CN111253473B - Preparation method of bacitracin impurity L based on photocatalysis - Google Patents
Preparation method of bacitracin impurity L based on photocatalysis Download PDFInfo
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- CN111253473B CN111253473B CN202010159151.7A CN202010159151A CN111253473B CN 111253473 B CN111253473 B CN 111253473B CN 202010159151 A CN202010159151 A CN 202010159151A CN 111253473 B CN111253473 B CN 111253473B
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Abstract
The invention provides a preparation method of bacitracin impurity L based on photocatalysis, which comprises the following steps: step S1, dissolving the bacitracin bulk drug into a solvent, and illuminating for 1-3 h at 50-60 ℃ to obtain a crude product containing bacitracin impurity L; and step S2, separating bacitracin impurity L and other components by adopting a preparation liquid phase, collecting bacitracin impurity L peak fraction, and evaporating the collected bacitracin impurity L peak fraction to dryness to obtain a bacitracin impurity L impurity reference substance. The technical scheme of the invention adopts a photocatalytic chemical conversion method to convert bacitracin A into an epimer of bacitracin A, namely impurity L, under the action of illumination, has simple and efficient steps and realizes the enrichment of low-cost crude impurities; simple post-treatment, low cost and little pollution.
Description
Technical Field
The invention belongs to the technical field of biochemistry, and particularly relates to a preparation method of a bacitracin impurity L based on photocatalysis.
Background
The bacitracin is obtained by fermenting Bacillus licheniformis (Bacillus licheni bacteria), has strong antibacterial effect on most gram-positive bacteria, is similar to penicillin in antibacterial effect, is also effective on penicillin-resistant staphylococcus aureus, and is mainly used for treating various infections caused by the drug-resistant staphylococcus aureus, such as septicemia, pneumonia, endocarditis and the like. The european pharmacopoeia EP9.6 contains A, B, C, D, E, F, G, H, I, J, K, L, M, N, O, P, Q related substances, wherein the limit of impurity L is specified to be 2.0%, and impurity L is D-alloisoleucine epimer of bacitracin a, whose chemical structure is shown below, which has an important influence on the safety and efficacy of the drug.
In recent years, various national drug administration departments gradually strengthen the supervision work of drug application, particularly the imitation drug application, especially put forward higher requirements on the quality of the imitation drug, and require pharmaceutical enterprises to carry out intensive research on various impurities affecting the safety and the effectiveness of the drugs, and a certain amount of impurity reference substances are obtained in the research process to carry out various pharmaceutical researches on the impurities, so that the preparation of the impurity reference substances is very important work.
It is described in the literature that Jon D.Epperson and Li-June Ming uses a traditional reversed phase HPLC method to directly separate bacitracin impurity L (the name in the literature: bacitracin A)2) The method adopts the fermentation sample to directly carry out separation preparation, the content of bacitracin impurity L in the sample is lower, the overall preparation efficiency is very low,the cost is very high, and the method is not suitable for preparing impurity reference substances.
Disclosure of Invention
Aiming at the technical problems, the invention discloses a preparation method of bacitracin impurity L based on photocatalysis, which realizes high-efficiency and low-cost separation and preparation of high-purity bacitracin impurity L reference substance meeting pharmacopoeia requirements.
In contrast, the technical scheme adopted by the invention is as follows:
a preparation method of bacitracin impurity L based on photocatalysis comprises the following steps:
step S1, dissolving the bacitracin bulk drug into a solvent, and illuminating for 1-3 h at 50-60 ℃ to obtain a crude product containing bacitracin impurity L; wherein the content of bacitracin impurity L in the crude product is more than 20%. Wherein, the content is the content characterized by an HPLC area normalization method, and is generally close to the real mass percentage content. The photocatalytic conversion route is as follows:
and step S2, separating bacitracin impurity L and other components by adopting a preparation liquid phase, collecting bacitracin impurity L peak fraction, and evaporating the collected bacitracin impurity L peak fraction to dryness to obtain a bacitracin impurity L impurity reference substance.
As a further improvement of the invention, in step S1, the illumination intensity is 4000-5000 Lux.
As a further improvement of the present invention, in step S1, the solvent is methanol or ethanol.
As a further improvement of the invention, the solvent contains a cosolvent, and the cosolvent is at least one of trifluoroacetic acid, formic acid or acetic acid.
In a further improvement of the present invention, in step S2, the bacitracin impurity L is separated from other components by eluting the crude product obtained in step S1 with an organic acid aqueous solution-acetonitrile system as a mobile phase and a C18 packing as a preparative column.
As a further development of the invention, the organic acid is trifluoroacetic acid, acetic acid or formic acid.
