Determination of Related Substances of Penicillin Sodium by Ultra-High Performance Liquid Chromatography

Abstract

The present invention belongs to the technical field of drug analysis, and specifically relates to an ultra-high performance liquid chromatography method for determining penicillin sodium related substances. The ultra-high performance liquid chromatography method for determining penicillin sodium related substances adopts ultra-high performance liquid chromatography, and the chromatographic column is C 18 The chromatographic column uses octadecylsilane bonded silica gel as a filler, a buffered salt solution as a mobile phase A, acetonitrile or methanol as a mobile phase B, and gradient elution. The determination method of the invention reduces the degradation degree of penicillin sodium in the mobile phase of the chromatographic column, avoids the misjudgment phenomenon of classifying impurities generated by degradation in the chromatographic column as sample impurities due to the limitation of analytical detection conditions, more truly reflects the actual quality status of the sample, shortens the analysis time, reduces the amount of solvent used, and reduces the analysis cost.

Description

Technical Field

[0001] The invention belongs to the technical field of drug analysis, and in particular relates to an ultra-high performance liquid chromatography determination method for sodium penicillin-related substances. Background Art

[0002] The chemical name of penicillin sodium is (2S, 5R, 6R)-3,3-dimethyl-6-(2-phenylacetylamino)-7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylic acid sodium salt, with a molecular weight of 356.38. The specific structural formula is as follows:

[0003]

[0004] There are many research reports on penicillin sodium related substances. For example, patent CN106404953A discloses a quality detection method for penicillin skin test freeze-dried powder, which uses high performance liquid chromatography, wherein the chromatographic column uses Nova pak C18, 5μm, 3.9mm×150nm; the chromatographic column temperature is 30℃, the mobile phase flow rate is 1.0ml / min, the detection wavelength is 235nm, the injection volume is 20μl, and the mobile phase is methanol-acetonitrile-water (63:12:25), and isocratic elution is performed. This detection method is mainly to ensure that the auxiliary materials lactose and mannitol can achieve good separation from penicillin and ensure the content detection of penicillin. It has not been studied whether the 12 impurities in the pharmacopoeias of various countries under the penicillin related substances can be effectively separated under this chromatographic condition.

[0005] In addition, the standards for penicillin sodium and penicillin sodium for injection are also included in the USP Pharmacopoeia, EP Pharmacopoeia, BP Pharmacopoeia, Indian Pharmacopoeia IP, and ChP. When the original pharmacopoeia method was used for testing, it was found that some EP impurities could not be effectively separated, and the liquid phase running time in the ChP2020 method was relatively long. The BP / EP pharmacopoeia method was used, and the column temperature was 50°C, which caused penicillin to degrade during the elution process in the chromatographic column (the baseline kept moving up from about 15 minutes to 24 minutes). The test solution had a large and obvious degradation unknown impurity (retention time was 23.952 minutes), as shown in the chromatogram. Figure 1 .

[0006] This impurity is not included in the pharmacopoeia. Through the preparation and structural analysis of this impurity, its structure confirmation and structural analysis speculate that this unknown obvious degradation impurity is a ring-opening impurity generated by hydrolysis of sodium penicillin. The phenylacetyl carbonyl of the ring-opening impurity attacks the five-membered ring amino group, further dehydrates to generate EP impurity D, and EP impurity D isomerizes to generate this impurity. This impurity is more likely to be generated in penicillin sodium solution under acid, high temperature and light conditions, but not easily generated in solid state.

