氨基糖苷類(lèi)抗生素分析

1、氨基糖苷類(lèi)抗生素分析氨基糖苷類(lèi)抗生素分析藥學(xué)院藥物分析教研室藥學(xué)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(lèi)抗生素代表藥物的體內(nèi)、掌握氨基糖苷類(lèi)抗生素代表藥物的體內(nèi)過(guò)程過(guò)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(qiáng)，血藥濃度高于毒副作用較強(qiáng)，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(zhǎng)，動(dòng)物食品殘留檢測(cè)較長(zhǎng)，動(dòng)物食品殘留檢測(cè)主要藥動(dòng)學(xué)特點(diǎn)主要藥動(dòng)學(xué)特點(diǎn)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(xì)胞外液、主要分布于細(xì)胞外液、可通過(guò)胎盤(pán)屏障，不易通過(guò)血腦屏障可通過(guò)胎盤(pán)屏障，不易通過(guò)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(gè)氨基糖，一般為右旋旋光特性含有多個(gè)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(jìn)行衍生化后進(jìn)行Solid-phase extraction SPENormal-phase or ion-exchangeSolid-phase micro-extraction SPME衍生化后進(jìn)行衍生化后進(jìn)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(duì)對(duì)OPA ：0.1 g/ml；其他則無(wú)影響；其他則無(wú)影響 Reaction time and temperature20、30、6060、40、10 min。OPA-MP在在30和和60時(shí)可產(chǎn)生干擾的熒光物質(zhì)，需用時(shí)可產(chǎn)生干擾的熒光物質(zhì)，需用CHCl3等提取后在測(cè)定等提取后在測(cè)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(jìn)行進(jìn)行HPLCHPLC分析。分析。方法學(xué)考察結(jié)果：方法學(xué)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