A method for detecting bacitracin in compound polymyxin B ointment

Abstract

The present invention discloses a method for detecting bacitracin in compound polymyxin B ointment. In the present invention, the compound polymyxin B ointment is heated and melted, n-hexane is added to disperse the ointment body evenly, then ammonia water solution is added for extraction, the extraction liquids are combined, the pH value is adjusted to 7.0, and then chromatographic detection is carried out under improved chromatographic conditions. For the pretreatment method of the present invention, the recovery rate of bacitracin is high, close to 100%, the types and contents of impurities have no difference from those of the reference substance, which can meet the detection requirements and can be used for the quality control of bacitracin in compound polymyxin B ointment.

Description

Technical Field

[0001] The invention relates to the technical field of drug detection, and in particular to a method for detecting bacitracin in compound polymyxin B ointment. Background Art

[0002] Compound polymyxin B ointment is a compound preparation composed of polymyxin B sulfate, neomycin sulfate, bacitracin and lidocaine hydrochloride. It is a safe and highly effective topical antibacterial drug used to prevent bacterial infections caused by minor cuts, abrasions and burns. Neomycin sulfate is an aminoglycoside antibiotic that can bind to the 30S subunit of the bacterial ribosome and interfere with the transcription of mRNA, thereby inhibiting the biosynthesis of bacterial proteins. Neomycin sulfate has good inhibitory activity against Gram-negative bacteria and a variety of Staphylococci. Due to the existence of neomycin-resistant Staphylococcus aureus, the use of neomycin sulfate alone to treat bacterial infections is insufficient. Bacitracin is a polypeptide antibiotic, and its antibacterial effect is achieved by inhibiting the synthesis of the diphosphate transport molecule-C, which is essential for the synthesis of bacterial cell wall peptidoglycan. 55 The dephosphorylation reaction of isoprene pyrophosphate is carried out. Bacitracin has a good inhibitory effect on Staphylococcus. The combined use of neomycin sulfate and bacitracin can play an antibacterial synergistic role, because bacitracin destroys the integrity of the bacterial cell wall, which is very beneficial to increase the effective concentration of neomycin in the bacterial cell. Polymyxin B sulfate also belongs to the class of polypeptide antibiotics. Its antibacterial effect is achieved by increasing the permeability of the bacterial cell wall, destroying the osmotic pressure balance of the cell wall, and causing cell lysis. Lidocaine hydrochloride accumulates in the cortical pain receptors and nerve endings to achieve the effect of local anesthesia, analgesia and itching in the cortex; it also reduces the synthesis and release of inflammatory mediators, interferes with the inflammatory cascade reaction, and achieves anti-inflammatory effects. Similar products of this compound preparation abroad are called Triple Antibiotic Ointment (TAO). Since 1953, it has been on the market in the United States as an OTC emergency medicine. In 1990, it was proposed by the Royal Pharmaceutical Society of Great Britain to be included in the preliminary list of eight drugs to be re-used as over-the-counter drugs.

[0003] At present, compound polymyxin B ointment is a conventional ointment and uses an appropriate amount of oily petrolatum as the matrix. Bacitracin has the characteristic of instability in aqueous solution, but it is very stable in the oily petrolatum matrix. Therefore, compound polymyxin B ointment adopts an oily matrix formulation instead of a cream or lotion form. The oily petrolatum matrix maintains the hydration of tissues. By forming an oily isolation film on the wound surface, it reduces the evaporation of wound skin moisture and keeps the wound moist. Treatment with topical antibiotic ointment can significantly reduce bacterial infection, accelerate wound healing, and reduce scar formation. However, when detecting bacitracin and related impurities, the ointment cannot be directly detected and needs to be pretreated, and the pretreatment operation will directly affect the quality control of bacitracin in the ointment. Summary of the Invention

[0004] In view of the above problems, the present invention provides a method for detecting bacitracin in compound polymyxin B ointment. The present invention heats and melts the compound polymyxin B ointment, adds n-hexane to disperse the ointment evenly, then adds an ammonia aqueous solution for extraction, and performs chromatographic detection under improved chromatographic conditions. The pretreatment method of the present invention has a high recovery rate of bacitracin, close to 100%, and the number and content of impurity types are no different from those of the reference substance, which can meet the detection requirements and can be used for the quality control of bacitracin in compound polymyxin B ointment.

