Method for detecting non-amino acid impurities in polymyxin B sulfate for injection

Abstract

The invention discloses a method for detecting non-amino acid impurities in polymyxin B sulfate for injection, and belongs to the technical field of medical analysis. According to the method, high performance liquid chromatography is adopted, and under the chromatographic conditions, a chromatographic column is an octadecyl silane bonded silica gel chromatographic column; the mobile phase comprises a mobile phase A and a mobile phase B, the mobile phase A is a potassium hexafluorophosphate buffer solution, the mobile phase B is acetonitrile, and the volume ratio of the mobile phase A to the mobile phase B is (50-60): (50-40). Potassium hexafluorophosphate is adopted as a mobile phase A, so that the peak trailing of impurities can be effectively improved, and the sensitivity of the method is improved; according to the method, a mobile phase with a high organic phase proportion is adopted, polymyxin sulfate B1, B2, B3 and B-1 and related amino acid impurities have peaks in dead volume, and detection of non-amino acid impurities is not interfered; the mobile phase system adopted by the invention has no ultraviolet absorption at 200 nm or above, so that the noise of the mobile phase is reduced, and the sensitivity and accuracy of the method are improved.

Description

Technical Field

[0001] The invention belongs to the technical field of pharmaceutical analysis, and particularly relates to a method for detecting non-amino acid impurities in polymyxin B for injection. Background Art

[0002] Polymyxin B sulfate is an antibiotic for treating extensively drug-resistant Gram-negative bacterial infections. It changes the permeability of bacterial cell membranes and causes cell death. It is effective against Gram-negative bacteria, especially multidrug-resistant (MDR) Gram-negative bacteria. Polymyxin B sulfate is a multi-component product, including four components: B1, B2, B3 and B1-I. Its structural formula is as follows (I):

[0003]

[0004] Polymyxin sulfate B1, B2, B3 and B1-I are products in which R, R', X and Y in formula (I) are selected from different groups, as shown in the following table:

[0005]

[0006] At the same time, according to the combination of R and R', there will be four degradation situations in the polymyxin B sulfate raw material. Since the structures of B1 and B1-1 are the same, there are only three non-amino acid impurities: impurity A, impurity B, and impurity C. The specific situation is described in the following table:

[0007]

[0008] Polymyxin B sulfate can usually be made into an injection preparation. Polymyxin B sulfate for injection shows certain efficacy in the treatment of urinary tract infections (UTI), especially for multi-drug resistant and extensively drug-resistant Gram-negative bacilli. There are reports on the detection methods of amino acid impurities D17 and D14 in polymyxin B sulfate for injection, but there are no reports on the corresponding detection methods of non-amino acid impurities. At present, the pharmacopoeias of various countries do not include the analytical detection methods of such impurities. Therefore, it should be controlled according to ICHQ3B (R2) <Impurities in New Drug Preparations>, and the limit should be controlled at ≤ 0.2%. The boiling points of the three non-amino acid impurities are all above 230°C, and they are difficult to vaporize and atomize, and cannot be detected using FID or CAD detectors. Considering that this type of impurities has weak ultraviolet absorption and low polarity, the use of ultraviolet detectors for detection requires extremely high detection sensitivity. The chromatographic conditions of polymyxin-related substances in existing reports cannot elute this type of impurities. Therefore, in order to effectively control the non-amino acid impurities in polymyxin B sulfate for injection, it is of great significance to develop a method with high detection sensitivity and strong elution ability. Summary of the invention

[0009] The purpose of the present invention is to establish a detection method which has good separation, high sensitivity, high accuracy, convenient operation, can stably and reliably detect non-amino acid impurities in polymyxin B sulfate for injection, and can better control product quality.

[0010] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0011] A method for determining non-amino acid impurities in polymyxin B sulfate for injection adopts a high performance liquid chromatography method. In the chromatographic conditions, the chromatographic column is an octadecylsilane bonded silica gel chromatographic column; the mobile phase comprises a mobile phase A and a mobile phase B, the mobile phase A is a potassium hexafluorophosphate buffer, the mobile phase B is acetonitrile, and the volume ratio of the mobile phase A to the mobile phase B is 50-60:50-40.

