Method for analyzing impurities of cefazolin sodium and cefazolin sodium polymer for injection

Abstract

The invention provides an impurity analysis method suitable for cefazolin sodium and a cefazolin sodium polymer for injection, which comprises the following steps of: performing qualitative and quantitative analysis on impurities by using a chromatographic method, and further confirming possible structures of the impurities by using a mass spectrum. The method has the advantages of high separation degree and high specificity, various impurities in the cefazolin sodium and the cefazolin sodium for injection can be better separated, the types of the impurities are defined, so that research on the process or toxicity of the cefazolin sodium is facilitated, and meanwhile, the purity of the cefazolin sodium and the purity of the cefazolin sodium are improved by separating the impurities. And the quality safety of the cefazolin sodium and the cefazolin sodium for injection can be controlled.

Description

Technical Field

[0001] The invention relates to the field of drug analysis, in particular to an analysis method for impurities in a cefazolin sodium polymer. Background Art

[0002] Allergic reactions to β-lactam antibiotics are common adverse reactions in clinical practice. They are mainly caused by polymer impurities generated during the production, transportation and storage of β-lactam antibiotics. Therefore, the control of polymer impurities is particularly important. Cefazolin sodium belongs to the first generation of cephalosporin antibiotics. It has the characteristics of strong bactericidal ability and low toxicity, and has good antibacterial activity against Gram-positive cocci. Since the 2005 edition, the Chinese Pharmacopoeia has added polymer control items to the quality standards of more and more types of antibiotics. With the development of chromatographic separation technology, the quality control methods of polymers have become more and more diversified. The methods currently reported mainly include: Sephadex G10 chromatography (Sephadex G10), high performance gel chromatography (HPSEC), reversed-phase high performance liquid chromatography (RP-HPLC), liquid chromatography-mass spectrometry, and column switching technology.

[0003] The analysis of polymer pointer impurities using reversed-phase high performance liquid chromatography (RP-HPLC) has been a hot topic in recent years. In the prior art, Zhang Xia et al. (Zhang Xia, Li Jin, Wang Chen, et al., Study on polymer impurities in cefazolin sodium raw materials and preparations [J]. Acta Pharmaceutica Sinica, 2021, 56(06): 1677-1682.) established an HPLC method: the chromatographic column was Shiseido Capcell Pak MG II (250 mm×4.6 mm, 5 μm), the mobile phase A was phosphate buffer (6.8 mg·mL-1 potassium dihydrogen phosphate solution, adjusted to pH 6.8 with 10 mol·L-1 sodium hydroxide solution), the mobile phase B was acetonitrile-phosphate solution (6.8 mg·mL-1 potassium dihydrogen phosphate solution) (1:1), the flow rate was 1.5 mL·min-1, the detection wavelength was 254 nm, the column temperature was 30°C, and the injection volume was 20 μL. Gradient elution program: At 0, 7, 15, 35, 45, 50, 55 and 65 min, the proportion of mobile phase A was 98%, 98%, 85%, 80%, 50%, 50%, 98% and 98%, respectively. The results showed that the separation of polymer 1 and 2 impurity peaks was poor under this chromatographic condition, e.g. Figure 8 As shown in the figure, peaks 10 and 11 are isolated, and the peaks around the polymer are rather messy. Therefore, a method with better separation is needed to achieve better separation of polymer impurities and higher specificity. Summary of the invention

[0004] The present invention aims to solve at least one of the technical problems existing in the prior art, and provides a RP-HPLC-HRMS analysis method suitable for the impurity analysis of cefazolin sodium and cefazolin sodium polymer for injection. The impurities are first qualitatively and quantitatively analyzed, and then the possible structure is further confirmed by mass spectrometry. The method has the advantages of high separation and high specificity, and can better control the quality and safety of cefazolin sodium and cefazolin sodium for injection.