[0005] The technical solution of the present invention is: a method for detecting bacitracin in compound polymyxin B ointment, characterized in that the compound polymyxin B ointment is heated and melted, n-hexane is added to disperse the ointment evenly, then an ammonia aqueous solution is added for extraction, the extraction liquids are combined, and the pH value is adjusted to 7.0; then chromatographic detection is carried out.

[0006] The above-mentioned bacitracin refers to the sum of bacitracin A, bacitracin B1, bacitracin B2 and bacitracin B3.

[0007] Preferably, after combining the extraction liquids, back-extraction is carried out with n-hexane, and the pH value of the aqueous phase is adjusted to 7.0. Preferably, the mass-volume ratio of compound polymyxin B ointment to back-extraction n-hexane is preferably 1 g: 3 mL.

[0008] Preferably, the mass-volume ratio of compound polymyxin B ointment to n-hexane is 1 g: 3 - 10 mL.

[0009] Preferably, the concentration of the ammonia aqueous solution is 10%.

[0010] Preferably, the mass-volume ratio of compound polymyxin B ointment to the ammonia aqueous solution is 1 g: 1 mL, and extraction is carried out 3 times.

[0011] Preferably, after adjusting the pH value to 7.0, water is added for dilution, shaken well, filtered, and chromatographic detection is carried out.

[0012] Furthermore, the chromatographic detection is as follows: octadecylsilane bonded silica gel is used as a filler; methanol-acetonitrile (25:5) is used as a mobile phase A, phosphate buffer (pH 6.0)-water (1:3) is used as a mobile phase B, and phase A and phase B are mixed in a volume ratio of 65:35 as the mobile phase; the column temperature is 30°C; the flow rate is 1 ml per minute; and the detection wavelength is 254 nm.

[0013] Beneficial effects of the present invention:

[0014] (1) The pretreatment method of the present invention has a high recovery rate of bacitracin, close to 100%, and the number and content of impurities are no different from those of the reference substance, which can meet the detection requirements and can be used for the quality control of bacitracin in compound polymyxin B ointment;

[0015] (2) The pretreatment method of the present invention uses a small amount of solvent. When the mass volume ratio of the paste to the solvent is 1:3, a very high recovery rate can be achieved. On the one hand, the amount of solvent is saved, which is convenient for subsequent solvent recovery and treatment. On the other hand, the amount of paste sample used during detection is saved, thereby improving research and development efficiency.

[0016] (3) The present invention improves the chromatographic detection conditions of bacitracin by changing the pretreatment conditions of the compound polymyxin B ointment, making it more suitable for the detection of bacitracin in the compound polymyxin B. The detection method has good repeatability, high sensitivity and good linear relationship. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 Liquid chromatogram of bacitracin and impurities in compound polymyxin B ointment after pretreatment (pretreatment 12);

[0018] Figure 2 Chromatogram of blank excipient solution of compound polymyxin B ointment;

[0019] Figure 3 is the linear relationship diagram of bacitracin A;

[0020] Figure 4 Liquid chromatogram of bacitracin reference substance;

[0021] Figure 5 The liquid chromatogram of the extraction phase of pretreatment method 1 (peak range: retention time 15-35min);

[0022] Figure 6 It is the liquid chromatogram of the extraction phase of pretreatment method 2 (peak range: retention time 20-35min);

[0023] Figure 7 It is the liquid chromatogram of the extraction phase of pretreatment method 11 (peak table range: retention time 20-35min);

[0024] Figure 8 It is the liquid chromatogram of the extraction phase of the pretreatment method 12 (peak table range: retention time 20 - 35 min). Specific embodiments

[0025] The present invention will be further described below in conjunction with specific embodiments and the accompanying drawings.

[0026] Example 1: Sample detection and methodological verification

[0027] The instruments, reference substances and reagents are shown in Tables 1 - 3 respectively.