[0012] Furthermore, the non-amino acid impurities include 6-methyloctanoic acid, 6-methylheptanoic acid and octanoic acid.

[0013] Furthermore, the concentration of the potassium hexafluorophosphate buffer is 5 to 15 mmol / L, preferably 10 mmol / L.

[0014] Furthermore, the pH value of the potassium hexafluorophosphate buffer is 2.0 to 2.6, preferably 2.3; in some embodiments, phosphoric acid is used to adjust the pH value of the potassium hexafluorophosphate buffer.

[0015] Further, the chromatographic column is a Sepax HP-C18 column. Preferably, the Sepax HP-C18 column has the following specifications: inner diameter × column length × particle size: 4.6 mm × 150 mm × 3 μm;

[0016] Furthermore, the HPLC chromatographic conditions also include:

[0017] The column temperature is 30-40°C, the flow rate is 0.8-1.2 ml / min; the detection wavelength is 208-212 nm; the injection volume is 50-100 μl;

[0018] Further, the column temperature is preferably 35°C;

[0019] Further, the flow rate is preferably 1.0 ml / min;

[0020] Further, the detection wavelength is preferably 210 nm;

[0021] Further: the injection volume is preferably 100 μl;

[0022] Furthermore, the method for determining non-amino acid impurities in polymyxin B sulfate for injection specifically comprises the following steps:

[0023] (1) Solution preparation: Prepare the solvent (blank solution), reference solution, and test solution separately;

[0024] (2) Detection method: Set the chromatographic conditions, wait until the system is stable, inject the solvent, reference solution, and test solution into the chromatograph, and record the chromatogram;

[0025] (3) Calculate the impurity content using the calculation formula, which is as follows:

[0026]

[0027] Where:

[0028] F—response factor of each impurity;

[0029] A t —The peak area of ​​each impurity peak in the test solution;

[0030] D t —Dilution multiple of the test solution;

[0031] W t —Weighing amount of the test sample, mg.

[0032] In the step (1), the solvent is a mixture of water and acetonitrile, and the volume ratio of water to acetonitrile is 85-75:15-25; in some embodiments, the volume ratio of water to acetonitrile is 80:20.

[0033] The reference substance solution in step (1) is a solution of reference substances of impurities A, B, and C dissolved in a solvent, wherein the concentration is about 24 μg to 36 μg of each of impurities A, B, and C per 1 ml.

[0034] In the step (1), the test solution is an appropriate amount of polymyxin B sulfate for injection, and its solubility is about 12 mg to 18 mg of polymyxin B sulfate per 1 ml.

[0035] Beneficial Effects

[0036] 1. The mobile phase system used in the present invention has no ultraviolet absorption above 200nm, which reduces the noise of the mobile phase and improves the sensitivity of the method; compared with the phosphoric acid system, the use of potassium hexafluorophosphate as mobile phase A can effectively improve the peak tailing of impurities and increase the sensitivity of the method.

[0037] 2. The present invention adopts a mobile phase with a relatively high proportion of organic phase, and polymyxin sulfate B1, B2, B3, B-1 and related amino acid impurities peak in the dead volume, which does not interfere with the detection of non-amino acid impurities; and the potassium hexafluorophosphate solution is miscible with acetonitrile, avoiding the risk of salting out caused by the mixing of sodium sulfate or phosphate system with acetonitrile, and is not easy to damage the chromatographic column.

[0038] 3. The method of the present invention can effectively detect non-amino acid impurities for injection, with strong specificity (no interference from blank solvent and test solution), high sensitivity (quantification limit of 0.04%, equivalent to 20% of the limit), good linearity, high accuracy (spike recovery rate of 98.3% to 102.7%), high precision and good durability. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 : Ultraviolet absorption diagram of impurity A in the method of the present invention in Example 1;

[0040] Figure 2 : Ultraviolet absorption diagram of impurity B in the method of the present invention in Example 1;

[0041] Figure 3 : Ultraviolet absorption diagram of impurity C in the method of the present invention in Example 1;

[0042] Figure 4 : Spectrum of reference solution of the method of the present invention in Example 1;

[0043] Figure 5 : Comparison of the solvent, test solution and reference solution of the method of the present invention in Example 1;