[0005] The present invention provides an impurity analysis method applicable to cefazolin sodium and cefazolin sodium polymer for injection, which is characterized by: adopting RP-HPLC chromatography method,

[0006] Wherein, the chromatographic conditions of RP-HPLC are:

[0007] Use ammonium acetate solution with a pH of 5.0±0.2-acetonitrile = 90-80:10-20 as mobile phase A;

[0008] Use ammonium acetate solution with a pH of 5.0±0.2-acetonitrile = 50-40:50-60 as mobile phase B,

[0009] Gradient elution;

[0010] The column temperature is 40℃±2℃.

[0011] Furthermore, the present invention provides an impurity analysis method applicable to cefazolin sodium and a cefazolin sodium polymer for injection, which is also characterized in that:

[0012] Octadecylsilane bonded silica gel is used as the chromatographic column or a chromatographic column with equivalent performance.

[0013] Furthermore, the present invention provides an impurity analysis method applicable to cefazolin sodium and a cefazolin sodium polymer for injection, which is also characterized in that:

[0014] The gradient elution is:

[0015]

[0016]

[0017] Furthermore, the present invention provides an impurity analysis method applicable to cefazolin sodium and a cefazolin sodium polymer for injection, which is also characterized in that:

[0018] The flow rate was 0.5-1.0 mL per minute.

[0019] Furthermore, the present invention provides an impurity analysis method applicable to cefazolin sodium and a cefazolin sodium polymer for injection, which is also characterized in that:

[0020] The injection volume is 10-20 μl.

[0021] Furthermore, the present invention provides an impurity analysis method applicable to cefazolin sodium and cefazolin sodium polymer for injection, which is also characterized by: using mass spectrometry to perform impurity structure analysis;

[0022] The mass spectrometry conditions are: spray voltage: 3.9KV;

[0023] Spray temperature: 320℃;

[0024] Ion transfer tube temperature: 320°C;

[0025] Sheath air: 60arb;

[0026] Auxiliary gas: 15arb;

[0027] Lens voltage: 55V;

[0028] Scan mode: Ful1 MS / dd MS 2 ;

[0029] Ionization mode: ESI+;

[0030] Primary scanning range: 200-1800m / z; primary resolution: 70000;

[0031] Secondary energy: NCE102030; secondary resolution: 17500; isolation width: 3.0m / z.

[0032] Based on the method of the present invention, an impurity of cefazolin sodium and a polymer of cefazolin sodium for injection is obtained, which is characterized by:

[0033]

[0034] Based on the method of the present invention, an impurity of cefazolin sodium and a polymer of cefazolin sodium for injection is obtained, which is characterized by:

[0035]

[0036] Functions and effects of the present invention:

[0037] The above analytical method can make the polymer impurities in cefazolin sodium and cefazolin sodium for injection better separated and more specific. At the same time, compared with the prior art, the present invention also has the advantages of reduced flow rate and reduced use of organic solvents, can better protect the chromatographic column, reduce the situation of salt precipitation in the mobile phase due to low temperature during use, and can save costs. At the same time, in order to further confirm the polymer structure, the present invention infers the possible structure of the polymer impurities through mass spectrometry analysis and fragment structure. Compared with the pharmacopoeia method, this method is more sensitive, has good specificity and high separation. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 , Typical diagram of Cefazolin Sodium for Injection polymer;

[0039] Figure 2 , Typical chromatogram mass spectrum of impurity POLYMER-1;

[0040] Figure 3 , Typical fragmentation mass spectrum of impurity POLYMER-1;

[0041] Figure 4 , Typical chromatogram mass spectrum of impurity POLYMER-2;

[0042] Figure 5 , Typical fragmentation mass spectrum of impurity POLYMER-2;

[0043] Figure 6 , system suitability map;

[0044] Figure 7 , spectrum of test solution;

[0045] Figure 8 , existing technology spectrum; DETAILED DESCRIPTION

[0046] This embodiment provides a RP-HPLC-HRMS analysis method suitable for the impurity analysis of cefazolin sodium and cefazolin sodium polymer for injection.