[0028] Table 1 Instrument list

[0029] Name Model Manufacturer Serial Number High Performance Liquid Chromatograph 2695 Waters Hplc-04 High Performance Liquid Chromatograph 2695 Waters Hplc-03 Electronic Balance BT125D Sartorius Tp-01 Electronic Balance TB-214 Sartorius Tp-02 pH Meter Jenco6173 Shanghai Renshi Electronic Instrument Co., Ltd. /

[0030] Table 2 Reference substance list

[0031] Name Batch Number Content (%) Source Impurity E 1-MMS-140-4 ～75 TRC Bacitracin A B015-01BL 98.637％ TOKU-E <![CDATA[Bacitracin B1]]> B016-01BL 95.066％ TOKU-E <![CDATA[Bacitracin B2]]> B017-01BL 92.680％ TOKU-E <![CDATA[Bacitracin B3]]> B060-01BL 94.427％ TOKU-E

[0032] Table 3 Reagent list

[0033] Name Batch Number Grade Source Methanol 0212180301 HPLC Shanghai Xingke Acetonitrile 0114180401 HPLC Shanghai Xingke Potassium Hydrogen Phosphate 20151201 AR Sinopharm Chemical Reagent Co., Ltd. Potassium Dihydrogen Phosphate 20100909 AR Tianjin Kemiou Ultra-pure Water / Ultra-pure Self-made

[0034] 1. Chromatographic conditions and system suitability confirmation

[0035] Preparation of the test solution: Take about 1 g of compound polymyxin B, weigh accurately, place it in a separatory funnel, heat to melt, add 3 mL of n - hexane, shake well to disperse the ointment evenly, add 1 mL of 10% ammonia aqueous solution for extraction, extract 3 times, combine the extraction solutions, add 3 mL of n - hexane for back - extraction, adjust the pH value of the extraction solution in the aqueous phase to 7.0 with hydrochloric acid, dilute with water, shake well, and filter.

[0036] Chromatographic conditions: Use octadecylsilane - bonded silica gel as the filler (Waters Xselect CSH C18 4.6×250 mm, 5 μm); use methanol - acetonitrile (25:5) as mobile phase A, and use phosphate buffer solution (adjust the pH value of the potassium hydrogen phosphate solution at 34.8 g / L to 6.0 with potassium dihydrogen phosphate at 27.2 g / L) - water (1:3) as mobile phase B, and mix mobile phase A and mobile phase B in a ratio of 65:35 as the mobile phase. The column temperature is 30 °C; the flow rate is 1 mL per minute; the detection wavelength is 254 nm.

[0037] Detection: Accurately measure the test solution and inject it into the liquid chromatograph, record the chromatogram until 3 times the retention time of the bacitracin A main peak. The chromatogram is shown in Figure 1 (Extract 10 g of ointment to obtain the test solution for related substances detection), Figure 8 (Extract 1 g of ointment to obtain the chromatographic peak of bacitracin); The chromatogram of the blank excipient solution is shown in Figure 2 (Extract 10 g of ointment).

[0038] As Figure 1 shown, the retention time of the main component bacitracin A of bacitracin is about 31.9 - 33.7 min, and the retention times of other components bacitracin B1, bacitracin B2, and bacitracin B3 are about 21.327 min, 22.222 min, and 25.792 min respectively, and the retention time of impurity E is about 76.312 min. After experimental verification, neither the blank solvent nor the blank excipients showed interference. Relative to bacitracin A, the RRT of bacitracin B1 is 0.64, and the RRT of bacitracin B3 is 0.78, which is consistent with the RRT of about 0.6 and 0.8 in the bacitracin standards of BP2013 and USP36. Impurity E was located using the peak location solution, and this method can fully control impurity E.

[0039] The peak purity of the sample was examined using a diode array detector (DAD). No other substances were contained in the main peak, indicating that the specificity of this method is good.

