An introduction to the use of ICP-MS in the clinical setting, that goes on to describe some potential new application areas for advanced instrumentation such as HPLC-ICP-MS, laser ablation-ICP-MS and immuno-tagging-ICP-MS for the measurement of biomolecules.

1. 1
Advanced Clinical Applications of
Inductively Coupled Plasma Mass
Spectrometry (ICP-MS)
“Bring a Little Sunshine into Your Laboratory”
Dr Chris Harrington
SAS Trace Element Laboratory
Guildford

2. 2
Overview of Presentation
• Background to ICP-MS
• Comparison to ESI-MS
• Clinical Applications
– Nutritional elements
– Orthopaedic applications
– Toxic metals
• Advanced ICP-MS
– ICP-MS as a chromatography detector
• Complementary ESI and ICP
• High accuracy IDMS
• New application areas
– Biomolecule measurement
– Applications in histopathology
– Applications to flow-cytometry

3. 3
Atmospheric Mass Spectrometry
Electrospray (ESI-MS)
“Soft ionisation” -
molecular and multiply-
charged ions produced.
Inductively coupled plasma
(ICP-MS)
“Hard ionisation” - elemental
ions produced.

4. 4
Comparison of Inorganic and Molecular MS
• Elemental MS
• Strengths
– Provides low LODs (pmol/L).
– Multi-elemental.
– Can be used with most
chromatographies
• requires retention time
standards for identification.
– High accuracy analysis (ID-
MS) is possible.
• Weaknesses
– Requires authentic standards
for identification.
– Acquisition and reporting
software not as well
developed as LC-MS.
• Molecular MS
• Strengths
– Provides low LODs (pmol/L).
– Can be used with most
chromatographies
• does not require standards for
identification., high mass
accuracy.
– High accuracy analysis (ID-
MS) is possible for low m/z
species.
• Weaknesses
– Ion generation sensitive to
matrix components.
– Ultimate LOD not as low as
for ICP-MS.

5. 5
Inductively Coupled Plasma
ICP-OES Plasma Cross Section of Plasma
• The plasma temperature is between 7000 and 10 000 K
• Same temperature as the surface of sun.
• Highly charged gas.

6. 6
Sunshine in Action: An Argon Gas Plasma

7. 7
ICP-MS: Detection Limits

8. 8
• Nutritional elements.
• Orthopaedic applications.
• Heavy metal toxicity.
Current Clinical Applications

9. 9
Nutritional Elements
• 26 essential, or suggested, essential elements for humans.
• NICE (2006) Clinical Guideline 32: Nutritional Support in
Adults measurement of Cu, Zn (baseline, 2-4 wks) and Se
(baseline, as required).
• Home PN patients monitored for these plus Mn.

10. 10
Orthopaedic Applications
Interpretation of Co Levels
•< 2 g/L: close to normal.
•2 - 5 g/L: equivocal result.
•5 - 10 g/L: high sensitivity
and specificity for abnormal
wear process.
•10 - 20 g/L:100%
specificity for abnormal
wear patient at greatly
increased risk of ARMD
(adverse reaction to metal debris).
•> 20 g/L: macroscopic
damage in periprosthetic
tissues, likely osteolysis.
MHRA Guidelines 2010:
Levels of Co and Cr > 7 µg/L
require further investigation
including imaging.

11. 11
Lead use dates back to the Romans, but still
used. Monitoring required for CLAW.
Tetra-ethyllead now banned, but
may have left lasting scars crime!
Blood lead > 0.48 umol/L in children requires
intervention. CDC level half this (>0.24 umol/L).
Mostly lead in old paint
Toxic Elements: As, Cd, Hg, Pb, Tl

12. 12
Advanced Clinical Applications
• ICP-MS as a chromatography detector.
– Measurement of drug biomarkers eg DNA-
adducts showing effect of cisplatin
– Metallomics eg identification of Cu-protein
interactions in Wilsons Disease.
– High accuracy measurements with traceability for
large biomolecules.

