Method for detecting N-methyl pyrrolidone in carfilzomib intermediate

Abstract

The invention discloses a method for detecting N-methyl pyrrolidone in a carfilzomib intermediate, which comprises the following steps: adopting a high performance liquid chromatography, using a ZORBAX Eclipse C18 ball porous filler chromatographic column to realize reverse phase separation, and using a diode array detector to perform quantitative analysis. Chromatographic conditions are as follows: a mobile phase A is water; a mobile phase B is methanol, and carrying out gradient elution on the phase A and the phase B; the flow velocity is 0.8 to 1.0 ml / min; the column temperature is 25-40 DEG C; the detection wavelength is 205 to 220 nm; the sample size is 10-20 [mu] l; and the diluent is a 50-70% methanol aqueous solution. The N-methyl pyrrolidone disclosed by the invention is relatively high in response, high in detection sensitivity, good in peak pattern and accurate and reliable in detection result; the high performance liquid chromatography is adopted, instruments, solvents and chromatographic columns used for detection are conventional and easy to obtain, samples and reference substances are easy to prepare and operate, and the method can be widely applied to detection of N-methyl pyrrolidone.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of pharmaceutical analysis, and particularly relates to a detection method of N-methyl pyrrolidone in a carfilzomib intermediate. BACKGROUND

[0002] Carfilzomib is a specific, irreversible proteasome-targeting inhibitor, which is suitable for treating multiple myeloma patients. (N-((1S)-3-methyl-1-((2R)-2-methyloxiranyl)carbonyl)butyl)-tert-butoxycarbamate) is a key intermediate in the synthesis of carfilzomib, and its molecular formula is: C 14 H 25 NO4, and its molecular weight is: 217.36, and its structural formula is as follows: .

[0003] N-methyl pyrrolidone is a solvent used in the synthesis reaction of the above-mentioned intermediate, which has certain reproductive toxicity to animals and human bodies, and its residue will affect the subsequent reaction, generate by-products, and affect the quality of carfilzomib finished products, so it is essential to control the residual amount of N-methyl pyrrolidone in the above-mentioned intermediate, and the accurate determination of the content of N-methyl pyrrolidone is particularly important.

[0004] At present, the detection methods of N-methyl pyrrolidone are mostly gas chromatography, and the detection sensitivity is not high due to the high boiling point of N-methyl pyrrolidone, and the chromatographic peak type is not good. SUMMARY

[0005] In order to solve the problems in the prior art, the purpose of the present application is to provide a detection method of N-methyl pyrrolidone in a carfilzomib intermediate.

[0006] In order to achieve the above-mentioned purpose and achieve the above-mentioned technical effect, the technical scheme adopted by the present application is: The detection method of N-methyl pyrrolidone in the carfilzomib intermediate comprises the following steps: The high performance liquid chromatography is used, the ZORBAX Eclipse C18 porous filler chromatographic column is used to realize the reversed phase separation, and the diode array detector is used for quantitative analysis; Chromatographic conditions: The mobile phase A is water, and the elution ratio is 20-60% (V / V); The mobile phase B is methanol, and the A phase and the B phase are gradient eluted; The flow rate is 0.8-1.0 ml / min; The column temperature is 25-40℃; The detection wavelength is 205-220 nm; The injection amount is 10-20 μl; The diluent is 50-70% (V / V) methanol aqueous solution.

[0007] Further, the gradient elution parameters are as follows: .

[0008] Further, the chromatographic column is Agilent ZORBAX Eclipse C18, the filler is spherical porous octadecylsilane bonded silica gel, the specification is 4.6*250mm, 5um.

[0009] Further, the flow rate is 1.0ml / min.

[0010] Further, the column temperature is 30℃.

[0011] Further, the detection wavelength is 210nm.

[0012] Further, the injection amount is 10ul.

[0013] Further, the diluent is 60% methanol aqueous solution (V / V).

