LC-MSMS - state of the art in endocrinology

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1 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

2 Quantification of marker molecules is essential in the diagnostic work-up of ALL endocrinological disorders.

3 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.

4 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

5 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

6 Potential roles of LC-MS/MS in endocrinology

7 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

8 Potential targets of LC-MS/MS in endocrinology

9 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

10 Protein quantification by mass-spectrometry

11 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

12 Small molecule hormone quantification by LC-MS/MS

13 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)

14 LC-MS/MS in endocrinology - status of application

15 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

16 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)

17 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

18 Small molecule analytes in endocrinology

19 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

20 Small molecule analytes in endocrinology 25-OH-Vitamin D3 (=hormone precursor) Plasma metanephrines Testosterone (total) Cortisol (urine (saliva, serum)) 17-OH-Progesteron Steroid profiles (Cortisol, cortison, 11-deoxycortisol, 21-deoxycortisol, corticosterone, 17-OH-P, androstendion, testosterone, progesteron, DHEA, DHEAS) routinely analyzed in a number of laboratories by LC-MS/MS analyzed in some routine laboratories Urinary 5-HIA / serotonin

21 Small molecule analytes in endocrinology 25-OH-Vitamin D3 (=hormone precursor) Plasma metanephrines Testosterone (total) Cortisol (urine (saliva, serum)) 17-OH-Progesteron Steroid profiles (Cortisol, cortison, 11-deoxycortisol, 21-deoxycortisol, corticosterone, 17-OH-P, androstendion, testosterone, progesteron, DHEA, DHEAS) routinely analyzed in a number of laboratories by LC-MS/MS analyzed in some routine laboratories Urinary 5-HIA / serotonin Free thyroid hormones (after ED / UF) reference methods second line routine

22 Small molecule analytes in endocrinology 25-OH-Vitamin D3 (=hormone precursor) Plasma metanephrines Testosterone (total) Cortisol (urine (saliva, serum)) 17-OH-Progesteron Steroid profiles (Cortisol, cortison, 11-deoxycortisol, 21-deoxycortisol, corticosterone, 17-OH-P, androstendion, testosterone, progesteron, DHEA, DHEAS) routinely analyzed in a number of laboratories by LC-MS/MS analyzed in some routine laboratories Urinary 5-HIA / serotonin Free thyroid hormones (after ED / UF) reference methods second line routine Aldosterone challenge! 1,25-dihydroxy vitamin D research 17-ß-Estradiol / estrogens, progesterone / gestagens

23 Small molecule analytes in endocrinology Analytes related to research :

24 Small molecule analytes in endocrinology Analytes related to research : e.g. Prostaglandins Endocannabinoids Endogenous digitalis-like substances Tamoxifen Bisphenol-A Metabolomics of stress-response

25 Problems and sources of errors

26 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)

27 Isobaric interferences 11-Deoxy X 21-Deoxy 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

28 Two examples of application of LC-MS/MS in endocrinology

29 25-OH vitamin D Kritik an der HPLC Messung von 25-OH Vitamin D mit IDS-RIA Ligandenbindungs-Assays IDS-EIA Nichols Liaison 1 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

30 Legro SR et al. J Clin Endocin Metabol 2010;95: 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?

31 Testosterone by LC-MS/MS

32 Testosterone by LC-MS/MS

33 Future perspectives of LC-MS/MS in endocrinology

34 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

35 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

36 Σ LC-MS/MS in endocrinology:

37 LC-MS/MS in endocrinology: powerful technology Σ

38 LC-MS/MS in endocrinology: powerful technology demanding technology Σ

39 Σ LC-MS/MS in endocrinology: powerful technology demanding technology technology under development

40 LC-MS/MS in endocrinology: powerful technology demanding technology Σ technology under development routine technology in many laboratories but has still a long way to a widespread standard technology

41 LC-MS/MS in endocrinology: powerful technology demanding technology Σ 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

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43 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

44 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