Method for detecting related substances in piperacillin sodium

Abstract

The invention provides a method for detecting related substances in piperacillin sodium. The octadecyl silane bonded silica gel chromatographic column is adopted for gradient elution, and quantitative detection of 25 impurities in piperacillin sodium is rapidly and accurately achieved by optimizing chromatographic column filler and specification, mobile phase concentration, gradient elution conditions and the like. Detection results show that all related substances are well separated from the main component piperacillin. Through methodology verification, the detection method provided by the invention has the advantages of high separation degree, accurate result and good stability, can realize detection of 25 impurities in piperacillin sodium within 33min, and can significantly improve the detection efficiency and reduce the detection cost. The 25 impurities cover related substances in the production, storage and stability test processes of piperacillin sodium. The detection method can be used as a stability indication method of piperacillin sodium, is used for quality control of piperacillin sodium, and has a wide application prospect.

Description

Technical Field

[0001] The invention relates to the technical field of analytical chemistry, in particular to a method for detecting impurities in drugs, and in particular to a method for detecting related substances in piperacillin sodium. Background Art

[0002] Piperacillin sodium is a penicillin antibiotic that kills bacteria by interfering with the synthesis of bacterial cell walls. It is widely used in the prevention and treatment of various infectious diseases caused by Gram-negative bacteria. Piperacillin sodium is a broad-spectrum penicillin antibiotic with a molecular formula of C 23 H 26 N5NaO7S, with a molecular weight of 539.54, has a chemical name of (2S,5R,6R)-3,3-dimethyl-6-[(R)-2-(4-ethyl-2,3-dioxo-1-piperazinecarboxamido)-2-phenylacetamido]-7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylic acid sodium salt, and has the following structural formula:

[0003]

[0004] ChP2020, USP-NF2024, and EP11 all contain the quality standards of piperacillin sodium. The chromatographic conditions for the determination of impurities related to piperacillin sodium in ChP2020 and USP-NF2024 are the same except for the wavelength. It is an isocratic elution method with a method duration of about 25 minutes, but the types of impurities included are few; there are only 2 specific impurities included in ChP2020, and only 4 specific impurities included in USP-NF2024. The method for the determination of related substances of piperacillin sodium included in EP11 is a gradient elution method, which can detect a total of 20 specific impurities, but the method duration is 85 minutes.

[0005] Document CN115469042A discloses a method for detecting related substances I in piperacillin sodium and tazobactam sodium for injection, which can effectively detect 10 impurities in piperacillin sodium, and the method takes 105 minutes. Document CN105911193B discloses a method for detecting related substances in piperacillin sodium and sulbactam sodium for injection, which can effectively detect 8 impurities in piperacillin sodium, and the method takes 10 minutes. Document (Yao Lan, Liu Yawei, Zhang Yajie, et al. Research on the determination method of related substances of piperacillin sodium / tazobactam sodium for injection [J]. Chinese Journal of Antibiotics, 2020, 45(03): 293-299.) discloses a method for determining related substances of piperacillin sodium / tazobactam sodium for injection, which can detect 13 known impurities in piperacillin sodium, and the method takes 40 minutes.

[0006] There are many types of impurities in piperacillin sodium, with large differences in polarity, which requires a high separation ability of the detection method. However, the existing detection methods have the defects of detecting few types of impurities or too long analysis time, which is not conducive to the quality control of piperacillin sodium. Therefore, the development of a fast and accurate method that can simultaneously detect multiple impurities in piperacillin sodium is of positive significance for ensuring the quality control of piperacillin sodium. Summary of the invention

[0007] The purpose of the present invention is to provide a method for detecting related substances in piperacillin sodium, so as to realize rapid quantitative detection of impurities in piperacillin sodium in a short time and provide a basis for quality control of piperacillin sodium.