[0007] Therefore, it is necessary to improve the analytical detection methods to reduce the degree of degradation of sodium penicillin in the mobile phase of the chromatographic column, to detect the related substances of sodium penicillin more truly, effectively and accurately, and to change the past phenomenon that the samples did not contain the corresponding impurities, but due to the limitations of analytical detection conditions, the impurities produced by degradation in the chromatographic column were misclassified as sample impurities, so as to more truly reflect the actual quality status of the samples. Summary of the invention

[0008] The technical problem to be solved by the present invention is to provide an ultra-high performance liquid chromatography determination method for sodium penicillin related substances, which reduces the degradation degree of sodium penicillin in the mobile phase of a chromatographic column, avoids the misjudgment phenomenon that impurities generated by degradation in the chromatographic column are classified as sample impurities due to the limitation of analytical detection conditions, more truly reflects the actual quality status of the sample, shortens the analysis time, reduces the amount of solvent used, and reduces the analysis cost.

[0009] The ultra-high performance liquid chromatography method for determining the related substances of sodium penicillin of the present invention adopts ultra-high performance liquid chromatography, and the chromatographic column is C 18 The chromatographic column uses octadecylsilane bonded silica gel as filler, a buffered salt solution as mobile phase A, acetonitrile or methanol as mobile phase B, and gradient elution.

[0010] Preferably, the C 18 The chromatographic column is YMC C18.

[0011] Preferably, the chromatographic column has specifications of 2.1×100 mm and 1.9 μm.

[0012] Preferably, the volume ratio of the mobile phase A to the mobile phase B is (100-0):(0-100); further preferably, the volume ratio of the mobile phase A to the mobile phase B is (95-50):(5-50).

[0013] Preferably, the gradient elution is a linear gradient elution, and the elution conditions are:

[0014] The first gradient elution time is 0 min, the mobile phase A accounts for 100-90%, and the mobile phase B accounts for 0-10%;

[0015] The second gradient elution time is 4.6 min, with mobile phase A accounting for 85-75% and mobile phase B accounting for 15-25%;

[0016] The third gradient elution time is 11.6 min, with mobile phase A accounting for 85-75% and mobile phase B accounting for 15-25%;

[0017] The fourth gradient elution time is 14.0 min, the mobile phase A accounts for 45-55%, and the mobile phase B accounts for 45-55%;

[0018] The fifth gradient elution time is 14.5 min, the mobile phase A accounts for 100-90%, and the mobile phase B accounts for 0-10%;

[0019] The fifth gradient elution time is 18.5 min, the mobile phase A accounts for 100-90%, and the mobile phase B accounts for 0-10%.

[0020] Further preferably, the gradient elution is a linear gradient elution, and the elution conditions are:

[0021] The first gradient elution time is 0 min, with mobile phase A accounting for 95% and mobile phase B accounting for 5%;

[0022] The second gradient elution time was 4.6 min, with mobile phase A accounting for 80% and mobile phase B accounting for 20%;

[0023] The third gradient elution time was 11.6 min, with mobile phase A accounting for 80% and mobile phase B accounting for 20%;

[0024] The fourth gradient elution time was 14.0 min, with mobile phase A accounting for 50% and mobile phase B accounting for 50%;

[0025] The fifth gradient elution time was 14.5 min, with mobile phase A accounting for 95% and mobile phase B accounting for 5%;

[0026] The fifth gradient elution time was 18.5 min, with mobile phase A accounting for 95% and mobile phase B accounting for 5%.

[0027] Preferably, the HPLC is detected using an ultraviolet detector with a detection wavelength of 210 to 254 nm, more preferably 225 nm.

[0028] Preferably, the buffered saline solution is a mixed aqueous solution of sodium dihydrogen phosphate and disodium hydrogen phosphate, or a mixed aqueous solution of dipotassium hydrogen phosphate and potassium dihydrogen phosphate; preferably a mixed aqueous solution of sodium dihydrogen phosphate and disodium hydrogen phosphate.

[0029] Preferably, the concentration of the buffered saline solution is 0.002-0.02 mol / L; more preferably 0.01 mol / L.

[0030] Preferably, the flow rate of the mobile phase is 0.25-0.35 ml / min; more preferably 0.3 ml / min.

