CN111220721A - Mupirocin ointment impurity qualitative positioning and testing method and application - Google Patents
Mupirocin ointment impurity qualitative positioning and testing method and application Download PDFInfo
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Abstract
The invention provides a mupirocin ointment impurity qualitative positioning and testing method and application. The method comprises the following steps: step S1, preparing mupirocin ointment sample into positioning test sample; step S2, detecting the liquid-mass linkage of the test sample; and step S3, analyzing the detection result, determining the structures of the impurities and the relative retention time of the impurities, and obtaining an impurity standard mass chromatogram and an impurity standard chromatogram. Compared with the prior art, the detection method has the beneficial effects that: when the impurity content is detected, the standard chromatogram of the product is directly compared, and reasonable chromatogram testing conditions are combined, so that the standard product does not need to be purchased for detection and comparison again, the operation is quicker, and the result is more precise and reliable.
Description
Technical Field
The invention belongs to the field of drug impurity inspection, and particularly relates to a mupirocin ointment impurity qualitative positioning and testing method and application.
Background
Mupirocin, also known as pseudomonic acid a, is a bacterial metabolite isolated from pseudomonas fluorescens in 1971, and is mainly used for preventing and treating gram-positive pathogenic bacteria, especially staphylococcus aureus, as a white-like solid produced by submerged fermentation of pseudomonas fluorescens. The metabolite produced by Pseudomonas fluorescens includes Pseudomonas acid A, B, C, D, E, F. The pseudomonosporanic acid A represents the main part of the activity of the pseudomonosporanic acid A, accounts for 90-95%, and other metabolites have no biological activity. Comprises methicillin-resistant staphylococcus aureus and skin bacterial infection caused by streptococcus, and the medicament is mainly ointment and is the first choice medicament for the topical antibacterial treatment of dermatology. Mupirocin ointment is not collected in the 2010 version of the Chinese pharmacopoeia, but mupirocin ointment is collected in the 23 rd volume of the new drug transfer standard of the national drug standards, the 35 th version of the United states Pharmacopeia (USP35), the 2012 th version of the British Pharmacopeia (BP2012) and the 16 th version of the Japanese pharmacopoeia (JP16) (the mupirocin calcium ointment is collected in JP 16).
The BP2009 mupirocin ointment also controls the bioactive-free metabolites as impurities, controls 3 known impurities, and the total impurities are not more than 20%. 9 known impurities are controlled in mupirocin ointment version USP34 published in 5 months of 2011, and the total impurities are less than 30%.
When mupirocin ointment impurities are detected, impurity standard substances are usually purchased and then compared after chromatographic detection, but not all impurity standard substances are easy to obtain, and a new impurity quantification method is needed to ensure simple, precise and comprehensive impurity control in order to better control the impurity content.
Disclosure of Invention
In order to solve the technical problem that the impurity detection of mupirocin ointment is difficult to obtain standard comparison when a new mupirocin ointment medicament is developed in the prior art, the invention provides a mupirocin ointment impurity qualitative positioning and testing method and application.
The qualitative impurity locating method for mupirocin ointment is characterized by comprising the following steps of:
step S1, preparing mupirocin ointment sample into positioning test sample;
step S2, detecting the liquid-mass linkage of the test sample;
step S3, analyzing the detection result, determining the structure of each impurity and the relative retention time of each impurity, and obtaining an impurity standard mass chromatogram and an impurity standard chromatogram;
in step S2, the chromatographic detection conditions are: adopting a C8 chromatographic column, wherein the column temperature is 33-37 ℃, the detection wavelength is 240nm, and the flow rate is as follows: 1ml/min, injection volume: performing gradient elution by using a mobile phase A and a mobile phase B, wherein the volume of the elution is 19-21 mul; the mobile phase A consists of 75 mass percent of 0.1mol/L ammonium acetate and 25 mass percent of tetrahydrofuran, and the mobile phase B consists of 70 mass percent of 0.1mol/L ammonium acetate and 30 mass percent of tetrahydrofuran;
in step S2, the mass spectrometry detection conditions are: primary mass spectrum: data are collected by adopting an ESI source and a Scan mode, scanning ions adopt a negative mode, Frag voltage is 135V, and a mass number scanning range is as follows: 300-700;
second-order mass spectrometry: the parent ion selection was 500.3, the Frag voltage was 135V, the collision voltage was 20mV, and the Production scan range was: 100-500.
further, in the step S1, after the mupirocin ointment sample is stood, the mupirocin ointment sample is weighed, and then tetrahydrofuran and sodium acetate are sequentially added to the mupirocin ointment sample for microfiltration, and the filtrate is collected to obtain the positioning test sample.
Compared with the prior art, the qualitative positioning method provided by the invention has the advantages that the research sample is directly prepared into the positioning test sample to be subjected to liquid-quality linkage detection, the test condition is reasonable, nine impurities of mupirocin can be controlled, the reference of the subsequent impurity content can be directly formed, and the impurity reference substance does not need to be purchased for comparison.
Further, in step S2, the chromatographic detection conditions are: using Zorbax Eclipse XDB-C8Chromatographic column, column temperature 35 ℃, detection wavelength 240nm, flow rate: 1ml/min, injection volume: 20 μ l.
Further, in step S2, the condition parameters of the gradient washing are:
| time (min) | Mobile phase A (%) | Mobile phase B (%) | Elution is carried out |
| 0 | 100 | 0 | Balancing |
| 0-6 | 100 | 0 | Single liquid isocratic |
| 6-35 | 100→0 | 0→100 | Linear gradient |
| 35-55 | 0 | 100 | Single liquid isocratic |
| 55-55.01 | 0→100 | 100→0 | Step gradient |
| 55.01-65 | 100 | 0 | Single liquid isocratic |
The mupirocin ointment impurity testing method is characterized by comprising the following steps of:
step A1, preparing mupirocin ointment sample into content test sample;
step A2, carrying out high performance color chromatography detection on the content test sample;
step A3, comparing the result of the step A2 with an impurity standard chromatogram to obtain the impurity content of the mupirocin ointment;
wherein the impurity standard chromatogram is obtained by detecting the mupirocin ointment impurity qualitative positioning method.
Further, the mupirocin ointment comprises the following components in percentage by weight: polyethylene glycol-400: 78.4%, polyethylene glycol-4000: 19.6% and mupirocin: 2 percent.
