氨基糖苷類抗生素分析分析解析

1、氨基糖苷類抗生素分析,藥學(xué)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,SM,GM,主要代表藥物,SisM,AK,◆,◆,◆,治療范圍窄 6～20μmol/l,毒副作用較強，血藥濃度高于30μmol /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(xué)特點,,Process in vivo,■,■,■,T1/2 與身體健康狀況密切相關(guān),Concentration

2、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,Characteristic,■,■,■,Nitrogen atom Alkali,-OH group water soluble polarity,Non-chromaphore for UV detection,,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,Analytical met

3、hods,Immune analysis IAUV or fluorescence analysisChromatography analysisLC/MS analysis,High-performance liquid chromatography,◆,On-line separation and detection,◆,Pre/Post-column derivation,◆,HPLC-MS/MS,Preparation

4、of the biological sample,Derivation,Analytical 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 mi

5、cro-extraction SPME衍生化后進行Column switching,Chemical Derivation,UV derivation reagent,Fluorescence derivation 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 FDNB,Optimization suitable derivation conditi

6、ons,Reaction solvent and pH value Methanol or ethanol 含醇量50％為好 pH=8.5~10.5,■,■,Concentration of derivation reagent對OPA ：0.1 μg/ml；其他則無影響,■,Reaction time and temperature20、30、60℃→60、40、10 min。OPA-β-MP在30和60℃時可產(chǎn)生干擾的熒光物質(zhì)

7、，需用CHCl3等提取后在測定,OPA-β-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: μBondapak C18Mobile phase : C

8、H3CN-H2O Flow rate:1.5 ml/min Derivation reagent : OPA Flow rate of derivation reagent : 0.8 ml/minTemperature: 50℃ Detection: fluorescence,,Optimization suitable conditions,Protein precipitation methanol 10000

9、rpm,,Sample preparation,Accuracy and precisionRecovery Linear range Limit detection LOQ LOD,,Evaluated data,,,,Derivation reagent,Mobile phase,Injection value,Chromatographic column,Reactor,Detector,Recorder,Waste,Pu

10、mp,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 (12.5×4 mm, I.D., 5 μm),Mobile-pha

11、se: Methanol-10%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 el

12、ution solution,,HPLC analysis,,SPE,IS,GM-C1,GM-C2,,,,分析方法的特異性高,Determination of KM in serum and urine by HPLC using pre-column derivation method,Column: Zorbax ODS (150×4.6 mm, I.D., 5 μm)Mobile-phase: methanol-wat

13、er=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