[0040] 2. Linear relationship

[0041] Take about 100 mg of the working reference substance of bacitracin A, weigh it accurately, place it in a 50 mL volumetric flask, dissolve it with water and make up to the mark, shake well, filter, and use it as the linear stock solution. Accurately pipette 5 mL of the linear stock solution into 10 mL and 50 mL volumetric flasks respectively, dilute to the mark with water, and shake well to obtain solutions with relative test solution concentrations of 50% and 10%; accurately pipette 5 mL of the 10% solution into a 50 mL volumetric flask, dilute to the mark with water, and shake well to obtain a solution with a relative test solution concentration of 1%; accurately pipette 5 mL of the 1% solution into 10 mL and 50 mL volumetric flasks respectively, dilute to the mark with water, and shake well to obtain solutions with relative test solution concentrations of 0.5% and 0.1%. Accurately pipette 100 μL of each of the above solutions and inject them into the liquid chromatograph, and record the chromatogram. Perform linear regression with the peak area of bacitracin A against the concentration C (μg / ml) to calculate the linear regression equation and the correlation coefficient. The test results are shown in Table 4 and Figure 3 as shown.

[0042] Table 4 Test results of the linear relationship of bacitracin A

[0043]

[0044] The test results show that bacitracin A has a good linear relationship in the concentration range of 2.03 μg / ml to 1013.7 μg / ml (relative to the test solution concentration of 0.1% to 50%).

[0045] 3. Detection limit

[0046] Under the determined chromatographic conditions, a series of solutions with different concentrations were prepared from the working reference substance of bacitracin A for detection, and the detection limit of bacitracin A was measured. The results are shown in Table 5.

[0047] Table 5 Results of Detection Limit of Bacitracin A

[0048]

[0049] 4. Repeatability

[0050] Take compound polymyxin B ointment and prepare the test solution according to the method under "Chromatographic Conditions and System Suitability Verification". Accurately measure 100 μl and inject it into the liquid chromatograph, and record the chromatogram. Repeat the preparation of the test solution 6 times for determination, calculate the amount of impurities by the area normalization method, and investigate the precision of this method. The test results are shown in Table 6.

[0051] Table 6 Results of Repeatability Test for the Determination of Related Substances of Bacitracin

[0052]

[0053] Conclusion: This product is an ointment compound preparation. Considering the relatively complex sample pretreatment process of this product, the total sum of impurities before the B1 peak and the RSD of impurity E are both less than 15.0%, indicating that the precision of this method is good.

[0054] 5. Intermediate Precision

[0055] To investigate the influence of random variation factors on precision, another analyst independently established a system and redetected by preparing 6 test solutions again according to the operation process under "Repeatability Test", using different instruments and on different dates. It is required to compare the precision results with the 12 determination results of the repeatability results to confirm good intermediate precision. The test results are shown in Table 7.

[0056] Table 7 Results of Intermediate Precision Test for the Inspection of Bacitracin

[0057]

[0058]

[0059] Test Conclusion: This product is an ointment compound preparation. Considering the relatively complex sample pretreatment process of this product, the total sum of impurities before the B1 peak and the RSD of impurity E are both less than 15.0%. From the above results, it can be seen that the precision of this method is good.

[0060] Example 2: Influence of Different Sample Pretreatment Methods on the Recovery Rate of Bacitracin

[0061] Under the chromatographic conditions of Example 1, select the best sample pretreatment method for compound polymyxin B ointment to solve the problem of the recovery rate of bacitracin, and then conduct subsequent total impurity detection and quality control.

[0062] The liquid chromatogram of the bacitracin reference substance is shown inFigure 4 It can be seen that the retention time of bacitracin A is about 33.716 min, and the retention times of other components, bacitracin B1, bacitracin B2, and bacitracin B3, are about 21.597 min, 22.574 min, and 26.283 min, respectively.

[0063] The external standard method was used in this invention to calculate the recovery rate of bacitracin:

[0064] Recovery rate % = A 样 × m 对 / A 对 × m 样 × 100%

[0065] A 样 : The sum of A in the sample 杆菌肽A+ A 杆菌肽B1+ A 杆菌肽B2+ A 杆菌肽B3 ;

[0066] A 对 : The sum of A in the reference substance 杆菌肽A+ A 杆菌肽B1+ A 杆菌肽B2+ A 杆菌肽B3 ;

[0067] m 对 : The weighed amount of the reference substance;

[0068] m 样 : The weighed amount of the sample.