[0031] Preferably, the temperature of the chromatographic column is 20-40°C; more preferably, it is 30°C.

[0032] Preferably, the injection volume is 2-5 μl; more preferably, it is 2.5 μl.

[0033] In a preferred experimental scheme, the determination method comprises the following steps:

[0034] (1) Preparation of impurity reference solution:

[0035] Impurity A reference solution: Take impurity A, dissolve it in methanol and dilute it with water to make a solution with a concentration of 8 μg / ml;

[0036] Impurity B reference solution: Take impurity B, dissolve it in methanol and dilute it with water to make a solution with a concentration of 20 μg / ml;

[0037] Impurity C reference solution: Take impurity C, dissolve it in methanol and dilute it with water to make a solution with a concentration of 8 μg / ml;

[0038] Impurity D reference solution: Take impurity D, dissolve it in methanol and dilute it with water to make a solution with a concentration of 8 μg / ml;

[0039] Impurity E reference solution: Take impurity E, dissolve it in methanol and dilute it with water to make a solution with a concentration of 80 μg / ml;

[0040] Impurity F reference solution: Take impurity F, dissolve it in methanol and dilute it with water to make a solution with a concentration of 40 μg / ml;

[0041] Impurity H reference solution: Take impurity H, dissolve it in methanol and dilute it with water to make a solution with a concentration of 8 μg / ml;

[0042] Impurity J reference solution: Take impurity J, dissolve it in methanol and dilute it with water to make a solution with a concentration of 8 μg / ml;

[0043] Impurity L reference solution: Take impurity L, dissolve it in methanol and dilute it with water to make a solution with a concentration of 8 μg / ml;

[0044] Impurity I reference solution: Take impurity I, dissolve it in methanol and dilute it with water to make a solution with a concentration of 8 μg / ml;

[0045] Impurity II reference solution: Take impurity II, dissolve it in methanol and dilute it with water to make a solution with a concentration of 8 μg / ml;

[0046] (2) Preparation of mixed solution:

[0047] Take 1.6 mg each of impurity A, impurity C, impurity D, impurity H, impurity J, impurity L, impurity I and impurity II, accurately weigh them, place them in 10 ml volumetric flasks, add methanol to dissolve them, dilute them to the mark with water, and shake them well; take 2 mg of impurity B, accurately weigh them, place them in 5 ml volumetric flasks, add methanol to dissolve them, dilute them to the mark with water, and shake them well; these are used as stock solutions of impurity A, impurity C, impurity D, impurity H, impurity J, impurity L, impurity I, impurity II and impurity B respectively;

[0048] Take 40 mg of sodium penicillin, 0.8 mg of impurity E, and 0.4 mg of impurity F and place them in the same 10 ml volumetric flask, and accurately add 0.5 ml of each of the stock solutions of impurity A, impurity C, impurity D, impurity H, impurity J, impurity L, impurity I, impurity II, and impurity B, shake to dissolve, add water to dilute to the scale, and shake well;

[0049] (3) Preparation of system suitability solution:

[0050] Take penicillin system suitability reference, dissolve it in methanol and dilute it with water to make a solution with a concentration of 5 mg / ml;

[0051] (4) Preparation of test solution:

[0052] Take the penicillin sodium test sample, dissolve it in water and dilute it to make a solution with a concentration of 4 mg / ml;

[0053] (5) Self-control solution:

[0054] Accurately measure 1 ml of the test solution, place it in a 100 ml volumetric flask, dilute to the mark with water, and shake well;

[0055] (6) The prepared sample was analyzed by ultra-high performance liquid chromatography, and the chromatographic conditions were as follows: chromatographic column, YMCC18, 2.1×100 mm, 1.9 μm, octadecylsilane bonded silica gel as filler; injection volume, 2-5 μl; detection wavelength, 210-254 nm; column temperature, 20-40° C.; flow rate, 0.25-0.35 mL / min; a mixed aqueous solution of disodium hydrogen phosphate and sodium dihydrogen phosphate as mobile phase A, acetonitrile as mobile phase B, and gradient elution;

[0056] Record the chromatogram, characterize the impurities by their retention time in the positioning solution and the mixed solution, and calculate the content of each impurity by the self-control method with correction factors.