[0008] The present invention is achieved in that:

[0009] A method for detecting related substances in piperacillin sodium, the method comprising the following steps:

[0010] (1) Prepare reference solution and test solution;

[0011] (2) Take the test sample solution and the reference sample solution for high performance liquid chromatography detection, obtain the chromatographic peaks and peak areas of the relevant substances, and calculate the impurity content;

[0012] The chromatographic conditions were as follows:

[0013] Chromatographic column: chromatographic column with octadecylsilane bonded silica gel as filler;

[0014] Mobile phase flow rate: 0.55~0.60mL / min;

[0015] Injection volume: 1.9 μL;

[0016] Detection wavelength: 220nm;

[0017] Column temperature: 40°C;

[0018] Mobile phase A: methanol-water-sodium dihydrogen phosphate dihydrate solution-tetrabutylammonium hydroxide solution;

[0019] Mobile phase B: methanol-water-sodium dihydrogen phosphate dihydrate solution-tetrabutylammonium hydroxide solution;

[0020] Mobile phase pH: 5.50 ± 0.02;

[0021] The gradient elution conditions were as follows:

[0022]

[0023] Furthermore, in the above step (2), the chromatographic column is Poroshell EC-C18.

[0024] Furthermore, in the above step (2), the filling specifications of the chromatographic column are 2.7 μm, 3.0*150 mm.

[0025] Furthermore, in the above step (2), the volume ratio of mobile phase A methanol-water-sodium dihydrogen phosphate dihydrate solution-tetrabutylammonium hydroxide solution is 275:621.7:100:3.3; the volume ratio of mobile phase B methanol-water-sodium dihydrogen phosphate dihydrate solution-tetrabutylammonium hydroxide solution is 615:281.3:100:3.7.

[0026] Furthermore, in the above step (2), the flow rate of the mobile phase is 0.55 mL / min.

[0027] Furthermore, in the above step (2), the concentration of the sodium dihydrogen phosphate dihydrate solution in the mobile phase is 31.2 g / L, and the molar concentration of the tetrabutylammonium hydroxide solution is 0.4 mol / L.

[0028] Furthermore, in the above step (2), the method for calculating the impurity content is the principal component self-comparison method with the addition of a correction factor.

[0029] Furthermore, the related substances include one or more of the following:

[0030]

[0031]

[0032] The present invention has the following beneficial effects:

[0033] (1) The method of the present invention has high separation, accurate results and good stability. The detectable impurities cover 25 impurities in the impurity spectrum of piperacillin sodium, including all specific impurities in ChP2020, USP-NF2024 and EP11 and impurities identified in non-pharmacopoeias. It is not only suitable for the detection of impurities actually existing in piperacillin sodium products, but also can be used for the detection of new impurities generated by drug degradation, and has a stability indication effect.

[0034] (2) The analysis time of the method of the present invention is only 33 minutes, which can significantly improve the detection efficiency.

[0035] (3) The method of the present invention also reduces reagent consumption and can effectively reduce detection costs.

[0036] The invention realizes the rapid detection of piperacillin sodium impurities, can be used for the quality control of piperacillin sodium, and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 It is the chromatogram of the impurity separation solution measured in Example 2.

[0038] Figure 2 It is the chromatogram of the test solution measured in Example 3.

[0039] Figure 3 It is the chromatogram of the identification solution measured in Comparative Example 1.

[0040] Figure 4 It is the chromatogram of the identification solution measured in Comparative Example 2.

[0041] Figure 5 This is the chromatogram of the identification solution measured in Comparative Example 3, corresponding to a concentration of sodium dihydrogen phosphate dihydrate solution in the mobile phase of 27.6 g / L.

[0042] Figure 6 This is the chromatogram of the identification solution measured in Comparative Example 3, corresponding to a concentration of sodium dihydrogen phosphate dihydrate solution in the mobile phase of 35.88 g / L.

[0043] Figure 7 This is the chromatogram of the impurity peak identification solution measured in Comparative Example 4. DETAILED DESCRIPTION

[0044] The present invention is further described below in conjunction with comparative examples and embodiments. The following embodiments are only for illustration and are not intended to limit the scope of protection of the present invention in any form.

[0045] Example 1

[0046] (1) Solution preparation

[0047] System suitability solution: Take 6 mg of piperacillin impurity A reference substance and 6 mg of piperacillin impurity I reference substance, place in a 20 mL volumetric flask, add mobile phase B to dissolve and dilute to scale, shake well. Take 2 mL of the above solution, place in a 10 mL volumetric flask, dilute to scale with mobile phase B, shake well. Prepare a mixed solution containing 0.06 mg of piperacillin impurity A and 0.06 mg of piperacillin impurity I per 1 mL.

