Detection Method of Related Substances Ⅱ in Piperacillin Sodium and Tazobactam Sodium for Injection

Abstract

The present invention belongs to the detection technology of pharmaceutical preparations, and specifically relates to a detection method for related substance II in piperacillin sodium and tazobactam sodium for injection, wherein high performance liquid chromatography (HPLC) is used to detect related substance II in piperacillin sodium and tazobactam sodium for injection, and the method comprises the steps of (1) HPLC detection conditions (2) solution preparation (3) determination, etc. The detection method can simultaneously and effectively detect piperacillin impurity U and piperacillin impurity V, has good impurity separation effect, and can accurately calculate the content of any impurity by peak area according to the main component external standard method of the added factor through the detection result, which is of great significance for product quality control.

Description

Technical Field

[0001] The present application relates to a method for detecting a pharmaceutical preparation, and in particular to a method for detecting a related substance II in piperacillin sodium and tazobactam sodium for injection. Background Art

[0002] Piperacillin belongs to the urea penicillin class, which has a broad antibacterial spectrum and strong antibacterial effect, especially against Pseudomonas aeruginosa. It is currently widely used at home and abroad and is considered to be the most valuable type of penicillin, but it is unstable to β-lactamase. Tazobactam is a new β-lactamase inhibitor, which belongs to the penicillin class and has a broad spectrum of enzyme inhibitory activity.

[0003] Piperacillin Sodium and Tazobactam Sodium for Injection is a compound preparation of piperacillin sodium and tazobactam sodium [the ratio of piperacillin to tazobactam labeled amount is 8:1]. Through the good synergistic effect of piperacillin and tazobactam, the antibacterial activity of piperacillin can be increased and its antibacterial spectrum can be expanded, making it effective against piperacillin and other β-lactamase-producing bacteria resistant to β-lactam antibiotics. In this way, piperacillin Sodium and Tazobactam Sodium for Injection has the dual characteristics of a broad antibacterial spectrum and inhibition of β-lactamase. It has good antibacterial activity in various tissues throughout the body and is one of the commonly used antibacterial drugs in clinical practice.

[0004] One of the main factors affecting the quality of drugs is drug impurities. By measuring its impurity components and content, the effective main component content can be obtained, which also plays an important role in drug quality control. In industrial production, piperacillin sodium and tazobactam sodium for injection will generate more impurities, including piperacillin impurity U and piperacillin impurity V. However, according to the Chinese Pharmacopoeia CP2020, the impurity detection method for related substances of piperacillin sodium and tazobactam sodium for injection is to limit the sum of the impurity peak areas, without the detection of a single impurity peak, and the prior art also has no method that can simultaneously detect piperacillin impurity U and piperacillin impurity V in piperacillin sodium and tazobactam sodium for injection, which cannot well control the quality of drugs and ensure the safety of medication.

[0005] Wherein piperacillin impurity U (piperacillin sulfoxide) is C 23 H 27 N 5 O 8 S, (2S, 5R, 6R) -6- [(R) -2- (4-ethyl-2,3-dioxapiperazine-1-carboxylic acid amide) -2-phenylacetamido] -3,3-dimethyl-7-oxo-4-thia-1-azabicyclo [3.2.0] heptane-2-carboxylic acid 4-oxide, the structural formula of which is as follows:

[0006]

[0007] Piperacillin impurity V (piperacillin methyl penicillin thiazole salt) is C 24 H 31 N 5 O 8 S, (2R, 4S)-2-{(R)-1-[(R)-2-(4-ethyl-2,3-dioxapiperazine-1-carboxamide)-2-phenylacetamide]-2-methoxy-2-oxoethyl}-5,5-dimethylthiazolidine-4-carboxylic acid, the structural formula of which is as follows:

[0008] Summary of the invention

[0009] The purpose of the present invention is to provide a method for detecting related substance II in piperacillin sodium and tazobactam sodium for injection, so as to solve the above-mentioned technical problems.

[0010] The technical solution adopted by the present invention is as follows:

[0011] Detection method of related substance II in piperacillin sodium and tazobactam sodium for injection: High performance liquid chromatography (HPLC) was used to detect related substance II in piperacillin sodium and tazobactam sodium for injection.