[0057] Preferably, in the above method, the solvent used to prepare the solution is water or a mixed solution of methanol and water in a volume ratio of 35:65.

[0058] The specific impurities are as follows:

[0059] Impurity A: (2S, 5R, 6R)-6-amino-3, 3-dimethyl-7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylic acid, molecular formula C 8 H 12 N 2 O 3 S, molecular weight 216.26, structural formula:

[0060]

[0061] Impurity B: Phenylacetic acid, molecular formula C 8 H 8 O 2 , molecular weight 136.15, structural formula:

[0062]

[0063] Impurity C: (2S, 5R, 6R)-6-[[(4-hydroxyphenyl)acetyl]amino]-3,3-dimethyl-7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylic acid, molecular formula C 16 H 18 N 2 O 5 S, molecular weight 350.39, structural formula:

[0064]

[0065] Impurity D: (3S,7R,7aR)-5-phenyl-2,2-dimethyl-2,3,7,7a-tetrahydroimidazo[5,1-b]thiazole-3,7-dicarboxylic acid, molecular formula C 16 H 18 N 2 O 4 S, molecular weight 334.39, structural formula:

[0066]

[0067] Impurity E: (4S)-2-[Carboxy[(phenylacetyl)amino]methyl]-5,5-dimethylthiazolidine-4-carboxylic acid, molecular formula C 16 H 20 N 2 O 5 S, molecular weight 352.40, structural formula:

[0068]

[0069] Impurity F: (2RS,4S)-2-[[(phenylacetyl)amino]methyl]-5,5-dimethylthiazolidine-4-carboxylic acid, molecular formula C 15 H20 N 2 O 3 S, molecular weight 308.40, structural formula:

[0070]

[0071] Impurity H: Potassium (2S,5R,6R)-6-(hexanoylamino)-3,3-dimethyl-7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylate, molecular formula C 14 H 21 KN 2 O 4 S, molecular weight 352.09, structural formula:

[0072]

[0073] Impurity J: 2-[Carboxy[(phenylacetyl)amino]methyl]-3-formyl-5,5-dimethylthiazolidine-4-carboxylic acid, molecular formula C 17 H 20 N 2 O 6 S, molecular weight 380.10, structural formula:

[0074]

[0075] Impurity L: 2-[Carboxy[(phenylacetyl)amino]methyl]-3-[2-[(phenylacetyl)amino]acetyl]-5,5-dimethylthiazolidine-4-carboxylic acid, molecular formula C 26 H 29 N 3 O 7 S, molecular weight 527.17, structural formula:

[0076]

[0077] Impurity I: 2-Hydroxy-5,5-dimethylthiazolidine-4-carboxylic acid, molecular formula C 6 H 14 NO 3 S, molecular weight 177.218, structural formula:

[0078]

[0079] Impurity II: 2-[(phenylacetyl)amino]-acetic acid, molecular formula C 10 H 11 NO 3 , molecular weight 193.07, structural formula:

[0080]

[0081] Compared with the prior art, the present invention has the following beneficial effects:

[0082] (1) The ultra-high performance liquid chromatography method for determining the related substances of sodium penicillin provided by the present invention adjusts the mobile phase to be neutral, lowers the column temperature, and shortens the detection time by using ultra-high performance liquid chromatography, thereby reducing the degree of degradation of sodium penicillin in the mobile phase of the chromatographic column, avoiding the phenomenon of misjudging the impurities generated by degradation in the chromatographic column as sample impurities due to the limitation of analytical detection conditions, more truly reflecting the actual quality status of the sample, shortening the analysis time, reducing the amount of solvent used, and reducing the analysis cost;

