LC-MS/MS in endocrinology
Transcription
LC-MS/MS in endocrinology
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. Quantification of marker molecules is essential in the diagnostic work-up of ALL endocrinological disorders. The interest of endocrinologists in high-quality analytics is essential. Quantification of marker molecules is essential in the Diagnostic work-up of ALL endocrinological disorders. The interest of endocrinologists in high-quality analytics is essential. Immunoassays (routine analyses) and GC-MS (reference methods): traditional players in endocrinological analytics Quantification of marker molecules is essential in the Diagnostic work-up of ALL endocrinological disorders. The interest of endocrinologists in high-quality analytics is essential. 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 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 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) 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) • 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 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 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 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 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 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 Free thyroid hormones (after ED / UF) reference methods second line routine 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 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 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 Small molecule analytes in endocrinology Analytes related to research : Small molecule analytes in endocrinology 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 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 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 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 LC-MS/MS in endocrinology: powerful technology Σ LC-MS/MS in endocrinology: powerful technology demanding technology Σ LC-MS/MS in endocrinology: powerful technology demanding technology technology under development Σ LC-MS/MS in endocrinology: powerful technology demanding technology technology under development routine technology in many laboratories but has still a long way to a widespread standard technology Σ LC-MS/MS in endocrinology: powerful technology demanding technology technology under development 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