[0012] (1) HPLC detection conditions are as follows:

[0013] Chromatographic column: Octadecylsilane bonded silica gel as filler

[0014] Mobile phase A: 3.12 g / L anhydrous sodium dihydrogen phosphate solution

[0015] Mobile phase B: methanol

[0016] The mobile phase was used for gradient elution, and the elution program was as follows:

[0017]

[0018] (2) Solution preparation: Prepare test sample solution and reference sample solution;

[0019] (3) Determination: Accurately measure 20 μl of the test solution and the reference solution respectively, inject them into the liquid chromatograph, determine according to the above chromatographic conditions, and record the chromatogram.

[0020] Optionally, the related substance II detected is piperacillin impurity U and piperacillin impurity V.

[0021] Optionally, the test solution is prepared by dissolving piperacillin sodium and tazobactam sodium for injection in mobile phase A, and the concentration of piperacillin in the test solution is 1.98-2.2 mg / ml, and the concentration of tazobactam is 0.23-0.27 mg / ml.

[0022] Optionally, the reference solution is prepared by dissolving piperacillin in methanol-mobile phase A solution (30:70), and the concentration of piperacillin in the reference solution is 0.01-0.03 mg / ml.

[0023] Optionally, the pH value of mobile phase A is adjusted to 3.65-3.75 with phosphoric acid.

[0024] Optionally, the particle size of the octadecylsilane bonded silica gel chromatographic column filler is 5 μm, the detection wavelength is 220 nm, the injection volume is 20 μl, and the flow rate is 1.0 ml / min.

[0025] Optionally, the column temperature is 27-33°C.

[0026] Optionally, the detection method can calculate the content of any impurity in the related substance II by peak area using the main component external standard method with an external correction factor based on the chromatogram of the detection record.

[0027] Optionally, the main component external standard method with an external correction factor is used to calculate the content of any impurity in the related substance II by peak area as follows:

[0028] Any impurity content in related substance II (%) = (A t-相应的杂质 / A s )×(C s / C t )×P×F×100%

[0029] In the formula, A t-相应的杂质 is the peak area of ​​any impurity peak in the chromatogram of the test solution;

[0030] A s is the peak area of ​​piperacillin in the chromatogram of the reference solution;

[0031] C t The concentration of piperacillin in the test solution;

[0032] C s is the concentration of piperacillin in the reference solution;

[0033] P is the content of piperacillin in the piperacillin reference substance;

[0034] F is the correction factor of each impurity relative to piperacillin.

[0035] Optionally, the correction factor for piperacillin impurity U is 1.00, and the correction factor for piperacillin impurity V is 1.44.

[0036] Optionally, the relative retention time of piperacillin impurity U is 0.72, the limit is 0.15%, and the relative retention time of piperacillin impurity V is 1.16, the limit is 0.2%.

[0037] In summary, due to the adoption of the above technical solution, the beneficial effects of the present invention are:

[0038] The invention ensures that the used mobile phase does not interfere with the detection of piperacillin impurity U and piperacillin impurity V in a sample, ensures that the separation between the chromatographic peaks of piperacillin, piperacillin impurity U and piperacillin impurity V meets the requirements, can effectively separate piperacillin impurity U and piperacillin impurity V in piperacillin sodium and tazobactam sodium for injection, is not interfered by other component peaks in the sample, and realizes the simultaneous detection of piperacillin impurity U and piperacillin impurity V.

[0039] The mobile phase composition of the invention is simple, only anhydrous sodium dihydrogen phosphate solution and methanol are used, and reagent consumption is small; the test solution is easy and fast to handle.

[0040] The method for calculating the content of any impurity in the related substance II by peak area through the main component external standard method with an external correction factor established in the present invention can ensure the accuracy of impurity quantification and simplify the detection procedure.

[0041] The results of the present invention show that the chromatographic separation of the related substance II in piperacillin sodium and tazobactam sodium for injection is good, and the verification results of the method of the present invention show that all verification items meet the requirements. The detection method provided by the present invention is used to determine the contents of piperacillin impurity U and piperacillin impurity V, and the results are accurate and reliable.