In a further improvement of the present invention, the volume concentration of the organic acid is 0.05 to 0.5%. Further, the volume concentration of the organic acid is 0.1%.
In a further modification of the present invention, in step S2, reduced pressure evaporation to dryness is performed using a rotary evaporator.
Compared with the prior art, the invention has the beneficial effects that:
the technical scheme of the invention adopts a photocatalytic chemical conversion method to convert bacitracin A into an epimer (namely impurity L) of bacitracin A under the action of illumination, the content of the impurity L in the obtained crude product is more than 20 percent, the steps are simple and efficient, and the low-cost impurity crude product enrichment is realized.
In addition, the technical scheme of the invention adopts the preparation liquid to carry out one-step separation preparation on the bacitracin acid destruction crude product, the adopted mobile phases are organic acid solution and acetonitrile, the preparation flow can be directly decompressed and evaporated to dryness, and the post-treatment is simple, low in cost and small in pollution.
Drawings
FIG. 1 is a high resolution mass spectrum of bacitracin impurity L prepared in example 2 of the present invention.
FIG. 2 shows bacitracin impurity L prepared in example 2 of the present invention1And (4) H spectrum atlas.
FIG. 3 shows bacitracin impurity L prepared in example 2 of the present invention13And (C) spectrum atlas.
Detailed Description
Preferred embodiments of the present invention are described in further detail below.
Example 1
The instrument comprises the following steps: preparation of the liquid phase (Waters 2525); rotary evaporator (shanghai asia rong); photochemical reaction apparatus (Shanghai PolyLai laboratory instruments Co., Ltd.).
Preparation of bacitracin impurity L crude product: dissolving 3g of bacitracin raw material medicine in 50ml of ethanol, adding 1ml of trifluoroacetic acid for assisting dissolution, and reacting for 2 hours at 50 ℃ in a photochemical reactor with the illumination intensity of 5000Lux to obtain a crude product with the impurity L content of 30%. The above contents are contents characterized by HPLC area normalization.
Preparing a column: the filler is Kromasil EternitylXT-10-C18, and the column tube is HB-DAC 50.
Preparation of liquid phase separation chromatographic conditions: performing isocratic elution by using trifluoroacetic acid water solution with the volume concentration of 0.1 percent and acetonitrile (70:30, V/V) as a mobile phase; the flow rate is 100 mL/min; the detection wavelength is 254 nm; the column temperature is room temperature; the sample is manually injected, and the sample injection amount is 2 ml.
Preparing a sample solution: concentrating the prepared bacitracin impurity L crude product to 10ml, and filtering with 0.45um organic filter membrane.
Collecting bacitracin impurity L fraction: and (3) after the system is balanced for 8 minutes under the condition of preparing the liquid phase separation chromatography, manually injecting a sample for elution and separation, starting to collect fractions when the bacitracin impurity L just comes out of a peak, ending at the tail part of the peak, and storing the bacitracin impurity L fractions at normal temperature. The instrument was repeated several times and the bacitracin impurity L fractions were pooled.
Fraction post-treatment of bacitracin impurity L: all fractions obtained by separation were evaporated to dryness at 30 ℃ under reduced pressure using a rotary evaporator to obtain bacitracin impurity L with a purity of 97.2% at 705 mg. The purity is characterized by HPLC area normalization and is generally close to the true mass percent.
The bacitracin impurity L obtained was analyzed and the results were as follows:
(1) LC-MS test:
the instrument comprises the following steps: agilecnt 1260HPLC-6530Q-TOF
The test method comprises the following steps: an ion source: dual AJS ESI ion sources; a positive ESI mode; temperature of the drying gas: 350 ℃; flow rate of drying gas: 12.0L/min; electrospray voltage: 4.5 kv.
And (3) testing results: the observed M/z1422.7554, M/z712.3582 and M/z475.2602 are [ M + H ] of the bacitracin impurity L obtained]+Ion, [ M +2H ]]2+Ion and [ M +3H]3+Ions. Calculating its molecular formula to be C66H103N17O16S, as with bacitracin A formula, the calculated relative deviation of theoretical values from actual measurements was 0.56 ppm.
(2) NMR measurement:
the instrument comprises the following steps: bruker AVANCE III HD 600 Switzerland 600 superconducting pulse Fourier transform nuclear magnetic resonance spectrometer.
The test method comprises the following steps: dissolving the sample in heavy water, and testing1H spectrum and13C-NMR spectrum, the obtained bacitracin epimer chemical structural formula is shown as the following formula, and NMR data are shown as the table 1.
