氨基糖苷類抗生素分析

1、氨基糖苷類抗生素分析氨基糖苷類抗生素分析藥學院藥物分析教研室藥學院藥物分析教研室【大綱要求大綱要求】1 1、掌握氨基糖苷類抗生素代表藥物的體內(nèi)、掌握氨基糖苷類抗生素代表藥物的體內(nèi)過程過程2 2、熟悉代表藥物的體內(nèi)分析方法、熟悉代表藥物的體內(nèi)分析方法OOOOHOHNNHNH2HNHNNH2OHOHCH3OHCHONHH3COHOHCH2OHOONHR3R2R1NH2HOH2NOOHOHNCH3OHCH3NH2SMGM主要代表藥主要代表藥物物OOH2NH2CNH2H2NHONH2OOHONHCH3OHCH3OOHNH2HOHOH2COOHNH2OOOHOHOHCH2NH2HNOOHH2NHSisM

2、AK治療范圍窄治療范圍窄 620mol/l 毒副作用較強，血藥濃度高于毒副作用較強，血藥濃度高于30mol /L 耳毒性和腎毒性耳毒性和腎毒性T1/2較長，動物食品殘留檢測較長，動物食品殘留檢測主要藥動學特點主要藥動學特點Process in vivoT1/2 與身體健康狀況密切相關(guān)與身體健康狀況密切相關(guān)Concentration 血藥濃度較低、幾乎完全血藥濃度較低、幾乎完全從腎臟排泄從腎臟排泄Tissue distribution 主要分布于細胞外液、主要分布于細胞外液、可通過胎盤屏障，不易通過血腦屏障可通過胎盤屏障，不易通過血腦屏障CharacteristicNitrogen atom A

3、lkali-OH group water soluble polarityNon-chromaphore for UV detection旋光特性含有多個氨基糖，一般為右旋旋光特性含有多個氨基糖，一般為右旋結(jié)合的苷鍵可發(fā)生水解結(jié)合的苷鍵可發(fā)生水解Analytical methodsImmune analysis IAUV or fluorescence analysisChromatography analysisLC/MS analysisHigh-performance liquid chromatographyOn-line separation and detectionPre/Pos

4、t-column derivationHPLC-MS/MSPreparation of the biological sampleDerivationAnalytical conditions條件摸索條件摸索Protein precipitation PPCH3CN CH3OH HClO4Liquid-liquid extraction LLE衍生化后進行衍生化后進行Solid-phase extraction SPENormal-phase or ion-exchangeSolid-phase micro-extraction SPME衍生化后進行衍生化后進行Column switching

5、Chemical DerivationUV derivation reagentFluorescence derivation reagent鄰苯二醛鄰苯二醛 OPAOPA丹酰氯丹酰氯 DNS-ClDNS-Cl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為好為好 pH=pH=8.510.5Co

6、ncentration of derivation reagent對對OPA ：0.1 g/ml；其他則無影響；其他則無影響 Reaction time and temperature20、30、6060、40、10 min。OPA-MP在在30和和60時可產(chǎn)生干擾的熒光物質(zhì)，需用時可產(chǎn)生干擾的熒光物質(zhì)，需用CHCl3等提取后在測定等提取后在測定OPA-MP RP-HPLC ChromatogramPre-column derivationSis MDetermination of GM using post-column derivation by RP-HPLC in plasmaAnal

7、ytical 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 preparationAccurac

8、y and precisionRecovery Linear range Limit detection LOQ LODEvaluated data %100(%)covReAddedknownTotaleryDerivation reagentMobile phaseInjection valueChromatographic columnReactorDetectorRecorderWastePumpWorkstation or softwareThree channelDetermination of GM in plasma and urine by RP-HPLC using sol

9、id 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

10、 solutionOPA elution solutionHPLC analysisSPEISGM-C1GM-C2分析方法的特異性高分析方法的特異性高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 Emiss

11、ion wavelength-425 nm Sample preparation: Sample preparation: 取血清取血清0.5 ml0.5 ml，加，加磷酸鹽緩沖液磷酸鹽緩沖液0.5 ml0.5 ml和乙腈和乙腈2 ml2 ml，窩旋混合，窩旋混合10 s10 s，高速離心（，高速離心（3000 rpm3000 rpm）15 min15 min，取上，取上清清0.5 ml 0.5 ml 于另乙試管中，加入于另乙試管中，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，迅速冷，迅速冷卻，取卻，取20l20l進行進行HPLCHPLC分析。分析。方法學考察結(jié)果：方法學考察結(jié)果：回收率回收率93.493.4104.8104.8日內(nèi)精密度日內(nèi)精密度3.23.23.8