[0042] The method of the invention is simple to operate and has a beautiful peak shape, and can efficiently and accurately detect and quantify the related substance II in piperacillin sodium and tazobactam sodium for injection, thereby ensuring the safety of medication and having important significance for product quality control. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] In order to more clearly illustrate the technical solutions in the embodiments of the present application, the drawings required for use in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments recorded in the present application. For ordinary technicians in this field, other drawings can also be obtained based on these drawings.

[0044] Figure 1 This is the chromatogram of the test solution in Example 1 of the present application;

[0045] Figure 2 is a chromatogram of the system suitability solution in Experimental Example 1 of this application;

[0046] Figure 3 This is the chromatogram of the sensitivity solution in Experimental Example 1 of this application; DETAILED DESCRIPTION

[0047] In order to enable those skilled in the art to better understand the solution of the present application, the technical solution in the embodiments of the present application will be clearly and completely described below in conjunction with the drawings in the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, not all of the embodiments. Based on the embodiments in the present application, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of this application.

[0048] Example 1

[0049] The method for detecting related substance II in piperacillin sodium and tazobactam sodium for injection comprises the following steps: using high performance liquid chromatography (HPLC) to detect related substance II in piperacillin sodium and tazobactam sodium for injection, wherein related substance II is piperacillin impurity U and piperacillin impurity V;

[0050] (1) HPLC detection conditions are as follows:

[0051] Chromatographic column: Octadecylsilane bonded silica gel as filler (YMC-Pack ODS-A column, 4.6mm×250mm, 5μm)

[0052] Mobile phase A: 3.12 g / L anhydrous sodium dihydrogen phosphate solution, adjusted to pH 3.7

[0053] Mobile phase B: methanol

[0054] Detection wavelength: 220nm, injection volume: 20μl, flow rate: 1.0ml / min, column temperature: 30℃

[0055] The mobile phase was used for gradient elution, and the elution program was as follows:

[0056]

[0057] (2) Prepare the test solution: accurately weigh piperacillin sodium and tazobactam sodium for injection, dissolve them in mobile phase A, and quantitatively dilute them into a solution containing 2 mg / ml piperacillin and 0.25 mg / ml tazobactam, and shake well;

[0058] Prepare reference solution: Accurately weigh an appropriate amount of piperacillin reference substance, dissolve it in methanol, and quantitatively dilute it with mobile phase A to make a solution containing approximately piperacillin (C 23 H 27 N 5 O 7 S) 0.02 mg of solution [methanol-mobile phase A (volume ratio 30:70)], shake well.

[0059] (3) Determination: Accurately measure 20 μl of the test solution and the reference solution, respectively, inject them into the liquid chromatograph, and determine them under the above chromatographic conditions. The chromatogram of the test solution is shown in Figure 1 According to the chromatogram, the peak area A of the corresponding impurity contained in the test solution is obtained. t-相应的杂质 ; That is, the piperacillin impurity U(A t-哌拉西林杂质U ), Tazobactam impurity V(A t-哌拉西林杂质V ).

[0060] (4) Calculation: If there are impurity peaks in the chromatogram of the test solution, calculate its content by peak area using the main component external standard method with an external correction factor, specifically:

[0061] Any impurity content in related substance II (%) = (A t-相应的杂质 / A s )×(C s / C t )×P×F×100%

[0062] In the formula, A t-相应的杂质 is the peak area of ​​any impurity peak in the chromatogram of the test solution;

[0063] A s is the peak area of ​​piperacillin in the chromatogram of the reference solution;

[0064] C t The concentration of piperacillin in the test solution;

[0065] C s is the concentration of piperacillin in the reference solution;

[0066] P is the content of piperacillin in the piperacillin reference substance;

[0067] F is the correction factor of each impurity relative to piperacillin;

[0068] The parameters corresponding to each impurity are shown in Table 1 below:

[0069] Table 1 List of parameters corresponding to each impurity

[0070] Impurity Name Relative retention time Correction Factor limit(%) Piperacillin Impurity U 0.72 1.00 0.15 Piperacillin Impurity V 1.16 1.44 0.2

[0071] Example 2

[0072] The difference between this embodiment and embodiment 1 is that the pH value of mobile phase A is 3.65.