[0044] Figure 6 : Spectrum of quantitative limit solution of the method of the present invention in Example 2;

[0045] Figure 7 : Spectrum of the reference solution of the method of the present invention in Comparative Example 1;

[0046] Figure 8 : Spectrum of the reference solution of the method of the present invention in Comparative Example 2;

[0047] Fig. 9 : Spectrum of the reference solution of the method of the present invention in Comparative Example 3;

[0048] Fig.10 : Spectrum of reference solution of the method of the present invention in Comparative Example 4. DETAILED DESCRIPTION

[0049] In order to enable those skilled in the art to better understand the technical solution of the present invention, the present invention is described in detail below through specific embodiments, but the content and scope of the present invention are not limited by the following embodiments.

[0050] The reagents used in the present invention can be purchased from the market or prepared by the method described in the present invention. The pH value of mobile phase A in each example is adjusted by phosphoric acid.

[0051] Example 1 Specificity Test

[0052] Solvent: Take 800 ml of water, add 200 ml of acetonitrile and mix well.

[0053] Preparation of test solution: Accurately weigh about 150 mg of polymyxin B sulfate for injection test sample, place it in a 10 ml volumetric flask, add solvent and sonicate to dissolve it, then dilute it to the scale with solvent and shake well.

[0054] Preparation of reference solution: Accurately weigh 30 mg of each impurity A, B, and C reference substance, place in a 100 ml volumetric flask, add solvent and sonicate to dissolve, then dilute to the scale with solvent and shake well; accurately transfer 5 ml, place in a 50 ml volumetric flask, dilute to the scale with solvent and shake well.

[0055] The solvent, reference solution, and test solution were injected respectively, and the detection conditions were as follows: chromatographic column: Sepax HP-C18 column; mobile phase: 10mmol / L potassium hexafluorophosphate buffer (pH2.3)-acetonitrile (volume ratio of 55:45), flow rate: 1.0ml / min; column temperature: 35℃; detection wavelength: 210nm; injection volume: 100μl. The test results are shown in Table 1 and Figures 1 to 5 As shown, Table 1 is the test results of the reference solution, Figure 1-3 They are the UV absorption diagrams of impurity A, impurity B, and impurity C, respectively. Figure 4 is the reference solution spectrum, Figure 5 It is a comparison chart of solvent, test solution and reference solution.

[0056] Table 1 Determination results of reference solution

[0057] name Retention time (min) Separation Impurity B 8.402 - Impurity C 9.124 3.1 Impurity A 13.135 12.7

[0058] The test results show that the impurities in the reference solution are well separated, and there are no impurity peaks in the solvent and the test solution to interfere with the detection of impurities A, impurity B, and impurity C. This method has good specificity.

[0059] Example 2 Detection Limit and Quantification Limit

[0060] Solvent: Take 800 ml of water, add 200 ml of acetonitrile and mix well.

[0061] Preparation of quantitative limit solution: Accurately weigh 30 mg of impurity A, B, and C reference substances, place in a 100 ml volumetric flask, add solvent and sonicate to dissolve, then dilute to the scale with solvent and shake well; accurately transfer 5 ml, place in a 50 ml volumetric flask, dilute to the scale with solvent and shake well; accurately transfer 5 ml again, place in a 25 ml volumetric flask, dilute to the scale with solvent and shake well.

[0062] Preparation of detection limit solution: Accurately pipette 3 ml of quantitation limit solution into a 10 ml volumetric flask, dilute to the mark with solvent, and shake well.

[0063] The quantitative limit solution and the detection limit solution were injected separately, and the detection conditions were as follows: chromatographic column: Sepax HP-C18 column; mobile phase: 10mmol / L potassium hexafluorophosphate buffer (pH2.3)-acetonitrile (volume ratio of 55:45), flow rate: 1.0ml / min; column temperature: 35℃; detection wavelength: 210nm; injection volume: 100μl. The test results, detection limit and quantitative limit results are shown in Table 2, and the quantitative limit solution spectrum is shown in Figure 6 shown.

[0064] Table 2 Results of detection limit and quantification limit

[0065]

[0066]

[0067] The test results show that the quantitative limit and detection limit of each impurity are equivalent to 0.04% and 0.012% of the test sample, the detection limit and quantitative limit are low, and the method has high sensitivity.

