LC-MS/MS in endocrinology

Transcription

LC-MSMS state of the art in endocrinology
Michael Vogeser
Institute of Clinical Chemistry
Hospital of the University of Munich – Germany
8 June 2011, Namur
Quantification of marker molecules is essential in the
diagnostic work-up of ALL endocrinological disorders.
diagnostic work-up of ALL endocrinological disorders.
The interest of endocrinologists in high-quality
analytics is essential.
Diagnostic work-up of ALL endocrinological disorders.
Immunoassays (routine analyses) and GC-MS (reference
methods): traditional players in endocrinological analytics
Diagnostic work-up of ALL endocrinological disorders.
Immunoassays (routine analyses) and GC-MS (reference
methods): traditional players in endocrinological analytics
LC-MS/MS: In use for >10 years now
Potential roles of LC-MS/MS in endocrinology
Potential roles of LC-MS/MS in endocrinology
 Classical reference methods
 Reference measurements in large series (applying ID)
 Research method (known analytes / new analytes)
 Routine analytics
Potential targets of LC-MS/MS in endocrinology
Potential targets of LC-MS/MS in endocrinology
 Small molecule hormones
 Proteo- / peptide-hormones
 Endocrine tumour markers
 Xenobiotics (endocrinological drugs; endocrine disruptors)
 Metabolites / patterns of metabolic hormone effects
Protein quantification by mass-spectrometry
Protein quantification by mass-spectrometry
 MS has a key role in proteomics → characterisation of proteins
 Quantification of proteins is a substantial challenge for LC-MS/MS
(low concentrations, heterogeneity of analytes)
 First reference methods described (e.g. HbA1c, insulin)
 Immunoassay-MS-hybrid assays (e.g., PTH, thyreoglobulin)
 Potential advantage: precise characterisation of sub-forms and
isoform-patterns
 No routine applications at present
Small molecule hormone quantification by LC-MS/MS
Small molecule hormone quantification by LC-MS/MS
Potential strengths of LC-MS/MS
 High potential specificity > reliable standardisation possible
 Isotope dilution internal standardisation >
compensation of matrix effects, high accuracy
(in particular when compared to competitive immunoassays)
 Good between-laboratory agreement, assay-independent reference
ranges (relevant for complex sub-populations in endocrinology)
 No interference from heterophilic antibodies
 Independence from antibody-reagent lots
 Cost efficiency (“biology is expensive, chemistry is cheap”)
 “Good” practicability, potentially high sample throughput (≠ GC-MS)
LC-MS/MS in endocrinology - status of application
University hospital laboratories / teaching hospitals:
widely used technology; routine / research combi-use
Commercial laboratories:
standard technology in core labs. TDM >> endocrinology
In-house methods / laboratory developed tests / home-brew
Kits (Chromsystems, Recipe, Biocrates, Perkin Elmer)
In-house methods / laboratory developed tests / home-brew
Kits (Chromsystems, Recipe, Biocrates, Perkin Elmer)
• Implementation of the technology, method development,
method maintenance is a substantial challenge !
• Poor support by the MS-manufacturer (applications)
• Routine use possible but not in a standard laboratory
Small molecule analytes in endocrinology
25-OH-Vitamin D3 (=hormone precursor)
Plasma metanephrines
Testosterone (total)
Cortisol (urine (saliva, serum))
17-OH-Progesteron
routinely analyzed
in a number of
laboratories
by LC-MS/MS
17-OH-Progesteron
routinely analyzed
in a number of
laboratories
by LC-MS/MS
Steroid profiles
analyzed in
some routine
laboratories
(Cortisol, cortison, 11-deoxycortisol, 21-deoxycortisol,
corticosterone, 17-OH-P, androstendion, testosterone,
progesteron, DHEA, DHEAS)
Urinary 5-HIA / serotonin
17-OH-Progesteron
routinely analyzed
in a number of
laboratories
by LC-MS/MS
Steroid profiles
analyzed in
some routine
laboratories
Free thyroid hormones (after ED / UF)
reference methods
second line routine
17-OH-Progesteron
routinely analyzed
in a number of
laboratories
by LC-MS/MS
Steroid profiles
analyzed in
some routine
