Method for measuring residual quantity of residual impurities DMAP and EDU in synthesis of antitumor drugs

Abstract

The invention discloses a method for measuring residual quantity of residual impurities DMAP and EDU in synthesis of antitumor drugs, and belongs to the technical field of drug analysis. According to the method, high performance liquid chromatography is adopted for detection; dimethyl sulfoxide is used as a solvent to dissolve the sample for detection; detection conditions of the high performance liquid chromatography are as follows: a chromatographic column is an octadecyl bonded silica gel column; the mobile phase A is a sodium perchlorate buffer solution or a potassium hexafluorophosphate buffer solution; the mobile phase B is acetonitrile; the detector is an ultraviolet detector; the detection wavelength for detecting the DMAP is 278 to 282 nm, and the detection wavelength for detecting the EDU is 198 to 202 nm; a gradient elution mode is adopted. The method is used for detecting DMAP and EDU residues in the raw material medicine, is high in sensitivity and accuracy, does not need a special column, can meet the control limit of the ICH guidance principle on genotoxic impurities and general impurities of the raw material medicine, provides reliable guarantee for impurity control of the raw material medicine, and further guarantees the safety of clinical medication.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical analysis technology, specifically relating to a method for determining the residual amounts of DMAP and EDU, which are synthetic impurities in antitumor drugs. Background Technology

[0002] Veneclare tablets are a B-cell lymphoma factor-2 inhibitor used to treat acute myeloid leukemia in adults. It was approved by the FDA on April 11, 2016. Its synthetic reaction formula is as follows:

[0003]

[0004] 4-Dimethylaminopyridine (DMAP) is a novel and highly efficient catalyst in chemical synthesis that can significantly improve the reaction rate and yield of chemical reactions. Currently, the pharmaceutical industry has successfully applied it to the synthesis process of chemical drugs, improving process conditions and reducing process costs. Its molecular formula is shown in Formula I.

[0005]

[0006] 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI), with a linear molecular structure and molecular formula shown in Formula II, is a common condensing and cross-linking agent in chemical synthesis and peptide condensation reactions, and has been widely used in the synthesis of polysaccharides, peptides, polymers, and chemical drugs. The Veneclare synthesis process employs hydration separation and multi-step water washing steps. EDCI is unstable in water, generating the byproduct 1-(3-dimethylaminopropyl)-3-ethylurea (EDU), with the molecular formula shown in Formula III. According to the Veneclare synthesis process, the condensing agent EDCI ultimately exists in the Veneclare active pharmaceutical ingredient in the form of EDU.

[0007]

[0008]

[0009] DMAP and EDU are two process impurities in veneclade active pharmaceutical ingredient (API). Therefore, developing a simple and accurate detection method is of great significance for ensuring the safety of veneclade API. DMAP has a genotoxicity warning structure, and according to ICH guidelines and the daily dosage and dosing cycle of veneclade, its control limit is 25 ppm. EDU is a general impurity, and its control limit is 0.1%.

[0010] Both DMAP and EDU are highly polar compounds and are not retained under typical reversed-phase chromatography conditions. Most existing literature reports suggest adding alkyl sulfonate ion-pairing reagents to the mobile phase and using a hydrophilic C18 column to increase the retention of these two compounds. CN115406979B discloses a method for detecting EDU, a hydrolysis product of EDCI, using a mobile phase of sodium heptanesulfonate ion-pairing reagent and triethylamine, and an Ultimate AQ-C18 column; CN114544841N discloses a method for detecting DMAP in pneumococcal polysaccharide-protein conjugate vaccines using a mobile phase of sodium heptanesulfonate ion-pairing reagent and a Sepax HP-C18 column; and patent 108267518A discloses a method for determining the DMAP content in abiraterone acetate using a mobile phase of alkyl sulfonate ion-pairing reagent.

