Method for detecting diethyl phosphate in afatinib maleate

Abstract

The invention discloses a method for detecting diethyl phosphate in afatinib maleate. The method has high system applicability, has incomparable advantages in specificity, quantitation limit, detection limit, linear range, repeatability and durability, and has high precision.

Description

Technical Field

[0001] The invention belongs to the field of chemical drug analysis and detection, and particularly relates to a method for detecting diethyl phosphate in afatinib maleate. Background Art

[0002] Afatinib maleate covalently binds to the kinase domains of EGFR (ErbB1), HER2 (ErbB2), and HER4 (ErbB4), irreversibly inhibiting tyrosine kinase autophosphorylation and leading to downregulation of ErbB signaling. It is primarily indicated for the treatment of: 1. Locally advanced or metastatic non-small cell lung cancer (NSCLC) with sensitizing mutations in the epidermal growth factor receptor (EGFR) gene who have not received prior EGFR tyrosine kinase inhibitor (TKI) treatment; 2. Locally advanced or metastatic squamous non-small cell lung cancer (NSCLC) with disease progression during or after platinum-based chemotherapy.

[0003]

[0004] Diethyl phosphate is commonly used as an extractant, solvent, and polymerization catalyst. It is irritating to the skin and respiratory tract, and severely irritating to the eyes. Therefore, developing a method for accurately detecting diethyl phosphate residues is of great practical significance. Currently, commonly used methods include traditional titration and ion chromatography. However, traditional titration has low sensitivity, while ion chromatography has poor specificity, long detection times, and high costs. Summary of the Invention

[0005] The purpose of the present invention is to provide a method for detecting diethyl phosphate in afatinib maleate, so that its specificity, quantitative limit, detection limit, linear range, repeatability, accuracy and other aspects fully meet the standards and have high precision.

[0006] To achieve the above object, the present invention provides the following technical solution: a method for detecting diethyl phosphate in afatinib maleate, comprising the following steps:

[0007] 1) Prepare the test solution

[0008] Take 2 mg of afatinib maleate sample and put it into a 10 mL volumetric flask. Add blank solution methanol and sonicate for 2 minutes to completely dissolve the sample. Then dilute to the mark with blank solution and shake well to prepare the test solution.

[0009] 2) Prepare reference solution

[0010] Take an appropriate amount of diethyl phosphate reference substance, dissolve it in methanol and quantitatively dilute it to make a solution containing about 200 ng per 1 ml as the reference substance solution.

[0011] 3) Accurately measure the test solution and reference solution respectively, inject them into the liquid chromatography-mass spectrometer, record the mass spectrum, and use the external standard method to calculate the content of diethyl phosphate in the test solution based on the peak area.

[0012] Specifically, the calculation formula in step 3) is:

[0013]

[0014] in:

[0015] Astd: average peak area of the analyte in the reference solution

[0016] Cstd: Concentration of reference solution (ng / ml)

[0017] As: peak area of the analyte in the sample solution

[0018] Cs: concentration of sample solution (mg / ml)

[0019] Specifically, the detection conditions of the LC-MS / MS are:

[0020]

[0021]

[0022] Preferably, the detection method can quantitatively detect diethyl phosphate with a minimum limit of 0.1% of the test sample, and the quantitative limit concentration is 5.301 ng / mL.

[0023] Beneficial Effects: This invention utilizes liquid chromatography-mass spectrometry (LC-MS) technology to significantly overcome the shortcomings of traditional titration methods, such as low sensitivity and ion chromatography, which suffer from insufficient specificity, long detection times, and high costs. This innovative approach not only achieves efficient and rapid detection, but also significantly improves detection accuracy and precision, demonstrating unparalleled technical advantages. This method exhibits excellent system applicability and achieves superior levels of performance across multiple key performance indicators, including specificity, limit of quantification, limit of detection, linear range, repeatability, and robustness, ensuring highly precise analytical results.

[0024] In rigorous limit of quantification testing, the method successfully lowered the detection limit of diethyl phosphate to 1.590 ng / mL, and the limit of quantification was determined to be 5.301 ng / mL. This data demonstrates that the method can accurately and quantitatively detect diethyl phosphate in test samples at concentrations as low as 0.1%, demonstrating its extremely high sensitivity and analytical capability. Furthermore, the recovery rate of diethyl phosphate remained stable between 80% and 115%, further validating the reliability and accuracy of this detection method. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 is a mass spectrum of a blank solvent in the method of the present invention;

[0026] Figure 2 is a mass spectrum of the reference solution in the method of the present invention;

[0027] Figure 3 is the mass spectrum of the test solution spiked with the standard limit in the method of the present invention;

[0028] Figure 4 is a mass spectrum of the test solution in the method of the present invention;

[0029] Figure 5 This is the standard curve of diethyl phosphate. DETAILED DESCRIPTION

[0030] In order to deepen the understanding of the present invention, the present invention will be further described in detail below with reference to embodiments and drawings. The embodiments are only used to explain the present invention and do not constitute a limitation on the scope of protection of the present invention.