[0069] Note: m 样 In this example, it is the usage amount of bacitracin in compound polymyxin B. One product (10 g) contains 5000 units (about 65.78 mg) of bacitracin.

[0070] The bacitracin reference substance used in this example was purchased from Huabei Pharmaceutical Huasheng Co., Ltd.; the compound polymyxin B ointment was purchased from Zhejiang Fenuo Pharmaceutical Co., Ltd.

[0071] Pretreatment method 1:

[0072] Take 1 g of compound polymyxin B ointment, dissolve it with 40 mL of ether, extract it 4 times with 40 mL of phosphate buffer solution (pH 6.0), combine the extraction solutions, dilute with water, shake well, filter, and perform chromatographic detection. The detection results are shown in Figure 5 , from Figure 5 it can be seen that there are many impurity peaks in the chromatogram. The recovery rate of bacitracin (bacitracin A + bacitracin B1 + bacitracin B2 + bacitracin B3) was calculated using the above recovery rate calculation formula. After calculation, its recovery rate was 74.25%, and the recovery rate was relatively low.

[0073] Pretreatment method 2: Change the extraction solution to ammonia water solution

[0074] Take 1 g of compound polymyxin B ointment, dissolve it with 40 mL of ether, extract it 4 times with 40 mL of 10% ammonia water solution, combine the extraction liquid, adjust the pH to 7.0 with hydrochloric acid, dilute it with water, shake well, filter, and conduct chromatographic detection. The detection results are shown in Figure 6 , compared with the first pretreatment method, the impurity peaks are reduced, but there is still 1 more impurity peak than the control. Using the above recovery rate calculation formula, the recovery rate of bacitracin (bacitracin A + bacitracin B1 + bacitracin B2 + bacitracin B3) is calculated. After calculation, its recovery rate is 75.61%, which is slightly increased but still on the low side and does not meet the test requirements.

[0075] Pretreatment method 3: Change to dissolve with n-hexanol

[0076] Take 1 g of compound polymyxin B ointment, dissolve it with 40 mL of n-butanol, extract it 4 times with 40 mL of 10% ammonia water solution, combine the extraction liquid, adjust the pH to 7.0 with hydrochloric acid, dilute it with water, shake well, filter, and conduct chromatographic detection. Using the above recovery rate calculation formula, the recovery rate of bacitracin (bacitracin A + bacitracin B1 + bacitracin B2 + bacitracin B3) is calculated. After calculation, the recovery rate of bacitracin is 94.1%, which is still on the low side and does not meet the test requirements.

[0077] Pretreatment method 4: Change to the final method of the present invention: heating and melting and n-hexane

[0078] Take about 1 g of compound polymyxin B, accurately weigh it, place it in a separating funnel, heat and melt it, add 40 mL of n-hexane, shake it to make the ointment evenly dispersed, add 13.3 mL of 10% ammonia water solution for extraction, extract 3 times, combine the extraction liquid, add hydrochloric acid to adjust the pH value of the extraction liquid to 7.0, dilute it with water, shake well, filter, and conduct chromatographic detection. Using the above recovery rate calculation formula, the recovery rate of bacitracin (bacitracin A + bacitracin B1 + bacitracin B2 + bacitracin B3) is calculated. After calculation, the recovery rate of bacitracin is 99.7%.

[0079] Pretreatment method 5: Adjust the solvent dosage

[0080] Take about 1 g of compound polymyxin B, accurately weigh it, place it in a separating funnel, heat and melt it, add 30 mL of n-hexane, shake it to make the ointment evenly dispersed, add 10 mL of 10% ammonia water solution for extraction, extract 3 times, combine the extraction liquid, add hydrochloric acid to adjust the pH value of the extraction liquid to 7.0, dilute it with water, shake well, filter, and conduct chromatographic detection. Using the above recovery rate calculation formula, the recovery rate of bacitracin (bacitracin A + bacitracin B1 + bacitracin B2 + bacitracin B3) is calculated. After calculation, the recovery rate of bacitracin is 99.4%.