Further, in the step A2, a C8 chromatographic column is adopted, the column temperature is 33-37 ℃, the detection wavelength is 240nm, and the flow rate is as follows: 1ml/min, injection volume: 20 mu l, and gradient elution is carried out by adopting a mobile phase C and a mobile phase D; the mobile phase C and the mobile phase D both consist of 0.1mol/L ammonium acetate and tetrahydrofuran; wherein, in the mobile phase C, the mass percent of 0.1mol/L ammonium acetate is 74-76%, and the mass percent of tetrahydrofuran is 24-26%; in the mobile phase D, the mass percent of 0.1mol/L ammonium acetate is 69-71%, and the mass percent of tetrahydrofuran is 29-31%.
Further, in the step A2, a C8 chromatographic column is adopted, the column temperature is 35 ℃, the detection wavelength is 240nm, and the flow rate is as follows: 1ml/min, injection volume: 20 mu l, and gradient elution is carried out by adopting a mobile phase C and a mobile phase D; the mobile phase C and the mobile phase D both consist of 0.1mol/L ammonium acetate and tetrahydrofuran; wherein, in the mobile phase C, the mass percent of 0.1mol/L ammonium acetate is 75%, and the mass percent of tetrahydrofuran is 25%; in the mobile phase D, the mass percent of 0.1mol/L ammonium acetate is 70%, and the mass percent of tetrahydrofuran is 30%.
Further, in the step A2, Zorbax Eclipse XDB-C is adopted8Chromatography column or Zorbax eclipse plus C8And (5) detecting by using a chromatographic column.
Further, in the step a2, the condition parameters of the gradient washing are:
| time (min) | Mobile phase C (%) | Mobile phase D (%) | Elution is carried out |
| 0 | 100 | 0 | Balancing |
| 0-6 | 100 | 0 | Single liquid isocratic |
| 6-35 | 100→0 | 0→100 | Linear gradient |
| 35-55 | 0 | 100 | Single liquid isocratic |
| 55-55.01 | 0→100 | 100→0 | Step gradient |
| 55.01-65 | 100 | 0 | Single liquid isocratic |
Compared with the prior art, the detection method has the beneficial effects that: when the impurity content is detected, the standard chromatogram of the product is directly compared, and reasonable chromatogram testing conditions are combined, so that the standard product does not need to be purchased for detection and comparison again, the operation is quicker, and the result is more precise and reliable.
Drawings
FIG. 1 is a typical chromatogram of a localized test sample;
FIG. 2 is a mupirocin ointment full scan map;
FIG. 3 is a first mass spectrum corresponding to the RRT0.36 chromatographic peak;
FIG. 4 is a first mass spectrum corresponding to the chromatographic peak RRT 0.62;
FIG. 5 is a first mass spectrum corresponding to the RRT0.65 chromatographic peak;
FIG. 6 is a first mass spectrum corresponding to the chromatographic peak RRT 0.77;
FIG. 7 is a first mass spectrum corresponding to the chromatographic peak RRT 0.93;
FIG. 8 is a first mass spectrum corresponding to the chromatographic peak RRT 1.12;
FIG. 9 is a first mass spectrum corresponding to the chromatographic peak RRT 1.24;
FIG. 10 is a first mass spectrum corresponding to the RRT1.96 chromatographic peak;
FIG. 11 is a first mass spectrum corresponding to the chromatographic peak RRT 2.13;
figure 12 is a mupirocin secondary mass spectrum;
FIG. 13 is a second-order mass spectrum corresponding to the RRT0.62 chromatographic peak;
FIG. 14 is a second-order mass spectrum corresponding to the RRT0.65 chromatographic peak;
FIG. 15 is a second-order mass spectrum corresponding to the chromatographic peak of RRT 1.12;
FIG. 16 is a second-order mass spectrum corresponding to the chromatographic peak RRT 1.24;
FIG. 17 is a typical chromatogram of a content test sample with a wavelength of 240 nm;
FIG. 18 typical chromatogram of a 240nm wavelength mupirocin control solution
FIG. 19 is a graph showing an ultraviolet absorption spectrum of each impurity of a content test sample;
FIG. 20 is a graph of the UV absorption spectrum of mupirocin in a content control solution;
FIG. 21 is a typical chromatogram of a content test sample with a wavelength of 220 nm;
FIG. 22 is a typical chromatogram of a content test sample with a wavelength of 230 nm;
FIG. 23 is a chromatogram representative of a sample tested for content at 240nm wavelength.
Detailed Description
The principles and features of this invention are described below in conjunction with the following drawings, which are set forth by way of illustration only and are not intended to limit the scope of the invention.
The inventive example tests the following weight percentages of the novel mupirocin ointment:
polyethylene glycol-400: 78.4%, polyethylene glycol-4000: 19.6% and mupirocin: 2 percent.
Example one
Qualitative localization of impurities
1.1 instruments
An Agilent 1200 high performance liquid chromatography system; agilent LC-MS/MS 6460, ESI source.
1.2 chromatographic and Mass Spectrometry conditions
1.2.1 chromatographic conditions
A chromatographic column: zorbax Eclipse XDB-C8(250mm×4.6mm，5μm)
Detection wavelength: 240nm
Flow rate: 1ml/min
Sample introduction volume: 20 μ l
Column temperature: 35 deg.C
Mobile phase:
solution A: 0.1M ammonium acetate: tetrahydrofuran 75: 25
Solution B: 0.1M ammonium acetate: tetrahydrofuran 70: 30
Gradiometer
| Time (min) | Mobile phase A (%) | Mobile phase B (%) | Elution is carried out |
| 0 | 100 | 0 | Balancing |
| 0-6 | 100 | 0 | Single liquid isocratic |
| 6-35 | 100→0 | 0→100 | Linear gradient |
| 35-55 | 0 | 100 | Single liquid isocratic |
| 55-55.01 | 0→100 | 100→0 | Step gradient |
| 55.01-65 | 100 | 0 | Single liquid isocratic |
1.2.2
Mass spectrum conditions:
primary mass spectrum: data are collected by adopting an ESI source and a Scan mode, scanning ions adopt a negative mode, Frag voltage is 135V, and a mass number scanning range is as follows: 300-700.
Second-order mass spectrometry: in view of the wide variety of species with molecular weight of 500.3, secondary mass spectrometry scans were performed to determine specific structures. The parent ion selection was 500.3, the Frag voltage was 135V, the collision voltage was 20mV, and the Production scan range was: 100-500.
1.3 positioning test sample preparation
The novel mupirocin ointment sample is placed in an environment with the temperature of 100 ℃ for 48 hours, a proper amount of the novel mupirocin ointment sample (about 50mg of mupirocin) is taken, precisely weighed, placed in a small beaker bottle, added with 5ml of tetrahydrofuran solution, stirred and dissolved, added with 5ml of sodium acetate solution, shaken evenly, filtered by a 0.5 mu m microporous membrane, and a subsequent filtrate is taken as a positioning test sample (each 1ml contains about 5mg of mupirocin).