[0031] Unless otherwise specified, the experimental methods used in the following examples are all conventional methods. Unless otherwise specified, the materials, reagents, etc. used in the following examples are all commercially available.

[0032] According to the detection method of the present invention, mainly for the detection of diethyl phosphate, the external standard method is used for calculation.

[0033] The information of the instruments and reagents used in the detection method of the present invention is shown in the following table:

[0034]

[0035] Example 1

[0036] This embodiment provides a method for detecting diethyl phosphate in afatinib maleate.

[0037] 1. LC-MS / MS conditions

[0038]

[0039]

[0040] 2. Detection Method

[0041] (1) Solvent

[0042] Blank solution: methanol

[0043] (2) Prepare the test solution

[0044] Take 2 mg of afatinib maleate sample and put it into a 10 mL volumetric flask, add blank solution, sonicate for 2 minutes to completely dissolve the sample, and dilute to the mark with blank solution, shake well, and use as the test solution.

[0045] (3) Preparation of reference solution

[0046] Take an appropriate amount of diethyl phosphate reference substance, dissolve it in methanol and quantitatively dilute it to make a solution containing about 2 μg per 1 ml, which serves as the reference substance stock solution.

[0047] Take 1 mL of diethyl phosphate reference stock solution and transfer it to a 10 mL volumetric flask. Add blank solution to the volume to prepare the reference solution.

[0048] (4) Prepare standard limit spiked test solution

[0049] Take 2 mg of afatinib maleate sample and put it into a 10 mL volumetric flask, add blank solution, sonicate for 2 minutes to completely dissolve the sample, add 1 mL of diethyl phosphate reference stock solution and dilute to the scale with blank solution, shake well, and use this as the standard limit spiked test solution.

[0050] (5) Accurately measure the test solution and reference solution, inject them into the liquid chromatography-mass spectrometer, record the mass spectrum, and use the external standard method to calculate the content of diethyl phosphate in the test solution based on the peak area.

[0051] 3. Calculation formula for diethyl phosphate content

[0052]

[0053] in:

[0054] Astd: average peak area of the analyte in the reference solution

[0055] Cstd: Concentration of reference solution (ng / ml)

[0056] As: peak area of the analyte in the sample solution

[0057] Cs: concentration of sample solution (mg / ml)

[0058] 4. Verification of the Method of the Invention

[0059] 1. Specificity test

[0060] (1) Blank solution: methanol.

[0061] (2) Reference solution: Take an appropriate amount of diethyl phosphate reference substance, dissolve it in methanol and quantitatively dilute it to make a solution containing about 2 μg per 1 ml, which serves as the reference substance stock solution.

[0062] Take 1 mL of diethyl phosphate reference stock solution and transfer it to a 10 mL volumetric flask. Add blank solution to the volume to prepare the reference solution.

[0063] (3) Test solution: 2 mg of afatinib maleate sample was transferred to a 10 mL volumetric flask, blank solvent was added, and the sample was completely dissolved by ultrasonication for 2 minutes. The volume was then adjusted to the mark with blank solvent and shaken to obtain the test solution.

[0064] (4) Standard limit spiked test solution: 2 mg of afatinib maleate sample was transferred to a 10 mL volumetric flask, blank solution was added, and the sample was completely dissolved by ultrasonication for 2 minutes. 1 mL of diethyl phosphate reference stock solution was added and the volume was adjusted to the mark with blank solution. The solution was shaken to obtain the standard limit spiked test solution.

[0065] According to the liquid chromatography-mass spectrometry conditions of Example 1, blank solution, reference solution, standard limit spiked test solution and test solution were injected respectively, and the mass spectra were recorded. Figures 1 to 4 .

[0066] The specificity test showed that the blank solution did not interfere with the determination of the analyte, and the analyte had no interference from adjacent peaks, indicating that the method had good specificity.

[0067] 2. Limit of Quantitation and Limit of Detection Tests

[0068] The limit of detection (LOD) and limit of quantification (LOQ) were determined using the signal-to-noise ratio method. The reference solution was serially diluted, and the measured signal was compared with the baseline noise to calculate the lowest concentration that could be reliably detected. The results are shown in Table 1.