[0068] Example 3 Linear Range

[0069] Solvent: Take 800 ml of water, add 200 ml of acetonitrile and mix well.

[0070] Preparation of linear stock solution: Accurately weigh 30 mg of impurity A, B, and C reference substances, place in a 100 ml volumetric flask, add solvent and sonicate to dissolve, then dilute to the scale with solvent and shake well.

[0071] Linear-200% concentration solution: Accurately pipette 5 ml of the linear stock solution into a 25 ml volumetric flask, dilute to the mark with solvent, and shake well.

[0072] Linear-100% concentration solution: Accurately pipette 5 ml of the linear stock solution into a 50 ml volumetric flask, dilute to the mark with solvent, and shake well.

[0073] Linear-80% concentration solution: Accurately pipette 4 ml of the linear stock solution into a 50 ml volumetric flask, dilute to the mark with solvent, and shake well.

[0074] Linear-50% concentration solution: Accurately pipette 2.5 ml of the linear stock solution into a 50 ml volumetric flask, dilute to the mark with solvent, and shake well.

[0075] Linearity-quantitation limit solution: Accurately pipette 1 ml of the linearity stock solution into a 50 ml volumetric flask, dilute to the mark with solvent, and shake well.

[0076] The above solutions were injected respectively, and the detection conditions were as follows: chromatographic column: Sepax HP-C18 column; mobile phase: 10mmol / L potassium hexafluorophosphate buffer (pH2.3)-acetonitrile (volume ratio of 55:45), flow rate: 1.0ml / min; column temperature: 35℃; detection wavelength: 210nm; injection volume: 100μl. The test results are shown in Table 3.

[0077] Table 3 Linear range results

[0078]

[0079]

[0080] The results show that the linear relationship of each impurity is good in the range of 0.04% to 0.4%.

[0081] Example 4 Accuracy Test

[0082] Solvent: Take 800 ml of water, add 200 ml of acetonitrile and mix well.

[0083] Preparation of accuracy stock solution: Accurately weigh 30 mg of impurity A, B, and C reference substances, place in a 100 ml volumetric flask, add solvent and sonicate to dissolve, then dilute to the scale with solvent and shake well.

[0084] Quantitative limit accuracy solution: Take 0.75g of polymyxin B sulfate for injection test sample, accurately weigh it, put it in a 50ml volumetric flask, add solvent and ultrasonicate to dissolve it, accurately add 1ml of accuracy stock solution, then dilute it to the scale with solvent and shake well.

[0085] 100% accuracy solution: Take 0.75g of polymyxin B sulfate injection test sample, accurately weigh it, put it in a 50ml volumetric flask, add solvent and ultrasonicate to dissolve it, accurately add 5ml of accuracy stock solution, then dilute it to the scale with solvent and shake well.

[0086] 150% accuracy solution: 0.75 g of polymyxin B sulfate injection test sample is accurately weighed and placed in a 50 ml volumetric flask. Solvent is added and ultrasonically dissolved. Accurately add 7.5 ml of accuracy stock solution, dilute to the scale with solvent, and shake well.

[0087] The above solutions were injected respectively, and the detection conditions were as follows: chromatographic column: Sepax HP-C18 column; mobile phase: 10mmol / L potassium hexafluorophosphate buffer (pH2.3)-acetonitrile (volume ratio of 55:45), flow rate: 1.0ml / min; column temperature: 35℃; detection wavelength: 210nm; injection volume: 100μl. The test results are shown in Table 4.

[0088] Table 4 Accuracy results

[0089]

[0090]

[0091] The results showed that the recoveries of each impurity at different concentration levels were between 98.3% and 102.7%, indicating that the method had high accuracy.

[0092] Example 5 Precision

[0093] Solvent: Take 800 ml of water, add 200 ml of acetonitrile and mix well.

[0094] Preparation of precision stock solution: Accurately weigh 30 mg of impurity A, B, and C reference substances, place in a 100 ml volumetric flask, add solvent and sonicate to dissolve, then dilute to the scale with solvent and shake well.