[0014] Compared with the prior art, the beneficial effects of the present application are: 1) The present application discloses a method for detecting N-methyl pyrrolidone in carfilzomib intermediate, which adopts high performance liquid chromatography, uses ZORBAX Eclipse C18 spherical porous filler chromatographic column to realize reverse phase separation, uses diode array detector for quantitative analysis, the response of N-methyl pyrrolidone is high, the detection sensitivity is high, the peak type is good, and the detection result is accurate and reliable. 2) The present application adopts high performance liquid chromatography, the instruments, solvents and chromatographic columns used for detection are conventional and easy to obtain, the sample and control preparation operation is simple, and the method can be universally applied to the detection of N-methyl pyrrolidone. BRIEF DESCRIPTION OF DRAWINGS

[0015] Figure 1 is the chromatogram of the blank solution in the embodiment of the present application; Figures 2-6 is the chromatogram of 5 groups of N-methyl pyrrolidone control solution in the embodiment of the present application; Figures 7-9 is the chromatogram of 3 batches of test solution (K230401, K230501, K230601) in the embodiment of the present application; Figures 10-12 is the chromatogram of 3 batches of spiked test solution (K2304011, K2305011, K2306011) in the embodiment of the present application. DETAILED DESCRIPTION

[0016] The present application will be described in detail below so that the advantages and features of the present application can be more easily understood by those skilled in the art, and the scope of protection of the present application can be more clearly defined.

[0017] The following presents a simplified summary of one or more aspects in order to provide a basic understanding of such aspects. This summary is not an extensive overview of all contemplated aspects, and is intended to neither identify key or critical elements of all aspects nor delineate the scope of any or all aspects. Its sole purpose is to present some concepts of one or more aspects in a simplified form as a prelude to the more detailed description that is presented later.

[0018] As shown in Figures 1-12 The present application discloses a method for detecting N-methyl pyrrolidone in carfilzomib intermediates, comprising the following steps: S1, instrument preparation: high performance liquid chromatograph; diode array detector; ZORBAX Eclipse C18 spherical porous packing chromatographic column; S2, mobile phase preparation: The mobile phase includes mobile phase A and mobile phase B, the mobile phase A is water, and the elution ratio is 20-60% (V / V), and the mobile phase B is methanol; S3, gradient elution parameter setting: The A phase and the B phase are subjected to gradient elution, and the gradient elution parameters are shown in Table 1; Table 1 The detection wavelength is 205-220 nm; the injection amount is 10-20 μl; the flow rate is 0.8-1.0 ml / min; and the column temperature is 25-40℃; S4, preparation of detection solution: The diluent is 50-70% (V / V) methanol aqueous solution; Preparation of carfilzomib intermediate test sample solution; Preparation of N-methyl pyrrolidone control sample solution; The above control sample solution and test sample solution are respectively injected for detection, and the chromatogram is recorded.

[0019] Example 1 As shown in Figures 1-12 The method for detecting N-methyl pyrrolidone in carfilzomib intermediates comprises the following steps: S1, instrument preparation: high performance liquid chromatograph: Agilent 1260; DAD detector; Agilent Openlab CDS2.3 software system; Chromatographic column: Agilent ZORBAX Eclipse C18, with spherical porous octadecylsilane-bonded silica gel as filler, 4.6 x 250 mm, 5 μm; S2, mobile phase preparation: The mobile phase comprises mobile phase A and mobile phase B, the mobile phase A is water, and the mobile phase B is methanol; S3, gradient elution parameter setting: The A phase and the B phase are subjected to gradient elution, and the gradient elution parameters are shown in Table 1; Table 1 The detection wavelength is 210 nm; the injection amount is 10 μl; the flow rate is 1.0 ml / min; and the column temperature is 30℃; S4, detection solution preparation: Blank / diluent: methanol: water = 6:4 (V / V); N-methylpyrrolidone stock solution I: 100 mg of N-methylpyrrolidone is weighed in a 10 ml volumetric flask, dissolved and diluted to the mark with the diluent, and shaken to obtain the stock solution; N-methylpyrrolidone stock solution II: 1 ml of N-methylpyrrolidone stock solution I is accurately weighed in a 100 ml volumetric flask, dissolved and diluted to the mark with the diluent, and shaken to obtain the stock solution; N-methylpyrrolidone stock solution III: 2 ml of N-methylpyrrolidone stock solution II is accurately weighed in a 20 ml volumetric flask, dissolved and diluted to the mark with the diluent, and shaken to obtain the stock solution; Reference solution: 2 ml of N-methylpyrrolidone stock solution III is accurately weighed in a 20 ml volumetric flask, dissolved and diluted to the mark with the diluent, and shaken to obtain the stock solution, 1 μg / ml; Test solution: 100 mg of the test sample is accurately weighed in a 10 ml volumetric flask, dissolved and diluted to the mark with the diluent, and shaken to obtain the test solution; Spiked test solution: 100 mg of the carfilzomib intermediate test sample is accurately weighed in a 10 ml volumetric flask, 1 ml of N-methylpyrrolidone stock solution III is accurately transferred, dissolved and diluted to the mark with the diluent, and shaken to obtain the spiked test solution, 1 μg / ml; The above blank solution, reference solution, test solution and spiked test solution are injected for detection, and the chromatogram is recorded.