[0048] Reference solution: Accurately weigh 30 mg of piperacillin reference substance, place in a 50 mL volumetric flask, add appropriate amount of methanol to dissolve, quantitatively dilute to scale with mobile phase B, and shake well; accurately measure 5 mL of the above solution, place in a 50 mL volumetric flask, quantitatively dilute to scale with mobile phase B, and shake well. 23 H 27 The concentration of N5O7S (calculated as 0.06 mg / mL) was 0.06 mg / mL.

[0049] (2) Chromatographic conditions

[0050] Instrument: High performance liquid chromatograph, Agilent 1260

[0051] Chromatographic column: Agilent Poroshell EC-C18 (2.7μm, 3.0*150mm)

[0052] Flow rate: 0.55mL / min

[0053] Injection volume: 1.9 μL

[0054] Detection wavelength: 220nm

[0055] Column temperature: 40°C

[0056] Mobile phase A: methanol-water-31.2 g / L sodium dihydrogen phosphate dihydrate solution-0.4 mol / L tetrabutylammonium hydroxide solution, volume ratio 275:621.7:100:3.3, pH value was adjusted to 5.50±0.02 with phosphoric acid.

[0057] Mobile phase B: methanol-water-31.2 g / L sodium dihydrogen phosphate dihydrate solution-0.4 mol / L tetrabutylammonium hydroxide solution, volume ratio 615:281.3:100:3.7, pH value was adjusted to 5.50±0.02 with phosphoric acid.

[0058] Gradient elution program:

[0059]

[0060]

[0061] (3) Impurity quantification method:

[0062]

[0063] Where: Bi——impurity content in the solution to be tested, %;

[0064] Ci——concentration of the solution to be tested, mg / mL;

[0065] Ai——peak area of ​​impurities in the solution to be tested;

[0066] Cr——concentration of piperacillin in the reference solution, mg / mL;

[0067] Ar——Peak area of ​​piperacillin in reference solution;

[0068] f——Correction factor of impurities (impurity A=1.3, impurity E=0.4, impurity I=3.2, other impurities 1.0)

[0069] The system suitability solution and the reference solution were injected respectively, and the chromatogram and data were recorded. The separation between the measured peaks of piperacillin impurity A and piperacillin impurity I was R = 4.01. The reference solution was injected 5 times continuously. The concentration of piperacillin in the reference solution was 0.06 mg / mL, the peak area measured was 284.8, the RSD of the main peak area was ≤0.5%, and the RSD of the main peak retention time was ≤0.1%. Thus, it was shown that the system conditions were good and analysis and detection could be carried out.

[0070] Example 2

[0071] (1) Solution preparation

[0072] Reference solution: same as in Example 1.

[0073] Impurity resolution solution: Take 12 mg of EDQM piperacillin peak identification standard (including piperacillin and impurities A, B, C, D, E, F, G, I, J, K, L, M, O, P, Q, R, S, T), add 0.5 mL of 0.8 mg / mL impurity H solution, 0.5 mL of 0.2 mg / mL impurity N solution, 0.5 mL of 0.4 mg / mL impurity U solution, and 0.5 mL of 0.8 mg / mL impurity V solution, and shake well. The concentration of the impurity resolution solution is 6.0 mg / mL.

[0074] (2) Execute the chromatographic conditions in Example 1, inject the reference solution and the impurity separation solution respectively, and record the chromatogram and data. The concentration of piperacillin in the reference solution is 0.06 mg / mL, and the peak area is 284.8. The chromatogram of the impurity separation solution is as follows: Figure 1 As shown, the content is calculated according to the impurity quantitative method of Example 1. The separation effect of each impurity in the impurity separation solution and the measured content are shown in Table 1. The results show that the separation effect of each impurity is good.

[0075] Table 1 Impurity separation effect and content of each impurity in the solution

[0076]

[0077]

[0078] Example 3

[0079] (1) Solution preparation

[0080] Reference solution: same as in Example 1.