[0095] Precision solution: Take 0.75 g of polymyxin B sulfate for injection test sample, accurately weigh it, put it in a 50 ml volumetric flask, add solvent and sonicate to dissolve it, accurately add 5 ml of precision stock solution, then dilute to the scale with solvent and shake well.

[0096] The above solutions were injected respectively, and the detection conditions were as follows: chromatographic column: Sepax HP-C18 column; mobile phase: 10mmol / L potassium hexafluorophosphate buffer (pH2.3)-acetonitrile (volume ratio of 55:45), flow rate: 1.0ml / min; column temperature: 35℃; detection wavelength: 210nm; injection volume: 100μl. The content of each impurity was calculated by the calculation formula, and the results are shown in Table 5.

[0097] Table 5 Repeatability results

[0098] Solution to be tested Impurity B(%) Impurity A(%) Impurity C(%) Precision Solution-1 0.19 0.21 0.20 Precision Solution-2 0.20 0.21 0.19 Precision Solution-3 0.19 0.21 0.20 Precision Solution-4 0.20 0.20 0.20 Precision Solution-5 0.19 0.21 0.20 Precision Solution-6 0.20 0.21 0.20 RSD 2.9％ 2.0％ 2.1％

[0099] The results showed that the RSDs of the repeatability of each impurity were 2.9%, 2.0% and 2.1% respectively, and the detection precision of this method was high.

[0100] Example 6 Durability Test

[0101] Solvent: Take 800 ml of water, add 200 ml of acetonitrile and mix well.

[0102] Preparation of impurity stock solution: Accurately weigh 30 mg of impurity A, B, and C reference substances, place in a 100 ml volumetric flask, add solvent and sonicate to dissolve, then dilute to the scale with solvent and shake well.

[0103] Reference solution: Accurately add 5 ml of impurity stock solution, place in a 50 ml volumetric flask, dilute to the scale with solvent, and shake well.

[0104] Spiked test solution: Take 0.75 g of polymyxin B sulfate for injection test sample, accurately weigh it, put it in a 50 ml volumetric flask, add solvent and ultrasonicate to dissolve it, accurately add 5 ml of impurity stock solution, then dilute to the scale with solvent and shake well.

[0105] Change the chromatographic conditions (flow rate, column temperature, buffer salt concentration, buffer salt pH, chromatographic column), inject and test the reference solution and the test solution, and record the results, as shown in Tables 6 and 7.

[0106] Table 6 Durability-Separation Results

[0107]

[0108] Table 7 Durability - Test Results

[0109]

[0110]

[0111] The results showed that the method had good robustness in the presence of slight changes in different chromatographic parameters.

[0112] Comparative Example 1

[0113] Solvent: Take 800 ml of water, add 200 ml of acetonitrile and mix well.

[0114] Preparation of reference solution: Accurately weigh 30 mg of each impurity A, B, and C reference substance, place in a 100 ml volumetric flask, add solvent and sonicate to dissolve, then dilute to the scale with solvent and shake well; accurately transfer 5 ml, place in a 50 ml volumetric flask, dilute to the scale with solvent and shake well.

[0115] The reference solution was injected for detection under the following conditions: chromatographic column: Sepax HP-C18 column; mobile phase: a mixed solution of mobile phase A and mobile phase B with a volume ratio of 55:45, mobile phase A is a 0.1% formic acid aqueous solution, and mobile phase B is acetonitrile; flow rate: 1.0 ml / min; column temperature: 35°C; CAD detector; injection volume: 100 μl. The detection spectrum is as follows Figure 7 shown.

[0116] The results show that there is no response to each impurity under the CAD detector.

[0117] Comparative Example 2

[0118] Solvent: Take 800 ml of water, add 200 ml of acetonitrile and mix well.

[0119] Preparation of reference solution: Accurately weigh 30 mg of each impurity A, B, and C reference substance, place in a 100 ml volumetric flask, add solvent and sonicate to dissolve, then dilute to the scale with solvent and shake well; accurately transfer 5 ml, place in a 50 ml volumetric flask, dilute to the scale with solvent and shake well.

[0120] The reference solution was injected for detection under the following conditions: chromatographic column: Sepax HP-C18 column; mobile phase: a mixed solution of mobile phase A and mobile phase B with a volume ratio of 55:45, mobile phase A is a 0.1% formic acid aqueous solution, and mobile phase B is acetonitrile; flow rate: 1.0 ml / min; column temperature: 35°C; detection wavelength: 210 nm; injection volume: 100 μl. The detection spectrum is as follows Figure 8 shown.

