高效毛细管电泳法和液质联用法分析测定替考拉宁
摘要
自从青霉素被发现并应用于临床后,抗生素为人类解除细菌性感染发挥了巨大的作用。但随着抗生素在临床上的广泛使用,各种耐药菌不断出现,细菌的耐药性问题逐渐成为关注的热点及研究的难点。糖肽类抗生素,尤其以万古霉素和替考拉宁为代表的抗生素,有效地解决了细菌耐药问题,成为抗耐药性感染的最后一道防线。
替考拉宁(teicoplanin)是Parenti等人于1978年在游动放线菌发酵产物中发现的一种糖肽类抗生素,它是继万古霉素之后用于治疗多重耐药菌感染的重要抗生素之一。
替考拉宁的分子结构、抗菌谱、抗菌活性均与万古霉素相似。它是由一组化学结构非常相似的化合物组成的抗生素混合物,其主要成分为:TA2-1、TA2-2、TA2-3、TA2-4、TA2-5、TA3-1等。由于替考拉宁分子结构上增加了脂肪酸侧链,相对分子质量增大,脂溶性增高,在药物代谢动力学上显示了良好特性。越来越多的证据表明,在治疗革兰氏阳性菌感染方面,替考拉宁比万古霉素更具有优势。
目的:建立替考拉宁的高效毛细管电泳分离方法并对其进行定量分析;建立替考拉宁的高效液相色谱分析方法并对其进行定量分析;应用液质联用法(LC-MS)对替考拉宁进行定性和定量分析。
方法:1.高效毛细管电泳法。(1)通过表面活性剂的浓度、缓冲液浓度及pH、温度及电压的优化,选择最佳的高效毛细管电泳分离条件,对建立的方法进行方法学验证。(2)系统适用性试验:在已确定的高效毛细管电泳条件下,以组分TA2-2考察系统理论板数,并考察相邻组分TA2-4与TA2-5的分离度。(3)精密度试验:取一份对照品溶液,重复进样6次,记录峰面积,计算峰面积的RSD值。(4)检测限的确定:将替考拉宁对照品溶液逐步稀释,测量最高噪音峰的峰高,使样品峰高为噪音峰高的3倍,此时对应的替考拉宁的量即为最低检测限。(5)样品测定:对三批替考拉宁样品按照已确定的分离条件进行测定。2.快速高效液相色谱法。(1)色谱条件的选择:选择不同流动相的组成、配比、流速及柱温,确定最佳的色谱分离条件。(2)系统适用性试验:在已确定的高效液相色谱条件下,以组分TA2-2考察系统理论板数,并考察与相邻组分TA2-3的分离度。(3)专属性试验:在合适的条件下,对替考拉宁样品进行热、碱、酸、氧化、光、湿的破坏。(4)精密度试验:取一份对照品溶液,重复进样6次,记录峰面积,计算峰面积的RSD值。(5)稳定性试验:取替考拉宁供试品溶液适量,室温下放置,分别于0、2、4、6、8、12小时,取样注入液相色谱仪,记录色谱图,计算峰面积的RSD值。(6)线性与范围:配制一系列浓度的替考拉宁对照品溶液,测定峰面积;以浓度为横坐标,以各个峰相应的峰面积为纵坐标,绘制标准曲线。(7)最低检测限的确定:将替考拉宁对照品溶液逐步稀释,使峰高为噪音峰高的3倍,此时对应的替考拉宁的量即为最低检测限。(8)样品测定:对三批替考拉宁样品按照已确定的色谱分离条件进行测定。3.液质联用法。(1)替考拉宁的一级质谱分析方法:采用高效液相色谱-电喷雾-四极杆质谱仪,通过质谱参数的优化选择,建立分析测定替考拉宁中主要组分的方法。(2)替考拉宁的二级质谱分析方法:采用高效液相色谱串联三重四极杆质谱仪,通过质谱参数的优化选择,采用母离子和子离子方式检测,建立分析替考拉宁中主要组分的方法.