[0011] When using alkyl sulfonate ion-pairing reagents, column equilibration is slow. New columns typically require overnight equilibration, and routine assays generally require at least two hours. Gradient programs are not recommended, as they may prevent the main component of the sample (active pharmaceutical ingredient or intermediate) from eluting. Alkyl sulfonate ion-pairing reagents can denature the chromatographic packing material. Generally, columns using alkyl sulfonate ion-pairing reagents must be dedicated to this purpose, increasing the cost of assays. Summary of the Invention

[0012] To address the problems of existing technologies in detecting residual highly polar impurities DMAP and EDU in drugs, this invention provides a method for determining the residual amounts of DMAP and EDU in the synthesis of antitumor drugs. This method is used to detect DMAP and EDU residues in veneclade active pharmaceutical ingredient (API). The method exhibits high sensitivity and accuracy, does not require a dedicated column, and meets the ICH guidelines for controlling genotoxic and general impurities in APIs. This provides a reliable guarantee for impurity control in veneclade API, thereby ensuring the safety of clinical use.

[0013] To achieve the above objectives, the present invention provides the following technical solution:

[0014] A method for determining the residual amounts of DMAP and EDU, residual impurities in the synthesis of antitumor drugs, employs high-performance liquid chromatography (HPLC); the sample is dissolved in dimethyl sulfoxide (DMSO) for detection.

[0015] The detection conditions for the high-performance liquid chromatography include:

[0016] The chromatographic column is an octadecyl bonded silica column;

[0017] Mobile phase A is sodium perchlorate buffer or potassium hexafluorophosphate buffer;

[0018] Mobile phase B is acetonitrile;

[0019] The detector is an ultraviolet detector;

[0020] The detection wavelength for DMAP is 278–282 nm, and the detection wavelength for EDU is 198–202 nm.

[0021] A gradient elution method is used; the elution program is set as follows:

[0022]

[0023] Furthermore, the detection conditions for the high-performance liquid chromatography also include:

[0024] The column temperature is 25-35℃, the injection volume is 5-10μl, and the flow rate is 0.8-1.2ml / min.

[0025] Furthermore, the chromatographic column is selected as a ZORBAX SB-C18 column. More preferably, the specifications of the ZORBAX SB-C18 column are: particle size × inner diameter × column length: 3.5μm × 4.6mm × 150mm.

[0026] Furthermore, the ultraviolet detector is a DAD detector or a multi-wavelength ultraviolet detector;

[0027] Furthermore, the column temperature is preferably 30°C;

[0028] Furthermore, the flow rate is preferably 1.0 ml / min;

[0029] Furthermore, the injection volume is preferably 5 μl;

[0030] Furthermore, the detection wavelength for DMAP is 280nm, and the detection wavelength for EDU is 200nm;

[0031] Furthermore, the concentration of the sodium perchlorate buffer or potassium hexafluorophosphate buffer is 15–25 mmol / L;

[0032] In some embodiments, the mobile phase A is selected as a sodium perchlorate buffer solution with a pH of 4 to 5; the acid used to adjust the pH can be perchloric acid.

[0033] In some embodiments, the mobile phase A is selected as a potassium hexafluorophosphate buffer solution with a pH of 2 to 3; the acid used to adjust the pH can be phosphoric acid.

[0034] Compared with the prior art, the present invention has the following beneficial effects:

[0035] 1. This invention enables the retention of highly polar impurities DMAP and EDU by eliminating the addition of alkyl sulfonate ion-pairing reagents to the mobile phase. This method abandons the addition of alkyl sulfonate ion-pairing reagents to the mobile phase and achieves the retention of DMAP and EDU on the reversed-phase C18 column through the liquid-liquid separation effect of the liquid-liquid separation agent, thus saving analysis time and improving detection efficiency.

[0036] 2. This method does not require a special hydrophilic chromatographic column; a regular reversed-phase C18 column can meet the detection requirements. Furthermore, it does not require a dedicated column and can share the same column with other detection items in the laboratory, thus reducing analysis costs.

[0037] 3. Gradient elution is used to achieve simultaneous detection of DMAP and EDU, which improves detection efficiency. At the same time, gradient elution allows for the elution of Vinecola, extending the life of the chromatographic column.

