A method for detecting DCC and DCU residues in docetaxel raw materials

Abstract

The invention relates to a detection method for residual DCC and DCU in a docetaxel raw material. The method uses a high performance liquid chromatography method to detect DCC and DCU in the docetaxel raw material. The method comprises the following steps: 1, preparing a test solution of the docetaxel raw material; 2, preparing a DCC reference solution and a DCU reference solution; 3, injecting samples, and detecting the test solution in step 1 and the reference solution in step 2 respectively according to the following conditions: chromatographic conditions: mobile phase: acetonitrile-water; gradient elution: the acetonitrile ratio in 0-5min is 15% isocratic washing; the acetonitrile ratio in 0-30min is gradually increased from 15% to 55%, and the acetonitrile ratio in 30min-60min is increased from 55% to 85%; the detection wavelength is 205nm; 4, the residual DCC and DCU in the test sample are calculated by using an external standard method based on the peak area of ​​the reference solution. The detection method for the content of DCC and DCU in the docetaxel raw material provided by the invention has low toxicity of the reagents used, less interference from impurities, and reliable and stable detection method.

Description

Technical Field

[0001] The present invention relates to the technical field of drug analysis, and in particular to a method for detecting DCC and DCU residues in docetaxel raw materials. Background Art

[0002] Docetaxel, also known as docetaxel, is an anti-tumor drug belonging to the taxane class of compounds. Its mechanism of action is to enhance microprotein polymerization and inhibit microtubule depolymerization, leading to the formation of stable non-functional microtubule bundles, thereby destroying the mitosis of tumor cells.

[0003] Currently, docetaxel is obtained by semi-synthesis from the natural extract 10-deacetylbaccatin III. In this synthesis process, DCC (dicyclohexylcarbodiimide) is used. Since DCC is unstable, it will degrade to form DCU (dicyclohexylurea). For safety reasons, it is very important to control the residues of DCC and DCU in docetaxel raw materials.

[0004] Currently, there is a Chinese patent with the publication number CN110749677A entitled A method for detecting N,N'-dicyclohexylurea and N,N'-dicyclohexylcarbodiimide. About 1g of sample is accurately weighed into a volumetric flask, extracted with methanol and diluted to the mark, and mixed. A portion of the supernatant is taken, diluted with acetone, filtered with an organic filter membrane, and analyzed according to the set gas chromatography conditions and mass spectrometry conditions. The retention time and qualitative ion are used for qualitative analysis, and the external standard method and quantitative ion are used for quantitative analysis. N,N'-dicyclohexylurea and N,N'-dicyclohexylcarbodiimide are qualitatively and quantitatively detected simultaneously.

[0005] However, docetaxel can only be slightly soluble in methanol. During the mass spectrometry analysis, a certain amount of docetaxel will precipitate, resulting in a decrease in docetaxel in the test solution, which in turn leads to an increase in the detection error of residual DCC and DCU in the docetaxel raw material. Summary of the invention

[0006] In view of the shortcomings of the above-mentioned prior art, the purpose of the present invention is to provide a method for detecting DCC and DCU residues in docetaxel raw materials, which has the advantages of good separation of DCC and DCU from impurities, strong specificity, high detection sensitivity, and avoiding excessive premature precipitation of docetaxel, thereby reducing detection errors.

[0007] The above technical objectives of the present invention are achieved through the following technical solutions:

[0008] A method for detecting DCC and DCU residues in docetaxel raw materials comprises the following steps:

[0009] Step S1, preparing a docetaxel API test solution;

[0010] Step S2, preparing DCU and DCC reference solutions respectively;

[0011] Step S3, performing chromatographic detection on the docetaxel API test solution, using a mobile phase of acetonitrile and water; gradient elution: isocratic elution with acetonitrile at a ratio of 15% from 0 to 5 minutes, isocratic elution with the ratio of acetonitrile gradually increasing from 15% to 55% from 5 to 30 minutes, and isocratic elution with the ratio of acetonitrile gradually increasing from 55% to 85% from 30 to 65 minutes; the column temperature range is 40 to 45° C.; the flow rate range is 1.0 to 1.4 ml / min; and the detection wavelength range is 200 to 210 nm.