结果:1.高效毛细管电泳法。(1)最佳的高效毛细管电泳分离条件为:未涂层石英毛细管67cm×50μm(有效长度60cm);缓冲液为40mmol·L-1硼砂缓冲液(含3.3%SDS,10mmol·L-1磷酸二氢钠,用硼酸调节PH 9.35);检测波长为214nm;电压为30kV;温度为30℃;在此条件下,替考拉宁的主要组分均获得了基线分离。(2)系统适用性:理论板数按组分A2-2计算为170000,组分TA2-4与TA2-5分离度大于1.5。(3)精密度:按上述条件平行测定6次,组分TA3-1、TA2-1、TA2-2、TA2-3、TA2-4、TA2-5各自峰面积的RSD分别为3.0%、1.7%、0.7%、1.3%、0.7%、0.8%,表明方法精密度良好。(4)最低检测限:以信噪比S/N=3为指标,测得本方法的最低检测限为0.2%。(5)样品测定结果:三批替考拉宁样品在已确定的分离条件下进行了测定,测得组分TA2-2的含量分别63.5%,65.1%,60.2%。2.快速高效液相色谱法。( 1 )色谱条件:色谱柱: HALOTM-C18色谱柱(4.6×50mm,2.7μm Agt公司);流动相:A相为15mmol/L乙酸铵(PH 6.0);B相为乙腈;流速为1.0ml/min;在0-0.5min:B相由18%到23%,在0.5-6min:B相由23%到23%,在6-6.5min:B相由23%到18%,线性梯度。柱温30℃。检测波长为214nm,进样量为20μl。(2)系统适用性试验:理论板数按组分TA2-2计算为23000,组分TA2-2与TA2-3分离度为1.7。(3)专属性试验:替考拉宁样品进行热、碱、酸、氧化、光、湿破坏,破坏的产物可与主要组分峰基线分离,表明该方法专属性良好。(4)精密度:按上述条件平行测定6次,组分TA3-1、TA2-1、TA2-2、TA2-3、TA2-4、TA2-5各自峰面积的RSD分别为1.1%,0.48%,0.95%,0.77%,0.44%,0.89%表明方法精密度良好。(5)溶液稳定性:取替考拉宁供试品溶液适量,室温下放置,分别于0、2、4、6、8、12小时,取20μl注入液相色谱仪,记录色谱图,组分TA3-1、TA2-1、TA2-2、TA2-3、TA2-4、TA2-5各自峰面积的RSD分别为0.58%、0.88%、0.79%、0.68%、0.82%、0.98%,表明样品溶液在12 h内稳定。(6)线性与范围:替考拉宁浓度在0.025-0.40mg/ml范围内与峰面积呈良好的线性,组分TA2-2的线性方程为y = 8.949×107x– 2400,相关系数r为0.9998。(7)最低检测限:以信噪比S/N=3为指标,测得本方法的最低检测量为0.8ng/ml,最低检测限为0.02%。(8)样品测定:三批替考拉宁样品进行了测定,测得组分TA2-2的含量分别62.7%、63.1%、59.3%。3.液质联用法。(1)一级质谱分析。LC-MS条件为:单级四极杆质谱,电喷雾电离源(ESI),正离子检测,电离电压3.0KV;锥孔电压30V;电喷雾接口干燥器(N2)流速280L/h,离子源温度115℃;脱溶剂气温度250℃。组分TA3-1、TA2-1、TA2-2、TA2-3、TA2-4和TA2-5的分子离子峰分别为M/Z 1563、M/Z 1877、M/Z 1879、M/Z 1879、M/Z 1893和M/Z 1893。(2)二级质谱分析。LC-MS-MS条件为:三重四极杆质谱,采用正离子模式,离子源温度设为105℃,离子源电离电压为3300V,雾化气流速为500L·hr-1。采集用母离子和子离子检测方式。组分TA2-1的母离子为M/Z 1877,产生的子离子为M/Z 1197和M/Z 314;组分TA2-2和TA2-3的母离子为M/Z 1879,产生的子离子为M/Z 1181和M/Z 316;组分TA2-4和TA2-5的母离子为M/Z 1893,产生的子离子为M/Z 1198和M/Z 330。
结论:本文建立了分析测定替考拉宁的高效毛细管电泳法、快速高效液相色谱法和液质联用法,并对新方法进行了方法学验证,对替考拉宁中各主要组分进行了测定。为该药的稳定性研究、质量控制提供了理论依据和技术支持。
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.