TABLE 1 of bacitracin impurity L13C and1h chemical shift data attribution
| No. | δH | δC | No. | δH | δC |
| 1 | 0.87(3H，t，J＝7.4Hz) | 10.7 | 33 | N/A | 174.6 |
| 2 | 1.28(1H，overlap)，1.42(1H，overlap) | 25.2 | 34 | 4.03(1H，d，J＝7.9Hz) | 58.4 |
| 3 | 1.97(1H，m) | 36.8 | 35 | 1.73(1H，overlap) | 35.7 |
| 4 | 0.90(3H，d，J＝7.0Hz) | 12.7 | 36 | 0.49(3H，d，J＝6.8Hz) | 14.4 |
| 5 | 4.24(1H，d，J＝4.3Hz) | 57.0 | 37 | 0.89(1H，overlap)，1.79(1H，m) | 24.4 |
| 6 | N/A | 173.2 | 38 | 0.66(3H，t，J＝7.4Hz) | 10.2 |
| 7 | 5.13(1H，t，J＝9.2Hz) | 77.2 | 39 | N/A | 173.2 |
| 8 | 3.52(1H，dd，J＝11.3，8.7Hz)，3.71(1H，m) | 36.2 | 40 | 4.54(1H，overlap) | 55.0 |
| 9 | N/A | 172.8 | 41 | 2.78(1H，m)，3.07(1H，overlap) | 37.1 |
| 10 | 4.36(1H，overlap) | 52.6 | 42 | N/A | 136.2 |
| 11 | 1.52(1H，overlap)，1.62(1H，overlap) | 40.0 | 43/43′ | 7.13(2H，d，J＝7.3Hz) | 129.0 |
| 12 | 1.52(1H，overlap) | 24.4 | 44/44′ | 7.24(2H，t，J＝7.4Hz) | 128.8 |
| 13 | 0.80(3H，d，J＝5.9Hz) | 20.9 | 45 | 7.18(1H，t，J＝7.3Hz) | 127.2 |
| 14 | 0.84(3H，d，J＝6.1Hz) | 22.0 | 46 | N/A | 172.5 |
| 15 | N/A | 174.4 | 47 | 4.71(1H，overlap) | 52.1 |
| 16 | 4.25(1H，overlap) | 53.5 | 48 | 2.88(1H，overlap)，3.14(1H，overlap) | 27.1 |
| 17 | 1.90(1H，m)，2.05(1H，m) | 26.2 | 49 | N/A | 128.1 |
| 18 | 2.35(2H，t，J＝7.3Hz) | 30.5 | 50 | 6.98(1H，s) | 117.3 |
| 19 | N/A | 177.4 | 51 | 8.54(1H，d，J＝1.2Hz) | 133.6 |
| 20 | N/A | 173.2 | 52 | N/A | 171.0 |
| 21 | 4.01(1H，d，J＝7.3Hz) | 58.4 | 53 | 4.57(1H，overlap) | 50.9 |
| 22 | 1.62(1H，overlap) | 35.8 | 54 | 2.65(1H，overlap)，2.72(1H，t，J＝5.4Hz) | 36.2 |
| 23 | 0.74(3H，d，J＝6.8Hz) | 14.7 | 55 | N/A | 175.3 |
| 24 | 1.03(1H，m)，1.28(1H，overlap) | 24.4 | 56 | N/A | 172.0 |
| 25 | 0.72(3H，t，J＝7.5Hz) | 10.0 | 57 | 4.57(1H，overlap) | 50.9 |
| 26 | N/A | 173.2 | 58 | 2.65(1H，overlap)，2.75(1H，m) | 37.1 |
| 27 | 4.20(1H，m) | 53.9 | 59 | N/A | 172.5 |
| 28 | N/A | 173.6 | 60 | N/A | 172.5 |
| 29 | 4.36(1H，overlap) | 52.7 | 61 | 3.10(1H，overlap)，3.14(1H，overlap) | 38.7 |
| 30 | 1.70(1H，overlap)，1.79(1H，m) | 28.5 | 62 | 1.42(2H，overlap) | 27.2 |
| 31 | 1.59(2H，overlap) | 23.3 | 63 | 1.20(1H，m)，1.28(1H，overlap) | 21.9 |
| 32 | 2.91(2H，overlap) | 38.9 | 64 | 1.64(1H，overlap)，1.73(1H，overlap) | 30.5 |
Example 2
The instrument comprises the following steps: preparation of the liquid phase (Waters 2525); rotary evaporator (shanghai asia rong); photochemical reaction apparatus (Shanghai PolyLai laboratory instruments Co., Ltd.).
Preparation of bacitracin impurity L crude product: 3g of bacitracin bulk drug is dissolved in 30ml of methanol, 1ml of trifluoroacetic acid is added, and the mixture reacts for 3 hours in a photochemical reactor with the illumination intensity of 4500Lux at 50 ℃ to obtain a crude product with the impurity L content of 35 percent. The above contents are contents characterized by HPLC area normalization.