[0069] Table 1: Limit of quantification and limit of detection results

[0070]

[0071] The detection limit concentration of diethyl phosphate is 1.590 ng / mL, and the signal-to-noise ratio of the chromatographic peak is not less than 3; the quantitative limit concentration is 5.301 ng / mL, and the signal-to-noise ratio of the chromatographic peak is not less than 10. After 6 consecutive injections, the RSD of the chromatographic peak area is 7.1%, which meets the detection requirements.

[0072] 3. Linearity and range detection

[0073] Take an appropriate amount of diethyl phosphate reference substance, dissolve it in methanol and quantitatively dilute it to make a solution containing about 2 μg per 1 ml, which serves as the reference substance stock solution.

[0074] Take each specification volumetric flask, accurately transfer a certain volume of diethyl phosphate reference stock solution into the volumetric flask, dilute to the mark with blank solution, shake well, and use them as each linear test solution. The preparation of each linear test solution is shown in Table 2 below. The linear relationship is plotted as a function of the measured peak area and the concentration of the analyte. The linear regression is performed using the least squares method. The value of the linear regression coefficient r should be no less than 0.990. The results are shown in Table 3 and Figure 5 .

[0075] Table 2: Linearity test solution preparation

[0076]

[0077] Table 3: Linearity determination results

[0078]

[0079] Note: Intercept ratio = linear equation intercept / 100% concentration peak area

[0080] From the results in the above table, it can be seen that the linear equation of diethyl phosphate is y=172.3666x+38.4678, its correlation coefficient is 0.9958, and the absolute value of the Y-axis intercept and the peak area percentage of the standard limit reference solution are 0.11%, indicating that this method has a good linear relationship.

[0081] 4. Precision test of reference solution injection

[0082] The reference solution of Example 1 was measured six times continuously to examine the relative standard deviation of the peak area. The results are shown in Table 4.

[0083] Table 4: Results of the injection precision test of the reference solution

[0084]

[0085]

[0086] As can be seen from the results in the above table, the detection method of the present invention has good precision in measuring the injection precision of the reference solution, with the peak area RSD being less than 10%.

[0087] 5. Stability testing

[0088] Take the reference solution of Example 1, the standard limit spiked test solution, and the test solution, and inject 1 μl at 0 h, 0.5 h, 2 h, 4 h, 5 h, 6.5 h, 8 h, 15 h, and 16 h, respectively. Record the chromatogram and calculate the relative standard deviation of the diethyl phosphate peak area. The test results are shown in Table 5.

[0089] Table 5: Solution stability test results

[0090]

[0091] As shown in the table above, at each time point over a 16-hour period at room temperature, the peak area RSDs for the diethyl phosphate standard limit reference solution were 6.0%, and the peak area RSDs for the standard limit spiked test solution were 5.5%. No test solution was detected. This indicates that the standard limit reference solution, test solution, and standard limit spiked test solution exhibited good stability over a 16-hour period at room temperature.

[0092] 6. Repeatability testing

[0093] The standard limit spiked test solution was taken and the test was repeated 6 times according to the detection method of Example 1 of the present invention to verify the good repeatability of the method. The results are shown in Table 6.

[0094] Table 6: Repeatability test results

[0095] serial number Recovery rate of standard limit spiked test solution (%) A 1 95.6 A 2 94.0 A 3 103.4 A 4 95.3 A 5 101.8 A 6 105.5 RSD 5.0％

[0096] As can be seen from the table above, the RSD of the recovery rate of diethyl phosphate after 6 detections was 5.0%, which proved that the method had good repeatability.

[0097] 7. Durability

[0098] The standard limit spiked test solution was taken. The differences in the recoveries of the analytes in the standard limit spiked test solution under different conditions are shown in Table 7 for the durability conditions and in Table 8 for the results.

[0099] Table 7: Durability conditions

[0100] Normal conditions See LC-MS / MS conditions Condition 1 The column flow rate was reduced to 0.58 mL / min, and the rest of the conditions were the same as normal. Condition 2 The column flow rate was increased to 0.62 mL / min, and the rest of the conditions were the same as normal. Condition 3 The proportion of water phase is reduced to 89%, and the rest are the same as normal conditions Condition 4 The proportion of water phase increased to 91%, and the rest was the same as normal conditions.

