Brig Dilshad Ahmed Khan MBBS MCPS FCPS MS FRCP PhD Professor of Pathology Armed Forces Institute of Pathology MS Principles ID: 775317
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Slide1
Clinical Applications of LC-MS/MS
Brig Dilshad Ahmed KhanMBBS, MCPS, FCPS, MS, FRCP, Ph.D Professor of Pathology Armed Forces Institute of Pathology
Slide2MS Principles
Different elements can be uniquely identified by their mass
Slide3Mass spectrometry
CH
3
COCH3
Sample Inlet
CH3+COCH3
Ionization
& adsorption of excess energy
Mass
analysis
CH
3
C
+
OCH
3
+
COCH
3
+
CH
3
+
COH
Fragmentation
(Dissociation)
Detection
3
Slide4What is LC MS/MS ?
Uses one LC and 2 mass analyzers in a single instrumentOne MS purifies the analyte ion from a mixture using a magnetic fieldThe other analyzes fragments of the analyte ion for identification and quantification
Ion source
MS-2
MS-1
Mixture of ions
Single ion
Fragments
4
Slide5MS vs. MS/MS
Inlet
Detect
Mass
Analyze
Ionize
MS
Inlet
Fragment
Mass Analyze
Ionize
Mass
Analyze
Detect
MS1
Collision
Cell
MS2
MS/MS
GC
HPLC
Separation
Identification
5
Slide6LC-MS/MS
Slide7Components of tandem mass spectrometer
Collision
Cell
Mass
s
pectrometer
Mass
Spectrometer
Detector
Ionization
source
ESI
APPIAPCI
ArgonXenon
Quatrupolemagnetic sector
Quatrupole
magnetic sector
Collision cell
MS1
MS2
Slide8Sample preparation
8
Slide9Ion sources make ions from sample molecules
High voltage applied
to metal sheath (~4 kV)
Sample
inlet
n
ozzle
(Lower
voltage
)
Charged droplets
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+
+
MH
+
MH
3
+
MH
2
+
Sample in solution
N
2
N
2
gas
Partial
vacuum
Electrospray ionization:
9
Slide10Analytical quadrupole
Slide11Quadrupole
Slide12MS/MS with triple quad
12
Slide13Ion trap
13
Slide14Operation modes
Slide15Static
Scanning
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.
Argon gas
Precursors
Product
Collision cell
MS1
MS2
Precursor ion scanning
Slide16Collision induced dissociation
Argon gas
Precursor ion
Product ions
Collision conditions
(fragmentation)
are controlled by altering:
The collision energy (
eV
)
16
Slide175eV
10
eV
30
eV
40 eV
20 eV
collision energy > fragmentation
Product ion scanning
Slide18[
M+H]
+
Mass
spectrum: progesterone
Full
s
can
a
cquisition
m
ode
Slide1919
Mass
spectrum
from MS1
Product ion spectrum from MS2
Product ions
Precursor
ion
Product Ion spectrum: progesterone
Product ion
scanning
Slide20Static(m/z 315.1)
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.
Argon gas
Precursors
Product
Collision cell
MS1
MS2
Product
ion scanning
Slide21Applications of LC/MS
Forensic toxicology / Drugs of abuseClinical toxicology/TDMClinical endocrinologyClinical chemistry Inborn errors of metabolism
21
Slide22Drugs screening/ confirmation
Broad spectrum drug screens acquires full scan spectraIdentification based on accurate massAccurate fragments mass analysis are important
Mass accuracy =
measured-theory theory
Resolution (FWHM) =
M
Mass s Δmass
22
Slide23Direct analysis of opiates
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Slide24LC/MS/MS
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Slide25Therapeutic Drug Monitoring
25
Slide26CSA
Tacrolimus
Sirolimus
Examples &
structures
Cyclosporin A (CyA)
Tacrolimus (TRL)Sirolimus (SRL)
26
Slide27Cyclosporine measurement
400µg/L spike – International
Cyclosporin PT Scheme
27
Slide28Cyclosporine Measurement
28
Slide29Clinical Endocrinology: Thyroid assay
Thyroid hormone testing is well served by immunoassays There needs to be a good correlation between FT4 and TSHLC/MS/MS for FT4 /T3 hormones?
29
Slide30NIH 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)
Handrick
et al. Clin Chem 2011; 57:122-7.
30
Slide31FT4/FT3 by LC/MS/MS
Slide32NIH 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).
Handrick
et al. Clin Chem. 2011; 57:122-7.
32
Slide33Thyroid testing
FT4/FT3 LCMS/MS method performed excellently and provide results which correlate well with TSHT4 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
Slide34Steroid
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
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Slide3535
Slide36Analyte
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%
Slide37Humans make vitamin D3 in the skin Plants and yeast make vitamin D2
Vitamin D Determination by LC/MS/MS
Slide38Measurement of Vitamin D
Slide39LC/MS/MS standard curves
6 point standard curve: (128, 64, 62, 16, 8, 4 ng/mL)
Slide4025OHD LC/MS/Ms validation
Reproducibility (%)Low(20-25 ng/mL)Medium(45-55 ng/mL)High (95-105 ng/mL)Intra-assay 25OHD2 25OHD39.011.26.97.58.18.5Intra-assay 25OHD2 25OHD39.713.511.610.78.88.4
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Slide4125OHD LC/MS/MS validation
Sensitivity (limit of quantification)
Slide42LC/MS/MS vs EIA
Roth HJ, et al. Ann Clin Biochem. 2008:45;153-159.
42
Slide4343
Proteomics in human diseases
Slide44Newborn Screening with LC-MS/MS
44
Slide45Inborn errors of metabolism
45
Slide46Amino acid profile (normal)
140
160
180
200
220
240
260
280
300
m/z, amu
50
% Intensity
100
INTERNE STANDARDS
Gly
Ala
Val
Leu
Met
Cit
Phe
Tyr
Glu
Gly
Ala
Ser
Pro
Val
Leu + Ile
Gln
Tyr
Phe
Glu
Asp
46
Slide47Phenylketonuria (PKU)
140
160
180
200
220
240
260
280
300
m/z, amu
50
% Intensity
100
INTERNAL STANDARDS
Gly
Ala
Val
Leu
Met
Cit
Phe
Tyr
Glu
Gly
Ala
Ser
Pro
Val
Leu + Ile
Gln
Tyr
Phe
Glu
Asp
47
Slide48m/z, amu
INTERNAL STANDARDS
Gly
Ala
Val
Leu
Met
Phe
Tyr
Glu
Gly
Ala
Ser
Pro
Val
Leu + Ile
Tyr
Phe
Glu
Asp
Amino Acid Profile from a Filter Paper Blood Spot:
MSUD
140
160
180
200
220
240
260
280
300
m/z, amu
50
% Intensity
100
48
Maple syrup urine disease (MSUD)
Slide49Summary
LC /MS/MS is the exciting instrument for clinical lab Better sensitivity and specificity Much less expensive then extraction assaysMultiplex – excellent efficiencyLC /MS/MS may replaces a few immunoassay in the reference clinical labs i.e vitamin D , drugs , hormones ?
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Slide50attention
Thanks
Slide51