A method for determining EDCI residue in cerutinib

Abstract

A method for determining EDCI residues in enolutinib belongs to the field of detection and analysis, and specifically relates to an analytical method for determining the residual content of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, a potential genotoxic impurity of the anti-tumor drug enolutinib. This method uses LC-MS / MS (liquid chromatography tandem mass spectrometry) to detect the residual 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride of the potential genotoxic impurity. The sensitivity of this method can be accurately quantified to 1.2ng / ml, with strong specificity, good repeatability, high sensitivity, and accurate results. The residual content of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride in enolutinib can be effectively detected, effectively ensuring the quality of the anti-tumor drug enolutinib.

Description

Technical Field

[0001] The invention belongs to the field of detection and analysis, and specifically relates to an analytical method for detecting the residual 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride of a potential genotoxic impurity by using liquid chromatography tandem mass spectrometry. Background Art

[0002] 1-Ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI) is a water-soluble condensing agent commonly used in synthesis, a general carbonyl activation reagent, used in the process of bonding amides and secondary amines; can react with phosphate groups; used in peptide synthesis; protein and nucleic acid crosslinking; preparation of immunocrosslinking products, under typical conditions, pH 4.0-6.0 and no buffer, under special conditions, amine and carboxylate buffers must be avoided; water-soluble peptide coupling agent; used for carbonyl modification in proteins; used for ester synthesis. It has been widely used in the field of chemical synthesis. The structural formula of 1-Ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride is as follows:

[0003]

[0004] 1-Ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride contains a carbodiimide structure, so we control it as a potential genotoxic impurity. On this basis, we established a method for determining the residue of 1-Ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride. The carbodiimide structure is as follows:

[0005] Summary of the invention

[0006] The present invention uses LCMSMS to detect the residue of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride. The specific test conditions include:

[0007] Mobile phase: A: 0.1% formic acid aqueous solution, B: acetonitrile;

[0008] MS detector: ESI source, SIM mode, scanning ion peak 174 (174.1950-174.2300);

[0009] Chromatographic column: octadecyl bonded silica gel as filler, 150*4.6mm, 5um;

[0010] Flow rate: 1.0ml / min;

[0011] Column temperature: 40°C;

[0012] Injection volume: 10 μL;

[0013] Gradient conditions:

[0014]

[0015] The residual detection of 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride is carried out by detecting the quasi-molecular ion [M+H]+=174.15 of the product after water derivatization to infer the residual 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride. The derivatization principle is as follows:

[0016]

[0017] Reference solution: Accurately weigh 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride reference substance and prepare it with water:acetonitrile = 1:1 to make 1 ml contain 6 ng 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride; mix thoroughly and let stand for 1 hour before use.

[0018] Aromatic acid sample solution: Accurately weigh the aromatic acid sample and mix it with water: acetonitrile = 1:1 to make 300ug aromatic acid in 1ml. See the full scan positioning diagram of the sample detection. Figure 1 , SIM diagram see Figure 2 .

[0019] Result calculation: Result of EDCI (ppm) = 10 6

[0020] A sample : Peak area of ​​EDCI in the sample

[0021] A WS : Peak area of ​​EDCI in reference solution

[0022] C WS : EDCI concentration in reference solution ng / ml

[0023] C sample : Concentration of test sample ng / ml

[0024] The quantitative limit of this method is 1.2 ng / ml, and the relative standard deviation of the peak area of ​​the 6-point reference solution is 1.3%.

[0025] The spike recovery rate at the level of 1.2ng / ml is between 95.1% and 106.4%, and the spike recovery rate at the level of 6ng / ml is between 106.9% and 109.8%. This shows that the benzene method has good sensitivity, repeatability and accuracy, and can meet the needs of detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 It is the positioning map of the full scan;

[0027] Figure 2 It is a SIM spectrum. DETAILED DESCRIPTION

[0028] Mobile phase: A: 0.1% formic acid aqueous solution, B: acetonitrile;

[0029] MS detector: ESI source, SIM mode, scanning ion peak 174;

[0030] Chromatographic column: octadecyl bonded silica gel as filler, 150*4.6mm, 5um;

[0031] Flow rate: 1.0ml / min;

[0032] Column temperature: 40°C;

[0033] Injection volume: 10 μL;

[0034] Gradient conditions:

[0035]

[0036] Reference solution: Accurately weigh 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride reference substance and prepare it with water:acetonitrile = 1:1 to make 1 ml contain 6 ng of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride; mix thoroughly and let stand for 1 hour before use.

[0037] Aromatic acid sample solution: accurately weigh the aromatic acid sample and prepare it with water:acetonitrile = 1:1 to make 300ug aromatic acid in 1ml.

[0038] Quantitation limit solution: dilute the control solution with acetonitrile:water = 1:1 to make 1 ml contain 1.2 ng of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride; mix thoroughly.

[0039] Quantitation limit spike solution: dilute the control solution with the enolutinib sample solution to make 1 ml contain 1.2 ng of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 300 ug of enolutinib, and mix thoroughly.

[0040] Reference level spike solution: Accurately weigh 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride reference substance and enolutinib sample, and prepare 1 ml containing 6 ng of 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride and 300 ug of enolutinib with water: acetonitrile = 1:1, and mix thoroughly. Leave for 1 hour before use.

[0041] The repeatability results of the control solutions are shown in Table 1. The peak area RSD of the six control solutions is 1.3%, which has good repeatability and can meet daily detection requirements.

[0042] Table 1: Repeatability of control solutions

[0043]

[0044] The results of spiked recovery are shown in Table 2. The recovery at the limit of quantitation level is between 95.1% and 106.4%, and the recovery at the control concentration level is between 106.9% and 109.8%, which have good recovery rates and meet the general requirements of accuracy.

[0045] Table 2: Accuracy test results

[0046]

Claims

1. A method for detecting the residual content of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride in cerutinib, characterized in that: The method is carried out using an LC-MS / MS combined detection method, wherein the chromatographic conditions are: The chromatographic column is filled with octadecyl bonded silica gel; the mobile phase consists of mobile phase A and mobile phase B, wherein mobile phase A is 0.1% formic acid aqueous solution and mobile phase B is acetonitrile; the elution method is gradient elution, and the conditions of the gradient elution are: The mass spectrometry conditions were: The ion source is ESI source, and positive ion detection and SIM mode are adopted; 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride in cerutinib remains in the mobile phase to produce water derivative products, and the corresponding detection ion peaks are 174.1950-174.2300; The ion source parameters are: The gas source pressure of the vaporization chamber is 50psi; Ion spray voltage 5500V; Temperature: 500°C; Ion source GS1 50PSI; Ion source GS2 60PSI; The flow rate of the mobile phase is 0.5 mL / min to 1.5 mL / min.

2. The method according to claim 1, characterized in that The column temperature of the chromatographic column is 30°C to 40°C.

3. The method according to claim 1, characterized in that include: The injection volume in the chromatographic conditions was 10uL; Accurately weigh 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride reference substance, and prepare it with water:acetonitrile = 1:1 to make 1 ml contain 6 ng 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride; mix thoroughly to make 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride reference substance solution, and let it stand for 1 hour; Accurately weigh the erlutinib sample and prepare it with water:acetonitrile = 1:1 to make 300ug erlutinib in 1ml as the erlutinib test solution; Pipette 10uL of the reference solution and the test solution into the LC-MS / MS liquid-mass spectrometer, perform detection according to the chromatographic conditions and mass spectrometry conditions, and record the chromatogram; According to the obtained chromatogram, the residual content of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride in the cerutinib raw material was calculated using the external standard method.

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