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Clinical Applications of LC-MS/MS Brig Dilshad Ahmed Khan MBBS, MCPS, FCPS, MS, FRCP, Ph.D Professor of Pathology Armed Forces Institute of Pathology MS Principles • Different elements can be uniquely identified by their mass Mass spectrometry +CH 3 CH3COCH3 CH3+COCH3 +COH CH3C+OCH3 +COCH 3 Sample Inlet Ionization & adsorption of excess energy Fragmentation (Dissociation) Mass analysis Detection 3 What is LC MS/MS ? • Uses one LC and 2 mass analyzers in a single instrument • One MS purifies the analyte ion from a mixture using a magnetic field • The other analyzes fragments of the analyte ion for identification and quantification Mixture of ions Ion source Single ion MS-1 Fragments MS-2 4 MS vs. MS/MS GC HPLC Inlet Mass Analyze Ionize MS Separation Inlet Ionize Detect Identification Mass Analyze Fragment Mass Analyze MS1 Collision Cell MS2 MS/MS Detect 5 LC-MS/MS Components of tandem mass spectrometer Ionization source ESI APPI APCI Mass spectrometer Collision Cell Quatrupole magnetic sector Argon Xenon MS1 Collision cell Mass Spectrometer Quatrupole magnetic sector MS2 Detector Sample preparation Online vs Offline LC often obviates need for derivatization LC compatible with on-line extraction LC compatible with direct injection 8 Ion sources make ions from sample molecules Partial vacuum Sample inlet nozzle (Lower voltage) Electrospray ionization: MH+ N2 Sample in solution N2 gas ++ + + ++ + + + + ++ ++ + + ++ + ++ + ++ + ++ + ++ + ++ + + + + + + + ++ + + + MH2+ + MH3+ High voltage applied to metal sheath (~4 kV) Charged droplets 9 Analytical quadrupole Quadrupole MS/MS with triple quad 12 Ion trap 13 Operation modes Precursor Ion Scanning • Analyzes charged precursors molecule Product Ion Scanning • Analyzes all products of a single precursor Multiple Reaction Monitoring • Analyzes for specific precursors producing specific products Precursor ion scanning MS1 Collision cell MS2 Argon gas Product Precursors Scanning Static 1st quadrupole mass analyzer : Scanning a mass range 2nd quadrupole : fixed, or Static, at the mass-to-charge ratio (m/z) of a product ion known to be common to the analytes in a mixture. Collision induced dissociation Argon gas O CH3 CH2 CH3 O CH3 CH2 O CH3 H3C O Precursor ion • CH3 Product ions Collision conditions (fragmentation) are controlled by altering: The collision energy (eV) 16 Product ion scanning 100 5eV % 0 100 10 eV % 0 100 20 eV % 0 100 30 eV % 0 100 40 eV % 0 20 40 60 80 100 120 140 160 180 200 collision energy > fragmentation 220 m/z Mass spectrum: progesterone Full scan acquisition mode [M+H]+ O 315.1 100 CH3 CH3 CH3 O % 316.1 0 200 220 240 260 280 300 320 340 360 380 m/z 400 Product Ion spectrum: progesterone Product ion scanning O 100 CH3 CH3 315.1 Mass spectrum from MS1 CH3 % 316.1 O Precursor ion 0 300 305 310 O CH2 97.0 100 109.0 315 320 325 m/z 330 CH3 CH2 O H3C CH3 % Product ions Product ion spectrum from MS2 0 100 125 150 175 200 225 250 275 300 19m/z 325 Product ion scanning MS1 Collision cell MS2 Argon gas Precursors Static(m/z 315.1) Product Scanning The first quadrupole mass analyzer is fixed at the mass-to-charge ratio (m/z) of the precursor ion to be interrogated while the second quadrupole is scanning over a user-defined mass range. Applications of LC/MS • Forensic toxicology / Drugs of abuse • Clinical toxicology/TDM • Clinical endocrinology • Clinical chemistry • Inborn errors of metabolism 21 Drugs screening/ confirmation • Broad spectrum drug screens acquires full scan spectra • Identification based on accurate mass • Accurate fragments mass analysis are important Mass accuracy = measured-theory theory Resolution (FWHM) = M Mass s Δmass 22 Direct analysis of opiates 23 LC/MS/MS 24 Therapeutic Drug Monitoring