液相層析質譜分析液相層析質譜分析 LC-MS Method development LC-MS Method development

and Analyte Identificationand Analyte Identification

授課教師：賴滄海教授授課教師：賴滄海教授03-12-200803-12-2008

IonizationIonization

ESIESI Sonic Spray ISonic Spray I APCIAPCI APPIAPPI APLIAPLI

Ion SeparationIon Separation

Mass rangeMass range Scan speed Scan speed Mass resolutionMass resolution Mass accuracyMass accuracy

Mass AccuracyMass Accuracy

An unknown compound appears aAn unknown compound appears at m/z 180.094. in the positive ion t m/z 180.094. in the positive ion mode.mode.

The mass accuracy of the spectroThe mass accuracy of the spectrometer is 2-5 ppm.meter is 2-5 ppm.

Assuming it contains C.H.O.N.P.S. F.Cl.BrAssuming it contains C.H.O.N.P.S. F.Cl.Br

Mass AnalyzerMass Analyzer QuadrupoleQuadrupole Ion trapIon trap Linear ion TrapLinear ion Trap Time of flightTime of flight

Steps in the quantification of drugsSteps in the quantification of drugs

SamplingSampling Sample preparationSample preparation Separation Separation DetectionDetection Data analysisData analysis

General ConsiderationsGeneral Considerations

What to measure ( drug/metabolites)What to measure ( drug/metabolites) Select matrix (blood,serum,plasma,tissuSelect matrix (blood,serum,plasma,tissu

e)e) Sample limitations (volume,sample size)Sample limitations (volume,sample size) Linear rangeLinear range Lower limit of quantificationLower limit of quantification Internal standardInternal standard

Problems with Deuterated IsProblems with Deuterated Is

[[1313C,DC,D33] rofecoxib exchange with ] rofecoxib exchange with

Hydrogen atoms on water molecularHydrogen atoms on water molecular

[J Chromatogr B 2002;767:117-129][J Chromatogr B 2002;767:117-129]

Positive identification with GC-MS(SIM)Positive identification with GC-MS(SIM)

1.1. 3 ions3 ions

2.2. Retention time the same as standardsRetention time the same as standards

3.3. Ion ratio Ion ratio correctcorrect4.4. S/N > 3S/N > 3

5.5. Sample processed to remove interferencesSample processed to remove interferences

Aspects to be considered when setting up Aspects to be considered when setting up LC-MS proceduresLC-MS procedures

1.1. Influence of the mobile phaseInfluence of the mobile phase

2.2. Influence of sodium and potassium on the Influence of sodium and potassium on the formation of adductsformation of adducts

3.3. Influence of the curtain gas flow Influence of the curtain gas flow

4.4. Reproducibility of mass spectraReproducibility of mass spectra

5.5. Influence of ion suppressionInfluence of ion suppression

6.6. GC-MS data to verify LC-MS resultsGC-MS data to verify LC-MS results

Mass spectrometryMass spectrometry

IonizationIonization Matrix effectMatrix effect Full scanFull scan MRMMRM

Figure 2.5 The influence of collision energy on the fragmentation of tacrolimus at (a)10, (b)20,(c)30,(d)40 and (e)50eV.

Sample preparationSample preparation

Sample dilution and protein Sample dilution and protein precipitationprecipitation

Liquid-liquid extractionLiquid-liquid extraction Solid phase extractionSolid phase extraction Two dimensional chromatography Two dimensional chromatography

(column switching)(column switching)