13. ICP-MS
Excellent sensitivity
and selectivity
Multi-elemental/isotopic
Quantification
Versatility and easy
coupling
Complementary information
to molecular MS techniques
but easier to interpret.
ESI-MS
MALDI-TOF
FT-ICR
ICP-MS as a Detector for Chromatography

14. 14
Metal Speciation and Metallomics
• Definition:
• ..the qualitative identification and the
quantitative determination, of the individual
chemical forms that comprise the total
concentration of a given trace element in a
sample.
(NATO Workshop on Speciation, 1989).
• Significance:
• The toxicity of metal(loid)s.
• The biogeochemistry of metal(loid)s.
• The functionality of biometallic species.

15. 15
Clinical Biomarkers of Chemotherapy
• Cisplatin and oxaliplatin are used for the
treatment of many cancers.
• Cisplatin is linked with cellular resistance and
side-effects.
• Both drugs form intrastrand crosslinks
between:
– the N7 positions of two adjacent guanines (GG),
– adjacent adenine-guanines (AG),
– guanines separated by an intervening nucleotide
(GNG).

16. 16
Instrumentation for Speciation Analysis
(b)
(a)
(a) Pt-DNA adduct
measurement system.
(b) API-MS/MS for
characterisation of
metalloproteins and Pt-
standards

17. 17
DNA Sample Preparation
Oasis HLB
• Wash 1 mL MeOH
• Wash 1 mL H2O
• Apply sample in 1 mL
4% MeOH
• Wash column with 1 mL
5% MeOH
• Elute 1mL MeOH
DNAse 1, Nuclease P1, Shrimp
Alkaline Phosphatase
Digestion
8 hours, 37ºC
Solid phase
extraction
-ve ESI-MS/MS
HPLC-ICP-MS
dGpdG + dApdG + dN
CisPt
Dry down and resuspend in
HPLC eluent
dGpdG + dApdG
CisPtCisPt
CisPt
DNA (100µg)
Parameter Conditions
Sample DNA extracted from
human blood
Column ACE5 C8
250 x 4.6mm i.d., 5µm
Mobile phase 1 mM TEAA (85%),
methanol (15%) (v/v)
Flow rate 1.0 ml min-1
Injection 20 µl
Interface PEEK tubing (5cm),
Micromist nebuliser.
Detector Q-ICP-MS
-ve ESI-MS/MS
Ion (m/z) 195, 198 ICP
750 - 850 ESI

18. 18
Characterisation of Cisplatin Standard
0
5000
10000
15000
20000
25000
30000
35000
40000
0 2 4 6 8 10
Time (min.)
Response(cps)
m/z 195
m/z 198
HPLC-ICP-MS
F10 ~50pg/ul @ 5ul/min
m/z
822 823 824 825 826 827 828 829 830 831 832 833
%
0
100
AM_20110203_014_F10 14 (0.240) Cm (4:30) TOF MS ES+
6.13e3823.91
822.91
823.41
824.91
824.41
825.91
825.41
826.92
826.41
827.88
827.40
828.86
829.87
830.87 831.89
823
825
824
827
+ve ESI-ToF-MS
[M + H]+ m/z 824

19. 19
Method Validation Data
Parameter
Value
Limit of detection as Pt 110 pmol L-1, 90 ng L-1, 2 pg absolute
Limit of quantitation as Pt 365 pmol L-1, 300 ng L-1, 15 pg absolute
Repeatability 3.4 % (area), 2.3 % (peak height)
Long term precision 7 % (area), 6 % (peak height)
Accuracy 98 %
Sensitivity 6408 cps/ppb (area),
400 cps/ppb (height)
Mean correlation
coefficient (n=5)
0.99991 (area), 0.9843 (height)

20. 20
Results: Cell-lines
Non-small cell lung cancer cell-lines:
A549 cisplatin resistant.
H23 cisplatin sensitive.
Intrastrand adducts repaired by Nucleotide Excision Repair
Cell-Line Adducts
0.00
1.00
2.00
3.00
4.00
5.00
6.00
7h 12h 24h
Time
AdductConc
H23Sensitive
A549Resistant
3.13
3.29
2.56
TIC
2.73