替考拉宁(teicoplanin)是Parenti等人于1978年在游动放线菌发酵产物中发现的一种糖肽类抗生素,它是继万古霉素之后用于治疗多重耐药菌感染的重要抗生素之一。
替考拉宁的分子结构、抗菌谱、抗菌活性均与万古霉素相似。它是由一组化学结构非常相似的化合物组成的抗生素混合物,其主要成分为:TA2-1、TA2-2、TA2-3、TA2-4、TA2-5、TA3-1等。由于替考拉宁分子结构上增加了脂肪酸侧链,相对分子质量增大,脂溶性增高,在药物代谢动力学上显示了良好特性。越来越多的证据表明,在治疗革兰氏阳性菌感染方面,替考拉宁比万古霉素更具有优势。
目的:建立替考拉宁的高效毛细管电泳分离方法并对其进行定量分析;建立替考拉宁的高效液相色谱分析方法并对其进行定量分析;应用液质联用法(LC-MS)对替考拉宁进行定性和定量分析。
方法:1.高效毛细管电泳法。(1)通过表面活性剂的浓度、缓冲液浓度及pH、温度及电压的优化,选择最佳的高效毛细管电泳分离条件,对建立的方法进行方法学验证。(2)系统适用性试验:在已确定的高效毛细管电泳条件下,以组分TA2-2考察系统理论板数,并考察相邻组分TA2-4与TA2-5的分离度。(3)精密度试验:取一份对照品溶液,重复进样6次,记录峰面积,计算峰面积的RSD值。(4)检测限的确定:将替考拉宁对照品溶液逐步稀释,测量最高噪音峰的峰高,使样品峰高为噪音峰高的3倍,此时对应的替考拉宁的量即为最低检测限。(5)样品测定:对三批替考拉宁样品按照已确定的分离条件进行测定。2.快速高效液相色谱法。(1)色谱条件的选择:选择不同流动相的组成、配比、流速及柱温,确定最佳的色谱分离条件。(2)系统适用性试验:在已确定的高效液相色谱条件下,以组分TA2-2考察系统理论板数,并考察与相邻组分TA2-3的分离度。(3)专属性试验:在合适的条件下,对替考拉宁样品进行热、碱、酸、氧化、光、湿的破坏。(4)精密度试验:取一份对照品溶液,重复进样6次,记录峰面积,计算峰面积的RSD值。(5)稳定性试验:取替考拉宁供试品溶液适量,室温下放置,分别于0、2、4、6、8、12小时,取样注入液相色谱仪,记录色谱图,计算峰面积的RSD值。(6)线性与范围:配制一系列浓度的替考拉宁对照品溶液,测定峰面积;以浓度为横坐标,以各个峰相应的峰面积为纵坐标,绘制标准曲线。(7)最低检测限的确定:将替考拉宁对照品溶液逐步稀释,使峰高为噪音峰高的3倍,此时对应的替考拉宁的量即为最低检测限。(8)样品测定:对三批替考拉宁样品按照已确定的色谱分离条件进行测定。3.液质联用法。(1)替考拉宁的一级质谱分析方法:采用高效液相色谱-电喷雾-四极杆质谱仪,通过质谱参数的优化选择,建立分析测定替考拉宁中主要组分的方法。(2)替考拉宁的二级质谱分析方法:采用高效液相色谱串联三重四极杆质谱仪,通过质谱参数的优化选择,采用母离子和子离子方式检测,建立分析替考拉宁中主要组分的方法.