[0012] Furthermore, in step S3, the chromatographic column is a pentafluorophenyl column.

[0013] Furthermore, in step S3, the specification of the chromatographic column is 250*4.6 mm, 3 μm.

[0014] Furthermore, in step S3, the injection volume is 20 μl.

[0015] Further, in step S3, the detection wavelength is 205 nm.

[0016] Furthermore, in step S1, the concentration of the injected test solution is 10 mg / ml.

[0017] Furthermore, in step S2, the concentration of the DCC reference solution is 3 μg / ml.

[0018] Further, in step S2, the concentration of the DCU reference solution is 3 μg / ml.

[0019] Furthermore, in step S1, the diluents used for the docetaxel API test solution are acetonitrile, tetrahydrofuran and water, and the ratio of acetonitrile:tetrahydrofuran:water is 1:0.1:1 (V / V / V).

[0020] Furthermore, in step S2, the diluents used for the DCC reference solution and the DCU reference solution are acetonitrile, tetrahydrofuran and water, and the ratio of acetonitrile:tetrahydrofuran:water is 1:0.1:1 (V / V / V).

[0021] In summary, the present invention has the following beneficial effects:

[0022] 1. The chromatographic column used is a pentafluorophenyl column, the mobile phase is acetonitrile and water, the chromatographic stationary phase is polar, the mobile phase is non-polar, and because other substances exist in the docetaxel raw material test solution in addition to DCC and DCU, a gradient elution method is adopted. In this process, the recording and analysis are completed after the DCC peak, and then the composition ratio of the mobile phase is changed to make DCU flow out faster, which not only improves the peak shape and column efficiency of each substance, but also improves the separation degree of DCC and DCU from impurities, makes the detection result error smaller, and greatly improves the detection sensitivity.

[0023] 2. Strictly control the chromatographic column specifications and injection volume, and strictly control the detection wavelength range, within which DCU and DCC are located.

[0024] 3. Docetaxel API test solution, DCU and DCC reference solutions are all used.

[0025] 4. The reagents used are of low toxicity. Acetonitrile vapor is mildly irritating and can cause respiratory irritation symptoms at high concentrations. Therefore, the column temperature is controlled in the range of 40-45°C to reduce the generation of acetonitrile vapor and the impact on staff while ensuring the smooth flow of the mobile phase and the test solution.

[0026] 5. The test solution and the reference solution use a combination of acetonitrile, tetrahydrofuran and water as diluents to make the test solution and the reference solution close to the liquid phase of the mobile phase, so that the solution and the mobile phase are better soluble in each other, which is beneficial to the separation of impurities. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 is the chromatogram of the blank solution in Example 1.

[0028] Figure 2 It is the chromatogram of the reference solution in Example 1.

[0029] Figure 3 It is the chromatogram of the test solution in Example 1.

[0030] Figure 4 It is the chromatogram of the blank solution in Example 2.

[0031] Figure 5 It is the chromatogram of the reference solution in Example 2.

[0032] Figure 6 It is the chromatogram of the test solution in Example 2. DETAILED DESCRIPTION

[0033] In order to make the purpose, technical solution and advantages of the present invention more clear, the solution proposed by the present invention is further described in detail below in conjunction with the accompanying drawings and specific implementation methods. According to the following description, the advantages and features of the present invention will be more clear.

[0034] Embodiment 1:

[0035] Chromatographic conditions and instrument conditions:

[0036] Agilent 1260, DAD detector, Agilent Openlab CDS2.3 software system, chromatographic column: pentafluorophenyl column, 250mm×4.6mm, 3um, detection wavelength 205nm, injection volume 20ul, flow rate 1.2ml / min, column temperature 45℃,

[0037] Mobile phase A was water and mobile phase B was acetonitrile. The proportion of acetonitrile was 15% during isocratic washing from 0 to 5 min. The proportion of acetonitrile gradually increased from 15% to 55% during 0 to 30 min. The proportion of acetonitrile increased from 55% to 85% during 30 to 65 min.