[0081] Pretreatment method 6: Adjust the solvent dosage

[0082] Take about 1 g of compound polymyxin B, accurately weigh it, place it in a separatory funnel, heat to melt, add 20 mL of n-hexane, shake to disperse the ointment evenly, add 6.7 mL of 10% ammonia water solution for extraction, extract 3 times, combine the extraction solutions, adjust the pH value of the extraction solution to 7.0 with hydrochloric acid, dilute with water, shake well, filter, and perform chromatographic detection. Calculate the recovery rate of bacitracin (bacitracin A + bacitracin B1 + bacitracin B2 + bacitracin B3) using the above recovery rate calculation formula. After calculation, the recovery rate of bacitracin is 100.15%.

[0083] Pretreatment method 7: Adjust the solvent dosage

[0084] Take about 1 g of compound polymyxin B, accurately weigh it, place it in a separatory funnel, heat to melt, add 10 mL of n-hexane, shake to disperse the ointment evenly, add 3.3 mL of 10% ammonia water solution for extraction, extract 3 times, combine the extraction solutions, adjust the pH value of the extraction solution to 7.0 with hydrochloric acid, dilute with water, shake well, filter, and perform chromatographic detection. Calculate the recovery rate of bacitracin (bacitracin A + bacitracin B1 + bacitracin B2 + bacitracin B3) using the above recovery rate calculation formula. After calculation, the recovery rate of bacitracin is 100.13%.

[0085] Pretreatment method 8: Adjust the solvent dosage

[0086] Take about 1 g of compound polymyxin B, accurately weigh it, place it in a separatory funnel, heat to melt, add 5 mL of n-hexane, shake to disperse the ointment evenly, add 1.7 mL of 10% ammonia water solution for extraction, extract 3 times, combine the extraction solutions, adjust the pH value of the extraction solution to 7.0 with hydrochloric acid, dilute with water, shake well, filter, and perform chromatographic detection. Calculate the recovery rate of bacitracin (bacitracin A + bacitracin B1 + bacitracin B2 + bacitracin B3) using the above recovery rate calculation formula. After calculation, the recovery rate of bacitracin is 100.12%.

[0087] Pretreatment method 9: Adjust the solvent dosage

[0088] Take about 1 g of compound polymyxin B, accurately weigh it, place it in a separatory funnel, heat to melt, add 3 mL of n-hexane, shake to disperse the ointment evenly, add 1 mL of 10% ammonia water solution for extraction, extract 3 times, combine the extraction solutions, adjust the pH value of the extraction solution to 7.0 with hydrochloric acid, dilute with water, shake well, filter, and perform chromatographic detection. Calculate the recovery rate of bacitracin (bacitracin A + bacitracin B1 + bacitracin B2 + bacitracin B3) using the above recovery rate calculation formula. After calculation, the recovery rate of bacitracin is 100.01%.

[0089] Pretreatment method 10: Adjust the solvent dosage

[0090] Take about 1 g of compound polymyxin B, accurately weigh it, place it in a separatory funnel, heat it to melt, add 2 mL of n-hexane, shake it to disperse the ointment evenly, add 0.67 mL of 10% ammonia water for extraction, extract 3 times, combine the extraction solutions, adjust the pH value of the extraction solution to 7.0 with hydrochloric acid, dilute it with water, shake well, filter, and perform chromatographic detection. Calculate the recovery rate of bacitracin (bacitracin A + bacitracin B1 + bacitracin B2 + bacitracin B3) using the above recovery rate calculation formula. After calculation, the recovery rate of bacitracin is 98.09%.