[0038] 4. Compared with LC-MS / MS and LC combined with evaporative light detection, this method has greater universality, lower analysis cost, and does not require the addition of triethylamine to the mobile phase to enhance EDU absorption. Attached Figure Description

[0039] Figure 1 The chromatogram (280 nm wavelength) of the DMAP reference solution under sodium perchlorate buffer conditions in Example 1 of the present invention.

[0040] Figure 2 The chromatogram (200 nm wavelength) of the EDU reference solution under sodium perchlorate buffer conditions in Example 1 of the present invention is shown.

[0041] Figure 3 The chromatogram (280 nm wavelength) of the spiked test solution under sodium perchlorate buffer conditions in Example 1 of the present invention is shown.

[0042] Figure 4 The chromatogram (200 nm wavelength) of the spiked test solution under sodium perchlorate buffer conditions in Example 1 of the present invention is shown.

[0043] Figure 5 This is a graph showing the linear relationship of DMAP under sodium perchlorate buffer conditions in Example 2 of the present invention.

[0044] Figure 6 This is a linear relationship graph of EDU under sodium perchlorate buffer conditions in Example 2 of the present invention;

[0045] Figure 7 The chromatogram (280 nm wavelength) of the DMAP reference solution under potassium hexafluorophosphate buffer conditions in Example 4 of the present invention is shown.

[0046] Figure 8The chromatogram (200 nm wavelength) of the EDU reference solution under potassium hexafluorophosphate buffer conditions in Example 4 of the present invention is shown.

[0047] Figure 9 The chromatogram (280 nm wavelength) of the DMAP reference solution in Comparative Example 1 is shown.

[0048] Figure 10 The chromatogram (200 nm wavelength) of the EDU reference solution in Comparative Example 1 is shown.

[0049] Figure 11 The chromatogram (280 nm wavelength) of the DMAP reference solution in Comparative Example 2 is shown.

[0050] Figure 12 The chromatogram of the EDU reference solution in Comparative Example 2 (200 nm wavelength) is shown. Detailed Implementation

[0051] To enable those skilled in the art to better understand the technical solutions of the present invention, the present invention will be described in detail below through specific embodiments, but the content and scope of protection claimed by the present invention are not limited to the following embodiments.

[0052] All reagents used in this invention are commercially available or can be prepared using the methods described herein. The DMAP and EDU reference standards were purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.

[0053] Example 1: Specificity Test

[0054] (1) Chromatographic conditions

[0055] Column: ZORBAX SB-C18, 4.6 mm × 150 mm, 3.5 μm

[0056] Mobile phase A: 20 mmol / L sodium perchlorate buffer

[0057] Mobile phase B: Acetonitrile

[0058] Column temperature: 30℃

[0059] Flow rate: 1.0 ml / min

[0060] Detector: DAD detector

[0061] Detection wavelengths: 280nm (DMAP), 200nm (EDU)

[0062] Injection volume: 5 μl

[0063] Gradient procedure:

[0064]

[0065]

[0066] (2) Solution preparation

[0067] Solvent: DMSO.

[0068] Mobile phase A: Weigh 2.45g of sodium perchlorate, add 1000ml of water to dissolve it, adjust the pH value to 4.5 with perchloric acid, and filter.

[0069] Mobile phase B: Take 1000 ml of acetonitrile and degas it by sonication.

[0070] DMAP reference stock solution: Weigh approximately 12.5 mg of DMAP reference standard accurately, place it in a 100 ml volumetric flask, add solvent, sonicate to dissolve and dilute to the mark, and shake well; accurately measure 5 ml of the above solution, place it in a 50 ml volumetric flask, and dilute to the mark with solvent.

[0071] EDU reference standard stock solution: Weigh approximately 25 mg of EDU reference standard accurately, place it in a 50 ml volumetric flask, add solvent, sonicate to dissolve and dilute to the mark, and shake well.