1.4 precisely measuring 20 mu l of test solution, injecting the solution into a liquid chromatograph, and recording a chromatogram and a mass spectrum, wherein a typical chromatogram, a mass spectrum and a positioning test sample full-scan chromatogram are as follows:
1.5 structural analysis of positioning test sample full-scan chromatogram
First order mass spectrometry
RRT0.36 chromatographic peak, [ M-H ] determined]-471.3, molecular weight 472.3. The impurity in control USP34 was pseudomonic acid F, formula C24H40O9The chemical name is: 7- ((E) -4- ((2S,3R,4R,5S) -3,4-Dihydroxy-5- (((2S,3S) -3- ((2S,3S) -3-hydroxybutan-2-yl) oxiran-2-yl) methyl) tetrahydro-2H-pyran-2-yl) -3-methylbutan-2-yloxy) heptanic acid. The corresponding primary mass spectrum 3 is shown, and the corresponding structural formula is shown as formula I.
RRT0.62 chromatographic Peak, [ M-H ] determined]-499.3 and molecular weight 500.3. The molecular weight is the same as the corresponding mupirocin impurity 1, mupirocin impurity 2, mupirocin impurity 3 and mupirocin impurity 4 in USP 34. Further secondary mass spectrometry studies were required. Corresponding to the primary mass spectrum 4.
RRT0.65 chromatographic Peak, [ M-H ] determined]-499.3 and molecular weight 500.3. The molecular weight is the same as the corresponding mupirocin impurity 1, mupirocin impurity 2, mupirocin impurity 3 and mupirocin impurity 4 in USP 34. Further secondary mass spectrometry studies were required. Corresponding to the primary mass spectrum 5.
RRT0.77 chromatographic Peak, [ M-H ] determined]-497.3 and 498.3 molecular weight. The impurity in control USP34 was pseudomonic acid D, formula C26H42O9The chemical name is: (E) -9- ((E) -4- ((2S,3R,4R,5S) -3,4-Dihydroxy-5- (((2S,3S) -3- ((2S,3S) -3-hydroxybutan-2-yl) oxiran-2-yl) methyl) tetrahydro-2H-pyran-2-yl) -3-methylbutan-2-oxyloxy) non-4-enoic acid. The corresponding primary mass spectrum 6 is shown as the formula II.
RRT0.93 chromatogram peak, [ M-H ] -515.3, molecular weight 516.3. The impurity in control USP34 is pseudomonic acid B, chemical formula C26H44O10, chemical name: 9- ((E) -3-Methyl-4- ((2S,3R,4S,5R) -3,4,5-trihydroxy-5- (((2S,3S) -3- ((2S,3S) -3-hydroxybutan-2-yl) oxairan-2-yl) Methyl) tetrahydro-2H-pyran-2-yl) but-2-oxyloxy) nonanic acid. The corresponding primary mass spectrum is shown as 7, and the structural formula is shown as formula III.
RRT1.12 chromatogram peak, measured [ M-H ] -499.3, molecular weight 500.3. The molecular weight is the same as the corresponding mupirocin impurity 1, mupirocin impurity 2, mupirocin impurity 3 and mupirocin impurity 4 in USP 34. Further secondary mass spectrometry studies were required. The corresponding primary mass spectrum 8 is shown.
RRT1.24 chromatogram peak, measured [ M-H ] -499.3, molecular weight 500.3. The molecular weight is the same as the corresponding mupirocin impurity 1, mupirocin impurity 2, mupirocin impurity 3 and mupirocin impurity 4 in USP 34. Further secondary mass spectrometry studies were required. The corresponding primary mass spectrum is shown as 9.
RRT1.96 chromatogram peak, found [ M-H ] -483.3, molecular weight 484.3. The impurity in control USP34 is pseudomonic acid C, formula C26H44O8, chemical name: 9- ((E) -4- ((2S,3R,4R,5S) -3,4-Dihydroxy-5- ((4R,5S, E) -5-hydroxy-4-methylhex-2-enyl) tetrahydro-2H-pyran-2-yl) -3-methyllbut-2-enyloxy) nonaic acid. The corresponding primary mass spectrum is shown as 10, and the structural formula is shown as formula IV.
RRT2.13 chromatogram peak, found [ M-H ] -527.3, molecular weight 528.3. The impurity in control USP34 is pseudomonic acid E, formula C28H48O9, chemical name: 11- ((E) -4- ((2S,3R,4R,5S) -3,4-Dihydroxy-5- (((2S,3S) -3-hydroxybutan-2-yl) oxoiran-2-yl) methyl) tetrahydro-2H-pyran-2-yl) -3-methyllbut-2-oyloxy) isocanoic acid. The corresponding primary mass spectrogram 11 is shown as a structural formula V.
Second order mass spectrometry
The secondary mass spectrum is shown in FIG. 12.
RRT0.62 chromatographic peak, fragment of [ M-H ] -173.2 and 325.2 was obtained in the secondary mass spectrum, see FIG. 13. The cracking process is shown in formula VI. According to its secondary mass fragmentation and fragmentation process analysis, the impurity in control USP34 was mupirocin impurity 1, of the formula C26H44O9, chemical name: 9- ((E) -4- ((2R,3aS,6S,7S,8aRS) -2- ((1RS,2S,3S) -1, 3-Dihydroxy-2-methylbutanyl) -7-hydroxyhexahydro-2H-furo (3,2-c) pyran-6-yl) -3-methylbutaut
RRT0.65 chromatogram peak, fragments of [ M-H ] -173.2, 229.2 and 325.2 were obtained in a secondary mass spectrum, see FIG. 14. The cleavage process is shown in formula VII. According to its secondary mass fragmentation and fragmentation process analysis, the impurity in control USP34 was mupirocin impurity 2, of the formula C26H44O9, chemical name: 9- ((E) -4- ((2R,3RS,4aS,7S,8S,8aR) -3,8-Dihydroxy-2- ((2S,3S) -3-hydroxybutan-2-yl) octahydropyrano (3,2-c) pyran-7-yl) -3-methyllbut-2-oxyloxy) nonaic acid.
Chromatographic peaks of RRT1.12 and RRT1.24, fragments of [ M-H ] -173.2 were obtained in the secondary mass spectrum, see FIG. 15, FIG. 16. The cracking process is shown in formula VIII.