[0047] In the method of the present embodiment, the impurities are first qualitatively and quantitatively analyzed by chromatography, and then mass spectrometry is used to further confirm their possible structures. The method has the advantages of high separation and high specificity, and can better separate various impurities in cefazolin sodium and cefazolin sodium for injection, clarify the type of impurities, and thus facilitate the study of its process or toxicity. At the same time, by separating each impurity, the quality safety of cefazolin sodium and cefazolin sodium for injection can also be controlled.

[0048] The specific method is as follows:

[0049] 1. RP-HPLC chromatographic conditions

[0050] According to the high performance liquid chromatography method (Chinese Pharmacopoeia 2020 Edition Part IV General Rules 0512), octadecylsilane bonded silica gel is used as the chromatographic column (Agilent 5HC-C18 (2), 250 mm × 4.6 mm, 5 um or a chromatographic column with equivalent performance); 10 mmol / L ammonium acetate solution (adjusted to pH 5.0 ± 0.2 with glacial acetic acid) - acetonitrile = 90-80: 10-20 is used as the mobile phase A, 10 mmol / L ammonium acetate solution (adjusted to pH 5.0 with glacial acetic acid) - acetonitrile = 50-40: 50-60 is used as the mobile phase B, and the gradient elution table is shown in the table below; the flow rate is 0.5-1.0 mL per minute; the column temperature is 40 ° C ± 2 ° C; the detection wavelength is 254 nm; the injection volume is 10-20 μl.

[0051] Gradient elution table

[0052]

[0053] 2. Mass spectrometry conditions

[0054] Instrument: Thermo Q Exactive high-resolution mass spectrometer

[0055] Spray voltage: 3.9KV; Spray temperature: 320℃; Ion transfer tube temperature: 320℃; Sheath gas: 60arb; Auxiliary gas: 15arb; Lens voltage: 55V

[0056] Scan mode: Ful1 MS / dd MS 2

[0057] Ionization mode: ESI+

[0058] Primary scanning range: 200-1800m / z; primary resolution: 70000

[0059] Secondary energy: NCE102030; Secondary resolution: 17500; Isolation width: 3.0m / z

[0060] Embodiment 1. Best embodiment

[0061] Mobile phase preparation:

[0062] Mobile phase A: 10mmol / L ammonium acetate solution (adjust pH to 5.0 with glacial acetic acid)-acetonitrile = 90:10

[0063] Mobile phase B: 10mmol / L ammonium acetate solution (adjust pH to 5.0 with glacial acetic acid)-acetonitrile = 50:50

[0064] Solution preparation:

[0065] Blank solution (diluent): ultrapure water.

[0066] Sensitivity solution: Take an appropriate amount of cefazolin sodium for injection, weigh accurately, dissolve in water and quantitatively dilute to make a solution containing about 0.2 μg per 1 ml, as the sensitivity solution (0.004%).

[0067] Culture medium: Take an appropriate amount of Cefazolin Sodium for Injection, weigh it accurately, dissolve it in water and quantitatively dilute it to make a solution containing about 50 mg per 1 ml, and let it stand at 20-25℃ for 24 hours.

[0068] Polymer impurity localization solution (method verification sample solution): Accurately measure 1 ml of culture solution, place in a 10 ml volumetric flask, add water to dilute to the mark, and shake well. (5 mg / ml)

[0069] Test solution: Take an appropriate amount of cefazolin sodium for injection, weigh accurately, dissolve in water and quantitatively dilute to make a solution containing about 5 mg per 1 ml.

[0070] Control solution: Accurately measure 1 ml of Cefazolin Sodium for Injection, place in a 100 ml volumetric flask, add water to dilute to the scale, shake well, and use as the control solution (1%).

[0071] Detection: Take 20 μL of each of the above solutions and inject them into the chromatograph respectively, analyze according to the following chromatographic conditions, and record the chromatogram. In the chromatogram of the sensitive solution, the signal-to-noise ratio of the cefazolin peak height should not be less than 10.

