactose, and lamivudine-lactose adduct).

[0020] This invention can overcome the problem of same m / z interference caused by excipients (such as PEG / polysorbate) in mass spectrometry detection, and avoid false positive results.

[0021] This invention enables simultaneous detection of multiple impurities, meeting the control requirements for trace impurities (≤0.05%) in drug quality research and release testing.

[0022] This invention has high specificity: by combining retention time with high-resolution mass spectrometry for precise mass number (±10 ppm), it can effectively distinguish target impurities from structural analogs or excipient interference peaks.

[0023] This invention has high sensitivity: the method limit of quantitation (LOQ) is 0.15 µg / mL (equivalent to 0.05% of the principal component), and the signal-to-noise ratio (S / N) is ≥10, meeting the requirements of ICH guidelines.

[0024] This invention enables simultaneous multi-component analysis: it can simultaneously perform qualitative and quantitative analysis of four Maillard reaction impurities in a single injection, thereby improving detection efficiency.

[0025] The method of this invention is robust and reliable: it has been fully verified in terms of linearity (r≥0.999), precision (RSD≤2%), accuracy (recovery rate 90-110%), and stability (14-18h), and is suitable for quality control under GMP environment.

[0026] Furthermore, the precise mass number + retention time + characteristic fragment method established in this paper can be extended to other drug systems containing primary amine APIs and reducing sugar excipients, providing a general technical platform for the early identification and control of meladard reaction risks in drug development. Attached Figure Description

[0027] The accompanying drawings are provided to further illustrate the invention and form part of the specification. They are used together with the embodiments of the invention to explain the invention and do not constitute a limitation thereof.

[0028] In the attached diagram: Figure 1 This is the spectrum of impurity 1 of the present invention.

[0029] Figure 2 This is the spectrum of impurity 2 in this invention.

[0030] Figure 3 This is the spectrum of impurity 3 of the present invention.

[0031] Figure 4 This is the spectrum of impurity 4 of the present invention.

[0032] Figure 5 This is a superimposed image of the detection results of a sample rich in impurity 2 and a sample without impurity 2 according to the present invention.

[0033] Figure 6 This is a superimposed image of the detection results of a sample rich in impurity 3 and a sample without impurity 3 according to the present invention.

[0034] Figure 7 This is a fragment ion diagram of impurity 1 of the present invention.

[0035] Figure 8 This is a fragment ion diagram of impurity 2 of the present invention.

[0036] Figure 9 This is a fragment ion diagram of impurity 3 of the present invention.

[0037] Figure 10 This is a fragment ion diagram of impurity 4 of the present invention. Detailed Implementation

[0038] The applicant will now describe the embodiments of the present invention in detail. It should be noted that the embodiments described below are exemplary and are only used to explain the present invention, and should not be construed as limiting the present invention. Furthermore, unless otherwise specified, all reagents used in the following embodiments are commercially available or can be synthesized by referring to existing literature or known methods. For reactions or test conditions not listed, they are all conventional techniques readily available to those skilled in the art. The terminology used in this invention is common in the art, and its meaning will be clearly understood by those skilled in the art; therefore, it will not be elaborated upon individually.

[0039] This invention provides an analytical method based on liquid chromatography-high resolution mass spectrometry (LC-HRMS) for detecting four Maillard reaction impurities in efamiti tablets. The specific steps are illustrated in different embodiments.

[0040] Example 1 The specific steps for sample preparation are as follows: Take one efamiti tablet, place it in a 100mL volumetric flask, add 20mL of water, shake thoroughly until disintegrated, add 60mL of methanol, sonicate for 30min, add water to the mark, and shake well; centrifuge at 13000G for 5min, take 5mL of the supernatant in a 10mL volumetric flask, add 60% methanol to the mark, shake well and wait for testing to obtain the test solution.

[0041] Example 2 The specific LC-HRMS conditions are as follows: Column: Waters Xselect HSS T3 C18 (3.5 μm, 4.6 × 100 mm) Mobile phase A: 10 mmol / L ammonium acetate aqueous solution Mobile phase B: Acetonitrile Injection volume: 5 μL Flow rate: 0.7 mL / min Column temperature: 35℃ Detection wavelength: 260nm Elution gradient (t(min) / B%): 0 / 0%, 5 / 0%, 23 / 60%, 24 / 95%, 25 / 95%, 26 / 0%, 30 / 0% Ion source: ESI Mode: Positive ion Capillary voltage: 3.8 kV Sheath flow velocity: 60 arbitrary units Collision energy: 20 eV Resolution: 70000 Capillary temperature: 320℃ Example 3 Qualitative and quantitative methods, as detailed below: (1) Qualitative analysis: Calculation of [M+H] based on the theoretical monoisotope molecular weight of each impurity. + The theoretical m / z values ​​were obtained (see Table 1 below). Ion chromatographic peaks were extracted within a ±10 ppm window, and the structure was confirmed by combining the secondary fragment ion map.