[0038] Experimental steps:

[0039] Blank solution / diluent: acetonitrile: tetrahydrofuran: water = 1:0.1:1 (V / V / V)

[0040] Control solution: Accurately weigh an appropriate amount of DCC reference substance and dilute it with a diluent solvent to a solution containing approximately 3 μg DCC per ml as the control solution.

[0041] Mixed control solution: Accurately weigh appropriate amounts of intermediate impurities IIIA, IIIB, IIIC, IIID, docetaxel intermediate II, and DCU, add diluent to dissolve and dilute to prepare a solution containing about 10 μg of each impurity per ml as a mixed control solution.

[0042] Spiked test solution: Weigh an appropriate amount of docetaxel sample (docetaxel crude product), add the reference solution to dissolve and dilute to a solution containing about 10 mg per 1 ml, as the spiked test solution.

[0043] Test solution: Weigh an appropriate amount of docetaxel sample, add blank solution to dissolve and dilute to a solution containing about 10 mg per 1 ml, as the test solution.

[0044] Take the blank solution, reference solution and test solution respectively and record the chromatogram. Figure 1 , Figure 2 , Figure 3 .

[0045] In the test results, the blank solution does not interfere with the detection, the RSD value of the retention time of DCC in the reference solution for 5 consecutive times is less than 1%, the RSD value of the peak area is less than 10%, and the signal-to-noise ratio of DCC in the reference solution is greater than 10; the mixed reference solution does not interfere with the detection of DCC; the separation degree of DCC and adjacent impurities in the spiked test solution is greater than 2.0, and the recovery rate of DCC in the spiked test solution is between 90% and 110%. This method can effectively detect 0.03% of DCC residues in docetaxel raw materials. The results are shown in Tables 1 and 2.

[0046] Solution type RT(min) Area S / N Reference solution 1 49.058 14.281 18 Reference solution 2 49.008 16.359 32 Reference solution 3 48.958 15.801 34 Reference solution 4 48.989 15.099 16 Reference solution 5 49.059 14.662 23 RSD(%) 0.1 5.6 N / A

[0047] Table 1 DCC test results in reference solution

[0048]

[0049]

[0050] Table 2 Recovery test results of DCC in spiked test solution

[0051] Embodiment 2:

[0052] Chromatographic conditions and instrument conditions:

[0053] Agilent 1260, DAD detector, Agilent Openlab CDS2.3 software system, chromatographic column: pentafluorophenyl column, 250mm×4.6mm, 3um, detection wavelength 205nm, injection volume 20ul, flow rate 1.2ml / min, column temperature 45℃,

[0054] Mobile phase A was water and mobile phase B was acetonitrile. The proportion of acetonitrile was 15% during isocratic washing from 0 to 5 min. The proportion of acetonitrile gradually increased from 15% to 55% during 0 to 30 min. The proportion of acetonitrile increased from 55% to 85% during 30 to 65 min.

[0055] Experimental steps:

[0056] Blank solution / diluent: acetonitrile: tetrahydrofuran: water = 1:0.1:1 (V / V / V)

[0057] Control solution: Accurately weigh an appropriate amount of DCU reference substance and dilute it with a diluent solvent to a solution containing approximately 3 μg DCU per ml as the control solution.

[0058] Mixed control solution: Accurately weigh appropriate amounts of IIIA, IIIB, IIIC, IIID, docetaxel intermediate II, and DCU, add diluent to dissolve and dilute to prepare a solution containing about 10 μg of each impurity per ml, as a mixed control solution.

[0059] Spiked test solution: Weigh an appropriate amount of docetaxel sample, add the reference solution to dissolve and dilute to a solution containing about 10 mg per 1 ml, as the spiked test solution.

[0060] Test solution: Weigh an appropriate amount of docetaxel sample, add blank solution to dissolve and dilute to a solution containing about 10 mg per 1 ml, as the test solution.