[0081] Test solution: Accurately weigh 150 mg of piperacillin sodium (commercially available from North China Pharmaceutical Group Xiantai Pharmaceutical Co., Ltd.), place in a 25 mL volumetric flask, dissolve and dilute to scale with mobile phase B, and shake well. The concentration of piperacillin in the test solution is 6.0 mg / mL.

[0082] (2) Execute the chromatographic conditions in Example 1, inject the reference solution and the test solution respectively, and record the chromatogram and data. The concentration of piperacillin in the reference solution is 0.06 mg / mL, and the peak area is 284.8. Figure 2 As shown, 9 impurities were detected in the test solution. The impurity content was obtained according to the impurity quantitative calculation method in Example 1, see Table 2.

[0083] Table 2 Types and contents of impurities measured in the test solution

[0084]

[0085]

[0086] Example 4

[0087] This example verifies the detection method of the present invention, and the sample tested is the test solution.

[0088] 4.1 Accuracy

[0089] Purpose: To examine the degree of closeness between the results of the method and the true value or reference value.

[0090] Method: Three batches of piperacillin sodium (commercially available from North China Pharmaceutical Group Xiantai Pharmaceutical Co., Ltd.) were prepared into test solutions according to the method in Example 1. The impurity content was detected according to the methods in Example 1 and European Pharmacopoeia EP11, and the test results were compared.

[0091] The specific method of Pharmacopoeia EP11 is as follows:

[0092] (I) Chromatographic conditions

[0093] Chromatographic column: Waters Xterra MS (C18, 5μm, 4.6mm*250mm)

[0094] Column temperature: 40°C;

[0095] Sample chamber temperature: 4°C;

[0096] Detector: UV-visible detector;

[0097] Detection wavelength: 220nm;

[0098] Flow rate: 1.0 mL / min;

[0099] Injection volume: 10 μL

[0100] (II) Solution preparation

[0101] Mobile phase A: a mixture of purified water-sodium dihydrogen phosphate dihydrate solution (27.6 g / L)-tetrabutylammonium hydroxide solution (320 g / L, equivalent to 10%)-methanol in a volume ratio of 622:100:3:275, and the pH was adjusted to 5.5 with phosphoric acid solution.

[0102] Mobile phase B: a mixture of purified water-sodium dihydrogen phosphate dihydrate solution (27.6 g / L)-tetrabutylammonium hydroxide solution (320 g / L, equivalent to 10%)-methanol in a volume ratio of 282:100:3:615, and the pH was adjusted to 5.5 with phosphoric acid solution.

[0103] (III) Gradient elution procedure:

[0104]

[0105]

[0106] Acceptable standard: for impurities with content less than 0.1%, the difference between the two methods is less than ±0.02%; for impurities with content greater than 0.1%, the RD value of the two methods is less than 10%. The impurity content measured by the two methods and the comparison results are shown in Table 3.

[0107] Table 3 Results of the accuracy test of the detection method

[0108]

[0109] As shown in the data in Table 3, for impurities with a content of less than 0.1% (impurities E, L, M, U, O, R, T), the maximum difference between the two methods is 0.015%, both < ± 0.02%; for impurities with a content of more than 0.1% (impurities B, D), the maximum RD value of the two methods is 7.0%, both < 10%. The results show that the detection method of the present invention has good accuracy.

[0110] 4.2 Precision

[0111] 4.2.1 Repeatability

[0112] Purpose: To examine the similarity between the results obtained from multiple samplings of the same batch of test products.

[0113] Method: Take 1 batch of piperacillin sodium (commercially available from Xiantai Pharmaceutical Co., Ltd., North China Pharmaceutical Group), prepare 6 test solutions according to the method in Example 1, inject samples continuously, record chromatograms and data, and calculate the RSD value of each impurity content. The results are shown in Table 4.

[0114] Acceptable standard: RSD of each impurity content (n=6) ≤ 15%.

[0115] Table 4 Results of the repeatability test of the detection method

[0116]

[0117]

[0118] As shown in the data in Table 4, the maximum RSD (n=6) of each impurity content is 2.4%, and all are ≤15%. The results show that the detection method of the present invention has good repeatability.