[0042] (2) Quantification: The test solution was used, and the external standard method was employed. Impurities 1, 2, and 3 were quantified using impurity 1 reference standard (due to their similar structures and the presence of trace amounts of impurity 2 and 3 in impurity 1 reference standard); impurity 4 was quantified using impurity 4 reference standard. Results are referenced. Figure 1-4 .

[0043] Table 1

[0044] This invention also provides Figure 5 and Figure 6 , Figure 5 and Figure 6 It is an overlay of the detection results of samples rich in impurities 2 and 3, and samples without impurities 2 and 3. It can be understood as an overlay of the actual detection results of different samples.

[0045] Example 4 The specific steps for eliminating interference are as follows: To address the interference peaks generated by excipients (such as PEG) around m / z 612 / 728, this method effectively distinguishes them by differences in retention time and fragment ion characteristics (e.g., impurity 2 contains characteristic fragments such as m / z 288 and 450, while PEG does not), thus avoiding misjudgment.

[0046] For mass spectrometry fragment ion maps of impurities 1-4, please refer to [link / reference]. Figure 7-10 By comparing the measured peak fragment ions with the graph, interference peaks can be distinguished.

[0047] Example 5 The signal-to-noise ratio results for the limit of detection and limit of quantitation solutions are shown in Table 2. Table 2

[0048] The results of the precision of the limit-of-quantitation solution injection are shown in Table 3. Table 3

[0049] The recovery results of the spiked sample solutions are shown in Table 4: Table 4

[0050] Note: No impurities 1 and 4 were detected in the background of the test solution.

[0051] The stability results of the reference solution are shown in Table 5: Table 5

[0052] The stability results of the spiked solution of the test sample are shown in Table 6: Table 6

[0053] Linear results, see Table 7: Table 7

[0054] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for detecting melatonin impurities in efamiti tablets based on LC-HRMS, characterized in that, Includes the following steps: Samples were prepared and qualitative and quantitative analyses were performed using LC-HRMS conditions, including: Chromatographic column: Waters Xselect HSS T3 C18, 3.5 μm, 4.6 × 100 mm; Mobile phase A: 10 mmol / L ammonium acetate aqueous solution; Mobile phase B: Acetonitrile; Detection wavelength: 260 nm; Ion source: ESI; Mode: Positive ion.

2. The method for detecting merrard reaction impurities in efamiti tablets based on LC-HRMS according to claim 1, characterized in that, LC-HRMS conditions also include: Injection volume: 5 μL; Flow rate: 0.7 mL / min; Column temperature: 35 °C; Capillary voltage: 3.8 kV; Sheath flow rate: 60 arbitrary units; Collision energy: 20 eV; Resolution: 70000; Capillary temperature: 320 °C.

3. The method for detecting meladard reaction impurities in efamiti tablets based on LC-HRMS according to claim 1, characterized in that, LC-HRMS conditions also include: Elution gradient: 0 minutes: Phase B ratio 0%; 5 minutes: Phase B ratio 0%; 23 minutes: Phase B ratio 60%; 24 minutes: Phase B ratio 95%; 25 minutes: Phase B ratio 95%; 26 minutes: Phase B ratio 0%; 30 minutes: Phase B ratio 0%.

4. The method for detecting merrard reaction impurities in efamiti tablets based on LC-HRMS according to claim 1, characterized in that, Sample preparation includes the following steps: Take efamiti tablets, shake with water to disintegrate them, add methanol, sonicate, add water to make up to volume, centrifuge, take the supernatant, dilute with methanol, and obtain the test solution as the sample.

5. The method for detecting merrard reaction impurities in efamiti tablets based on LC-HRMS according to claim 1, characterized in that, Qualitative analysis includes the following steps: Based on the theoretical monoisotopic molecular weight calculations of each impurity, [M+H] is calculated. + The theoretical m / z value was determined, and ion chromatographic peaks were extracted within a ±10 ppm window. The structure was then confirmed using a secondary fragment ion map.

6. The method for detecting merrard reaction impurities in efamiti tablets based on LC-HRMS according to claim 1, characterized in that, Quantitative analysis includes the following steps: The external standard method was used to quantify impurities 1, 2, and 3 using impurity 1 reference standard; and impurity 4 was quantified using impurity 4 reference standard.

7. The method for detecting meladard reaction impurities in efamiti tablets based on LC-HRMS according to claim 6, characterized in that, Impurity 1 is C 31 H 50 N5O 20 P, impurity 2 is C 21 H 34 N5O 14 P.

8. The method for detecting merrard reaction impurities in efamiti tablets based on LC-HRMS according to claim 6, characterized in that, Impurity 3 is C 26 H 42 N5O 17 P, impurity 4 is C 20 H 31 N3O 13 S.

9. The method for detecting meladard reaction impurities in efamiti tablets based on LC-HRMS according to claim 1, characterized in that, Interference can also be eliminated using LC-HRMS conditions, including the following steps: Interference peaks caused by excipients can be effectively distinguished by differences in retention time and fragment ion characteristics, thus avoiding misjudgment.

10. The method for detecting merrard reaction impurities in efamiti tablets based on LC-HRMS according to claim 9, characterized in that, The excipients include PEG.

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