[0083] (2) The determination method of the present invention can quickly and effectively detect the related substances of penicillin sodium, not only reducing the degree of degradation of penicillin sodium during the detection process, but also having better separation between impurities, good system applicability, good linear relationship, high precision, good accuracy, good repeatability, and more accurate and true results. BRIEF DESCRIPTION OF THE DRAWINGS

[0084] Figure 1 This is a typical spectrum of BP / EP related substances;

[0085] Figure 2 A blank solvent spectrum is detected for the method of the present invention;

[0086] Figure 3 It is a localization map of impurity A detected by the method of the present invention;

[0087] Figure 4 It is a localization map of impurity C detected by the method of the present invention;

[0088] Figure 5 It is a localization map of impurity D detected by the method of the present invention;

[0089] Figure 6 It is a localization map of impurity E detected by the method of the present invention;

[0090] Figure 7 It is a localization map of impurity F detected by the method of the present invention;

[0091] Figure 8 It is a localization map of impurity H detected by the method of the present invention;

[0092] Fig. 9 It is a localization map of impurity J detected by the method of the present invention;

[0093] Fig.10 It is a localization map of impurity L detected by the method of the present invention;

[0094] Fig.11 It is a localization map of impurity I detected by the method of the present invention;

[0095] Fig.12 It is a localization map for detecting impurity II by the method of the present invention;

[0096] Fig.13 A mixed solution spectrum is detected by the method of the present invention;

[0097] Fig.14 It is a graph showing the suitability of the detection system of the method of the present invention;

[0098] Fig.15 The method of the present invention detects the sample solution spectrum;

[0099] Fig.16 The present invention is a method for detecting a self-control solution spectrum. DETAILED DESCRIPTION

[0100] The present invention is further described below in conjunction with embodiments, but the protection scope of the present invention is not limited thereto, and all changes made to the technical solution of the present invention by professionals in the field should fall within the protection scope of the present invention.

[0101] The penicillin sodium raw material used in the examples comes from: Ruiyang Pharmaceutical Co., Ltd., batch number 892101102. Other reagents are commercially available conventional raw materials unless otherwise specified.

[0102] The determination method used in the examples includes: sample preparation, sample determination and impurity content calculation, as shown in Table 1.

[0103] Table 1 Determination method

[0104]

[0105] Example 1

[0106] Specificity test:

[0107] The specificity of the method is examined by injecting each impurity location solution and a mixed solution containing impurities as a specific solution. The specificity test is used to determine the relative retention time and separation degree of the potential impurity under the assay conditions and to determine whether the impurity of concern can be effectively separated.

[0108] The specificity test results are shown in Table 2.

[0109] Table 2 Results of specificity test for relevant substances

[0110]

[0111] From Table 2, it can be concluded that the minimum separation degree between penicillin and adjacent impurities is 1.283 (not less than 1.2), which meets the requirements. All known impurities can be effectively separated with strong specificity.

[0112] Example 2

[0113] Detection limit and quantification limit test:

[0114] Take appropriate amount of penicillin sodium and various impurity reference substances, and use stepwise dilution method. The concentration when the signal-to-noise ratio S / N ≥ 3 is taken as the detection limit concentration; when the signal-to-noise ratio S / N ≥ 10, the concentration is taken as the quantification limit concentration. The RSD of the retention time of 6 injections of the quantification limit solution should not be greater than 1%, and the RSD of the peak area should not be greater than 10%.

[0115] The results of the detection limit and quantification limit tests are shown in Tables 3 and 4.