[0072]

[0073] The flow rate was 1.0 mL per minute; the column temperature was 40°C ± 2°C; the detection wavelength was 254 nm; and the injection volume was 20 μl.

[0074] Test results of polymers of 3 batches of Cefazolin Sodium for Injection

[0075]

[0076] *Polymer 1 and 2 are produced when the solution of Cefazolin Sodium for Injection is left at 20-25°C for more than 24 hours. Therefore, undetectable results may appear in the newly prepared product.

[0077] Example 2. Comparative experimental conditions:

[0078] Example 2.1. Test results at different temperatures (50 mg / ml solution of Cefazolin Sodium for Injection was placed at 20-25°C for 24 hours)

[0079] Column temperature 38℃ result table

[0080]

[0081] Column temperature 42℃ result table

[0082]

[0083]

[0084] Example 2.2. Detection results at different pH values

[0085] Mobile phase pH 4.8 result table

[0086]

[0087] Mobile phase pH 5.2 durability results table

[0088]

[0089] The possible structures and degradation mechanisms of the two polymer impurities are as follows:

[0090] ① Impurity POLYMER-1 (tR = 34.08min)

[0091] The POLYMER-1 chromatographic peak was directly detected by RP-HPLC-HRMS. Figure 2 As shown. The quasi-molecular ion peak of POLYMER-1 is m / z649.05249 [M+H]. Using the high accuracy of high-resolution mass spectrometry (error less than 5ppm), the molecular formula of the quasi-molecular ion peak of POLYMER-1 impurity is calculated to be C through the even electron rule and nitrogen rule, isotope peak abundance and the structure of cefazolin. 22 H 21 N 10 O6S4, indicating that this impurity is a polymer impurity with a molecular weight of 648Da. The main fragment m / z489.08 structure shows that the cefazolin structure on the polymer impurity is not ring-opened, and the polymerization site is on the carboxyl group of the second carbon of cefazolin. Cefazolin is easily hydrolyzed to lose tetrazolylacetic acid, and the CS bond of the third side chain is broken to lose thiothiadiazole, which is further cyclized to lactone, so this polymer impurity is formed by the dehydration polymerization of lactone degradation products and cefazolin. The preliminary inferred structure is shown in the following structural formula, and the molecular weight accuracy and fragments support the structural inference. The polymer impurity is named cefazolin dimer derivative II.

[0092]

[0093] like Figure 3 The following is a typical fragmentation mass spectrum of impurity POLYMER-1. Based on this mass spectrum, the following cleavage process can be inferred:

[0094]

[0095] The mechanism of its production can be inferred as follows: Cefazolin is easily hydrolyzed to lose tetrazolylacetic acid and thiadiazole on the third-side chain, which is further cyclized to form a lactone. The lactone dehydrates and polymerizes with cefazolin to form a polymer impurity.

[0096]

[0097] ② Impurity POLYMER-2 (tR = 38.20min)

[0098] The chromatographic peaks of POLYMER-2 were directly detected by RP-HPLC-HRMS and the following results were obtained: Figure 4 As shown in the figure, the quasi-molecular ion peak of POLYMER-2 is m / z601.04051[M+H]. Using the high accuracy of high-resolution mass spectrometry (error less than 5ppm), the molecular formula of the quasi-molecular ion peak of POLYMER-2 impurity is calculated to be C through the even electron rule and nitrogen rule, isotope peak abundance and cefazolin structure. 19 H 21 N8O7S4, indicating that the POLYMER-2 impurity is a polymer impurity with a molecular weight of 600Da. The polymer impurity is judged to have a relatively complete cefazolin structure by the fragments m / z100.05 and m / z468.055. The impurity structure is inferred by molecular formula calculation that the MMTD side chain at position 3 of cefazolin is replaced by a hydroxyl group, and the carbon at position 2 and the sulfur at position 5 are directly connected to form a compound after breaking, and the degradation compound is esterified with cefazolin to form a polymer impurity. Therefore, the structure is preliminarily inferred to be as shown in the following structural formula, and the molecular weight accuracy and fragments support the structural inference. The polymer impurity is named cefazolin dimer derivative I.