[0073] Example 3

[0074] The difference between this embodiment and embodiment 1 is that the pH value of mobile phase A is 3.75.

[0075] Example 4

[0076] The difference between this embodiment and embodiment 1 is that the concentration of piperacillin in the test solution is 1.98 mg / ml, and the concentration of tazobactam is 0.23 mg / ml.

[0077] Example 5

[0078] The difference between this embodiment and embodiment 1 is that the concentration of piperacillin in the test solution is 2.2 mg / ml, and the concentration of tazobactam is 0.27 mg / ml.

[0079] Example 6

[0080] The difference between this embodiment and embodiment 1 is that the column temperature is 28°C.

[0081] Comparative Example 1

[0082] The difference between this comparative example and Example 1 is that the mobile phase elution procedure is different.

[0083] The elution procedure is shown in Table 2 below:

[0084] Time (minutes) Mobile phase A (%) Mobile phase B (%) 0 90 10 5 85 15 10 65 35 35 55 45 60 35 65 85 15 85 86 90 10 100 90 10

[0085] Comparative Example 2

[0086] The difference between this comparative example and Example 1 is that the pH value of mobile phase A is 3.5.

[0087] The main peak retention time, theoretical plate number, tailing factor, separation degree, and S / N of the above Examples 1 to 5 and Comparative Examples 1 to 2 were tested, and the test results are shown in Table 3 below:

[0088] Table 3 Detection results of piperacillin main peak in the test solution of Examples 1 to 6 and Comparative Examples 1 to 2

[0089] Retention time Theoretical plates Tailing Factor Separation Example 1 29.922 18751 3.87 17.03 Example 2 29.988 18651 3.65 16.99 Example 3 30.001 18675 3.76 17.12 Example 4 29.906 18961 3.51 18.01 Example 5 30.026 18311 3.95 16.99 Example 6 33.379 17212 2.92 14.70 Comparative Example 1 34.998 16240 3.96 12.65 Comparative Example 2 35.076 15557 4.02 10.58

[0090] As can be seen from the above table, 3.12 g / L of anhydrous sodium dihydrogen phosphate solution is used as mobile phase A, methanol is used as mobile phase B, the concentration of the test solution and the chromatographic conditions are limited, and after gradient elution, the retention time of Examples 1 to 6 is detected to be in the range of 29.900 to 33.400 min, the theoretical plate number is in the range of 17100 to 18900, the tailing factor is in the range of 2.90 to 4.00, and the separation degree is in the range of 13.50 to 18.10; compared with Comparative Examples 1 and 2, it is shown that when the method of the present invention is used to detect piperacillin impurity U and piperacillin impurity V, the main peak retention time is moderate, the theoretical plate number and the separation degree are good, and the tailing factor meets the requirements.

[0091] The contents of piperacillin impurity U and piperacillin impurity V measured in the above Examples 1 to 6 were calculated by peak area using the main component external standard method with an external correction factor. The calculation results are shown in Table 4 below:

[0092] Table 4 Calculation results of impurity content

[0093] Piperacillin impurity U content (%) Piperacillin impurity V content (%) Example 1 0.009 0.096 Example 2 0.009 0.094 Example 3 0.009 0.090 Example 4 0.008 0.086 Example 5 0.009 0.100 Example 6 0.009 0.095

[0094] Experimental Example 1

[0095] The following is the methodological verification of the detection method of related substance II in piperacillin sodium and tazobactam sodium for injection of the present invention:

[0096] 1. System suitability test

[0097] Take appropriate amount of piperacillin impurity H and tazobactam impurity A reference substances, add mobile phase A to dissolve and quantitatively dilute to prepare a system suitability solution containing approximately 10 μg of piperacillin impurity H and 6 μg of tazobactam impurity A per 1 ml.

[0098] The chromatographic conditions in Example 1 were used to perform HPLC detection, and the chromatogram of the obtained system suitability solution was shown in Figure 2 As can be seen from the figure, the peak order is tazobactam impurity A, piperacillin impurity H. The separation between the peak of piperacillin impurity H and the peak of tazobactam impurity A is not less than 1.5, and the system applicability is good; the number of theoretical plates meets the requirements; the tailing factor of piperacillin in the reference solution is not greater than 2.0.

