The Added Value of Mass Spectrometryin Clinical Chemistry

Jaak Billen and Steven Pauwels

The ImmunoassayA workhorse with some flaws

Sandwich assay• Two epitopes• Excess capture antibody• Excess detection antibody

• Most sensitive immunoassays• Large dynamic range• High specificity

Competitive immunoassay• Only one epitope• One antibody• Competion for antibody

• Less sensitive• Less specific• Small dynamic range

Immuno-assaypro contra• Relatively sensitive• Easy• Fully automated• Direct measurement

• Lack of accuracy• Lack of sensitivity• Lack of specificity• Limited dynamic range• Expensive (kit) reagents• Black box

Mass SpectrometryA reference method for hormones

1) Sample preparation• Protein precipitation (methanol, acetonitrile, ZnSO4)

• Liquid liquid extraction• Solid phase extraction

2) HPLC

HPLC in practice

- Pumps- Fluids- Tubings- Sample loader- Complex valves- Column

3) Tandem Mass Spectrometry

Mass Spectrometryin practice- Ion source- Switching valves- Tubings- Vacuum pump- Nitrogen generator

Mass Spectrometryin practice- Spray probe- Heating probes- Curtain plate- Curtain plate aperture- Orifice

LC-MS/MS in EndocrinologyAnalytical Superiority

Added value LC-MS/MS: Accuracy

0

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0 100 200 300 400 500Androsteendion_RIA_extraction_

Andr

oste

endi

one_

LCM

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y = -15.2 + 0.65 x n = 97

van den Ouweland JMW, Kema IP. J Chromatography B 2012

Added LC-MS/MS: Sensitivity

Cortisol Prednisolon

Cross reactivity!

Added value LC-MS/MS: Specificity

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???

Kruisreactie van Elecsys Estradiol tests en fulvestrant (Fosladex)

ROCHE diagnostics – maart 2016

Het onjuiste niveau van estradiol kan leiden tot een verkeerde interpretatie van het hormonaal statuut en daardoor kan het gebruik van fulvestrant gewijzigd worden.

Bovendien kan de doeltreffendheid van de anti-estrogeen behandeling ondergewaardeerd worden en dat kan leiden tot een medisch risico voor postmenopauzale vrouwen die behandeld worden met fulvestrant.

Als de behandeling met fulvestrant gewijzigd of onderbroken werd als gevolg van schijnbaar hoge estradiolresultaten, zou een ander procedé moeten worden gebruikt zoals LC-MS.

Roche Estradiol II: exemestane interference

Patient E2 MS ng/L (pmol/L) E2 ECLIA ng/L (pmol/L)

1 <1.3 (<4.8)) 21 (77.2)

2 <1.3 (<4.8) 7 (25.7)

3 <1.3 (<4.8) 12 (44.1)

4 <1.3 (<4.8) 33 (121.3)

5 <1.3 (<4.8) 15 (55.1)

Results for E2 via ECLIA and MS for all 5 patients treated with exemestane (Aromasin)

Added value LC-MSMS: steroid profiling

Added value LC-MS/MS: pro and contraAdvantages

• Analytical superiority• “Cheap” reagents

o Organic solventso Deuterated internal standardso Robust columso Calibrator setso Pipetting tips and plates

• High troughput possible• Develop new assays

Disadvantages

• Expensive instruments• Batch mode• Skilled labor required• LIS interface• Regulartory uncertainty• Ruggedness

The Added Value of Mass Spectrometryin Clinical Chemistry

Jaak Billen and Steven PauwelsClinical Chemists

University Hospitals LeuvenPentalfa March 24th 2016

Mass SpectrometryWhat’s in a Name?