[0091] Pretreatment method 11: n-hexane back extraction

[0092] Take about 1 g of compound polymyxin B, accurately weigh it, place it in a separatory funnel, heat it to melt, add 3 mL of n-hexane, shake it to disperse the ointment evenly, add 1 mL of 10% ammonia water for extraction, extract 3 times, combine the extraction solutions, add 3 mL of n-hexane for back extraction, adjust the pH value of the extraction solution in the aqueous phase to 7.0 with hydrochloric acid, dilute it with water, shake well, filter, and perform chromatographic detection. The detection results are as Figure 7 shown. Calculate the recovery rate of bacitracin (bacitracin A + bacitracin B1 + bacitracin B2 + bacitracin B3) using the above recovery rate calculation formula. After calculation, the recovery rate of bacitracin is 100.18%.

[0093] Pretreatment method 12: Increase the amount of ointment taken

[0094] Compared with pretreatment method 11, increase the amount of ointment taken to verify the pretreatment method (increase from 1 g to 10 g).

[0095] Take about 10 g of compound polymyxin B, accurately weigh it, place it in a 125 mL separatory funnel, heat it to melt, add 30 mL of n-hexane, shake it to disperse the ointment evenly, add 10 mL of 10% ammonia water for extraction, extract 3 times, combine the extraction solutions, add 30 mL of n-hexane for back extraction, adjust the pH value of the extraction solution in the aqueous phase to 7.0 with hydrochloric acid, dilute it with water, shake well, filter, and perform chromatographic detection. The detection results are as Figure 8 shown. Calculate the recovery rate of bacitracin (bacitracin A + bacitracin B1 + bacitracin B2 + bacitracin B3) using the above recovery rate calculation formula. After calculation, the recovery rate of bacitracin is 100.27%. It can meet the detection requirements, and its recovery rate, the percentage of the peak area of bacitracin obtained, the number and content of impurity types are all no different from those of pretreatment method 11.

[0096] In summary, compared with using other extraction solvents (pretreatment methods 1 - 3), the pretreatment method (pretreatment methods 4 - 12) of the present invention using n-hexane + 10% ammonia water has a high recovery rate of bacitracin, close to 100%, the number and content of impurity types are no different from those of the reference substance, can meet the detection requirements, and can be used for the quality control of bacitracin in compound polymyxin B ointment.

[0097] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited by the embodiments. Any other changes, modifications, combinations, substitutions, and simplifications made without departing from the spirit and principle of the present invention shall be equivalent replacement methods and are all included in the protection scope of the present invention.

Claims

1. A method for detecting bacitracin in compound polymyxin B ointment, characterized in that, Heat and melt compound polymyxin B ointment, add n-hexane to disperse the ointment evenly, then add 10% ammonia aqueous solution for extraction. Combine the extraction solutions and adjust the pH value to 7.0; then perform chromatographic detection; The bacitracin mentioned refers to the sum of bacitracin A, bacitracin B1, bacitracin B2, and bacitracin B3; The chromatographic detection is as follows: Use octadecylsilane-bonded silica gel as the filler; use methanol-acetonitrile with a volume ratio of 25:5 as mobile phase A, and use pH 6.0 phosphate buffer-water with a volume ratio of 1:3 as mobile phase B. Mix mobile phase A and mobile phase B in a volume ratio of 65:35 as the mobile phase; the detection wavelength is 254 nm.

2. The detection method according to claim 1, characterized in that, After combining the extraction solutions, use n-hexane for back-extraction, and adjust the pH value of the aqueous phase to 7.

0.

3. The detection method according to claim 2, characterized in that, The mass-volume ratio of compound polymyxin B ointment to back-extraction n-hexane is 1 g: 3 mL.

4. The detection method according to claim 1, characterized in that The mass-volume ratio of the compound polymyxin B ointment to n-hexane is 1 g: 3 - 10 mL.

5. The detection method according to claim 1, characterized in that, The mass-volume ratio of the compound polymyxin B ointment to ammonia aqueous solution is 1 g: 1 mL, and extract 3 times.

6. The detection method according to claim 1, characterized in that, After adjusting the pH value to 7.0, add water for dilution, shake well, filter, and perform chromatographic detection.

7. The detection method according to claim 1, characterized in that, The column temperature for chromatographic detection is 30 °C; the flow rate is 1 ml per minute.

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