| Since the penicilin was found and applied to the clinic, the antibiotics made a great effort to relief people from the bacterial infection. However, due to the universal use of the antibiotics, the research of drug resistance has gradually became both the focus owing to its applicability and the nodus as a result of its complicacy. The glycopeptides antibiotics which were applied to the clinic first, such as vancomycin and teicoplanin, successfully solved the problem of the drug resistance and became last line of defense for infection of drug-resistance bacteria.Teicoplanin was found in 1978 and was produced by an actinoplanes. Followed vancomycin, teicoplanin was another very important antibiotic that could be used for the multi-drug- resistant bacterial infection in the clinic.Compared with, the structure, the antibacterial spectrum and the antibacterial infection in the clinic.As for the structure, the antibacterial spectrum and the antibacterial activity, teicoplanin was similar to those of vancomycin. It has six components with very similar chemical structure: TA2-1, TA2-2, TA2-3, TA2-4, TA2-5 and its de-acylgroup glucoseamine TA3-1. Owing to the fatty acid side chains in its molecular structure, teicoplanin was more lipophilic and had better characteristics in the pharmacokinetic. More and more clinical data showed that, compared to vancomycin, teicoplanin was the better choice on treating the Gram-positive bacteria infection.Objective: To qualitative and quantitative analysis of teicoplanin ,three new methodes were established ,i.e. the HPCE method, the HPLC method and the LC-MS method.Method: 1. HPCE method. (1) By optimizing factors which affect the separation, such as the concentration of surfactant, the pH value , concentration of buffers, the supplied voltage and temperature, the optimum conditions for separation were selected. (2) System suitablity test: On the optimized chromatographic separation condition, the resolution of TA2-4 and TA2-5,and the theoretical plate of TA2-2 were determinated.(3)Precision test : The sample solution was analyzed for six times; the peaks areas of teicoplanin were determinaed, and relative standard deviation was calculated(.4)Limit of detection (LOD) test: Dilute the reference solution until the ratio of signal and noise ( S/N )was not less than 3.The limit of detection was determinaed.(5) Sample analysis:Determination the content of three batchs of teicoplanin. 2. Fast HPLC method.(1) optimization chromatographic condition: the best separation condition was chosen by optimizing different columns, adjusting solvent proportion of mobile phase and column temperature. (2) System suitability test: On the optimized chromatographic separation condition, the resolution of TA2-2 and TA2-3,and the theoretical plate of TA2-2 were determinated. (3) Specificity test: By treat with heating, base, acid, hydrogen peroxide (H2O2), strong light and high moisture, the sample of teicoplanin were analyzed. (4) Precision test: The sample solution was analyzed for six times; the peaks areas of teicoplanin were determinaed, and relative standard deviation was calculated.(5) Stability test: By determinaed sample solutions at different time on the room temperature, the stability of the sample solution was determined. (6) Linearity and range of calibration curve: Prepared a series of the reference solutions and determined peaks areas, then calibration curve was obtained by the contents of teicoplanin and the peaks areas. (7) Limit of detection test: Dilute the reference solution until the ratio of signal and noise ( S/N )was not less than 3.The limit of detection was determinaed. (8) Sample analysis:Determination the content of three batchs of teicoplanin. 3.HPLC-MS method.(1) To identify the main components of teicoplanin, a HPLC-MS was established to analyze each component . (2) A high performance liquid chromatography–tandem mass spectrometric method (HPLC-MS-MS) was established for the analysis of teicoplanin. Mass spectrometric detection was operated on a triple quadrupole tandem mass spectrometer equipped with electrospray ionization (ESI) source in positive mode.Results: 1. HPCE method. (1)Using sodium tetraborate buffer containing 3.3%SDS, the base-line separation of the six components of teicoplanin was achieved and the new method was validated. (2)System suitablitily test: On the optimized separation condition, the resolution between TA2-4 and TA2-5 was more than 1.5,and the theoretical plate of TA2-2 were was about 170,000. (3) Precision test: The Precision of six components at six times were good and the RSD of the peaks areas of TA3-1,TA2-1,TA2-2,TA2-3,TA2-4 and TA2-5 were 3.0%, 1.7%, 0.7%, 1.3%,0.7% and 0.8% respectively.(4) Limit of detection (LOD) test: The limit of detection of teicoplanin was 0.2%.(5) Sample analysis: The content of TA2-2 for three batchs were 63.5%,65.1% and 60.2% respectively. 2 Fast HPLC method. (1)The HPLC separation was performed by gradient elution on a HALOTM-C18 analytical column , with a mobile phase consisting of acetonitrile and 10 mmol ammonium acetate(PH 6.0)at the flow rate of 2.5 ml/min. The detection wavelength was 214 nm. The column temperature was set at 30℃. Injection volume was 20μl.(2)System suitablitily test: On the optimized chromatographic separation condition, the resolution bewteen TA2-2 and TA2-3 was more than 1.7,and the theoretical plate of TA2-2 were about 230000. (3) Specificity test: By analyzed accelerate samples,the specificity was proved.(4) Precision test: The Precision of six components at six times were good and the RSD of the peaks areas of TA3-1,TA2-1,TA2-2,TA2-3,TA2-4 and TA2-5 were 1.1%, 0.48%, 0.95%, 0.77%, 0.44% and 0.89% respectively.(5) Stability test: The RSD of the peaks areas of TA3-1,TA2-1,TA2-2,TA2-3,TA2-4 and TA2-5 was 0.58%,0.88%,0.79%,0.68%,0.82% and 0.98% respectively.The test solution was stable in 12 hours. (6) Linearity and range of calibration curve:The linear range for teicoplanin was 0.025-0.4mg/ml. The calibration curve was y = 8.949×107x– 2400,(TA2-2:r=0.9998). (7) Limit of detection test: The detection limit of teicoplanin was 0.8ng/ml. (8) Sample analysis: The content of TA2-2 was 54.0%,55.1% and 56.2% respectively. 3.HPLC-MS method.(1)By using electrospray ionzation and positive ion monitoring, the main components of teicoplanin were analyzed. The temperature of the desolvation and the source block were set at 250℃and 115℃respectively. The flow rate of cone gas(N2) was 280L/h. The capillary voltage and cone voltage were 3.0KV and 30KV respectively. The molecular ions(M+H) of TA3-1,TA2-1,TA2-2,TA2-3,TA2-4 and TA2-5 were 1563,1877,1879,187,1893 and 1893 respectively.(2) The triple quadrupole mass spectrometer with a electrospray ion source was used in the positive ion mode . The temperature of the source block was set at 105℃and the ionization voltage was 3300V. The daughter ion of parent ion (M/Z1877) of TA2-1 were M/Z 1197and 314. The daughter ion of parent ion (M/Z1879) of TA2-2 and TA2-3 were M/Z 1181 and 316. The daughter ion of parent ion (M/Z1893) of TA2-4 and TA2-5 were M/Z 1198 and 330.Conclusion: Three new methodes were established, i.e. the HPCE method, the HPLC method and the LC-MS method. By validation, the three new methods were proved to be specific , accurate and sensitive. They may be used to qualitative and quantitative analysis of teicoplanin in the new drug development for quality control and stability study.
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