Clindamycin isomer, analytical preparation method and application thereof
A technology of clindamycin and isomers, applied in the field of pharmaceutical analytical chemistry and analytical chemistry, can solve the problems of lack of understanding of importance, little consideration of the adverse effects of impurities on drug safety, and insufficient consideration of impurity limits, etc. question
Inactive Publication Date: 2013-05-29
SHANGHAI INST OF PHARMA IND +1
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AI-Extracted Technical Summary
Problems solved by technology
 2. Reaction raw materials that exist due to incomplete reaction, reaction initial complexes, synthetic intermediate products, by-products and other substances related to the synthesis process;
 5. Except for degradation products and toxic impurities, the impurities that have been controlled in the raw materials are generally no longer controlled in the preparation;
From the analysis of new drug declarations in recent years, there are many problems in the research and limit determination of impurities, mainly as follow...
The invention provides a clindamycin isomer which has a structure shown in the formula II. The invention further provides an analytical preparation method of the clindamycin isomer. The method is characterized in that the clindamycin raw materials are analyzed and the clindamycin isomer is separated and prepared from the raw materials. The method comprises the following steps of: a) determining the clindamycin raw materials by the LC-MS (liquid chromatography-mass spectrometry) method, and determining the clindamycin isomer in the raw materials according to relative retaining time and/or molecular weight of analyzed components; b) determining conditions of column chromatography according to the relative retaining time and/or molecular weight of the clindamycin isomer, obtained in the step a, and enriching analyzed components corresponding to the relative retaining time and/or molecular weight by positive phase silica gel column chromatography; and c) determining condition of the preparation liquid phase method according to chromatographic retention behavior displayed by the relative retaining time of the clindamycin isomer, obtained in the step a, and collecting the analyzed components corresponding to the relative retaining time by the liquid phase method.
Antibacterial agentsOrganic active ingredients +4
ChemistryColumn chromatography +5
- Experimental program(3)
 Example 1
 LC-MS Determination of Clindamycin API and Crude Product
 Liquid Mass Spectrometry: HPLC Waters 2486, MS Waters micromass ZQ 4000. Chromatographic column: Diamonsil C 18 (5μ250×4.6mm); mobile phase is acetonitrile-tetrahydrofuran-water-formic acid (18%: 3%: 79%: 0.2%), the pH value of ammonia is adjusted to 5.45; the column temperature is room temperature; detection Wavelength 210nm; flow rate 1.0mL/min, split into mass spectrometer. The mass spectrometry conditions were electrospray ionization source positive ion (ESI+) detection mode; source temperature 80°C; cone voltage 35v.
 LC-MS detection of APIs
 Dissolve the mobile phase for bulk pharmaceuticals with batch number 090303×7 into a solution with a concentration of 2 mg/mL, and the injection volume is 20 μL. LC-MS test results are as figure 1 Shown.
 Six related substances except clindamycin in the Clindamycin Hydrochloride API were detected by the liquid quality. According to the retention time, they are related substance 1 (3.95min), related substance 2 (4.20min), and related substance 3 (12.39min), related substance 4 (21.89min), related substance 5 (23.25min), clindamycin (28.24min, main component), related substance 6 (32.62min).
 Table 1. LC-MS analysis of Clindamycin Hydrochloride API
 In order to investigate the situation of related substances in different batches of APIs, take 081002×5 batches, 060901×5 batches and 060902×5 batches of APIs respectively, and dissolve them into a solution with a concentration of 2mg/mL in the mobile phase. The injection volume is 20μL. . Perform LC-MS detection, and the results are shown in Figure 2.
 Table 2. LC-MS analysis results of three batches of APIs
 It can be seen from Table 2 that the related substances detected in the three batches of APIs are the same as 090303×7 batches of APIs, and the content of each related substance TIC chart integral is similar to the content of 090303×7 batches of API TIC chart integration.
