氨基糖苷类抗生素分析分析解析.ppt

1、氨基糖苷类抗生素分析,药学院药物分析教研室,【大纲要求】,1、掌握氨基糖苷类抗生素代表药物的体内过程2、熟悉代表药物的体内分析方法,SM,GM,主要代表药物,SisM,AK,治疗范围窄 620mol/l,毒副作用较强，血药浓度高于30mol /L 耳毒性和肾毒性,T1/2较长，动物食品残留检测,主要药动学特点,Process in vivo,T1/2 与身体健康状况密切相关,Concentration 血药浓度较低、几乎完全从肾脏排泄,Tissue distribution 主要分布于细胞外液、可通过胎盘屏障，不易通过血脑屏障,Characteristic,Nitrogen atom Alka

2、li,-OH group water soluble polarity,Non-chromaphore for UV detection,旋光特性含有多个氨基糖，一般为右旋结合的苷键可发生水解,Analytical methods,Immune analysis IAUV or fluorescence analysisChromatography analysisLC/MS analysis,High-performance liquid chromatography,On-line separation and detection,Pre/Post-column derivation,HP

3、LC-MS/MS,Preparation of the biological sample,Derivation,Analytical conditions,条件摸索,Protein precipitation PPCH3CN CH3OH HClO4Liquid-liquid extraction LLE衍生化后进行Solid-phase extraction SPENormal-phase or ion-exchangeSolid-phase micro-extraction SPME衍生化后进行Column switching,Chemical Derivation,UV derivati

4、on reagent,Fluorescence derivation reagent,邻苯二醛 OPA丹酰氯 DNS-Cl茚三酮,2,4,6-三硝基苯磺酸 TNBS1-氟-2,4-二硝基苯 FDNB,Optimization suitable derivation conditions,Reaction solvent and pH value Methanol or ethanol 含醇量50为好 pH=8.510.5,Concentration of derivation reagent对OPA ：0.1 g/ml；其他则无影响,Reaction time and temperature2

5、0、30、6060、40、10 min。OPA-MP在30和60时可产生干扰的荧光物质，需用CHCl3等提取后在测定,OPA-MP RP-HPLC Chromatogram,Pre-column derivation,Sis M,Determination of GM using post-column derivation by RP-HPLC in plasma,Analytical conditions:Analytical column: Bondapak C18Mobile phase : CH3CN-H2O Flow rate:1.5 ml/min Derivation reage

6、nt : OPA Flow rate of derivation reagent : 0.8 ml/minTemperature: 50 Detection: fluorescence,Optimization suitable conditions,Protein precipitation methanol 10000 rpm,Sample preparation,Accuracy and precisionRecovery Linear range Limit detection LOQ LOD,Evaluated data,Derivation reagent,Mobile phase

7、,Injection value,Chromatographic column,Reactor,Detector,Recorder,Waste,Pump,Workstation or software,Three channel,Determination of GM in plasma and urine by RP-HPLC using solid column derivation technique,Analytical conditions,Column: Lichrosorb RP-18 (12.54 mm, I.D., 5 m),Mobile-phase: Methanol-10

8、%HAcFlow rate: 1.0 ml/minDetection: excitation wavelength 340 nm, emission wavelength 418 nm,Sample solution,IS added,Complex solution,Silica gel column chromatography,Ethanol elution solution,OPA elution solution,HPLC analysis,SPE,IS,GM-C1,GM-C2,分析方法的特异性高,Determination of KM in serum and urine by H

9、PLC using pre-column derivation method,Column: Zorbax ODS (1504.6 mm, I.D., 5 m)Mobile-phase: methanol-water=72: 28Flow rate: 1.0 ml/minDetector: FD Excitation wavelength-335 nm Emission wavelength-425 nm,Sample preparation: 取血清0.5 ml，加磷酸盐缓冲液0.5 ml和乙腈2 ml，窝旋混合10 s，高速离心（3000 rpm）15 min，取上清0.5 ml 于另乙试管中，加入OPA 0.25 ml，再混合10 s，60 水浴保持15 min，迅速冷却，取20l进行HPLC分析。,方法学考察结果：回收率93.4104.8日内精密度3.23.8日间精密度4.25.4检测限440g/ml,【复习题】,1、简述氨基糖苷类抗生素的结构及体内过程特点。2、简述氨基糖苷类抗生素的体内药物分析常用到的分析技术？,