[0116] Table 3 Test results of detection limit and quantitative limit of relevant substances-1

[0117]

[0118] Table 4 Test results of detection limit and quantitative limit of relevant substances-2

[0119]

[0120]

[0121] From Table 3-4, it can be concluded that the maximum detection concentration of the quantitative limit of penicillin and each impurity is equivalent to 0.0203% (not more than 0.05%) of the test sample concentration, which meets the requirements. The maximum RSD of the retention time of the 6-shot quantitative limit solution of penicillin and each impurity is 0.40% (not more than 1%), and the maximum RSD of the peak area is 8.02% (not more than 10%). The quantitative limit and detection limit are good.

[0122] Example 3

[0123] Linearity and range tests:

[0124] The linearity of the method was achieved by measuring the linearity of 5 to 8 solutions of different concentrations, and the peak area was plotted against the concentration, showing good linearity (linear correlation coefficient ≥ 0.99), and the range of the determination was determined at the same time.

[0125] The results of the penicillin linearity test are shown in Table 5.

[0126] Table 5 Results of the linear relationship test of penicillin in the inspection of related substances

[0127]

[0128]

[0129] The results of the linearity test of impurity A are shown in Table 6.

[0130] Table 6 Results of linear relationship test of impurity A in related substance inspection

[0131]

[0132] The results of the linearity test of impurity B are shown in Table 7.

[0133] Table 7 Results of linear relationship test of impurity B in related substance inspection

[0134]

[0135] The results of the linearity test of impurity C are shown in Table 8.

[0136] Table 8 Results of linear relationship test of impurity C in related substance inspection

[0137]

[0138]

[0139] The results of the linearity test of impurity D are shown in Table 9.

[0140] Table 9 Results of linear relationship test of impurity D in related substance inspection

[0141]

[0142] The results of the linearity test of impurity E are shown in Table 10.

[0143] Table 10 Results of linear relationship test of impurity E in related substance inspection

[0144]

[0145] The results of the linearity test of impurity F are shown in Table 11.

[0146] Table 11 Results of linear relationship test of impurity F in related substance inspection

[0147]

[0148] The results of the impurity H linearity experiment are shown in Table 12.

[0149] Table 12 Results of linear relationship test of impurity F in related substance inspection

[0150]

[0151] The results of the impurity J linearity test are shown in Table 13.

[0152] Table 13 Results of linear relationship test of impurity J in related substance inspection

[0153]

[0154]

[0155] The results of the impurity L linearity test are shown in Table 14.

[0156] Table 14 Results of linear relationship test of impurity L in related substance inspection

[0157]

[0158] According to the above linear test results, the relative correction factor (f = k 主 / k 杂质 ), the specific results are shown in Table 15.

[0159] Table 15 Test results of correction factors for impurities in related substances

[0160] name Relative correction factor penicillin / Impurity A 1.4 Impurity B 0.9 Impurity C 0.5 Impurity D 0.8 Impurity E 1.7 Impurity F 1.8 Impurity H 1.9 Impurity J 0.9 Impurity L 0.8

[0161] From Table 5-15, it can be concluded that the correlation coefficients between the linearity and range of penicillin and its impurities are all greater than 0.99, and the linear relationship is good.

[0162] Example 4

[0163] Accuracy test:

[0164] Accuracy refers to the degree of closeness between the method and the true value, expressed as recovery rate, and is achieved by examining the accuracy of adding impurities of different concentrations to the sample. This method requires that the average recovery rate of each impurity is within the range of 85%-110%; RSD < 10%.

[0165] The accuracy test results of each impurity are shown in Table 16.

[0166] Table 16 Results of the accuracy test of relevant substances

[0167] name Average recovery rate (%) RSD(%) Impurity A 99.25 3.37 Impurity B 103.9 1.01 Impurity C 99.16 0.98 Impurity D 106.2 2.06 Impurity E 94.67 1.03 Impurity F 97.81 1.37 Impurity H 98.06 0.89 Impurity J 92.05 3.25 Impurity L 95.78 2.24 Impurity I 96.49 3.63 Impurity II 102.5 2.53

[0168] From Table 16, it can be concluded that the average values ​​of the impurity recovery rates are all within the range of 85%-110%, and the maximum RSD of the average values ​​of the impurity recovery rates is 3.63% (RSD<10%), which meets the requirements and has good accuracy.