结果:1.高效毛细管电泳法。(1)最佳的高效毛细管电泳分离条件为:未涂层石英毛细管67cm×50μm(有效长度60cm);缓冲液为40mmol·L-1硼砂缓冲液(含3.3%SDS,10mmol·L-1磷酸二氢钠,用硼酸调节PH 9.35);检测波长为214nm;电压为30kV;温度为30℃;在此条件下,替考拉宁的主要组分均获得了基线分离。(2)系统适用性:理论板数按组分A2-2计算为170000,组分TA2-4与TA2-5分离度大于1.5。(3)精密度:按上述条件平行测定6次,组分TA3-1、TA2-1、TA2-2、TA2-3、TA2-4、TA2-5各自峰面积的RSD分别为3.0%、1.7%、0.7%、1.3%、0.7%、0.8%,表明方法精密度良好。(4)最低检测限:以信噪比S/N=3为指标,测得本方法的最低检测限为0.2%。(5)样品测定结果:三批替考拉宁样品在已确定的分离条件下进行了测定,测得组分TA2-2的含量分别63.5%,65.1%,60.2%。2.快速高效液相色谱法。( 1 )色谱条件:色谱柱: HALOTM-C18色谱柱(4.6×50mm,2.7μm Agt公司);流动相:A相为15mmol/L乙酸铵(PH 6.0);B相为乙腈;流速为1.0ml/min;在0-0.5min:B相由18%到23%,在0.5-6min:B相由23%到23%,在6-6.5min:B相由23%到18%,线性梯度。柱温30℃。检测波长为214nm,进样量为20μl。(2)系统适用性试验:理论板数按组分TA2-2计算为23000,组分TA2-2与TA2-3分离度为1.7。(3)专属性试验:替考拉宁样品进行热、碱、酸、氧化、光、湿破坏,破坏的产物可与主要组分峰基线分离,表明该方法专属性良好。(4)精密度:按上述条件平行测定6次,组分TA3-1、TA2-1、TA2-2、TA2-3、TA2-4、TA2-5各自峰面积的RSD分别为1.1%,0.48%,0.95%,0.77%,0.44%,0.89%表明方法精密度良好。(5)溶液稳定性:取替考拉宁供试品溶液适量,室温下放置,分别于0、2、4、6、8、12小时,取20μl注入液相色谱仪,记录色谱图,组分TA3-1、TA2-1、TA2-2、TA2-3、TA2-4、TA2-5各自峰面积的RSD分别为0.58%、0.88%、0.79%、0.68%、0.82%、0.98%,表明样品溶液在12 h内稳定。(6)线性与范围:替考拉宁浓度在0.025-0.40mg/ml范围内与峰面积呈良好的线性,组分TA2-2的线性方程为y = 8.949×107x– 2400,相关系数r为0.9998。(7)最低检测限:以信噪比S/N=3为指标,测得本方法的最低检测量为0.8ng/ml,最低检测限为0.02%。(8)样品测定:三批替考拉宁样品进行了测定,测得组分TA2-2的含量分别62.7%、63.1%、59.3%。3.液质联用法。(1)一级质谱分析。LC-MS条件为:单级四极杆质谱,电喷雾电离源(ESI),正离子检测,电离电压3.0KV;锥孔电压30V;电喷雾接口干燥器(N2)流速280L/h,离子源温度115℃;脱溶剂气温度250℃。组分TA3-1、TA2-1、TA2-2、TA2-3、TA2-4和TA2-5的分子离子峰分别为M/Z 1563、M/Z 1877、M/Z 1879、M/Z 1879、M/Z 1893和M/Z 1893。(2)二级质谱分析。LC-MS-MS条件为:三重四极杆质谱,采用正离子模式,离子源温度设为105℃,离子源电离电压为3300V,雾化气流速为500L·hr-1。采集用母离子和子离子检测方式。组分TA2-1的母离子为M/Z 1877,产生的子离子为M/Z 1197和M/Z 314;组分TA2-2和TA2-3的母离子为M/Z 1879,产生的子离子为M/Z 1181和M/Z 316;组分TA2-4和TA2-5的母离子为M/Z 1893,产生的子离子为M/Z 1198和M/Z 330。
结论:本文建立了分析测定替考拉宁的高效毛细管电泳法、快速高效液相色谱法和液质联用法,并对新方法进行了方法学验证,对替考拉宁中各主要组分进行了测定。为该药的稳定性研究、质量控制提供了理论依据和技术支持。
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.