[0099] 2. Sensitivity

[0100] Accurately measure an appropriate amount of the reference solution prepared in Example 1, and quantitatively dilute it with mobile phase A to prepare a sensitivity solution containing approximately 1 μg of piperacillin per 1 ml.

[0101] The chromatographic conditions in Example 1 were used to perform HPLC detection, and the chromatogram of the obtained sensitive solution was shown in FIG. Figure 3 The signal-to-noise ratio of the piperacillin peak height should be greater than 10.

[0102] 3. Solution stability test

[0103] The test solution and reference solution prepared in Example 1 were accurately measured and stored at 4°C. The stability of the solutions was investigated after 0 h, 4 h, 8 h, and 12 h. The results are shown in Table 5 below.

[0104] Table 5 Solution stability test results

[0105] Solution type 0h 4h 8h 12h Peak area RSD Test solution 1089.866 1090.648 1089.452 1089.127 0.06％ Reference solution 10.361 10.334 10.337 10.313 0.19％

[0106] The test results show that: at 4°C, the RSD of the peak area of ​​the test solution measured after 0h, 4h, 8h, and 12h is 0.06%, and the RSD of the peak area of ​​the reference solution measured after 0h, 4h, 8h, and 12h is 0.19%, indicating that the method of the present invention has good solution stability.

[0107] 4. Accuracy test

[0108] Accurately measure an appropriate amount of the test solution prepared in Example 1, and add three mixed impurity reference solutions containing 50%, 100% and 150% of the known impurity limits, respectively. Prepare 3 portions of each concentration in parallel, for a total of 9 portions, to obtain 9 portions of accuracy solution. Take the above 9 portions of accuracy solution, perform HPLC detection using the chromatographic conditions of Example 1, and calculate the amount and recovery rate of each impurity in the test sample.

[0109] The recovery rate (%) formula = (measured amount - original amount of each impurity in the test sample) / added amount * 100%.

[0110] The recovery results obtained were: recovery rate of piperacillin impurity U: 103.3%, recovery rate of piperacillin impurity V: 97.4%.

[0111] Accuracy recovery rate standard: If in a 100% recovery solution, 0.1% ≤ impurity content < 1%, the recovery rate of the impurity content should be within the range of 90% to 108%.

[0112] Therefore, the recoveries of piperacillin impurity U and piperacillin impurity V in the present invention all meet the accuracy recovery standard.

[0113] 5. Precision test

[0114] Accurately measure an appropriate amount of the test solution prepared in Example 1, add mixed impurity reference solutions containing known impurity concentrations, prepare 6 portions in parallel, and obtain 6 precision solutions. Take the above 6 precision solutions, perform HPLC detection using the chromatographic conditions of Example 1, and calculate the RSD of the amount of each impurity in the test sample.

[0115] RSD results: RSD of piperacillin impurity U: 0.9%, RSD of piperacillin impurity V: 1.7%.

[0116] Precision RSD standard: When the impurity content in 6 precision solutions is 0.1%≤<1%, the RSD of the impurity content should be no more than 3%.

[0117] Therefore, the RSDs of piperacillin impurity U and piperacillin impurity V in the present invention both meet the precision RSD standard.

[0118] 6. Linear regression of piperacillin impurity U and piperacillin impurity V

[0119] Accurately weigh the piperacillin reference substance, the piperacillin impurity U reference substance, and the piperacillin impurity V reference substance, and prepare 6 reference substance solutions with different concentrations according to the sensitivity to a certain concentration range (2 times the impurity limit concentration), and measure the peak area of ​​each component according to the chromatographic conditions of Example 1, and perform linear regression analysis with concentration as the abscissa (X) and the average peak area as the ordinate (Y).