 According to ICH regulations, it is necessary to describe the structure of impurities whose content is greater than one thousandth. The liquid quality test detected that the content of five related substances in the API except for related substance 2 exceeded one thousandth, and the structure of these five related substances needed to be identified. Reference British Pharmacopoeia  , The structures of three related substances are known, which are determined by comparing their molecular weights: related substance 1 is lincomycin, and related substance 3 is clindamycin B. Name related substance 4 as impurity 1, related substance 5 as impurity 2, and related substance 6 as impurity 3. The present invention focuses on impurity 2, which is the clindamycin isomer represented by formula II.
 LC-MS detection of crude product
 Since the content of other related substances in the bulk drug except the main component is very small, it is not easy to enrich the target impurity, so the sample used in the study of the target impurity is the crude clindamycin hydrochloride (batch number: S090701). Same as the detection method of the raw material, the result of LC-MS detection of the crude product is as follows image 3 Shown.
 Table 3 LC-MS analysis of crude clindamycin hydrochloride
 In addition to the six related substances detected in the crude drug, there are also related substances 7 and 8 in the crude product. Pay attention to whether these two related substances will affect the enrichment of the target impurities during the study. The content of two target impurities was slightly increased: the content of impurity 1 increased to 7.27%, and the content of impurity 2 increased to 1.26%. The content of impurity 3 is still low.
 Normal phase silica gel column chromatography to enrich the target impurity-the clindamycin isomer represented by formula II
 In order to preliminarily enrich the target impurity-the clindamycin isomer represented by formula II, according to the structural characteristics of the clindamycin isomer, normal phase silica gel column chromatography was used for the study.
 The separation principle of silica gel chromatography is based on the different adsorption of substances on silica gel. Generally speaking, substances with greater polarity are easily adsorbed by silica gel, and substances with weaker polarity are not easily adsorbed by silica gel. The entire chromatography process That is the process of adsorption, desorption, re-adsorption, and re-desorption.
 Use different ratios of ethyl acetate and methanol as the eluent to select the ratio that can achieve the best separation and enrichment effect.
 Chromatography column: 5×100cm;
 Pretreatment: Because clindamycin hydrochloride contains multiple hydroxyl groups in its structure, it is relatively active. Therefore, the silica gel should be deactivated before use. The specific method is: weigh 100g of 100-200 mesh crude silica gel, soak in industrial-grade methanol overnight, drain the methanol from the Buchner funnel, and place it in a 70℃ water bath to make the silica gel The residual methanol was evaporated to dryness, and then packed into a column.
 Sample loading: Weigh 1g of crude clindamycin hydrochloride (batch number: S090701), dissolve in methanol, add dropwise to a crucible containing 1g of silica gel, place it in a 60°C water bath, mix the sample, and apply dry method to the column.
 The conditions of normal phase column chromatography are: sample: silica gel = 1:50, and the elution sequence is as follows: (ethyl acetate: methanol 9:1) 1800 mL, (ethyl acetate: methanol 6:1) 1680 mL, (ethyl acetate: Methanol 5:1) 600 mL, (ethyl acetate: methanol 4: 1) 600 mL, (ethyl acetate: methanol 3:1) 600 mL, (ethyl acetate: methanol 2: 1) 600 mL, (ethyl acetate: methanol 1 :1) 600mL, 600mL of methanol. Among them, the part that can enrich impurity 3 is ethyl acetate: methanol 9:1, the part that can enrich impurity 2 is ethyl acetate: methanol 6:1, and the part that can enrich impurity 1 is (ethyl acetate: methanol 5: 1) ~ The elution position of methanol.
 Separation and Preparation of Target Impurities-Clindamycin Isomers of Formula II by HPLC
 After the three target impurities are preliminarily enriched by normal phase column chromatography, the target impurities are further separated and purified by the preparative liquid method to obtain the target impurities with higher purity for structural identification.