21. 21
Results: Human Blood
237ndPatient 8
257ndPatient 7
252ndPatient 6
1245ndPatient 5
113ndPatient 4
214ndPatient 3
343ndPatient 2
146ndPatient 1
After
Treatment
(ng l-1)
Before Treatment
(ng l-1)
Identification Human blood sample (10 ml).
Taken 1 hr after drug infusion
Left for 7h prior to DNA
extraction.
GG-CP adduct conc 257 ng L-1.
AG-CP adduct below LOD.
3.13
3.29
2.73
TIC

22. 22
Isotopic Adduct Standard
ESI-MS scan of
enriched
standard.
ESI-MS scan
of natural
standard.
HPLC-ICP-MS analysis of isotopically
enriched cisplatin 198Pt.
This can be used for high accuracy
IDMS measurements.
m/z 195
m/z 198

23. 23
• Autosomal recessive disorder (1 : 30000 in UK).
• Defect in the gene encoding the ATP7B transporter.
• Leads to accumulation of Cu in liver and brain.
• Diagnosed by low serum Cu (<4umol/L), low Cp
(<0.25 g/L), raised Cu urine excretion, Kayser-
Fleischer rings, high liver Cu.
• Clinical presentation hepatic, neurological and
psychiatric.
• Fatal but treatable with Cu chelators.
• Diagnosis hampered by poor specificity Cp assay
lacking differentiation of apo and halo Cp forms.
• Free Cu calculations unreliable.
Cu-Proteins in Wilson’s Disease

24. 24
Cu-Proteins in Wilson’s Disease
Ceruloplasmin (CP)
Albumin (Alb)
• Overcomes
problems found
with current CP
assays.
• Apo-CP is not
measured.

25. 25
Metalloprotein Isotopic Enrichment
• Rusticyanin is a small (16 614 Da) copper containing
protein isolated from Acido-thiobacillus ferroxidans.
It was chosen for this study
because:
1) It is well characterised
(XAFS).
2) It has an established
amino acid sequence.
3) It can be generated in
large quantities by a cell
culture and protein
expression system.

26. 26
Standard Production
(a) The expression vector contains the lac promoter
and its neighbouring lacZ gene encoding b-
galactosidase.
(b) If lacZ is replaced by the gene encoding for Rc,
IPTG will stimulate the expression of Rc.

27. 27
HPLC-ICP-MS Analysis: Natural-Cu Rc
0
5000
10000
15000
20000
0 10 20 30
Time (min)
Response(cps)
Enriched-Cu Rc
•An enriched Cu isotope is
inserted into the protein.
•63Cu 0.4 atom % and 65Cu
99.6 atom%.
•This can be used for high
accuracy IDMS
measurements.
0
5000
10000
15000
20000
0 10 20 30
Time (min)
Response(cps) Natural-Cu Rc
•Shown in red is the
response for 63Cu and in
blue for 65Cu.
•These are in the ratio 2.2:1
reflecting the natural
abundance of Cu.
0
5000
10000
15000
20000
0 10 20 30
Time (min)
Response(cps)

28. 28
Why Not Organic IDMS?
• No difference was observed for the two proteins using
ESIMS. Tris pH 7.0 buffer.
•Would require an instrument with a resolution of 17000.
•By using elemental MS a quadrupole instrument with unit
mass resolution can be used.
• Natural-Cu Rc • Enriched-Cu Rc

29. 29
• High resolution spatial analysis using laser
ablation ICP-MS.
• Metal tagging methods to make molecules
visible to ICP-MS measurement.
• Flow-cytometry for the identification of cell-
types.
New Application Areas

30. 30
• Solid analysis with high spatial resolution.
• Uses:
• Analysis of electrophoresis gels.
• Analysis of metal localization in cells, nails,
hair, brain etc.
• Histopathological analysis of thin-sections
after staining with metal containing ligands.
Laser Ablation-ICP-MS

31. Quadrupole ICPMS
(Agilent 7500ce/cs)
Laser-Ablation
(RESOlution M-50
Excimer laser 193 nm (ns-pulses)
+
Laser Ablation-ICP-MS
Schematics of
‘Laurin’ two-volume cell

32. LA-ICPMS data: TOENAIL
164 µm, 3 Hz, ~4 J/cm2,
193 nm, ≤900 s depth-
profiling.
time [s]
Clear peaks in As and
Pb resolved by slow
drilling
Hair
44 m 74 m
Iceman
finger nail
• Calibration difficult.
• Involves pressing
disks from similar
materials.
• S can be used as
internal standard.