With reference to USP34 mupirocin cream quality standards, the relative retention times of known impurities were used to qualitatively locate the impurities, corresponding to the impurities shown in table 1 below:
TABLE 1 impurity location table
| Name (R) | Relative retention time |
| Pseudomonic acid F | 0.36 |
| Mupirocin impurity 1 | 0.6 |
| |
0.63 |
| Pseudomonic acid D | 0.75 |
| Pseudomonic acid B | 0.9 |
| Mupirocin | 1.0 |
| |
1.15 |
| |
1.23 |
| Pseudomonic acid C | 2.03 |
| Pseudomonic acid E | 2.24 |
Example two
Chromatographic test condition determination process.
Mupirocin control: china institute for drug and biological products, lot No. 130568-200501.
Test samples: the novel mupirocin ointment is developed by Hubei Tantian pharmaceutical corporation.
Specification: 5g of mupirocin and 0.1g of mupirocin.
2.1 determination of the detection wavelength
The method comprises the steps of controlling 9 known impurities in the USP34 mupirocin cream quality standard, qualitatively locating the impurities by adopting the relative retention time of the impurities, adopting a diode array detector to scan within the wavelength of 200-400 nm to extract an ultraviolet absorption spectrogram of each impurity peak, adopting a mupirocin reference solution to obtain an ultraviolet absorption diagram of mupirocin, and determining the detection wavelength.
2.1.1 instruments
An Agilent 1200 high performance liquid chromatography system; an Agilent 1200Chemstation workstation; G1315C DAD detector; G1367D autosampler
2.1.2 chromatographic conditions
The chromatographic conditions were as follows:
a chromatographic column: zorbax C8(250 mm. times.4.6 mm, 5 μm)
Detection wavelength: 240nm
Flow rate: 1ml/min
Sample introduction volume: 20 μ l
Column temperature: 35 deg.C
Mobile phase:
mobile phase C: 0.1M ammonium acetate: tetrahydrofuran 75: 25
Mobile phase D: 0.1M ammonium acetate: tetrahydrofuran 70: 30
| Time (min) | Mobile phase C (%) | Mobile phase D (%) | Elution is carried out |
| 0 | 100 | 0 | Balancing |
| 0-6 | 100 | 0 | Single liquid isocratic |
| 6-35 | 100→0 | 0→100 | Linear gradient |
| 35-55 | 0 | 100 | Single liquid isocratic |
| 55-55.01 | 0→100 | 100→0 | Step gradient |
| 55.01-65 | 100 | 0 | Single liquid isocratic |
2.1.3 test article solution
Mupirocin ointment (lot: 090501) is placed in 100 deg.C environment for 48 hr, and an appropriate amount (about equivalent to 50mg of mupirocin) of the ointment is taken, precisely weighed, placed in a small beaker bottle, added with 5ml of tetrahydrofuran solution, stirred to dissolve, added with 5ml of sodium acetate solution, shaken well, filtered through a 0.5 μm microporous membrane, and the subsequent filtrate is taken as a test solution (each 1ml contains about 5mg of mupirocin).
2.1.4 control solutions
Taking 10mg of mupirocin reference substance, precisely weighing, placing in a 10ml measuring flask, adding 5ml of tetrahydrofuran solution, ultrasonically dissolving, adding sodium acetate solution to scale, and shaking up; 1ml of the solution is precisely measured, placed in a 10ml measuring flask, diluted to the scale with a mixed solution of sodium acetate and tetrahydrofuran, and shaken up to serve as a control solution (containing 100 mug of mupirocin per 1 ml).
2.1.5 testing
Respectively measuring 20 mul of test solution and reference solution, injecting into a liquid chromatograph, scanning within 200-400 nm wavelength, recording chromatogram, and extracting ultraviolet absorption spectrogram of each impurity peak and main peak. The ultraviolet absorption spectra of mupirocin and known impurities are shown in fig. 17-23.
The results of the ultraviolet absorption spectrograms show that mupirocin and known impurities have maximum absorption at the wavelength of 221nm and are close to the ultraviolet end, and the absorption of tetrahydrofuran which is a chromatographic reagent is larger as the tetrahydrofuran is closer to the ultraviolet end, so that the chromatographic baseline is easy to drift. Comparing chromatograms at wavelengths of 220nm, 230nm and 240nm, the number of impurities is the same. In order to reduce the effect of baseline drift, the wavelength of 240nm was selected as the detection wavelength of the substance of interest, with reference to USP34 mupirocin cream quality standard.
2.2 System suitability test
2.2.1 instruments
Elett 1201 liquid chromatograph
2.2.2 chromatographic conditions
Chromatographic conditions are as follows: see 2.1.2.
2.2.3 solution preparation
2.2.3.1 test solution
Taking a proper amount (about 50mg equivalent to mupirocin), precisely weighing, placing in a small beaker, adding 5ml of tetrahydrofuran solution, stirring for dissolving, adding 5ml of sodium acetate solution, shaking, filtering with 0.5 μm microporous membrane, and collecting the filtrate as test solution (each 1ml contains about 5mg of mupirocin).
2.2.3.2 control solution
1ml of the test solution is precisely measured, placed in a 50ml measuring flask, diluted to the scale with a mixed solution of sodium acetate and tetrahydrofuran, and shaken up to serve as a control solution (containing 100 mug of mupirocin per 1 ml).
2.2.3.3 blank adjuvant solution
Taking about 2.5g of blank matrix, and preparing a blank auxiliary material solution according to the preparation method of the test solution.
2.3.4 System suitability test
Precisely measuring 20 μ l of the control solution, injecting into a liquid chromatograph, and adjusting the sensitivity of the apparatus to make the main peak height of the control solution 20% of the full-scale range. The column effect of mupirocin is not less than 7000, the tailing factor of mupirocin peak is not more than 1.75, the sample introduction is repeated for 5 times, and the relative standard deviation of mupirocin is less than 2%. Under the chromatographic conditions, 20 mul of sample solution is precisely measured and injected into a liquid chromatograph, and the separation degree of the pseudomonic acid D and the mupirocin is not less than 3. Table 2.
TABLE 2 results of systematic suitability test
And (4) conclusion: under the condition, the retention time of mupirocin is about 20min, in a chromatogram of a control solution, the number of theoretical plates is 50488 according to mupirocin, the tailing factor of a mupirocin peak is 1.16, sample injection is repeated for 5 times, the relative standard deviation of the mupirocin peak area is 0.94%, in a chromatogram of a test solution, the separation degree of pseudomonic acid D and mupirocin is 4.12, the separation degree of impurities is more than 1.0, and the system adaptability requirement is met.
2.3.5 blank adjuvant interference test
Precisely measuring 20 mu l of blank auxiliary material solution, injecting the blank auxiliary material solution into a liquid chromatograph, recording a chromatogram, and inspecting the interference condition of the blank auxiliary material on measurement. The blank auxiliary material has an absorption peak close to the solvent peak before 4min, and has good separation with the mupirocin main peak and the impurity peak due to the fact that the positions of the solvent peak and the substrate peak are close to the front and the peak shape is sharp, and the impurity determination is not interfered.