 Separation and preparation of impurity 1 and impurity 2
 Impurity 1 and Impurity 2 are both isomers of the main component clindamycin, and the separation conditions for the two are relatively strict. Due to the influence of the injection volume, it takes a long time to obtain pure impurity 1 and impurity 2 through one preparation, and the cost is high. Therefore, the second preparation method is adopted to obtain a large amount of impurity 1 through the first preparation. The mixture of impurity and impurity 2 is completely separated by the second preparation. Experiments have proved that the method of secondary preparation is feasible, and the purity of the prepared impurity 1 and impurity 2 both meet the standard of structure identification.
 First preparation conditions
 Apparatus: Waters 510 semi-preparative liquid phase, Waters 484 detector, μBondapakTM C18 (7.8×300mm) for chromatographic column, detection wavelength at 210nm, flow rate at 1.0×2.25mL/min.
 The mobile phase conditions are 20% acetonitrile, 1.25% tetrahydrofuran, 78.75% water, 0.2% formic acid, and ammonia to adjust the pH to about 5.58. Such as Figure 4 As shown, the peak at 29.525 min is collected to obtain a mixed component of impurity 1 and impurity 2.
 After a period of preparation, the obtained mixed components of impurity 1 and impurity 2 are detected by LC-MS, such as Figure 5 Shown. Compared with the LC of the crude product, the retention time is 19.62min is impurity 1, the retention time is 20.87min is impurity 2, and the retention time is 25.53min is clindamycin. The contents of the three components are: impurity 1: 68.67%, impurity 2: 16.39%, and clindamycin: 14.94%.
 Second preparation conditions
 Instrument: HP 1100, equipped with Waters 2695Separation Module, Waters 2487DualλAbsorbance Detector Waters. Chromatographic column: Sepax HP-C18 (5μm 10.0×250mm).
 The second preparation of Impurity 1 and Impurity 2 uses a binary pump, and the conditions of the mobile phase are determined as follows: Pump A: 21% acetonitrile, 3% tetrahydrofuran, 76% water, 0.2% formic acid, pH of about 5.20; Pump B: acetonitrile; A:B=95:5. Detection wavelength: 210nm, flow rate: 1.5mL/min, column temperature: 35°C. Preparation of liquid phase diagram such as Image 6 As shown, the prepared sample is a mixture of impurity 1 and impurity 2 obtained in the first preparation. Impurity 1 is obtained at 25.90 min in the collection figure, and impurity 2 is obtained at 27.68 min.
 Purity detection of impurity 1 and impurity 2 obtained by secondary preparation
 After the second preparation according to the conditions of 22.214.171.124, impurity 1 and impurity 2 were obtained respectively. The two components were placed in a water bath at 50°C under reduced pressure and the mobile phase was evaporated to obtain a white solid (containing ammonium formate). After the methanol was dissolved, the LC-MS detection was performed, and the result is shown in Figure 7. After comparison with the crude product and identification by mass spectrometry, the peak with a retention time of 18.78 min in Figure 3-14B was identified as impurity 1, with a content of 95.00%; the peak in Figure 3-14C with a retention time of 21.27 minutes was identified as impurity 2, with a content of 94.61 %.
 Example 2
 Target impurity-structure identification of clindamycin isomer represented by formula II
 The elemental composition of the obtained pure clindamycin isomers was determined by high-resolution mass spectrometry, and then clindamycin was determined by hydrogen nuclear magnetic resonance spectrum, carbon spectrum and two-dimensional spectrum DEPT, HMBC, HMQC, COSY and NOESY spectra. The structure of the isomer. The instruments used are Micromass Q-TOF mass spectrometer and American Varian nuclear magnetic resonance spectrometer (400MHz). The following structural formula is the structure of clindamycin, and Table 4 is the data of clindamycin NMR spectrum.
 Table 4. Clindamycin 1 H-NMR spectrum, 13 C-NMR spectrum, HMQC spectrum, COSY spectrum,
 NOESY spectrum attribution
 There are four chiral centers in the structure of clindamycin, and their configurations are 6S, 7S, 1'S, 3'R.