[0119] 4.2.2 Intermediate precision

[0120] Objective: To investigate the impact of random variation factors (including different time, different inspectors, and different instruments) on the inspection.

[0121] Method: Take 1 batch of piperacillin sodium (sold by North China Pharmaceutical Group Xiantai Pharmaceutical Co., Ltd.), and prepare 3 test solutions according to the method in Example 1 by different inspectors at different times, use different instruments to measure, record chromatograms and data, and calculate the content and RSD value of each impurity. The results are shown in Table 5.

[0122] Acceptable standard: RSD of each impurity content (n=6) ≤ 15%.

[0123] Table 5 Results of precision investigation of detection method

[0124]

[0125] As shown in the data in Table 5, the maximum RSD (n=6) of each impurity content is 6.3%, and all are ≤15%. The results show that the detection method of the present invention has good intermediate precision.

[0126] 3.3 Durability

[0127] Purpose: To investigate the robustness of the method when slight changes in chromatographic conditions occur.

[0128] Method: Take 1 batch of piperacillin sodium (sold by North China Pharmaceutical Group Xiantai Pharmaceutical Co., Ltd.), prepare 3 test solutions according to the method of Example 1, and examine the RSD values ​​of the impurity contents when the mobile phase pH is ± 0.02, the flow rate is + 0.05 mL / min, and the other chromatographic conditions are the same as those in Example 1. The results are shown in Tables 6 and 7.

[0129] Acceptable standard: RSD of each impurity content of different variables of the same factor ≤ 15%.

[0130] Table 6 Results of the mobile phase pH tolerance test for the detection method

[0131]

[0132] Table 7 Results of the mobile phase flow rate tolerance test for the detection method

[0133]

[0134] As shown in the data in Tables 6 and 7, when the pH is 5.48, 5.50, and 5.52, the maximum RSD (n=9) of each impurity content is 7.9%, all ≤15%; when the flow rate is 0.55mL / min and 0.60mL / min, the maximum RSD (n=6) of each impurity content is 5.6%, all ≤15%. The results show that the detection method of the present invention is durable when the mobile phase pH is ±0.02 and the flow rate is +0.05mL / min.

[0135] In the following comparative examples, the tested solution is the piperacillin impurity peak identification solution (EDQM piperacillin peak identification standard is taken and prepared into a 6.0 mg / mL solution with mobile phase B, containing piperacillin and impurities A, B, C, D, E, F, G, I, J, K, L, M, O, P, Q, R, S, T).

[0136] Comparative Example 1: Investigation of chromatographic column packing and specifications

[0137] The chromatographic column Waters ACQUITY UPLC HSS C18 (1.8 μm, 2.1×75 mm) was selected to examine the piperacillin impurity peak identification solution, and the flow rate was adjusted to 0.3 mL / min, the injection volume was 0.6 μL, and the gradient elution program was as follows according to the chromatographic column specifications. Other detection conditions were the same as in Example 1. See the chromatogram Figure 3 .

[0138]

[0139] The results showed that when the chromatographic column Waters ACQUITY UPLC HSS C18 was used, the separation degree between impurity I and impurity A was 0.65, and impurity F and impurity K were combined into one peak (see Figure 3 ), the separation effect is worse than that of the detection method in Example 1.

[0140] Comparative Example 2: Investigation of chromatographic column packing and specifications

[0141] The chromatographic column Waters ACQUITY Premier BEH C18 (1.7 μm, 2.1×100 mm) was selected to investigate the piperacillin impurity peak identification solution, and the flow rate was adjusted to 0.25 mL / min, the injection volume was 0.8 μL, and the gradient elution program was as follows according to the chromatographic column specifications. Other detection conditions were the same as in Example 1. The chromatogram is shown in Figure 4 .

[0142]

[0143] The results showed that when the chromatographic column Waters ACQUITY Premier BEH C18 was used, the separation degree between impurity I and impurity A was 0.75, and impurity F and impurity K were combined into one peak (see Figure 4 ), the separation effect is worse than that of the detection method in Example 1.