引文
1 中国药典委员会.中华人民共和国药典 2005 年版,化学工业出版社.
2 Gholizadeh Y, Couralin Patrice. Acquired and intrinsic glycopeptide resistance in enterococci.International Journal of Antimicrobial Agents,2000, 16: S11-S17
3 Somma S,Gastaldo L,Corti A.Teicoplanin,a new antibiotic from Actinoplanes teichomyceticus nov.Sp.Antimicrobiall Agents & Chemotherapy,1984,26(6):917- 923
1 中国药典委员会.中华人民共和国药典 2005 年版,化学工业出版社.
2 夏振强,古勇,官家发.糖肽类抗生素替考拉宁研究进展[J].天然产物研究与开发.2005,17(2):247-252.
3 杨红,秦川萍.HPLC 法测定替考拉宁组分和有关物质[J].中国抗生素杂志.2006,31(3):184-192.
1 McCormick M H,Mcguire J M,Pittenger G E,et al.Vancomycin,a new antibiotic,Chemical and biologic,properties [J].Antibiot Annu,1955-1956,3:606
2 Smith T L,Pearson M L,Wilcox K R,et al.Emergence of vancomycin resistance in Staphylococcus aureus, glycopeptide-intermediate Staphylococcus aureus[J].New Eng J Med,1999,340(7):493
3 俞文和,杨红根.抗生素生产工艺学[M].第 1 版,沈阳辽宁科学技术出版社,1988:305
4 皮博睿,俞云松.万古霉素不敏感革兰阳性菌感研究进展中国处方药,2006,9(54):10-13
5 夏振强,古勇,官家发.糖肽类抗生素替考拉宁研究进展[J].天然产物研究与开发.2005,17(2):247-252
6 Parenti F Structure and mechanism of action of teicoplanin[J]. HospInfect,1986,7:79
7 李扬,傅得兴,糖肽类抗生素-替考拉宁的药理及临床评价. 中国医药导刊,2000,2(4):51-52
8 纪淳安.替考拉宁的抗菌活性和临床应用.国外医药抗生素分册,2001,22(3):122-124
9 Sloos JH, Van-de-Klundert JA, Dijkshoorn L, et al.Changing susceptibilities of coagusase-negative staphylococci to teicoplanin in a teaching hospital. J Antimicrob Chemother, 1998,42(6):787-79
10 吴晓平,马富艳,华春珍.金黄色葡萄球菌对万古霉素耐药性 的 研 究 进 展 [J]. 杭 州 师 范 学 院 学 报 ( 医 学版),2006,1,47-50
11 尚新艳,阮丽军,许激扬,陈代杰.第二代糖肽类抗生素的研究进展.中国抗生素杂志,2007,5(32):263-267
12 Gu Z I,Bian C F,Zhang Y D,et al.Medical pharmcology[M]. Beijing:People,s Publish House,1998,144-150
13 Armstrong D W,Tang Y B,Chen s s,Zhou Y W,Bagwill C,Chen J R.Anal chem,1994,66(9):1473
14 F.Huil,K.H.Ekborg-Ott,D.W.Armstrong.High-performance liquid chromatographic and capillary elec-trophoretic enantioseparation of plant growth regulators and related indole compounds using macrocyclic antibi-otics as chiral selectors.Journal of Chromatography A,7001,906:91-103
15 丁国生,唐安娜,王俊德,丛润滋,包建民. 替考拉宁键合手性固定相高效液相色谱法直接拆分泮托拉唑钠对映体.色谱,2006,24(1):46-48
16 ChenY,Wang SY,Hu GY.Determination of teicoplanin in injection by bioassay. Chinese Pharmaceutical Journal,2002. 4,37(4):30-304
17 付靖,刘菘,暴娜,郑煜.高效液相色谱法检测替考拉宁组分及其有关物质.河北化工,2006,29(8):62-63
2 Gholizadeh Y, Couralin Patrice. Acquired and intrinsic glycopeptide resistance in enterococci.International Journal of Antimicrobial Agents,2000, 16: S11-S17
3 Somma S,Gastaldo L,Corti A.Teicoplanin,a new antibiotic from Actinoplanes teichomyceticus nov.Sp.Antimicrobiall Agents & Chemotherapy,1984,26(6):917- 923
1 中国药典委员会.中华人民共和国药典 2005 年版,化学工业出版社.