2.3.6 Special Property test
The novel mupirocin ointment is subjected to strong acid, strong base, oxidation, strong light and high temperature destructive tests, and whether the proposed chromatographic conditions can effectively separate various degradation products is investigated.
2.3.6.1 destructive sample solution preparation
① sample solution is destroyed by acid, 10ml of sample solution is taken, 1ml of 1mol/L hydrochloric acid is added, the mixture is placed for 1 day, 1ml of 1mol/L sodium hydroxide is added, the mixture is shaken up and filtered, and at the same time, 10ml of blank auxiliary material solution is taken for blank test.
② alkali destroyed sample solution, 10ml of sample solution is taken, 1ml of 1mol/L sodium hydroxide is added, the mixture is placed for 1 day, 1ml of 1mol/L hydrochloric acid is added, the mixture is shaken up and filtered, and simultaneously 10ml of blank auxiliary material solution is taken for blank test.
③ oxidizing and destroying the sample solution, taking 10ml of the sample solution, adding 1ml of 30% hydrogen peroxide, standing for 2 days, shaking up, filtering, and simultaneously taking 10ml of blank adjuvant solution for blank test.
④ destroying the sample solution by light irradiation, taking 10ml of the sample solution, standing for 2 days under the condition of 4500Lx light irradiation, shaking up, filtering, and simultaneously taking 10ml of blank adjuvant solution for blank test.
⑤ destroying the sample solution at high temperature, taking 10ml of the sample solution, standing at 105 deg.C for 1 day, shaking, filtering, and taking 10ml of blank adjuvant solution for blank test.
2.3.6.2 test results
And (3) respectively injecting 20 mu l of the destructive sample solution into a liquid chromatograph, and recording a chromatogram, wherein the test result is shown in tables 3-7.
TABLE 3 results of acid destructive testing
TABLE 4 results of the alkaline Destruction test
| Name of the component | Retention time (min) | Relative retention time | Degree of separation |
| Destruction of impurity 1 by alkali | 3.88000 | 0.17 | ---- |
| Destruction of |
4.72167 | 0.21 | 4.34 |
| Destruction of impurities by |
4.97750 | 0.22 | 1.15 |
| Base destroying impurity 4 (pseudomonic acid F) | 7.61000 | 0.34 | 9.30 |
| Destruction of impurities by |
9.60000 | 0.43 | 4.98 |
| Destruction of impurities 6 by alkali | 10.16750 | 0.46 | 1.28 |
| Alkali destruction of |
12.90083 | 0.58 | 6.96 |
| Base destroying impurity 8 (mupirocin impurity 1) | 13.36333 | 0.60 | 0.91 |
| Base destroying impurity 9 (mupirocin impurity 2) | 14.29417 | 0.64 | 1.40 |
| Base destroying impurity 10 (pseudomonic acid D) | 16.77750 | 0.76 | 3.74 |
| Destruction of impurities 11 by alkali | 21.08917 | 0.95 | 6.59 |
| Major peak of mupirocin | 22.19500 | 1.00 | 1.25 |
| Base destroying impurity 12 (mupirocin impurity 3) | 25.65250 | 1.16 | 3.55 |
| Base destroying impurity 13 (mupirocin impurity 4) | 27.27333 | 1.23 | 2.00 |
| Base destroying impurity 14 (pseudomonic acid C) | 42.20583 | 1.90 | 17.07 |
TABLE 5 high temperature destructive test results
| Name of the component | Retention time (min) | Relative retention time | Degree of separation |
| High temperature destruction of impurities 1 | 4.70917 | 0.22 | --- |
| High temperature destruction of |
4.95917 | 0.23 | 1.19 |
| High temperature destruction of impurities 3 (Pseudomonas acid F) | 7.56667 | 0.35 | 9.12 |
| High temperature destruction of |
9.53167 | 0.44 | 5.08 |
| High temperature destruction of |
10.08917 | 0.47 | 1.28 |
| High temperature destruction of impurities 6 | 11.50500 | 0.54 | 3.36 |
| High temperature destruction of |
12.08333 | 0.56 | 1.47 |
| High temperature destruction of impurities 8 | 12.62750 | 0.59 | 1.33 |
| High temperature disruption of impurity 9 (mupirocin impurity 1) | 13.18417 | 0.61 | 1.06 |
| High temperature disruption of impurities 10 (mupirocin impurities 2) | 13.97250 | 0.65 | 1.16 |
| High temperature destruction of impurities 11 | 15.47833 | 0.72 | 2.44 |
| High temperature destruction of impurity 12 (pseudomonic acid D) | 16.55583 | 0.77 | 1.86 |
| High temperature destruction of impurities 13 | 18.27083 | 0.85 | 2.96 |
| High temperature destruction of impurities 14 | 18.92333 | 0.88 | 1.23 |
| High temperature destruction of impurities 15 (pseudomonic acid B) | 19.74417 | 0.92 | 1.26 |
| High temperature destruction of |
20.54583 | 0.96 | 1.25 |
| Major peak of mupirocin | 21.50333 | 1.00 | 1.00 |
| High temperature disruption of impurity 17 (mupirocin impurity 3) | 23.94917 | 1.11 | 2.34 |
| High temperature destruction of impurities 18 | 25.34583 | 1.18 | 1.85 |
| High temperature disruption of impurity 19 (mupirocin impurity 4) | 26.84833 | 1.25 | 1.83 |
| High temperature destruction of impurity 20 (pseudomonic acid C) | 41.93667 | 1.95 | 16.43 |
| High temperature destruction of impurities 21 (pseudomonic acid E) | 47.56500 | 2.21 | 4.32 |
TABLE 6 oxidative destruction test results
| Name of the component | Retention time (min) | Relative retention time | Degree of separation |
| Oxidative destruction of impurity 1 (Pseudomonas acid F) | 7.24500 | 0.35 | ---- |
| Oxidative destruction of |
9.11083 | 0.45 | 5.15 |
| Oxidative destruction of |
9.62583 | 0.47 | 1.29 |
| Oxidation damage impurity 4 (mupirocin impurity 1) | 13.02417 | 0.64 | 7.21 |
| Oxidation destroy impurities 5 (mupirocin impurities 2) | 13.70333 | 0.67 | 1.24 |
| Oxidative destruction of impurities 6 | 14.93000 | 0.73 | 2.14 |
| Oxidative destruction of impurities 7 (Pseudomonas acid D) | 15.82833 | 0.77 | 1.48 |
| Oxidative destruction of impurities 8 | 17.60167 | 0.86 | 3.07 |
| Oxidative destruction of impurities 9 | 18.26833 | 0.89 | 1.31 |
| Oxidative destruction of impurities 10 (pseudomonic acid B) | 18.96000 | 0.93 | 1.30 |
| Oxidative destruction of impurities 11 | 19.64917 | 0.96 | 1.34 |