[0121] The results showed that in this system, the baseline noise was large, which affected the sensitivity of detection.

[0122] Comparative Example 3

[0123] Solvent: Take 800 ml of water, add 200 ml of acetonitrile and mix well.

[0124] Preparation of reference solution: Accurately weigh 30 mg of each impurity A, B, and C reference substance, place in a 100 ml volumetric flask, add solvent and sonicate to dissolve, then dilute to the scale with solvent and shake well; accurately transfer 5 ml, place in a 50 ml volumetric flask, dilute to the scale with solvent and shake well.

[0125] The reference solution was injected for detection under the following conditions: chromatographic column: Sepax HP-C18 column; mobile phase: a mixed solution of mobile phase A and mobile phase B with a volume ratio of 55:45, mobile phase A is a 0.1% phosphoric acid aqueous solution, and mobile phase B is acetonitrile; flow rate: 1.0 ml / min; column temperature: 35°C; detection wavelength: 210 nm; injection volume: 100 μl. The detection spectrum is as follows Fig. 9 shown.

[0126] The results show that under this system, the tailing factor of impurity B is 2.2, which does not meet the requirements.

[0127] Comparative Example 4

[0128] Solvent: Take 800 ml of water, add 200 ml of acetonitrile and mix well.

[0129] Preparation of reference solution: Accurately weigh 30 mg of each impurity A, B, and C reference substance, place in a 100 ml volumetric flask, add solvent and sonicate to dissolve, then dilute to the scale with solvent and shake well; accurately transfer 5 ml, place in a 50 ml volumetric flask, dilute to the scale with solvent and shake well.

[0130] The reference solution was injected for detection under the following conditions: chromatographic column: Sepax HP-C18 column; mobile phase: 30mmol / L sodium sulfate solution (pH adjusted to 2.3 with phosphoric acid)-acetonitrile (volume ratio 70:30), flow rate: 1.0ml / min; column temperature: 35℃; detection wavelength: 210nm; injection volume: 100μl. The detection spectrum is as follows Fig.10 shown.

[0131] The results showed that in this system, the method for related substances of Polymyxa sulfate B for injection in the Chinese Pharmacopoeia was used as a reference, and the proportion of acetonitrile in the mobile phase was increased from 20% to 30%, but impurities C and A were still not eluted.

Claims

1. A method for determining non-amino acid impurities in polymyxin B sulfate for injection, characterized in that: The high performance liquid chromatography method is adopted. Among the chromatographic conditions, the chromatographic column is an octadecylsilane bonded silica gel chromatographic column; the mobile phase includes mobile phase A and mobile phase B, mobile phase A is potassium hexafluorophosphate buffer, mobile phase B is acetonitrile, and the volume ratio of mobile phase A to mobile phase B is 50-60:50-40.

2. The measuring method according to claim 1, characterized in that The non-amino acid impurities include 6-methyloctanoic acid, 6-methylheptanoic acid and octanoic acid.

3. The measuring method according to claim 1, characterized in that The concentration of the potassium hexafluorophosphate buffer is 5-15 mmol / L.

4. The measuring method according to claim 3, characterized in that The pH value of the potassium hexafluorophosphate buffer is 2.0-2.

6.

5. The measuring method according to claim 1, characterized in that The chromatographic column is a Sepax HP-C18 column.

6. The measuring method according to claim 5, characterized in that The specifications of the Sepax HP-C18 column are inner diameter×column length×particle diameter: 4.6 mm×150 mm×3 μm.

7. The assay method according to any one of claims 1 to 6, characterized in that The chromatographic conditions of the high performance liquid chromatography method also include: column temperature of 30-40° C., flow rate of 0.8-1.2 ml / min; detection wavelength of 208-212 nm; injection volume of 50-100 μl.

8. The measuring method according to claim 7, characterized in that: The chromatographic conditions of the high performance liquid chromatography method also include: column temperature of 35° C., flow rate of 1.0 ml / min; detection wavelength of 210 nm; injection volume of 100 μl.

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