[0072] Reference stock solution: Accurately measure 5 ml each of the above DMAP reference stock solution and EDU reference stock solution, place them in the same 50 ml volumetric flask, dilute to the mark with solvent, and shake well.

[0073] Reference solution: Accurately measure 5 ml of the above reference stock solution and place it in a 50 ml volumetric flask. Dilute to the mark with solvent.

[0074] Spiked test solution: Weigh approximately 250 mg of Veneclair raw material accurately, place it in a 50 ml volumetric flask, add an appropriate amount of solvent and sonicate to dissolve, add 5 ml of reference stock solution, dilute to the mark with solvent, and shake well.

[0075] (3) Measurement methods and test results

[0076] The solvent, reference solution, and spiked test solution were injected into the chromatograph, respectively, and the chromatograms were recorded. The chromatograms of the DMAP reference solution, EDU reference solution, spiked test solution (DMAP), and spiked test solution (EDU) are shown below. Figure 1 , Figure 2 , Figure 3 and Figure 4 As shown.

[0077] Under these chromatographic conditions, although the elution times of DMAP and EDU are close, EDU does not show a peak at 280 nm, and DMAP does not show a peak at 200 nm. Therefore, DMAP and EDU do not interfere with each other, and the matrix of the test solution does not interfere with the detection of DMAP and EDU. The method has good specificity.

[0078] Example 2: Limit of Detection, Limit of Quantitation, and Linear Correlation Test

[0079] (1) Chromatographic conditions: Same as in Example 1.

[0080] (2) Solution preparation

[0081] The solvent, mobile phase A, mobile phase B, stock solutions of each reference standard, and reference standard solutions are the same as in Example 1.

[0082] Detection limit solution (10% of the limit concentration): Accurately measure 0.5 ml of the reference stock solution and place it in a 50 ml volumetric flask. Dilute to the mark with solvent.

[0083] Limit of Quantitation Solution (30% of the limit concentration): Accurately measure 1.5 ml of the reference standard stock solution, place it in a 50 ml volumetric flask, and dilute to the mark with solvent.

[0084] Linear solutions: Accurately measure 2.5 ml, 4 ml, 5 ml, 6 ml and 8 ml of the reference stock solution respectively, place them in 50 ml volumetric flasks, and dilute to the mark with solvent.

[0085] (3) Measurement methods and test results

[0086] The limit of detection solution, limit of quantitation solution, and each linear solution were injected into the chromatograph, and the chromatograms were recorded. The DMAP linearity chromatogram and EDU linearity chromatogram are shown below. Figure 5 and Figure 6 As shown, DMAP and EDU exhibit good linearity, with correlation coefficients r of 1.00 and 0.997, respectively. The S / N ratios of both the detection limit solution and the quantitation limit solution meet the standards, indicating high sensitivity of this method.

[0087] Table 1. Experimental results for limit of quantitation and limit of detection.

[0088] Limit of Quantification Solution S / N Detection limit solution S / N Reference solution S / N DMAP 33 13 135 EDU 18 5 56

[0089] Example 3 Accuracy Test

[0090] (1) Chromatographic conditions: Same as in Example 1.

[0091] (2) Solution preparation

[0092] The solvent, mobile phase A, mobile phase B, stock solutions of each reference standard, and reference standard solutions are the same as in Example 1.

[0093] Quantitation limit accuracy solution: Accurately weigh approximately 250 mg of veneclade active pharmaceutical ingredient and place it in a 50 ml volumetric flask. Add an appropriate amount of solvent and sonicate to dissolve. Accurately add 1.5 ml of the reference stock solution and dilute to the mark with solvent. Prepare three parallel solutions.

[0094] 50% accuracy solution: Accurately weigh approximately 250 mg of veneclade raw material, place it in a 50 ml volumetric flask, add an appropriate amount of solvent, and sonicate to dissolve. Accurately add 2.5 ml of the reference stock solution, and dilute to the mark with solvent. Prepare three parallel solutions.