[0169] Example 5

[0170] Durability test:

[0171] By changing different chromatographic conditions, the system applicability meets the standard requirements, the determination results of relevant substances under different conditions are consistent, and the durability is good.

[0172] The results obtained under different chromatographic conditions are shown in Table 17.

[0173] Table 17 Related material inspection durability test results

[0174]

[0175]

[0176] From Table 17, it can be concluded that by changing different chromatographic conditions, the separation degree of impurity E and its isomers in the system suitability solution is as low as 1.391 (>1.2), which meets the requirements, the maximum single impurity detection is basically consistent, and the RSD of the total impurity change is 6.66% (RSD <10%), which meets the requirements. This method has good durability.

[0177] It can be known from the above verification results that the determination method of the present invention uses a neutral mobile phase, reduces the column temperature, shortens the detection time, can effectively reduce the degradation degree of penicillin sodium in the chromatographic column, changes the past sample does not contain corresponding impurities, due to the limitation of analytical detection conditions, the impurities produced by degradation in the chromatographic column are classified as sample impurities, and can more truly reflect the actual quality of the sample. In addition, the method has high sensitivity, strong specificity, high accuracy, good durability, strong applicability, and stable and reliable results, so it can be used for the quality control of penicillin sodium, provides effective guarantee for the quality of the final product, and fills the gap in the detection of penicillin sodium related substances using liquid chromatograph.

