1

Advanced Clinical Applications of

Inductively Coupled Plasma Mass

Spectrometry (ICP-MS)

“Bring a Little Sunshine into Your Laboratory”

Dr Chris HarringtonSAS Trace Element Laboratory

Guildford

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

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

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

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

Sunshine in Action: An Argon Gas Plasma

7

ICP-MS: Detection Limits

8

• Nutritional elements.

• Orthopaedic applications.

• Heavy metal toxicity.

Current Clinical Applications

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

Orthopaedic Applications

Interpretation of Co Levels

•< 2 µg/L: close to normal.

•2 - 5 µg/L: equivocal result.

•5 - 10 µg/L: high sensitivity

and specificity for abnormal

wear process.

•10 - 20 µg/L:100%

specificity for abnormal

wear patient at greatly

increased risk of ARMD (adverse reaction to metal debris).

•> 20 µg/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

Lead use dates back to the Romans, but still

used. Monitoring required for CLAW.

Tetra-ethyllead now banned, but

may have left lasting scarsFcrime!

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

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.

ICP-MSExcellent 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

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

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

Instrumentation for Speciation Analysis

(b)

(a)

(a) Pt-DNA adduct

measurement system.

(b) API-MS/MS for

characterisation of

metalloproteins and Pt-

standards

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

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/z822 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

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

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

Adduct

Conc

H23 Sensitive

A549 Resistant

3.13

3.29

2.56

TIC

2.73

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

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

• 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

Cu-Proteins in Wilson’s Disease

Ceruloplasmin (CP)

Albumin (Alb)• Overcomes

problems found

with current CP

assays.

• Apo-CP is not

measured.

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

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

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

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

• 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

• 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

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

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 µm 74 µm

Iceman

finger nail

• Calibration difficult.

• Involves pressing

disks from similar

materials.

• S can be used as

internal standard.

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

• 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

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

moleculeNanoparticle

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

• 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

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™

Replace fluorophores

and fluorescence =

0

10

20

30

40

50

60

70

80

90

100380

403

426

449

472

495

518

541

564

587

610

633

656

679

702

725

748

771

794

wavelength

inte

nsity

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

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

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!!