[0020] Experimental results: The chromatogram of the blank solution is shown in Figure 1 .

[0021] The chromatogram of the N-methylpyrrolidone reference solution is shown in Figures 2-6 .

[0022] The chromatograms of the three batches of test sample solutions (K230401, K230501, K230601) are shown in Figures 7-9 .

[0023] The chromatograms of the three batches of spiked test sample solutions (K2304011, K2305011, K2306011) are shown in Figures 10-12 .

[0024] The system suitability results of the reference sample solutions are shown in Table 2 below.

[0025] Table 2 In Table 2, the results of five injections of the same reference sample solution for Reference Sample Solution-1 to Reference Sample Solution-5.

[0026] The detection results of the three batches of test samples (K230401, K230501, K230601, which are the results of three injections of the same test sample solution) are shown in Table 3 below.

[0027] Table 3 The detection results of the three batches of spiked test samples (K2304011, K2305011, K2306011, which are the results of three injections of the same spiked test sample solution) are shown in Table 4 below.

[0028] Table 4 Result analysis: The blank solution does not interfere with the detection, the RSD of the retention time of five consecutive injections of the reference sample solution is less than 1.0%, the RSD of the peak area is less than 10.0%, and the signal-to-noise ratio is greater than 20, the system suitability is good, and the sensitivity is good; N-methylpyrrolidone is not detected in the three batches of carfilzomib intermediates, and the recovery rate of N-methylpyrrolidone in the three batches of spiked test sample solutions is 99.43-103.10%, which meets the requirement of 80-120%, and the accuracy is good.

[0029] The above results prove that at a limit concentration of 1 μg / ml (100 ppm), the detection sensitivity of N-methylpyrrolidone meets the requirements, and the detection results are accurate and reliable.

[0030] Parts or structures not specifically described in the present application can be implemented using existing technologies or existing products, which will not be described here.

[0031] The above merely illustrates the embodiments of the present application, and does not limit the patent scope of the present application, and any equivalent structure or equivalent process transformation, or direct or indirect application in other related technical fields, are also included in the patent protection scope of the present application.

Claims

1. A method for the detection of N-methylpyrrolidone in a carfilzomib intermediate, characterized in that, The method comprises the following steps: High performance liquid chromatography is used to realize reverse phase separation by using ZORBAX Eclipse C18 porous ball filler chromatographic column, and quantitative analysis is carried out by using a diode array detector; Chromatographic conditions: The mobile phase A is water, and the elution ratio is 20-60% (V / V); The mobile phase B is methanol, and A phase and B phase are gradient eluted; The flow rate is 0.8-1.0 ml / min; The column temperature is 25-40 DEG C; The detection wavelength is 205-220 nm; The injection amount is 10-20 ul; The diluent is 50-70% (V / V) methanol aqueous solution.

2. The method for detecting N-methylpyrrolidone in a castanospermine intermediate according to claim 1, characterized in that, The gradient elution parameters are: 。 3. The method for detecting N-methylpyrrolidone in a castanospermine intermediate according to claim 1, wherein, The chromatographic column is Agilent ZORBAX Eclipse C18, the filler is porous octadecylsilane bonded silica gel, the specification is 4.6*250 mm, 5 um.

4. The method of claim 1, wherein the intermediate of carfilzomib is characterized by the presence of N-methylpyrrolidone. The flow rate is 1.0 ml / min.

5. The method of claim 1, wherein the intermediate of carfilzomib is characterized by the presence of N-methylpyrrolidone. The column temperature is 30 DEG C.

6. The method of claim 1, wherein the detection of N-methylpyrrolidone in a candidate intermediate of carfilzomib is characterized by, The detection wavelength is 210 nm.

7. The method of claim 1, wherein the intermediate of carfilzomib is characterized by the presence of N-methylpyrrolidone. The injection amount is 10 ul.

8. The method of claim 1, wherein the detection of N-methylpyrrolidone in a casozol intermediate is characterized by, The diluent is 60% methanol aqueous solution (V / V).

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