[0095] 100% accuracy solution: Accurately weigh approximately 250 mg of veneclade raw material, place it in a 50 ml volumetric flask, add an appropriate amount of solvent, and sonicate to dissolve. Accurately add 5 ml of the reference stock solution, and dilute to the mark with solvent. Prepare 3 parallel solutions.

[0096] 150% accuracy solution: Accurately weigh approximately 250 mg of veneclade raw material, place it in a 50 ml volumetric flask, add an appropriate amount of solvent, and sonicate to dissolve. Accurately add 7.5 ml of the reference stock solution, and dilute to the mark with solvent. Prepare three parallel solutions.

[0097] (3) Measurement methods and test results

[0098] The reference solution and each accuracy solution were injected into the chromatograph, and the chromatograms were recorded. Tables 2 and 3 show the accuracy test results for DMAP and EDU, respectively. The spiked recoveries of DMAP and EUD were both between 95% and 105%, with RSDs of 1.93% and 1.31%, respectively, indicating high accuracy of the methods.

[0099] Table 2. Experimental Results of DMAP Accuracy

[0100]

[0101] Table 3. Experimental Results of EUD Accuracy

[0102]

[0103] Example 4 Potassium hexafluorophosphate buffer experiment

[0104] (1) Chromatographic conditions

[0105] Column: ZORBAX SB-C18, 4.6 mm × 150 mm, 3.5 μm; Mobile phase A: 20 mmol / L potassium hexafluorophosphate buffer.

[0106] Mobile phase B: Acetonitrile

[0107] Column temperature: 30℃

[0108] Flow rate: 1.0 ml / min

[0109] Detector: DAD detector

[0110] Detection wavelength: 280nm (DMAP), 200nm (EDU) Injection volume: 5μl

[0111] Gradient procedure:

[0112]

[0113]

[0114] (2) Solution preparation

[0115] Solvent: DMSO.

[0116] Mobile phase A: Weigh 3.68g of potassium hexafluorophosphate, add 1000ml of water to dissolve it, adjust the pH to 3.0 with phosphoric acid, and filter.

[0117] Mobile phase B: Take 1000 ml of acetonitrile and degas it by sonication.

[0118] The stock solutions and reference solutions of each reference standard are the same as in Example 1.

[0119] (3) Measurement methods and test results

[0120] The solvent and reference solution were injected into the chromatograph separately, and the chromatograms were recorded. The chromatograms of the DMAP reference solution and the EDU reference solution are shown below. Figure 7 and Figure 8 As shown.

[0121] Under potassium hexafluorophosphate buffer chromatographic conditions, both DMAP and EDU were retained on the C18 column without interference, and the sensitivity met the standard.

[0122] Comparative Example 1

[0123] (1) Chromatographic conditions

[0124] Column: Hypersil GOLD AQ, 4.6 mm × 150 mm, 5 μm

[0125] Mobile phase: 20 mmol / L potassium dihydrogen phosphate solution

[0126] Column temperature: 35℃

[0127] Flow rate: 1.0 ml / min

[0128] Detector: DAD detector

[0129] Detection wavelengths: 280nm (DMAP), 200nm (EDU)

[0130] Injection volume: 5 μl

[0131] (2) Solution preparation

[0132] Solvent: Take 200ml of acetonitrile and 800ml of water, and mix well.

[0133] DMAP reference solution: Accurately weigh approximately 12.5 mg of DMAP reference standard, place it in a 100 ml volumetric flask, add solvent, sonicate to dissolve and dilute to the mark, and shake well; accurately measure 1 ml of the above solution, place it in a 50 ml volumetric flask, and dilute to the mark with solvent. Take 2 ml of the above solution, place it in a 10 ml volumetric flask, and dilute to the mark with solvent.

[0134] EDU reference solution: Weigh approximately 12.5 mg of EDU reference standard accurately, place it in a 100 ml volumetric flask, add solvent, sonicate to dissolve and dilute to the mark, and shake well.

[0135] Mobile phase: Weigh 2.72g of potassium dihydrogen phosphate, add 1000ml of water to dissolve, adjust the pH to 3.0 with phosphoric acid, and filter.