33. LA-ICP-MS in Histology
• Haematoxylin and eosin are frequently used histological
stains containing Br and Al.
• LA-ICP-MS has resolution down to 10 µm.
• Elemental distribution maps of C, Al, Br & Pt in human
esophageal tumor sections after Cisplatin therapy.

34. 34
• Large molecules can be labelled with trace
elements.
• Antibodies can be tagged with metals
• Aptamers
• Proteins can be derivatized with and then
labelled with metals
Tagging and Labelling
metal-tagged antibody
S N
O
O
R N
n
SS N
O
O
R N
O
O
*
n
N
O
O
R N
O
O
*
n

35. 35
Labelling/Tagging Molecules with Metals
1) Add analyte
2) Add 2nd NP-
tagged antibody
Response cps
m/z
QUANTITATIVE DATA
LA-ICP-MS
HPLC-ICP-MS
m/z
Response cps
STRUCTURAL INFO
MALDI-MS
Recognition
molecule
Nanoparticle
Molecular mass
spectrometry
LC-ESI, DESI
STRUCTURAL INFO
Solid support
Nanoparticles include:
Au, Pt, REE (60 atoms).
Analytes include:
peptides; proteins;
DNA-adducts.
Recognition molecules
include: antibodies;
DNA structures.

36. 36
• A variation of conventional flow cytometry
using metal tagged antibodies instead of
fluorochromes
• Detection by ToF-MS of discrete masses of the
metal tags.
• The lack of any significant mass spectral
overlap allows analysis up to 100 parameters
simultaneously on single cells.
• Mass cytometry has applications in
immunology, haematology and oncology.
ICP-TOF-MS Mass Cytometry

37. Many unique labels available
Cd
5
Se
5
Te
5
24 elements: 67 stable isotopes
La
1
Hf
5
Re
2
Ru
6
Rh
1
Ir
2
Pd
4
Pt
4
Ag
2
Au
1
In
2
Ce
1
Pr
1
Nd
5
Sm
6
Eu
2
Gd
5
Tb
1
Dy
4
Ho
1
Er
4
Tm
1
Yb
5
Lu
1
13 lanthanides: 37 stable isotopes
metal-tagged antibody
S N
O
O
R N
n
SS N
O
O
R N
O
O
*
n
N
O
O
R N
O
O
*
n
37
Flow Cytometry ICP-MS: CyCyCyCyTOF™

38. Replace fluorophores
and fluorescence
0
10
20
30
40
50
60
70
80
90
100
380
403
426
449
472
495
518
541
564
587
610
633
656
679
702
725
748
771
794
wavelength
intensity
Flow Cytometry ICP-MS: CyCyCyCyTOF™
138 143 148 153 158 163 168 173 178
100
90
80
70
60
50
40
30
20
10
0
with metals and
atomic mass spectrometry
• Abundant tags of similar
intensity
• Discrete signals: minimal
overlap
• No compensation required
• Background cellular
signal: Zero

39. Concluding Remarks
• ICP-MS has developed from use in the geosciences to
the biosciences.
• Offers blank limited LODs and multi-elemental
capabilities.
• Versatile detector for various separation modes.
• Primary method when used for ID-MS.
• Measurement of S and P offers the potential for high
accuracy analysis of proteins and DNA.
• Tagging molecules with metals opens up many more
possibilities for measurement of biomolecules.

40. 40
Acknowledgements
•Andrew Taylor, Director of SAS Trace Elements
Laboratory, for help, advice and encouragement.
•David Langton, Stockton-Upon-Tees, orthopaedic
studies.
•Andy Duncan, Glasgow Royal Infirmary, Wilsons
Patient samples.
•Wolfgang Muller, Royal Holloway University of
London, LA-ICP-MS of nails.
•Scott Tanner, University of Toronto, CEO CyCyCyCyTOF™.
•MRC and BBSRC for funding.
Thanks for Listening!!