| Major peak of mupirocin | 20.42500 | 1.00 | 0.80 |
| Oxidation destructive impurities 12 (mupirocin impurity 3) | 23.91292 | 1.17 | 3.40 |
| Oxidation destroy impurities 13 (mupirocin impurities 4) | 25.40083 | 1.24 | 2.69 |
| Oxidative destruction of impurities 14 | 25.92500 | 1.27 | 1.03 |
| Oxidation of impurities 15 (pseudomonic acid C) | 40.47667 | 1.98 | 19.37 |
| Oxidation of impurity 16 (Pseudomonas acid E) | 46.01417 | 2.25 | 5.08 |
TABLE 7 photo destructive test results
| Name of the component | Retention time (min) | Relative retention time | Degree of separation |
| Photo-destructive impurity 1 (pseudomonic acid F) | 7.53583 | 0.36 | ---- |
| Photo-damage of |
9.47833 | 0.45 | 4.93 |
| Photo-damage of |
10.05417 | 0.47 | 1.34 |
| Photo disruption impurities 4 (mupirocin impurity 1) | 13.54917 | 0.64 | 7.29 |
| Photo disruption impurities 5 (mupirocin impurities 2) | 14.24583 | 0.67 | 1.26 |
| Photo-damage of impurities 6 | 15.52250 | 0.73 | 2.12 |
| Photo-destruction of impurities 7 (pseudomonic acid D) | 16.46667 | 0.78 | 1.47 |
| Photo-damage of impurities 8 | 17.49500 | 0.82 | 1.81 |
| Photo-damage of impurities 9 | 18.16917 | 0.86 | 1.35 |
| Photo-damage of |
18.88750 | 0.89 | 1.35 |
| Photo-destructive impurities 11 (pseudomonic acid B) | 19.65583 | 0.93 | 1.43 |
| Photo-damage to the foreign matter 12 | 20.42833 | 0.96 | 1.48 |
| Major peak of mupirocin | 21.22750 | 1.00 | 0.76 |
| Photo disruption impurities 13 (mupirocin impurities 4) | 25.40250 | 1.20 | 3.49 |
| Photo-damage to foreign matter 14 | 27.01333 | 1.27 | 2.18 |
| Photo damage impurity 15 (pseudomonic acid C) | 42.16667 | 1.99 | 17.79 |
| Illumination destroys impurity 16 (pseudomonic acid E) | 47.81250 | 2.25 | 5.41 |
2.3.6.3 conclusion of the test
According to the test results, mupirocin is obviously degraded under the conditions of strong acid, strong base, oxidation, strong light and high temperature, and all degradation products can be well separated from the main peak of mupirocin under the formulated chromatographic strip.
2.3.7 detection Limit determination
Precisely measuring 1ml of reference solution, sequentially diluting to 101, 102 and 103 times by using a mixed solution of sodium acetate and tetrahydrofuran, precisely measuring 20 μ l of each dilution level of reference solution, sequentially injecting into a liquid chromatograph, and recording chromatogram, wherein the injection amount when the signal-to-noise ratio is 3: 1 is used as a detection limit. The minimum detectable amount was 40 ng.
2.3.8 durability test
The durability was examined by varying certain parameters under chromatographic conditions. Injecting 20 mul of sample solution into a liquid chromatograph, wherein the separation degree of pseudomonic acid D and mupirocin is not less than 3, the separation degree between impurity peaks is not less than 1.0, and the separation degree between an impurity peak adjacent to a mupirocin main peak and the main peak is not less than 0.75.
2.3.8.1 changing the type of chromatographic column
①Zorbax Eclipse XDB-C8(250mm×4.6mm，5μm)
②Zorbax Eclipse Plus C8(250mm×4.6mm，5μm)
③VARIAN Pursuit 5C8(250mm×4.6mm，5μm)
④COSMOSIL 5C8-MS(250mm×4.6mm，5μm)
⑤Purospher STAR RP-8e(250mm×4.6mm，5μm)
The test results are shown in Table 8.
TABLE 8 chromatographic column durability test results
And (4) conclusion: five chromatographic columns C8(250mm multiplied by 4.6mm, 5 mu m) with different models are used for durability examination, the separation degrees of the pseudomonic acid D and the mupirocin are respectively 6.99, 6.44, 7.35, 8.65 and 6.99, the separation degrees of all impurity peaks are all more than 1.0, the separation degrees of the impurity peaks adjacent to the main peak of the mupirocin are all more than 0.75, and the durability requirement is met. The relative retention times of the 9 known impurities substantially corresponded to USP34, but the relative retention times of the unknown impurities differed considerably, only the chromatograms of Zorbax Eclipse XDB-C8, Zorbax Eclipse Plus C8 were in agreement. The column used in earlier studies on localization of known impurities (liquid chromatography) was Zorbax Eclipse XDB-C8(250 mm. times.4.6 mm, 5 μm), so the column was identified as Zorbax C8(250 mm. times.4.6 mm, 5 μm).
2.3.8.2 changing the pH of the mobile phase
① pH5.7, ② pH5.6, ③ pH5.8, Table 9.
Table 9 mobile phase pH durability test results
And (4) conclusion: it is not recommended to change the pH ± 0.1, since lowering the pH leads to a decrease in the number of impurities between mupirocin and pseudomonic acid and raising the pH leads to a decrease in the number of impurities between pseudomonic acid D and mupirocin. The optimal pH of the buffer was 5.7.
2.3.8.3 changing the ratio of mobile phase
① mobile phase C0.1M ammonium acetate tetrahydrofuran 75: 25
Mobile phase D: 0.1M ammonium acetate: tetrahydrofuran 70: 30
② mobile phase C0.1M ammonium acetate tetrahydrofuran 77: 23
Mobile phase D: 0.1M ammonium acetate: tetrahydrofuran 72: 28
③ mobile phase C0.1M ammonium acetate tetrahydrofuran 73: 27
Mobile phase D: 0.1M ammonium acetate: tetrahydrofuran 68: 32
④ mobile phase C0.1M ammonium acetate tetrahydrofuran 76: 24
Mobile phase D: 0.1M ammonium acetate: tetrahydrofuran 71: 29
⑤ mobile phase C0.1M ammonium acetate tetrahydrofuran 74: 26
Mobile phase D: 0.1M ammonium acetate: tetrahydrofuran 69: 31
The test results are shown in 10
TABLE 10 mobile phase ratio durability test results
And (4) conclusion: changing the mobile phase ratio by 2% results in a significant change in relative retention time, an increase of 2% in the mobile phase ratio results in a reduction in the number of impurities between pseudomonic acid D and mupirocin, and a decrease of 2% in the mobile phase ratio results in a reduction in the number of impurities between mupirocin and pseudomonic acid C. The mobile phase composition can only change by 1%.