[0061] Take the blank solution, reference solution and test solution respectively and record the chromatogram. Figure 4 , Figure 5 , Figure 6 .

[0062] In the test results, the blank solution did not interfere with the detection, the RSD value of the retention time of DCU in the reference solution for 5 consecutive times was less than 1%, the RSD value of the peak area was less than 10%, and the signal-to-noise ratio of DCU in the reference solution was greater than 10; the mixed reference solution did not interfere with the detection of DCU; the separation degree of DCU and adjacent impurities in the spiked test solution was greater than 2.0, and the recovery rate of DCC in the spiked test solution was between 90% and 110%. Detailed results are shown in Tables 3 and 4 below.

[0063] Solution type RT(min) Area S / N Reference solution 1 25.756 18.563 48 Reference solution 2 25.769 17.891 62 Reference solution 3 25.771 18.380 75 Reference solution 4 25.763 18.755 52 Reference solution 5 25.750 17.960 66 RSD(%) 0.1 2.1 N / A

[0064] Table 3 DCU test results in reference solution

[0065] Solution type Area Measured concentration C (μg / ml) Recovery rate (%) Spiked test solution 1 18.380 2.9628 100.42 Spiked test solution 2 18.731 3.0194 102.33 Spiked test solution 3 18.350 2.9580 100.25 RSD(%) N / A N / A 1.2

[0066] Table 4 DCU test results in reference solution

[0067] The technical features of the above-described embodiments may be arbitrarily combined. To make the description concise, not all possible combinations of the technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0068] The above-mentioned embodiments only express several implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. A method for detecting DCC and DCU residues in docetaxel API, characterized in that: The following steps are involved: Step S1, preparing a docetaxel API test solution; Step S2, preparing DCU and DCC reference solutions respectively; Step S3, performing chromatographic detection on the docetaxel API test solution, using a mobile phase of acetonitrile and water; gradient elution: isocratic elution with acetonitrile at a ratio of 15% from 0 to 5 minutes, isocratic elution with the ratio of acetonitrile gradually increasing from 15% to 55% from 5 to 30 minutes, and isocratic elution with the ratio of acetonitrile gradually increasing from 55% to 85% from 30 to 65 minutes; the column temperature range is 40 to 45° C.; the flow rate range is 1.0 to 1.4 ml / min; the detection wavelength range is 200 to 210 nm; In step S3, the chromatographic column is a pentafluorophenyl column; In step S3, the specifications of the chromatographic column are 250*4.6 mm, 3 μm.

2. The method for detecting DCC and DCU residues in docetaxel bulk drug according to claim 1, characterized in that: In step S3, the injection volume is 20 μl.

3. The method for detecting DCC and DCU residues in docetaxel bulk drug according to claim 1, characterized in that: In step S3, the detection wavelength is 205 nm.

4. The method for detecting DCC and DCU residues in docetaxel bulk drug according to claim 1, characterized in that: In step S1, the concentration of the injected test solution is 10 mg / ml.

5. The method for detecting DCC and DCU residues in docetaxel bulk drug according to claim 1, characterized in that: In step S2, the concentration of the DCC reference solution is 3 μg / ml.

6. The method for detecting DCC and DCU residues in docetaxel bulk drug according to claim 1, characterized in that: In step S2, the concentration of the DCU reference solution is 3 μg / ml.

7. The method for detecting DCC and DCU residues in docetaxel bulk drug according to claim 1, characterized in that: In step S1, the diluents used for the docetaxel API test solution are acetonitrile, tetrahydrofuran and water, and the volume ratio of acetonitrile:tetrahydrofuran:water is 1:0.1:

1.

8. The method for detecting DCC and DCU residues in docetaxel bulk drug according to claim 1, characterized in that: In step S2, the diluents used for the DCC reference solution and the DCU reference solution are acetonitrile, tetrahydrofuran and water, and the volume ratio of acetonitrile:tetrahydrofuran:water is 1:0.1:1.

Images