[0144] Comparative Example 3 Investigation of the concentration of sodium dihydrogen phosphate dihydrate in the mobile phase

[0145] The concentrations of sodium dihydrogen phosphate dihydrate solution in the mobile phase were adjusted to 27.6 g / L and 35.88 g / L respectively. Other detection conditions were the same as those in Example 1. The piperacillin impurity peak identification solutions were investigated respectively. Figure 5 , Figure 6 , the separation degree of related impurities is shown in Table 8.

[0146] Figure 5 , 6 The results in Table 8 show that the concentration of sodium dihydrogen phosphate dihydrate solution has a significant effect on the separation of impurity F and impurity K, impurity U and piperacillin, and impurity M and adjacent unidentified impurities. When the concentration is 27.6 g / L, impurity F and impurity K are not separated (see Figure 5 ); when the concentration is 35.88 g / L, the separation degree between impurity U and piperacillin is 1.06, and impurity M and the adjacent unidentified impurities are combined into one peak (see Figure 6 ). When the concentration of sodium dihydrogen phosphate dihydrate solution in the mobile phase is 27.6 g / L and 35.88 g / L, the overall separation effect is worse than that of the detection method in Example 1.

[0147] Table 8 Separation effect under different concentrations of sodium dihydrogen phosphate solution

[0148]

[0149] Comparative Example 4 Gradient Elution Procedure Investigation

[0150] The gradient elution program was adjusted, and other detection conditions were the same as in Example 1, and the piperacillin impurity peak identification solution was investigated.

[0151] The gradient elution procedure is as follows. See the chromatogram for details. Figure 7 .

[0152]

[0153] The results showed that the analysis time was the same as in Example 1, but the impurity D with the longest retention time was not eluted and could not be detected (see Figure 7 ). Under the elution procedure of this comparative example, the detection ability is worse than that of the detection method in Example 1.

Claims

1. A method for detecting related substances in piperacillin sodium, characterized in that: The method comprises the following steps: (1) Prepare reference solution and test solution; (2) Take the test sample solution and the reference sample solution for high performance liquid chromatography detection, obtain the chromatographic peaks and peak areas of the relevant substances, and calculate the impurity content; The chromatographic conditions were as follows: Chromatographic column: chromatographic column with octadecylsilane bonded silica gel as filler; Mobile phase flow rate: 0.55~0.60mL / min; Injection volume: 1.9 μL; Detection wavelength: 220nm; Column temperature: 40°C; Mobile phase A: methanol-water-sodium dihydrogen phosphate dihydrate solution-tetrabutylammonium hydroxide solution; Mobile phase B: methanol-water-sodium dihydrogen phosphate dihydrate solution-tetrabutylammonium hydroxide solution; Mobile phase pH: 5.50 ± 0.02; The gradient elution conditions were as follows:

2. The method for detecting related substances in piperacillin sodium according to claim 1, characterized in that: The chromatographic column in step (2) is Poroshell EC-C18.

3. The method for detecting related substances in piperacillin sodium according to claim 1, characterized in that: In step (2), the filling specifications of the chromatographic column are 2.7 μm, 3.0*150 mm.

4. The method for detecting related substances in piperacillin sodium according to claim 1, characterized in that: In step (2), the volume ratio of the mobile phase A, methanol-water-sodium dihydrogen phosphate dihydrate solution-tetrabutylammonium hydroxide solution, is 275:621.7:100:3.3; the volume ratio of the mobile phase B, methanol-water-sodium dihydrogen phosphate dihydrate solution-tetrabutylammonium hydroxide solution, is 615:281.3:100:3.

7.

5. The method for detecting related substances in piperacillin sodium according to claim 1, characterized in that: In step (2), the flow rate of the mobile phase is 0.55 mL / min.

6. The method for detecting related substances in piperacillin sodium according to claim 1, characterized in that: In step (2), the concentration of the sodium dihydrogen phosphate dihydrate solution in the mobile phase is 31.2 g / L, and the molar concentration of the tetrabutylammonium hydroxide solution is 0.4 mol / L.

7. The method for detecting related substances in piperacillin sodium according to claim 1, characterized in that: In step (2), the method for calculating the impurity content is the principal component self-comparison method with the addition of a correction factor.

8. The method for detecting related substances in piperacillin sodium according to claim 1, characterized in that: The relevant substances include one or more of the following:

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