2.8.4 Change the column temperature
① column temperature 35 ℃;
② column temperature 40 deg.C
③ column temperature 30 DEG C
④ column temperature 37 deg.C
⑤ column temperature 33 deg.C
The test results are shown in Table 11
TABLE 11 column temperature durability test results
And (4) conclusion: the column temperature cannot be changed by 5 ℃, and the increase of the column temperature by 5 ℃ leads to the reduction of the number of impurities between the pseudomonic acid D and the mupirocin. A 5 ℃ decrease in column temperature resulted in a reduction in the number of impurities between mupirocin and pseudomonic acid C and a separation of the main peak from the previous impurity peak of < 0.75. The column temperature can be varied by 2 ℃.
EXAMPLE III
The novel mupirocin ointment products are detected in three batches, namely the first batch, the second batch and the third batch.
The detection steps are as follows:
step A1, preparing mupirocin ointment sample into content test sample;
the mupirocin ointment sample is placed in an environment at 100 ℃ for 48 hours, a proper amount (about 50mg of mupirocin) of the mupirocin ointment sample is taken, precisely weighed, placed in a small beaker bottle, added with 5ml of tetrahydrofuran solution, stirred and dissolved, added with 5ml of sodium acetate solution, shaken evenly, filtered by a 0.5 mu m microporous membrane, and a subsequent filtrate is taken as a positioning test sample (5 mg of mupirocin is contained in every 1 ml).
Step A2, carrying out high performance color chromatography detection on the content test sample;
the chromatographic conditions were as follows:
a chromatographic column: zorbax C8(250 mm. times.4.6 mm, 5 μm)
Detection wavelength: 240nm
Flow rate: 1ml/min
Sample introduction volume: 20 μ l
Column temperature: 35 deg.C
Mobile phase:
mobile phase C: 0.1M ammonium acetate: tetrahydrofuran 75: 25
Mobile phase D: 0.1M ammonium acetate: tetrahydrofuran 70: 30
Gradiometer
| Time (min) | Mobile phase C (%) | Mobile phase D (%) | Elution is carried out |
| 0 | 100 | 0 | Balancing |
| 0-6 | 100 | 0 | Single liquid isocratic |
| 6-35 | 100→0 | 0→100 | Linear gradient |
| 35-55 | 0 | 100 | Single liquid isocratic |
| 55-55.01 | 0→100 | 100→0 | Step gradient |
| 55.01-65 | 100 | 0 | Single liquid isocratic |
Step A3, comparing the result of the step A2 with an impurity standard chromatogram to obtain the impurity content of the mupirocin ointment;
the impurity standard chromatogram is obtained by detecting the impurity qualitative positioning method of mupirocin ointment in the embodiment, and the test results are shown in table 12.
TABLE 12 mupirocin ointment impurity test results
Example four
The novel mupirocin ointment products are detected in three batches, namely the first batch, the second batch and the third batch.
The detection steps are as follows:
step A1, preparing mupirocin ointment sample into content test sample;
the mupirocin ointment sample is placed in an environment at 100 ℃ for 48 hours, a proper amount (about 50mg of mupirocin) of the mupirocin ointment sample is taken, precisely weighed, placed in a small beaker bottle, added with 5ml of tetrahydrofuran solution, stirred and dissolved, added with 5ml of sodium acetate solution, shaken evenly, filtered by a 0.5 mu m microporous membrane, and a subsequent filtrate is taken as a positioning test sample (5 mg of mupirocin is contained in every 1 ml).
Step A2, carrying out high performance color chromatography detection on the content test sample;
the chromatographic conditions were as follows:
a chromatographic column: zorbax C8(250 mm. times.4.6 mm, 5 μm)
Detection wavelength: 240nm
Flow rate: 1ml/min
Sample introduction volume: 20 μ l
Column temperature: 33 deg.C
Mobile phase:
mobile phase C: 0.1M ammonium acetate: tetrahydrofuran 74: 26
Mobile phase D: 0.1M ammonium acetate: tetrahydrofuran 69: 31
Gradiometer
| Time (min) | Mobile phase C (%) | Mobile phase D (%) | Elution is carried out |
| 0 | 100 | 0 | Balancing |
| 0-6 | 100 | 0 | Single liquid isocratic |
| 6-35 | 100→0 | 0→100 | Linear gradient |
| 35-55 | 0 | 100 | Single liquid isocratic |
| 55-55.01 | 0→100 | 100→0 | Step gradient |
| 55.01-65 | 100 | 0 | Single liquid isocratic |
Step A3, comparing the result of the step A2 with an impurity standard chromatogram to obtain the impurity content of the mupirocin ointment;
wherein the standard impurity chromatogram is obtained by detecting the mupirocin ointment impurity qualitative positioning method in the embodiment.
EXAMPLE five
The novel mupirocin ointment products are detected in three batches, namely the first batch, the second batch and the third batch.
The detection steps are as follows:
step A1, preparing mupirocin ointment sample into content test sample;
the mupirocin ointment sample is placed in an environment at 100 ℃ for 48 hours, a proper amount (about 50mg of mupirocin) of the mupirocin ointment sample is taken, precisely weighed, placed in a small beaker bottle, added with 5ml of tetrahydrofuran solution, stirred and dissolved, added with 5ml of sodium acetate solution, shaken evenly, filtered by a 0.5 mu m microporous membrane, and a subsequent filtrate is taken as a positioning test sample (5 mg of mupirocin is contained in every 1 ml).
Step A2, carrying out high performance color chromatography detection on the content test sample;
the chromatographic conditions were as follows:
a chromatographic column: zorbax C8(250 mm. times.4.6 mm, 5 μm)
Detection wavelength: 240nm
Flow rate: 1ml/min
Sample introduction volume: 20 μ l
Column temperature: 37 deg.C
Mobile phase:
④ mobile phase C0.1M ammonium acetate tetrahydrofuran 76: 24
Mobile phase D: 0.1M ammonium acetate: tetrahydrofuran 71: 29
Gradiometer
Step A3, comparing the result of the step A2 with an impurity standard chromatogram to obtain the impurity content of the mupirocin ointment;
wherein the standard impurity chromatogram is obtained by detecting the mupirocin ointment impurity qualitative positioning method in the embodiment.