[0136] (3) Measurement methods and test results

[0137] The DMAP and EDU reference solutions were injected into the chromatograph, respectively, and the chromatograms were recorded. The chromatograms of the DMAP and EDU reference solutions are shown below. Figure 9 and Figure 10 As shown, under these chromatographic conditions, neither DMAP nor EDU is retained, and detection is affected by solvent peaks, making simultaneous detection impossible.

[0138] Comparative Example 2

[0139] (1) Chromatographic conditions

[0140] Column: Hypersil GOLD AQ, 4.6 mm × 150 mm, 5 μm

[0141] Mobile phase: 5 mmol / L heptanesulfonate sodium buffer-acetonitrile (15:85)

[0142] Column temperature: 35℃

[0143] Flow rate: 1.0 ml / min

[0144] Detector: DAD detector

[0145] Detection wavelengths: 280nm (DMAP), 200nm (EDU)

[0146] Injection volume: 5 μl

[0147] (2) Solution preparation

[0148] 5 mmol / L heptanesulfonate sodium buffer: Weigh 1.1 g of heptanesulfonate sodium and 2.72 g of potassium dihydrogen phosphate, add 1000 ml of water to dissolve, adjust the pH to 3.0 with phosphoric acid, and filter.

[0149] Mobile phase: Take 850 ml of sodium heptanesulfonate buffer and 150 ml of acetonitrile, shake and mix repeatedly, and degas by sonication.

[0150] The solvent, stock solutions of each reference standard, and reference standard solutions are the same as in Example 1.

[0151] (3) Test Results

[0152] Inject the reference solution into the chromatograph and record the chromatogram. The chromatograms of the DMAP reference solution and the EDU reference solution are shown below. Figure 11 and Figure 12 As shown. Under these chromatographic conditions, both DMAP and EDU were retained on the column, but due to isocratic elution, the peaks broadened, resulting in a low peak response for DMAP. The S / N ratio of the reference solution peak was only 9.8, which did not meet the sensitivity standard.

Claims

1. A method for determining the residual amounts of DMAP and EDU, residual impurities in the synthesis of antitumor drugs, characterized in that, High-performance liquid chromatography (HPLC) was used for detection; the sample was dissolved in dimethyl sulfoxide (DMSO) for analysis. The detection conditions for the high-performance liquid chromatography include: The chromatographic column is an octadecyl bonded silica column; Mobile phase A is sodium perchlorate buffer or potassium hexafluorophosphate buffer; Mobile phase B is acetonitrile; The detector is an ultraviolet detector; The detection wavelength for DMAP is 278–282 nm, and the detection wavelength for EDU is 198–202 nm. A gradient elution method is used, and the elution program is set as follows:

2. The determination method according to claim 1, characterized in that, The detection conditions for the high-performance liquid chromatography also include: column temperature of 25-35℃, injection volume of 5-10μl, and flow rate of 0.8-1.2ml / min.

3. The determination method according to claim 1, characterized in that, The chromatographic column was a ZORBAX SB-C18 column.

4. The determination method according to claim 3, characterized in that, The chromatographic column has the following specifications: particle size × inner diameter × column length: 3.5μm × 4.6mm × 150mm.

5. The determination method according to claim 1, characterized in that, The ultraviolet detector is a DAD detector or a multi-wavelength ultraviolet detector.

6. The determination method according to claim 2, characterized in that, The column temperature was 30℃, the flow rate was 1.0 ml / min, and the injection volume was 5 μl.

7. The determination method according to claim 1, characterized in that, The detection wavelength for DMAP is 280nm, and the detection wavelength for EDU is 200nm.

8. The determination method according to claim 1, characterized in that, The concentration of the sodium perchlorate buffer or potassium hexafluorophosphate buffer is 15–25 mmol / L.

9. The determination method according to claim 8, characterized in that, The pH value of the sodium perchlorate buffer solution is 4-5.

10. The determination method according to claim 8, characterized in that, The pH value of the potassium hexafluorophosphate buffer solution is 2-3.

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