Preparing a column: the filler is Kromasil EternitylXT-10-C18, and the column tube is HB-DAC 50.
Preparation of liquid phase separation chromatographic conditions: performing isocratic elution by using trifluoroacetic acid water solution with the volume concentration of 0.1 percent and acetonitrile (70:30, V/V) as a mobile phase; the flow rate is 100 mL/min; the detection wavelength is 254 nm; the column temperature is room temperature; the sample is manually injected, and the sample injection amount is 6 ml.
Preparing a sample solution: and (3) passing the prepared bacitracin impurity L crude product through a 0.45um organic filter membrane.
Collecting bacitracin impurity L fraction: and (3) after the system is balanced for 8 minutes under the condition of preparing the liquid phase separation chromatography, manually injecting a sample for elution and separation, starting to collect fractions when the bacitracin impurity L just comes out of a peak, ending at the tail part of the peak, and storing the bacitracin impurity L fractions at normal temperature. The instrument was repeated several times and the bacitracin impurity L fractions were pooled.
Fraction post-treatment of bacitracin impurity L: all fractions obtained by separation were evaporated to dryness at 30 ℃ under reduced pressure using a rotary evaporator to obtain 765mg bacitracin impurity L with purity of 97.1%. The purity is characterized by HPLC area normalization and is generally close to the true mass percent.
Analyzing the obtained bacitracin impurity L, wherein the high resolution mass spectrum of the bacitracin impurity L is shown in figure 1,1the spectrum of the H spectrum is shown in figure 2,13the spectrum of the spectrum C is shown in fig. 3, and it can be seen that bacitracin impurity L obtained in the present example has the same molecular formula as bacitracin A, meets the requirements of pharmacopoeia, and has little deviation between the calculated theoretical value and the actual measured value.
The foregoing is a more detailed description of the invention in connection with specific preferred embodiments and it is not intended that the invention be limited to these specific details. For those skilled in the art to which the invention pertains, several simple deductions or substitutions can be made without departing from the spirit of the invention, and all shall be considered as belonging to the protection scope of the invention.
Claims (6)
1. A preparation method of bacitracin impurity L based on photocatalysis is characterized by comprising the following steps:
step S1, dissolving the bacitracin bulk drug into a solvent, and illuminating for 1-3 h at 50-60 ℃ to obtain a crude product containing bacitracin impurity L; the solvent contains a cosolvent, and the cosolvent is at least one of trifluoroacetic acid, formic acid or acetic acid;
step S2, separating bacitracin impurity L and other components by adopting a preparation liquid phase, collecting bacitracin impurity L peak fraction, evaporating the collected bacitracin impurity L peak fraction to dryness, and obtaining bacitracin impurity L impurity reference substance;
in step S1, the illumination intensity is 4000-5000 Lux.
2. The method of claim 1 for preparing a bacitracin impurity L based on photocatalysis, comprising: in step S1, the solvent is methanol or ethanol.
3. The method of any one of claims 1-2, wherein the step of preparing bacitracin impurity L comprises: in step S2, the crude product obtained in step S1 is eluted and separated with an organic acid aqueous solution-acetonitrile system as a mobile phase and a C18 filler as a preparative column.
4. The method of claim 3 for preparing the bacitracin impurities L based on photocatalysis, wherein: the organic acid is trifluoroacetic acid, acetic acid or formic acid.
5. The method of claim 4 for preparing bacitracin impurities L based on photocatalysis, wherein: the volume concentration of the organic acid is 0.05-0.5%.
6. The method of claim 3 for preparing the bacitracin impurities L based on photocatalysis, wherein: in step S2, the mixture is evaporated to dryness under reduced pressure using a rotary evaporator.
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| CN102153633A (en) * | 2010-12-27 | 2011-08-17 | 江苏九阳生物制药有限公司 | Method for extracting and separating bacitracin |
| CN104844557B (en) * | 2014-02-18 | 2017-03-15 | 天津药物研究院有限公司 | A kind of preparation method of sofalcone known impurities |
| CN105504020B (en) * | 2016-01-23 | 2019-12-20 | 雅赛利(台州)制药有限公司 | Pharmaceutical grade bacitracin and preparation device thereof |
| CN106039766B (en) * | 2016-05-17 | 2018-09-21 | 辽宁大学 | A method of separation gambogicacid epimer |
| US10426834B1 (en) * | 2016-08-11 | 2019-10-01 | Akorn, Inc. | Methods of sterilizing drugs |
| CN110156670B (en) * | 2019-06-21 | 2022-08-16 | 重庆华邦胜凯制药有限公司 | Method for synthesizing a plurality of atorvastatin impurities at one time and application thereof |
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