Comparative example 1
The samples of the same batch in the example are detected, and the detection steps are as follows:
mupirocin ointment (about 50mg equivalent to mupirocin) was added with an appropriate amount of 0.1mol/L sodium dihydrogen phosphate buffer (pH adjusted to 6.3 with 1mol/L sodium hydroxide solution) to make a solution containing about 5mg of mupirocin per 1 ml.
Performing chromatographic detection, and testing chromatographic conditions according to BP standard
Comparison after testing:
BP2009 mupirocin ointment controlled 3 known impurities, mupirocin impurity C (relative mupirocin retention time about 0.65), mupirocin impurity D (relative mupirocin retention time about 0.5), mupirocin impurity E (relative mupirocin retention time about 0.55), but in the known impurity localization hplc studies the flow rate was changed to 1ml/min, resulting in a relative retention time of the known impurities that differs from the BP2009 mupirocin ointment specification by about 0.1, thus revising the relative retention times of the 3 known impurities as: mupirocin impurity C (about 0.75% relative to mupirocin retention time), mupirocin impurity D (about 0.6 relative to mupirocin retention time), mupirocin impurity E (about 0.64 relative to mupirocin retention time), means that impurities in the form of the label are controlled as other unknown impurities, with a limit of 1.5%. The test results are shown in table 13.
TABLE 13 mupirocin ointment impurity test results
According to the method, no impurity reference substance is adopted for comparison, 9 impurities can be controlled by adopting reasonable chromatographic mass spectrometry conditions, and subsequent reasonable chromatographic tests can be directly compared, so that a new thought is provided for impurity detection and control in the process of developing mupirocin ointment. The comparative example detected 15 impurities, and the method of the present invention detected 16 impurities.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.
Claims (10)
1. A mupirocin ointment impurity qualitative positioning method is characterized by comprising the following steps:
step S1, preparing mupirocin ointment sample into positioning test sample;
step S2, detecting the liquid-mass linkage of the test sample;
step S3, analyzing the detection result, determining the structure of each impurity and the relative retention time of each impurity, and obtaining an impurity standard mass chromatogram and an impurity standard chromatogram;
in step S2, the chromatographic detection conditions are: adopting a C8 chromatographic column, wherein the column temperature is 33-37 ℃, the detection wavelength is 240nm, and the flow rate is as follows: 1ml/min, injection volume: performing gradient elution by using a mobile phase A and a mobile phase B, wherein the volume of the elution is 19-21 mul; the mobile phase A consists of 75 mass percent of 0.1mol/L ammonium acetate and 25 mass percent of tetrahydrofuran, and the mobile phase B consists of 70 mass percent of 0.1mol/L ammonium acetate and 30 mass percent of tetrahydrofuran;
in step S2, the mass spectrometry detection conditions are: primary mass spectrum: data are collected by adopting an ESI source and a Scan mode, scanning ions adopt a negative mode, Frag voltage is 135V, and a mass number scanning range is as follows: 300-700;
second-order mass spectrometry: the parent ion selection was 500.3, the Frag voltage was 135V, the collision voltage was 20mV, and the Product ion scan range was: 100-500.
2. the method for qualitatively locating mupirocin ointment impurities as claimed in claim 1, wherein in step S1, after standing the mupirocin ointment sample, weighing the mupirocin ointment sample, sequentially adding tetrahydrofuran and sodium acetate, performing microfiltration, and collecting the filtrate to obtain the location test sample.
3. The method for qualitatively locating mupirocin ointment impurities as claimed in claim 1, wherein in step S2, the chromatographic detection conditions are: using Zorbax Eclipse XDB-C8Chromatographic column, column temperature 35 ℃, detection wavelength 240nm, flow rate: 1ml/min, injection volume: 20 μ l.
4. The method for qualitatively locating mupirocin ointment impurities as claimed in claim 1, wherein in step S2, the condition parameters of the gradient washing are:
5. A mupirocin ointment impurity testing method is characterized by comprising the following steps:
step A1, preparing mupirocin ointment sample into content test sample;
step A2, carrying out high performance color chromatography detection on the content test sample;
step A3, comparing the result of the step A2 with an impurity standard chromatogram to obtain the impurity content of the mupirocin ointment;
wherein the standard impurity chromatogram is obtained by detecting the mupirocin ointment impurity qualitative localization method of any one of claims 1 to 4.
6. Use of the test method of claim 5 for testing impurities in a mupirocin ointment, wherein the mupirocin ointment comprises the following components in weight percent: polyethylene glycol-400: 78.4%, polyethylene glycol-4000: 19.6% and mupirocin: 2 percent.
7. The use according to claim 6, wherein in step A2, a C8 chromatographic column is used, the column temperature is 33-37 ℃, the detection wavelength is 240nm, and the flow rate: 1ml/min, injection volume: 20 mu l, and gradient elution is carried out by adopting a mobile phase C and a mobile phase D; the mobile phase C and the mobile phase D both consist of 0.1mol/L ammonium acetate and tetrahydrofuran; wherein, in the mobile phase C, the mass percent of 0.1mol/L ammonium acetate is 74-76%, and the mass percent of tetrahydrofuran is 24-26%; in the mobile phase D, the mass percent of 0.1mol/L ammonium acetate is 69-71%, and the mass percent of tetrahydrofuran is 29-31%.
8. The use according to claim 6, wherein in step A2, C8 chromatographic column is used, the column temperature is 35 ℃, the detection wavelength is 240nm, and the flow rate is as follows: 1ml/min, injection volume: 20 mu l, and gradient elution is carried out by adopting a mobile phase C and a mobile phase D; the mobile phase C and the mobile phase D both consist of 0.1mol/L ammonium acetate and tetrahydrofuran; wherein, in the mobile phase C, the mass percent of 0.1mol/L ammonium acetate is 75%, and the mass percent of tetrahydrofuran is 25%; in the mobile phase D, the mass percent of 0.1mol/L ammonium acetate is 70%, and the mass percent of tetrahydrofuran is 30%.
9. The use according to claim 6, wherein in step A2, Zorbax Eclipse XDB-C is used8Chromatography column or Zorbax Eclipse Plus C8And (5) detecting by using a chromatographic column.
10. The use according to claim 6, wherein in step A2, the condition parameters of gradient washing are:
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| CN114740102A (en) * | 2022-03-14 | 2022-07-12 | 广东省药品检验所（广东省药品质量研究所、广东省口岸药品检验所） | Dipyridamole impurity positioning solution and preparation method and application thereof |
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