2 夏振强,古勇,官家发.糖肽类抗生素替考拉宁研究进展[J].天然产物研究与开发.2005,17(2):247-252.
3 杨红,秦川萍.HPLC 法测定替考拉宁组分和有关物质[J].中国抗生素杂志.2006,31(3):184-192.
1 McCormick M H,Mcguire J M,Pittenger G E,et al.Vancomycin,a new antibiotic,Chemical and biologic,properties [J].Antibiot Annu,1955-1956,3:606
2 Smith T L,Pearson M L,Wilcox K R,et al.Emergence of vancomycin resistance in Staphylococcus aureus, glycopeptide-intermediate Staphylococcus aureus[J].New Eng J Med,1999,340(7):493
3 俞文和,杨红根.抗生素生产工艺学[M].第 1 版,沈阳辽宁科学技术出版社,1988:305
4 皮博睿,俞云松.万古霉素不敏感革兰阳性菌感研究进展中国处方药,2006,9(54):10-13
5 夏振强,古勇,官家发.糖肽类抗生素替考拉宁研究进展[J].天然产物研究与开发.2005,17(2):247-252
6 Parenti F Structure and mechanism of action of teicoplanin[J]. HospInfect,1986,7:79
7 李扬,傅得兴,糖肽类抗生素-替考拉宁的药理及临床评价. 中国医药导刊,2000,2(4):51-52
8 纪淳安.替考拉宁的抗菌活性和临床应用.国外医药抗生素分册,2001,22(3):122-124
9 Sloos JH, Van-de-Klundert JA, Dijkshoorn L, et al.Changing susceptibilities of coagusase-negative staphylococci to teicoplanin in a teaching hospital. J Antimicrob Chemother, 1998,42(6):787-79
10 吴晓平,马富艳,华春珍.金黄色葡萄球菌对万古霉素耐药性 的 研 究 进 展 [J]. 杭 州 师 范 学 院 学 报 ( 医 学版),2006,1,47-50
11 尚新艳,阮丽军,许激扬,陈代杰.第二代糖肽类抗生素的研究进展.中国抗生素杂志,2007,5(32):263-267
12 Gu Z I,Bian C F,Zhang Y D,et al.Medical pharmcology[M]. Beijing:People,s Publish House,1998,144-150
13 Armstrong D W,Tang Y B,Chen s s,Zhou Y W,Bagwill C,Chen J R.Anal chem,1994,66(9):1473
14 F.Huil,K.H.Ekborg-Ott,D.W.Armstrong.High-performance liquid chromatographic and capillary elec-trophoretic enantioseparation of plant growth regulators and related indole compounds using macrocyclic antibi-otics as chiral selectors.Journal of Chromatography A,7001,906:91-103
15 丁国生,唐安娜,王俊德,丛润滋,包建民. 替考拉宁键合手性固定相高效液相色谱法直接拆分泮托拉唑钠对映体.色谱,2006,24(1):46-48
16 ChenY,Wang SY,Hu GY.Determination of teicoplanin in injection by bioassay. Chinese Pharmaceutical Journal,2002. 4,37(4):30-304
17 付靖,刘菘,暴娜,郑煜.高效液相色谱法检测替考拉宁组分及其有关物质.河北化工,2006,29(8):62-63