Chromatography and Mass Spectrometry

Gas Chromatography(GC)

Liquid Chromatography (LC) ESIAPCI

EI

Time-of-Flight (TOF)OrbiTrap (OT)

Quadrupole (Q)Iontrap (IT)

Pressure (U/HPLC)Buffers

Column1D or 2D

Multi/Hybrids(eg QQQ, TOFTOF, QTOF )

LRMS

HRMS

Chromatography• Gas Chromatography

o Easier link with MSo First in clinical routine (GC-MS)

• Liquid Chromatographyo Difficult link with MS (Nobel prize for ESI)o More universalo Easy sample preparation possible

Q IT TOF FT OT FT ICR

Resolution - +/- + ++ +++

Mass Accuracy (ppm) - - ++ ++ +++

Acquisition Speed (Hz) + + +++ +/- -

m/z range - + +++ + ++

Linearity ++ + + +/- +/-Price ++ ++ +/- +/- -

Types of Mass SpectrometersLRMS HRMS

Mass Accuracy and ResolutionHRMS (TOF)Measured m/z

476.1913

Carbon Isotopes

Theoretical m/z476.1915

LRMS (Q)Measured m/z

475.9

High resolution andMass Acuracy come

at a price!

Holcapek et al. J Chromatography B 2012

=LC-QQQ (tandem MS or MS/MS) (ESI)*Most Applications Described*Sensitivity in MRM mode*Linearity*Ease of Use*Price

‘Mass Spectrometry’ in Clinical Lab

Liquid Chromatography (LC)

Figures courtesy of AB Sciex

Mass Spectrometry (Q)

Cortisol Prednisolon362 Da 360 Da

++

+MS1

+

Tandem Mass Spectrometry (QQQ)

++

+++

+

MS1 MS2

+++

Importance of Chromatography

Mr 400.6

Standard with 25(OH) D+epi’s

Added Value in Toxicology

Difficulties in Toxicology• Immunoassays: Fast and easy but…

o Cross Reactivity (known and unknown)o Clinical Cutoffs in Urine?o Limited Coverage (eg synthetic opioids)

• GC-MS: Specific with broader coverage but…o Volitales (eg no glucuronides)o Labour-Intensiveo Specialized Knowledge

Difficulties in Toxicology

Genetic Variation

Absorption Variation

Excretion variation (polyuria,…)

Co-medication

XTC

Also MDA and amphetamine!Main Metabolites CR???

DoseFrequency

Difficulties in Toxicology

Cross reactivityRitalinic acid?

Trazodon(Trazolan)

mCPP

Difficulties in Toxicology

Cross reacts withAmphetamineImmunoassay

Added Value in ToxicologyImmunoassay Opiates= Positive

Could be…Heroine?Morphine?Codeine?

Differentiation:*Ratio Codeine/Morphine*Impurities

Added Value in Toxicology*Dilute 100 µL urine 1 in 10*Inject on LC-MS systems

Codeine

Codeine-glucuronide

Morphine-glucuronide

Morphine

Added Value in TDM

• Immunoassayso Most often relatively performanto Sometimes not (yet) available (eg Voriconazole)

• LC-QQQo Relatively easy Method Development (eg Voriconazole)o Arbiter for Discordant Immunoassay Results

Difficulties in TDM

LC-QQQ as Arbiter

Oyaert et al. Clin Chim Acta. 2015 20;441:63-70.

Routine-Research Interaction

Routine-Research Interaction

*Relevant Drug metaboliteDiscovery (LC-QTOF)*New Designer Drugs

Research

*Introduction in Routine Method (LC-QQQ)

Routine

*Abnormal or Interfering Peaks*Strong Clinical Suspicion with

negative screen

Examination of Sample with eg QTOF, other

Chromatogram (LC-QTOF)

Desmethyl-Papaverine-Glucuronide (4 isomers) Papaverine

Desmethyl-Papaverine-Glucuronide

Desmethyl-Papaverine

Fragments (TOF) at 5.01 min

Chromatogram (LC-QTOF)

Fragments (QTOF) at 8.42 min

Zuclopenthixol-sulfoxide-Glucuronide

Chlorine Isotope

Limitations• Standardization• Matrix Effects• Automation• Interpretation/Development

‘People >> Software >> Hardware Performance, especially for HRMS’

Future Perspectives• Automation• Miniaturization• Service from Companies• Peptides (in routine)• Proteins