氨基糖苷类抗生素分析氨基糖苷类抗生素分析药学院药物分析教研室药学院药物分析教研室【大纲要求大纲要求】1 1、掌握氨基糖苷类抗生素代表药物的体内、掌握氨基糖苷类抗生素代表药物的体内过程过程2 2、熟悉代表药物的体内分析方法、熟悉代表药物的体内分析方法SMGM主主要要代代表表药药物物SisMAK治疗范围窄治疗范围窄 620mol/l 毒副作用较强，血药浓度高于毒副作用较强，血药浓度高于30mol /L 耳毒性和肾毒性耳毒性和肾毒性T1/2较长，动物食品残留检测较长，动物食品残留检测主主要要药药动动学学特特点点Process in vivoT1/2 与身体健康状况密切相关与身体健康状况密切相关Concentration 血药浓度较低、几乎完全血药浓度较低、几乎完全从肾脏排泄从肾脏排泄Tissue distribution 主要分布于细胞外液、主要分布于细胞外液、可通过胎盘屏障，不易通过血脑屏障可通过胎盘屏障，不易通过血脑屏障CharacteristicNitrogen atom Alkali-OH group water soluble polarityNon-chromaphore for UV detection旋光特性含有多个氨基糖，一般为右旋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结合的苷键可发生水解Analytical methodsImmune analysis IAUV or fluorescence analysisChromatography analysisLC/MS analysisHigh-performance liquid chromatographyOn-line separation and detectionPre/Post-column derivationHPLC-MS/MSPreparation of the biological sampleDerivationAnalytical conditions条件摸索条件摸索Protein precipitation PPCH3CN CH3OH HClO4Liquid-liquid extraction LLE衍生化后进行衍生化后进行Solid-phase extraction SPENormal-phase or ion-exchangeSolid-phase micro-extraction SPME衍生化后进行衍生化后进行Column switchingChemical DerivationUV derivation reagentFluorescence derivation reagent邻苯二醛邻苯二醛 OPAOPA丹酰氯丹酰氯 DNS-DNS-ClCl茚三酮茚三酮2,4,6-2,4,6-三硝基苯磺酸三硝基苯磺酸 TNBSTNBS1-1-氟氟-2,4-2,4-二硝基苯二硝基苯 FDNBFDNBOptimization suitable derivation conditions Reaction solvent and pH value Methanol or ethanol 含醇量含醇量5050为好为好 pH=pH=8.510.5Concentration of derivation reagent对对OPA ：0.1 g/ml；其他则无影响；其他则无影响 Reaction time and temperature20、30、6060、40、10 min。OPA-MP在在30和和60时可产生干扰的荧光物质，需用时可产生干扰的荧光物质，需用CHCl3等提取后在测定等提取后在测定OPA-MP RP-HPLC ChromatogramPre-column derivationSis MDetermination of GM using post-column derivation by RP-HPLC in plasmaAnalytical conditions:Analytical column: Bondapak C18Mobile phase : CH3CN-H2O Flow rate:1.5 ml/min Derivation reagent : OPA Flow rate of derivation reagent : 0.8 ml/minTemperature: 50 Detection: fluorescenceOptimization suitable conditionsProtein precipitation methanol 10000 rpmSample preparationAccuracy and precisionRecovery Linear range Limit detection LOQ LODEvaluated data Derivation reagentMobile phaseInjection valueChromatographic columnReactorDetectorRecorderWastePumpWorkstation or softwareThree channelDetermination of GM in plasma and urine by RP-HPLC using solid column derivation techniqueAnalytical conditionsColumn: Lichrosorb RP-18 (12.54 mm, I.D., 5 m)Mobile-phase: Methanol-10%HAcFlow rate: 1.0 ml/minDetection: excitation wavelength 340 nm, emission wavelength 418 nmSample solutionIS addedComplex solutionSilica gel column chromatographyEthanol elution solutionOPA elution solutionHPLC analysisSPEISGM-C1GM-C2分析方法的特异性高分析方法的特异性高Determination of KM in serum and urine by HPLC using pre-column derivation methodColumn: 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: Sample preparation: 取血清取血清0.5 ml0.5 ml，加，加磷酸盐缓冲液磷酸盐缓冲液0.5 ml0.5 ml和乙腈和乙腈2 ml2 ml，窝旋混合，窝旋混合10 s10 s，高速离心（，高速离心（3000 rpm3000 rpm）15 min15 min，取上，取上清清0.5 ml 0.5 ml 于另乙试管中，加入于另乙试管中，加入OPA 0.25 mlOPA 0.25 ml，再混合，再混合10 s10 s，60 60 水浴保持水浴保持15 min15 min，迅速，迅速冷却，取冷却，取20l20l进行进行HPLCHPLC分析。分析。方法学考察结果：方法学考察结果：回收率回收率93.493.4104.8104.8日内精密度日内精密度3.23.23.83.8日间精密度日间精密度4.24.25.45.4检测限检测限4 440g/ml40g/ml【复习题复习题】1 1、简述氨基糖苷类抗生素的结构及体内、简述氨基糖苷类抗生素的结构及体内过程特点。过程特点。2 2、简述氨基糖苷类抗生素的体内药物分、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？析常用到的分析技术？