氨基糖苷類抗生素分析課件

1、氨基糖苷類抗生素分析藥學院藥物分析教研室【大綱要求】1、掌握氨基糖苷類抗生素代表藥物的體內(nèi)過程2、熟悉代表藥物的體內(nèi)分析方法SMGM主要代表藥物SisMAK治療范圍窄 620mol/l 毒副作用較強，血藥濃度高于30mol /L 耳毒性和腎毒性T1/2較長，動物食品殘留檢測主要藥動學特點Process in vivoT1/2 與身體健康狀況密切相關Concentration 血藥濃度較低、幾乎完全從腎臟排泄Tissue distribution 主要分布于細胞外液、可通過胎盤屏障，不易通過血腦屏障CharacteristicNitrogen atom Alkali-OH group water

2、 soluble polarityNon-chromaphore for UV detection旋光特性含有多個氨基糖，一般為右旋結合的苷鍵可發(fā)生水解Analytical methodsImmune analysis IAUV or fluorescence analysisChromatography analysisLC/MS analysisHigh-performance liquid chromatographyOn-line separation and detectionPre/Post-column derivationHPLC-MS/MSPreparation of the

3、 biological sampleDerivationAnalytical conditions條件摸索Protein precipitation PPCH3CN CH3OH HClO4Liquid-liquid extraction LLE衍生化后進行Solid-phase extraction SPENormal-phase or ion-exchangeSolid-phase micro-extraction SPME衍生化后進行Column switchingChemical DerivationUV derivation reagentFluorescence derivation

4、 reagent鄰苯二醛 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 FDNBOptimization suitable derivation conditions Reaction solvent and pH value Methanol or ethanol 含醇量50為好 pH=8.510.5Concentration of derivation reagent對OPA ：0.1 g/ml；其他則無影響Reaction time and temperature20、30、6060、40、10 min。OPA-MP在30和60時可產(chǎn)生干擾的

5、熒光物質(zhì)，需用CHCl3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 plasmaAnalytical conditions:Analytical column: Bondapak C18Mobile phase : CH3CN-H2O Flow rate:1.5 ml/min Derivation reagent : OPA Flow rate of derivation reagent : 0

6、.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 columnReactorDetect

7、orRecorderWastePumpWorkstation 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 n

8、m, emission wavelength 418 nmSample solutionIS addedComplex solutionSilica gel column chromatographyEthanol elution solutionOPA elution solutionHPLC analysisSPEISGM-C1GM-C2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 Emission wavelength-425 nm Sample preparation: 取血清0.5 ml，加磷酸鹽緩沖液0.5 ml和乙腈2 ml，窩旋混合10 s，高速離心（3000 rpm）15 min，取上清0.5 ml 于另乙試管中，加入OPA 0.25 ml，再混合10 s，6