Claims

1. A method for determining the related substances of sodium penicillin by ultra-high performance liquid chromatography, characterized in that: The steps include: (1) Preparation of impurity reference solution: Impurity A reference solution: Take impurity A, dissolve it in methanol and dilute it with water to make a solution with a concentration of 8 μg / ml; Impurity B reference solution: Take impurity B, dissolve it in methanol and dilute it with water to make a solution with a concentration of 20 μg / ml; Impurity C reference solution: Take impurity C, dissolve it in methanol and dilute it with water to make a solution with a concentration of 8 μg / ml; Impurity D reference solution: Take impurity D, dissolve it in methanol and dilute it with water to make a solution with a concentration of 8 μg / ml; Impurity E reference solution: Take impurity E, dissolve it in methanol and dilute it with water to make a solution with a concentration of 80 μg / ml; Impurity F reference solution: Take impurity F, dissolve it in methanol and dilute it with water to make a solution with a concentration of 40 μg / ml; Impurity H reference solution: Take impurity H, dissolve it in methanol and dilute it with water to make a solution with a concentration of 8 μg / ml; Impurity J reference solution: Take impurity J, dissolve it in methanol and dilute it with water to make a solution with a concentration of 8 μg / ml; Impurity L reference solution: Take impurity L, dissolve it in methanol and dilute it with water to make a solution with a concentration of 8 μg / ml; Impurity I reference solution: Take impurity I, dissolve it in methanol and dilute it with water to make a solution with a concentration of 8 μg / ml; Impurity II reference solution: Take impurity II, dissolve it in methanol and dilute it with water to make a solution with a concentration of 8 μg / ml; (2) Preparation of mixed solution: Take 1.6 mg each of impurity A, impurity C, impurity D, impurity H, impurity J, impurity L, impurity I, and impurity II, accurately weigh them, place them in 10 ml volumetric bottles, add methanol to dissolve them, dilute them to the mark with water, and shake them well; take 2 mg of impurity B, accurately weigh them, place them in 5 ml volumetric bottles, add methanol to dissolve them, dilute them to the mark with water, and shake them well; these are used as stock solutions of impurity A, impurity C, impurity D, impurity H, impurity J, impurity L, impurity I, impurity II, and impurity B respectively; Take 40 mg of sodium penicillin, 0.8 mg of impurity E, and 0.4 mg of impurity F and place them in the same 10 ml volumetric flask, and accurately add 0.5 ml of each of the stock solutions of impurity A, impurity C, impurity D, impurity H, impurity J, impurity L, impurity I, impurity II, and impurity B, shake to dissolve, add water to dilute to the scale, and shake well; (3) Preparation of system suitability solution: Take penicillin system suitability reference, dissolve it in methanol and dilute it with water to make a solution with a concentration of 5 mg / ml; (4) Preparation of test solution: Take the penicillin sodium test sample, dissolve it in water and dilute it to make a solution with a concentration of 4 mg / ml; (5) Self-control solution: Accurately measure 1 ml of the test solution, place it in a 100 ml volumetric flask, dilute to the mark with water, and shake well; (6) The prepared sample was analyzed by ultra-high performance liquid chromatography, and the chromatographic conditions were as follows: chromatographic column, YMCC18, 2.1×100 mm, 1.9 μm, octadecylsilane bonded silica gel as filler; injection volume, 2-5 μl; detection wavelength, 210-254 nm; column temperature, 20-40° C.; flow rate, 0.25-0.35 mL / min; a mixed aqueous solution of disodium hydrogen phosphate and sodium dihydrogen phosphate as mobile phase A, acetonitrile as mobile phase B, and gradient elution; the concentration of the mixed aqueous solution of disodium hydrogen phosphate and sodium dihydrogen phosphate was 0.002-0.02 mol / L; Record the chromatogram, characterize the impurities by the retention time of the positioning solution and the mixed solution, and calculate the content of each impurity by the self-control method with correction factors; The gradient elution is a linear gradient elution, and the elution conditions are: The first gradient elution time is 0 min, the mobile phase A accounts for 100-90%, and the mobile phase B accounts for 0-10%; The second gradient elution time is 4.6 min, with mobile phase A accounting for 85-75% and mobile phase B accounting for 15-25%; The third gradient elution time is 11.6 min, with mobile phase A accounting for 85-75% and mobile phase B accounting for 15-25%; The fourth gradient elution time is 14.0 min, the mobile phase A accounts for 45-55%, and the mobile phase B accounts for 45-55%; The fifth gradient elution time is 14.5 min, the mobile phase A accounts for 100-90%, and the mobile phase B accounts for 0-10%; The sixth gradient elution time is 18.5 min, the mobile phase A accounts for 100-90%, and the mobile phase B accounts for 0-10%; in, The structural formula of impurity A is as follows: The structural formula of impurity B is as follows: The structural formula of impurity C is as follows: The structural formula of impurity D is as follows: The structural formula of impurity E is as follows: The structural formula of impurity F is as follows: The structural formula of impurity H is as follows: The structural formula of impurity J is as follows: The structural formula of impurity L is as follows: The structural formula of impurity I is as follows: The structural formula of impurity II is as follows:

2. The method for determining the related substances of sodium penicillin by ultra-high performance liquid chromatography according to claim 1, characterized in that: The gradient elution is a linear gradient elution, and the elution conditions are: The first gradient elution time is 0 min, with mobile phase A accounting for 95% and mobile phase B accounting for 5%; The second gradient elution time was 4.6 min, with mobile phase A accounting for 80% and mobile phase B accounting for 20%; The third gradient elution time was 11.6 min, with mobile phase A accounting for 80% and mobile phase B accounting for 20%; The fourth gradient elution time was 14.0 min, with mobile phase A accounting for 50% and mobile phase B accounting for 50%; The fifth gradient elution time was 14.5 min, with mobile phase A accounting for 95% and mobile phase B accounting for 5%; The sixth gradient elution time was 18.5 min, with mobile phase A accounting for 95% and mobile phase B accounting for 5%.

3. The method for determining the related substances of sodium penicillin by ultra-high performance liquid chromatography according to claim 1, characterized in that: The injection volume for UPLC was 2.5 μl.

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