Upload
others
View
2
Download
0
Embed Size (px)
Citation preview
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
50
100
150
200
250
300
350
400
450
0 100 200 300 400 500Androsteendion_RIA_extraction_
Andr
oste
endi
one_
LCM
S
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
???
???
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