[0120] Table 6 Linearity test results

[0121]

[0122] The test results show that the solution concentration (μg / ml) of piperacillin impurity U in the range of 0.440 to 3.900 has a good linear relationship with its peak area, the linear equation is: y=0.5775x+0.0038, and the linear correlation coefficient R=0.9999;

[0123] The solution concentration (μg / ml) of piperacillin impurity V in the range of 0.400 to 1.600 and its peak area show a good linear relationship, the linear equation is: y=0.3924x+0.0106, and the linear correlation coefficient R=0.9983; the correction factors of piperacillin impurity U and piperacillin impurity V calculated according to the linear equation of piperacillin is: y=0.5636x+0.0357 are 1.00 and 1.44 respectively, if the calculated value is between 0.9 and 1.1, the correction factor is calculated as 1; if the calculated value is between 0.2 and 5.0, the impurity content needs to be included in the correction factor calculation; if the correction factor is outside 0.2 to 5.0, the impurity content should be calculated using the external standard method.

[0124] As described above, the above embodiments are only used to illustrate the technical solutions of the present application, rather than to limit them. Although the present application has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present application.

Claims

1. A method for detecting related substance II in piperacillin sodium and tazobactam sodium for injection, characterized in that: The method comprises the following steps: using high performance liquid chromatography to detect the related substance II in piperacillin sodium and tazobactam sodium for injection; The HPLC detection conditions are as follows: Chromatographic column: Octadecylsilane bonded silica gel as filler Mobile phase A: 3.12 g / L anhydrous sodium dihydrogen phosphate solution; the mobile phase A is adjusted to a pH value of 3.65 to 3.75 with phosphoric acid Mobile phase B: methanol The mobile phase was used for gradient elution, and the elution program was as follows: Solution preparation: prepare test solution and reference solution; Determination: Accurately measure 20 μl of the test solution and the reference solution respectively, inject them into the liquid chromatograph, determine according to the above-mentioned HPLC detection conditions, and record the chromatogram; The related substance II is piperacillin sulfoxide and piperacillin methyl penicillin thiazole salt.

2. The method for detecting related substance II in piperacillin sodium and tazobactam sodium for injection according to claim 1, characterized in that: The test solution is prepared by dissolving piperacillin sodium and tazobactam sodium for injection in the mobile phase A. The piperacillin concentration in the test solution is 1.98-2.2 mg / ml, and the tazobactam concentration is 0.23-0.27 mg / ml.

3. The method for detecting related substance II in piperacillin sodium and tazobactam sodium for injection according to claim 1, characterized in that: The reference solution is prepared by dissolving piperacillin in methanol-mobile phase A solution at a volume ratio of 30:

70. The concentration of piperacillin in the reference solution is 0.01-0.03 mg / ml.

4. The method for detecting related substance II in piperacillin sodium and tazobactam sodium for injection according to claim 1, characterized in that: In the high performance liquid chromatography detection conditions, the particle size of the octadecylsilane bonded silica gel chromatographic column filler is 5 μm, the detection wavelength is 220 nm, and the flow rate is 1.0 ml / min.

5. The method for detecting related substance II in piperacillin sodium and tazobactam sodium for injection according to claim 1, characterized in that: The column temperature is 27-33°C.

6. The method for detecting related substance II in piperacillin sodium and tazobactam sodium for injection according to any one of claims 1 to 5, characterized in that: The detection method calculates the content of any impurity in the related substance II by peak area through the main component external standard method with an external correction factor.

7. The method for detecting related substance II in piperacillin sodium and tazobactam sodium for injection according to claim 6, characterized in that: The main component external standard method with the added correction factor is used to calculate the content of any impurity in the related substance II by peak area. The calculation formula is as follows: Any impurity content in related substance Ⅱ / % = A t-相应的杂质 / A s ×C s / C t ×P×F×100% In the formula, A t-相应的杂质 is the peak area of ​​any impurity peak in the chromatogram of the test solution; A s is the peak area of ​​piperacillin in the chromatogram of the reference solution; C t The concentration of piperacillin in the test solution; C s is the concentration of piperacillin reference substance in the reference substance solution; P is the content of piperacillin in the piperacillin reference substance; F is the correction factor of each impurity relative to piperacillin.

8. The method for detecting related substance II in piperacillin sodium and tazobactam sodium for injection according to claim 7, characterized in that: The correction factor for piperacillin sulfoxide was 1.00 and the correction factor for piperacillin methylthiazolidine was 1.44.

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