[0099]

[0100] like Figure 5 The following is a typical fragmentation mass spectrum of impurity POLYMER-2. Based on this mass spectrum, the following pyrolysis process can be inferred:

[0101]

[0102] The mechanism of its production is speculated to be: the four-membered ring and the six-membered ring of cefazolin are easily cleaved and opened at high temperature. At this time, the thiol group is unstable and easily demethylthiothiazolidine is replaced by a hydroxyl group. The degradation products formed after the cleavage of the six-membered ring react with the dehydrated polymer of the cefazolin ring opening product to form a new polymer impurity.

[0103]

[0104] The following polymer impurity chromatographic peaks and inferred structures of polymer impurities were obtained through mass spectrometry analysis and fragment structure inference, and were named POLYMER-1 and POLYMER-2 according to the order of polymer impurity peaks.

[0105] Comparative Example 1

[0106] The determination method of the standard polymer of cefazolin sodium in Part II of the 2020 edition of the Chinese Pharmacopoeia is consistent with that in the molecular exclusion chromatography (General Rules 0514 of Part IV of the 2020 edition of the Chinese Pharmacopoeia), and the chromatogram is recorded as follows: Figure 6-7 shown.

[0107] Test results of three batches of cefazolin sodium polymers

[0108]

[0109] Comparative Example 2

[0110] The results of the 6-month stability polymer detection of cefazolin sodium for injection using the ChP2020 method and reversed-phase HPLC are as follows. It can be seen that the reversed-phase chromatography method can detect multiple polymer impurities of cefazolin sodium, and the separation degree of each impurity is good, indicating that the reversed-phase chromatography method for analyzing polymer impurities of cefazolin sodium has good specificity.

[0111] Summary table of polymer stability test results

[0112]

[0113] Note: *C18 method.

Claims

1. A method for analyzing impurities of cefazolin sodium and cefazolin sodium polymer for injection, characterized in that: Using RP-HPLC chromatography method, Among them, the chromatographic conditions of RP-HPLC are: Use ammonium acetate solution with a pH of 5.0±0.2-acetonitrile = 90-80:10-20 as mobile phase A; Use ammonium acetate solution with a pH of 5.0±0.2-acetonitrile = 50-40:50-60 as mobile phase B, Gradient elution; The column temperature is 40℃±2℃.

2. The impurity analysis method applicable to cefazolin sodium and cefazolin sodium polymer for injection as claimed in claim 1, characterized in that: Octadecylsilane bonded silica gel is used as the chromatographic column or a chromatographic column with equivalent performance.

3. The impurity analysis method applicable to cefazolin sodium and cefazolin sodium polymer for injection as claimed in claim 1, characterized in that: The gradient elution is: 。 4. The impurity analysis method applicable to cefazolin sodium and cefazolin sodium polymer for injection as claimed in claim 1, characterized in that: The flow rate was 0.5-1.0 mL per minute.

5. The impurity analysis method applicable to cefazolin sodium and cefazolin sodium polymer for injection as claimed in claim 1, characterized in that: The injection volume is 10-20 μl.

6. The impurity analysis method applicable to cefazolin sodium and cefazolin sodium polymer for injection as claimed in claim 1, characterized in that: Use mass spectrometry for impurity structure analysis; The mass spectrometry conditions are: spray voltage: 3.9KV; Spray temperature: 320℃; Ion transfer tube temperature: 320°C; Sheath air: 60arb; Auxiliary gas: 15arb; Lens voltage: 55V; Scan mode: Ful1 MS / dd MS 2 ; Ionization mode: ESI+; Primary scanning range: 200-1800m / z; primary resolution: 70000 Secondary energy: NCE102030; secondary resolution: 17500; isolation width: 3.0m / z.

7. An impurity of cefazolin sodium and a polymer of cefazolin sodium for injection, characterized in that:

8. An impurity of cefazolin sodium and a polymer of cefazolin sodium for injection, characterized in that:

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