Download Document

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