Immunosuppressants • Avoid organ rejection after transplants • Extremely narrow therapeutic window • Have to be taken & monitored lifelong 25 Examples & structures Tacrolimus CSA Sirolimus Cyclosporin A (CyA) Tacrolimus (TRL) Sirolimus (SRL) 26 Cyclosporine measurement Ciclosporin (µg/L) 500 400 300 H P LC H -U P LC V -M S R A IA xS Y M TD x E M C IT E D IA A + C M IA 200 27 400µg/L spike – International Cyclosporin PT Scheme Cyclosporine Measurement 28 Clinical Endocrinology: Thyroid assay Thyroid hormone testing is well served by immunoassays There needs to be a good correlation between FT4 and TSH LC/MS/MS for FT4 /T3 hormones? 29 NIH study 2010 n=109, Siemens Immulite 2500 • Inverse log-linear relationship: Immune assay-FT4 and TSH r = 0.45 (95% CI 0.29 - 0.59) 30 Handrick et al. Clin Chem 2011; 57:122-7. FT4/FT3 by LC/MS/MS NIH study 2010 n=109 Siemens Immulite 2500 • Inverse log-linear relationship: LCMS/MS-FT4 and log TSH • r = 0.84 (95% CI 0.77- 0.88). 32 Handrick et al. Clin Chem. 2011; 57:122-7. Thyroid testing • FT4/FT3 LCMS/MS method performed excellently and provide results which correlate well with TSH • T4 testing by immunoassay appears to be reliable except during pregnancy? • We recommended performing a protein separation step using UF at 37oC for FT4/FT3 testing. Quantification can then be performed employing either IA or MS/MS • Potential to report thyroid profiles e.g. T4, T3, rT3, 3,5-T2,3- 33 Steroid • Steroid measurement by tandem mass spectrometry is reliable, fast and relatively inexpensive • Profile testing permits a deeper insight into endocrine disorders allowing improved patient diagnosis and management • Teaching hospital laboratories can provide for reliable measurement of 5-12 steroids with single good MS/MS 34 35 Analyte IE (CV) LCMS/MS (CV) Corticosterone 10% 9% DHEAS 9% 4% 11-Deoxcortisol 16% 7% Aldosterone 12% 6-9% 25-Hydroxyvitamins D3 12% 6% DHEA 12% 6% Progesterone 12% 3-9% Vitamin D Determination by LC/MS/MS • Humans make vitamin D3 in the skin • Plants and yeast make vitamin D2 Measurement of Vitamin D 2 forms of 25Hydroxyvitamin need to be measured to obtain total 25OHD concentration D2 D3 LC/MS/MS simultaneous measures 25OHD2 and 25OHD3 LC/MS/MS standard curves 6 point standard curve: (128, 64, 62, 16, 8, 4 ng/mL) 25OHD LC/MS/Ms validation Reproducibility (%) Low (20-25 ng/mL) Medium (45-55 ng/mL) High (95-105 ng/mL) Intra-assay 25OHD2 25OHD3 9.0 11.2 6.9 7.5 8.1 8.5 Intra-assay 25OHD2 25OHD3 9.7 13.5 11.6 10.7 8.8 8.4 40 25OHD LC/MS/MS validation Sensitivity (limit of quantification) LC/MS/MS vs EIA 42 Roth HJ, et al. Ann Clin Biochem. 2008:45;153-159. Proteomics in human diseases 43 Newborn Screening with LC-MS/MS Organic acidemias Fatty acid oxidation def Urea cycle defects Amino acidurias 44 Inborn errors of metabolism 45 Amino acid profile (normal) 100 Pro INTERNE STANDARDS % Intensity Ala Phe 50 Leu + Ile Phe Leu Gln Val Ala Gly Gly 140 Ser Val 160 180 Tyr Tyr Cit Met 200 220 m/z, amu Glu Asp Glu 240 260 280 300 46 Phenylketonuria (PKU) Phe 100 % Intensity Pro INTERNAL STANDARDS Ala 50 Phe Leu + Ile Gly Gly 140 Gln Val Ala Ser 160 Val 180 Leu Cit Met 200 220 m/z, amu Tyr Glu Asp Glu Tyr 240 260 280 300 47 Maple syrup urine disease (MSUD) Amino Acid Profile from a Filter Paper Blood Spot: MSUD Leu + Ile 100 Pro % Intensity INTERNAL STANDARDS 50 Ala Val Ala Gly Gly 140 Ser Phe Leu Phe Val Met 160 180 200 220 m/z, amu m/z, amu Glu Tyr Tyr Asp 240 Glu 260 280 300 48 Summary • LC /MS/MS is the exciting instrument for clinical lab • Better sensitivity and specificity • Much less expensive then extraction assays • Multiplex – excellent efficiency • LC /MS/MS may replaces a few immunoassay in the reference clinical labs i.e vitamin D , drugs , hormones ? 49 attention Thanks