 Impurity 2-Structural identification of clindamycin isomer represented by formula II
 Instruments: Micromass Q-TOF mass spectrometer; American Varian nuclear magnetic resonance spectrometer (400MHz);
 High resolution mass spectrometry detection of impurity 2 [M+Na] + The mass-to-charge ratio is 447.1694, and the element composition is determined to be C 18 H 33 ClN 2 O 5 S, the impurity is also an isomer of clindamycin. The structure of impurity 2 identified by the full set of nuclear magnetic resonance spectra is shown in Formula II below.
 Table 6, Impurity 2 1 H-NMR spectrum, 13 C-NMR spectrum, HMBC spectrum, HMQC spectrum, COSY spectrum, NOESY spectrum attribution
 Impurity 2 is the isomer of clindamycin hydrochloride, so the two should have the same core structure, but the configuration of the chiral carbon is different. Carbon spectrum and DEPT spectrum display, δ C 171.1 is quaternary carbon, which is classified as C-10. Proton spectrum four CH 3 Attribution is as follows: δ H 0.66 (3H, t, J=7.2Hz) is classified as H-8’, δ H 1.22 (3H, d, J=7.2Hz) is classified as H-8, δ H 1.98 (3H, s) is classified as H-9, δ H 2.24 (3H, s) is classified as H-5’, corresponding to the HMQC spectrum, δ C 13.5 Attributable to C-8’, δ C 21.9 Attributable to C-8, δ C 12.9 Attributable to C-9, δ C 40.9 is assigned to C-5'.
 COSY spectrum shows that H-8’ and δ H 1.12~1.04 (2H, m) are related and are classified as H-7'. H-8 and δ H 4.36~4.43 (1H, m) are related and are classified as H-7. According to the HMQC spectrum, δ C 20.8 attributable to C-7’, δ C 58.3 Attributable to C-7.
 Reference clindamycin hydrochloride and impurity 1, δ H 5.18 (1H, d, J = 5.6 Hz) is classified as H-1, δ C 88.1 is assigned as C-1. H-1 and δ in COSY spectrum H 3.91 (1H, dd, J = 5.6, 10.2 Hz) is related, and is classified as H-2. H-2 and δ H 3.44 (1H, dd, J=3.2, 10.2 Hz) is related, and is classified as H-3. H-3 and δ H 3.64 (1H, d, J=3.2 Hz) correlation, attributable to H-4. Control HMQC spectrum, δ C 68.0 attributable to C-2, δ C 70.6 attributable to C-3, δ C 68.4 attributable to C-4.
 HMBC spectrum shows that H-8’ and δ C 20.8 and δ C 36.9 related. δ C 20.8 has been classified as C-7’, and δ C 36.9 is CH on the DEPT spectrum 2 Signal, therefore δ C 36.9 is assigned as C-6'. Control HMQC spectrum, δ H 1.19 to 1.14 (2H, m) are classified as H-6'. H-8 and δ C 52.6 and δ C 58.3 correlation, δ C 58.3 has been classified as C-7, δ C 52.6 is assigned as C-6. Control HMQC spectrum, δ H 4.21 (1H, dd, J=10.0, 1.6 Hz) is classified as H-6. From the coupling constant, δ H 4.10 (1H, d, J=10.4Hz) is assigned as H-5, compared with HMQC spectrum, δ C 69.4 is assigned as C-5.
 DEPT spectrum display, δ C 37.4 and δ C 61.0 for CH 2 Signal peak, attributable to δ by chemical structure C 37.4 is C-2, δ C 61.0 is C-4'. According to the HMQC spectrum, H-2’ and H-4’ are split into two groups of peaks, δ H 2.33~2.26 (1H, m) and 1.36~1.29 (1H, m) are classified as H-2’, δ H 2.77 to 2.75 (1H, m) and 2.58 to 2.53 (1H, m) are classified as H-4'. H-2’ and δ in HMBC spectrum C 69.6 related, since C-1’ has stronger de-shielding effect than C-3’, it belongs to δ C 69.6 is C-1'. Finally δ C 36.3 Attributable to C-3'.