laboratories
Free thyroid hormones (after ED / UF)
reference methods
second line routine
Aldosterone
1,25-dihydroxy vitamin D
17-ß-Estradiol / estrogens, progesterone / gestagens
challenge !
research
Analytes related to research :
Analytes related to research :
e.g.
Prostaglandins
Endocannabinoids
Endogenous digitalis-like substances
Tamoxifen
Bisphenol-A
Metabolomics of stress-response
…
Problems and sources of errors
Problems and sources of errors
 Practicability – full commitment of experts required on site
(method implementation, handling of problems)
 Gross handling errors (e.g. errors in labelling, data transfer to LIMS)
 Ion suppression and matrix effects on ionisation →
stable isotope labelled internal standards available
 Appropriate calibration materials (→ kits)
 Sensitivity (cortisol vs. aldosteron !)
 Isomers / isobars of the target analytes
(→ chromatographic separation)
Isobaric interferences
21-Deoxy
11-Deoxy
X
X
X 17-Deoxy
• 3-epi 25-OH-vitamin D3
• epi-testosterone
• 2-AG / 1-AG
• structurally unrelated isobars?
→ sufficient degree of
chromatography is
essential !
Dr. Seger, Innsbruck 2009
Two examples of application of LC-MS/MS in endocrinology
25-OH vitamin D
Kritik an der
Messung von 25-OH Vitamin D mit
HPLC
IDS-RIA
Ligandenbindungs-Assays
Nichols
Liaison 1
IDS-EIA
Liaison 2
Roche
Bias-Plot (y-Achse LC-MS/MS)
Roth et al., Ann Clin Biochem 2008;45:153-9
→ large scale validation of routine methods by LC-MS/MS
Legro SR et al.
J Clin Endocin Metabol 2010;95:5305-13
Blinded send-out of paired samples to an
academic laboratory and to a commercial laboratory
for testosterone quantification by LC-MS/MS and RIA
Sub-set of paired samples in the same laboratory
→ correlation and reproducibility ?
Testosterone by LC-MS/MS
Testosterone by LC-MS/MS
Future perspectives of LC-MS/MS in endocrinology
Probable:
 Widespread application of good kit solutions
 Automation of sample preparation
 Better standardisation of instrument configurations and handling
 Increasing percentage of MS-analyses in endocrine testing
Probable:
 Widespread application of good kit solutions
 Automation of sample preparation
 Better standardisation of instrument configurations and handling
 Increasing percentage of MS-analyses in endocrine testing
Desirable but uncertain:
 Development of plug-and-play closed MS/MS-based analyzers by
the IVD-industry
 Transformation from a routine technology in specialised laboratories
to a routine technology for standard endocinology labs
(as realized for immunoassays)
 Metabolomic profiling approaches to endocrinological investigations
Σ
LC-MS/MS in endocrinology:
Σ
powerful technology
Σ
powerful technology
demanding technology
Σ
powerful technology
technology under development
Σ
powerful technology
routine technology in many laboratories but has still a long way
to a widespread standard technology
Σ
powerful technology
routine technology in many laboratories but has still a long way
to a widespread standard technology
probably has the potential to improve clinical care by analyses
of superior quality compared to standard techniques
Hospital of the University of Munich
~ 2,300 beds
~ 9,900 co-workers
~ 5,000 students
2 main locations: Campus Grosshadern and Campus Inner City
Institute of Clinical Chemistry
~ 200 co-workers
4 affiliates
~ 8 mio. analyses per years
Clinical chemistry, haematology, coagulation, immunology and
endocrinology testing, therapeutic drug monitoring, human genetics
www.klinikum.uni-muenchen.de/Institut-fuer-Klinische-Chemie/de
Mass spectrometry in the Institute of Clinical Chemistry
Introduction of GC-MS in 1975
(urinary catecholamine metabolites, organic acids,
stable isotope metabolic-turn-over studies, etc.)
Introduction of LC-MS/MS in 2000
At present analysis of tacrolimus, cyclosporin, everolimus, sirolimus,
busulfan, itraconazole, posaconazole; research applications (e.g., steroids)
~ 200 samples per day
4 instruments (Waters)
10 technicians trained

LC-MS/MS in endocrinology

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