[0101] Table 8: Recovery results of diethyl phosphate under different conditions

[0102] condition Recovery rate of diethyl phosphate (%) Normal conditions 94.8 Column flow rate 0.58 mL / min 89.4 Column flow rate 0.62 mL / min 92.7 The initial proportion of the aqueous phase is 89% 92.7 The initial proportion of the aqueous phase is 91% 88.4 RSD (%) 2.9

[0103] As can be seen from the table above, the RSD of the recoveries of the analytes in the standard limit spiked test solution measured under different conditions was 2.9%, indicating that the method has good robustness.

[0104] 8. Accuracy test

[0105] The sample addition recovery method was used to determine the ratio (recovery rate) between the actual measured amount and the theoretical amount of diethyl phosphate in the spiked test sample. The recovery rate was expressed as a percentage. The recovery rate was required to be between 80% and 115% to confirm that the method had good accuracy. The results are shown in Table 9.

[0106] Table 9: Accuracy test results

[0107]

[0108] As can be seen from the above table, the average recovery rate of diethyl phosphate is 96.9%, which meets the validation requirements (80% to 115%), confirming that the method has good accuracy; the recovery rate RSD value is less than 10%, indicating good accuracy.

[0109] In summary, in the specificity test, the blank solvent, the main component of the test sample, and the adjacent peaks did not interfere with the detection of diethyl phosphate.

[0110] In the quantitative limit test, the detection limit concentration of diethyl phosphate was 1.590 ng / mL, and the chromatographic peak signal-to-noise ratio was not less than 3; the quantitative limit concentration was 5.301 ng / mL, and the chromatographic peak signal-to-noise ratio was not less than 10. After 6 consecutive injections, the chromatographic peak area RSD was 7.1%.

[0111] In the linear range test, the linear equation of diethyl phosphate was y=172.3666x+38.4678, the correlation coefficient was 0.9958, and the absolute value of the Y-axis intercept and the peak area percentage of the standard limit reference solution were 0.11%.

[0112] In the accuracy test, the recovery of diethyl phosphate was between 80% and 115%, with RSD ≤ 10%.

[0113] In the repeatability test, the recoveries of diethyl phosphate in the six spiked samples all met the validation requirements (80% to 115%).

[0114] In the robustness test, the RSD of the recoveries of the analytes in the standard limit spiked test solution measured under different conditions was ≤10%.

[0115] Therefore, the present invention demonstrates that the liquid chromatography-mass spectrometry method for detecting diethyl phosphate in afatinib maleate has high system applicability. Furthermore, it demonstrates unparalleled advantages in specificity, limit of quantification, limit of detection, linear range, durability, stability, and repeatability, with high precision.

[0116] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the invention.

Claims

1. A liquid chromatography-mass spectrometry method for determining diethyl phosphate in afatinib maleate, characterized in that: The specific steps include: 1) Prepare the test solution Take afatinib maleate sample into a volumetric flask, add blank solution to dissolve and quantitatively dilute to make a solution containing approximately 0.2 mg per 1 ml, shake well, and use it as the test solution; 2) Prepare reference solution Take an appropriate amount of diethyl phosphate reference substance, dissolve it in blank solution and quantitatively dilute it to make a solution containing about 200 ng per 1 ml as the reference solution; 3) Accurately measure the test solution and reference solution respectively, inject them into the liquid chromatography-mass spectrometer, record the mass spectrum, and use the external standard method to calculate the content of diethyl phosphate in the test solution based on the peak area.

2. The method according to claim 1, characterized in that The preparation method of the test solution is: Take 2 mg of afatinib maleate sample and put it into a 10 mL volumetric flask, add blank solution, sonicate for 2 minutes to completely dissolve the sample, and dilute to the mark with blank solution, shake well, and use as the test solution.

3. The method according to claim 1, characterized in that The preparation method of the reference solution is: Take an appropriate amount of diethyl phosphate reference substance, dissolve it in blank solution and quantitatively dilute it to make a solution containing about 2 μg per 1 ml as the reference substance stock solution; Take 1 mL of diethyl phosphate reference stock solution and transfer it to a 10 mL volumetric flask. Add blank solution to the volume to prepare the reference solution.

4. The method according to claims 1-3, characterized in that The blank solution is methanol.

5. The method according to claim 1, wherein The detection conditions of the liquid chromatography are:

6. The method according to claim 5, characterized in that During gradient elution, the elution program is:

7. The method according to claim 1, characterized in that The mass spectrometry parameters are:

8. The method according to claim 1, characterized in that The minimum quantitative limit of diethyl phosphate in this detection method is 5.301 ng / mL.

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