 The NOESY spectrum shows that the H-1' of clindamycin is related to H-5', while the H-1' and H-5' of impurity 2 have no correlation peaks, indicating that H-1' and H- of clindamycin The 5'position is closer, and the H-1' and H-5' positions in impurity 2 are farther away. Therefore, it is inferred that the difference between impurity 2 and clindamycin is mainly in the configuration of the 1'-position carbon atom. The 1'-position carbon atom of clindamycin is in the S configuration, and the 1'-position carbon atom of the impurity 2 is in the R configuration.
 Therefore, the target impurities all have the same core structure as clindamycin, and the molecular weight of impurity 2 is the same as that of clindamycin, which is an isomer of clindamycin.
 There are four chiral carbons in the structure of clindamycin, namely C-6, C-7, C-1' and C-3', and the configuration is 6S, 7S, 1'S, 3'R, respectively. Impurity 2 with clindamycin 1 HNMR and 13 The CNMR data is similar. The structure difference from clindamycin is at the 1'position. The four chiral carbon configurations of impurity 2 are 6S, 7S, 1'R, 3'S, respectively. Impurity 2 is not included in the pharmacopoeia and literature.
 Example 3
 Bacteriostatic experiment of clindamycin hydrochloride and impurity 2 of the present invention
 The test used to determine the effectiveness of antibacterial drugs in inhibiting bacterial growth in vitro is called bacteriostatic test. This experiment investigated the antibacterial activity of clindamycin isomers with an apparent content of more than 0.1% in clindamycin hydrochloride raw materials. The pure clindamycin isomers obtained were the same as clindamycin hydrochloride. The antibacterial activity of the raw material drug was compared.
 Preparation of test solution
 Clindamycin Hydrochloride (090303×7 batch, Zhejiang Tiantai Pharmaceutical Co., Ltd.): 1.091mg, dissolved in 1mL water;
 Impurity 2: 1.200mg, dissolved in 0.5mL water;
 Experimental strain
 Staphylococcus aureus (Gram-positive bacteria), Bacillus subtilis (bacteria), Candida albicans (fungi); provided by the Department of Biology, Shanghai Pharmaceutical Industry Research Institute.
 Medium preparation
 Candida albicans medium (%)
 Glucose 0.1 Yeast extract 0.25
 KCl 0.18 NaAc 0.82
 Agar 1.5 pH 7.0
 Sterilize at 121℃ for 30min
 Staphylococcus aureus and Bacillus subtilis (%)
 Peptone 0.6 Beef extract 0.15
 Yeast extract 0.6 Glucose 0.1
 Agar 1.5 pH 6.5
 Sterilize at 121℃ for 30min
 Preparation of filter paper
 Choose high-quality filter paper with strong water absorption, use a punch to make a circular filter paper sheet with a diameter of 6mm, and sterilize it by dry heat for later use.
 experimental method
 Agar Diffusion Disc Method  :Suck 0.1mL of bacterial solution and spread it evenly on the M-H agar watch glass. Add 10 μL of clindamycin hydrochloride and clindamycin isomer solutions evenly onto the sterilized paper sheet. After drying, use sterile tweezers to pick up the paper sheets and place them on a watch glass containing bacteria at equal distances. . Cover the watch glass and place it flat in a 37°C incubator for 24 hours and then take it out. Observe the antibacterial effect.
 Experimental results
 Clindamycin hydrochloride and clindamycin isomers have antibacterial effects on Staphylococcus aureus and Bacillus subtilis. The antibacterial effect on Staphylococcus aureus is as follows Figure 8 As shown, the zone of inhibition of impurity 2 is smaller than that of clindamycin hydrochloride. Therefore, the antibacterial effect of impurity 2 is worse than that of clindamycin hydrochloride. The antibacterial effects of Bacillus subtilis are as follows Picture 9 As shown, the bacteriostatic zone of the four is relatively large, indicating that clindamycin hydrochloride and clindamycin isomers have a good inhibitory effect on Bacillus subtilis. Picture 10 There was no inhibition zone around the filter paper sheet showing clindamycin hydrochloride and clindamycin isomers, indicating that neither had antibacterial activity against Candida albicans.
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