Quan / Qual Analyses in Clinical Research using a Second Generation Exactive High Resolution Mass Spectrometer

Transcription

1 Quan / Qual Analyses in Clinical Research using a Second Generation Exactive High Resolution Mass Spectrometer Bertrand Rochat, PhD quantitative Mass Spectrometry Facility Lake of Geneva, Lausanne, Switzerland

2 Why a High Resolution in Clinical Research? Present Situation at the CHUV QF in Lausanne: 7 triple quadrupole installed, 1 ion trap recently replaced by an Exactive Plus About 8-9% of all analyses performed by ion transitions (SRM) for 1. Therapeutic drug research and 2. endogenous biomarker research Quantitative analyses using Triple Quadrupole (TQ-) still representing the gold standard

3 Why a High Resolution in a Research? «Quan / Qual» analyses by High Resolution Full Scan Acquisition (HR-FS) a much better «picture» of the samples (troubleshooting, adducts, charge states...) retrospective data treatment identification of unknowns semi-quantitative metabolite phenotyping Time saving no waste of time for setting-up ion transitions direct transfer of an analysis from HR- to another HR- Also a Shift of Paradigm in Clinical Research? HR- replacing TQ- for Quan analyses? Key Criteria : Quantitative analyses. Are HR- as good as triple quadrupole for quantification?

4 The Goal of our work Is the Exactive Plus HR- able to perform robust quantitative analyses in busy and productive clinical research labs? AS N 2 oven Quan/Qual analyses ESI LC

5 Exactive Plus HR-, a second generation ALL can be replaced by a quadrupole improved resolution R max : Exactive 1K, 1Hz Exactive Plus 14K, 1.8 Hz partial capabilities of ion selection improved sensitivity

6 Criteria to evaluate the Exactive Plus HR- to perform Quan analyses 1. detection selectivity in HR full scan 2. accuracy and precision of mass determination 3. dynamic range and linearity of Calibration curves 4. level accuracy of UNK samples in comparison to the quantification performed with our TQ- analyses 5. ease of use and bottleneck in a clinical research environment

7 Molecules determined by Exactive and Exactive Plus in our matrix samples for research o Antifungal drugs : caspofungin, fluconazole, itraconazole, posaconazole andv oriconazole o Anticancer drugs : dasatinib, imatinib, nilotinib, sorafenib sunitinib o Immunosupressive drugs : cylosporine, everolimus, sirolimus and tacrolimus o Vitamines D : 25-hydroxy-vitamin D3 o Amino acids : 3 amino acids determined in metabolism research m/z ranging from 76 (glycine) to 122 (cyclosporine) Sample type and sample prep. o Human plasma or serum o protein precipitation, SPE and Liquid Solid Extraction, alone or in combination LC conditions o usual LC- mobile phases, 1D-LC or column switching (immunosuppressants) o C18, CN or HILIC and UHPLC or HPLC stationary phases o 2.1mm inner diameter, 3-1mm length

8 Criteria for evaluation 1. detection selectivity in HR full scan 2. accuracy and precision of mass determination 3. dynamic range and linearity of Calibration curves 4. level accuracy of UNK samples in comparison to the quantification performed with our TQ- analyses 5. ease of use and bottleneck in a clinical research environment

9 How much Resolution is needed in HR - full scan to quantify compounds in matrix samples? Key parameters to consider : o m/z value o biomatrix and its clean up o chromatographic conditions o concentration of the analyte o mass extraction window (MEW) Resolution of 2, to <, : HR-FS with a 5-1ppm MEW SRM Resolution of, : HR-FS with a 5-1 ppm MEW SRM selectivity Xia YQ et al., Rapid Commun Mass Spectrom. 211 Kaufmann A et al., Anal. Chim. Acta. 21 Kellmann M et al, J. Am. Soc. Mass Spectrom. 29 Van der Heeft E et al., J. Am. Soc. Mass Spectrom. 29

10 1. Detection Selectivity: Resolution of the Exactive Plus Resolution (m/ m FWHM ) 18, 16, 14, 12, Mass accuracy of caffeine at m/z N=1; 1 min acquisition time just post mass calibration Resolution set at mass accuracy [ppm] precision (SD) [ppm] 14, , , , , 8, Resolution set at m/z 2 6, 4, 2, ' 1'2 1'4 1'6 1'8 2' m/z 14, 7, 35, 17,

11 Measured Mass Resolution and Acquisition Rates on Exactive Plus Resolution set at m/z 2 m/z 14' 7' 35' 17' '27 74'32 39'73 19' '625 54'7 26'884 13' '52 39'996 19'948 1' '686 34'532 17'53 8'68 Acq. Rate (Hz) HR-FS Immunosupressants Vitamine D Hepcidin HR-FS HR- ALL 2.9 Hz Compatible with Quan/Qual UPLC analyses

12 1. Detection selectivity in HR full scan LC - Exactive - chromatograms of LLOQ extracted serum and plasma samples 1 Ciclosporine A 1 Fluconazole Relative Abundance 1 1 Everolimus Sirolimus Relative Abundance 1 1 Posaconazole Voriconazole 1 Tacrolimus 1 Itraconazole Time [min] Time [min]

13 1. Detection selectivity in HR full scan 1 LC - Exactive - chromatograms of LLOQ extracted plasma samples Dasatinib 1 LC - Exactive Plus chromatograms of ULOQ extracted plasma samples 25-OH-Vit-D epi-25-OH-Vit-D Relative Abundance Imatinib Nilotinib Sorafenib Time [min] IS : D 3-25-OH-Vit-D3 24,25-OH-Vit-D3 Time [min] 25-OH-Vit-D2 IS : D 6-3-epi-25-OH-Vit-D3

14 Quantitative amino acid & acylcarnitine analysis in plasma using HILIC coupled to an Exactive Plus high resolution mass spectrometer Selected amino acids from LC-HR chromatograms from a plasma extract Relative Abundance min min min Relative Abundance b-ala Relative Abundance RT: SM: 5B E Tau _ E6 Phe _ E6 Leu Ile _ E6 Tyr _ E4 Met _11 1.8E6 Val _ E5 Thr _ Time (min) RT: SM: 5B E5 Ser _ E5 Ala Sar _ E Hyp _ E Pro _ E Gly _ E Cit _ E Glu _ Time (min) RT: SM: 5B 1 5.1E6 Arg _ E Orn 1242_ E Pip _ E Lys _ E Car _ E Ans _ Time (min) RT: SM: 5B E F: C FT + p ESI Full ms [.-7.] 12418_15 Relative Abundance C1 Labelled ISTD acylcarnitines spiked in plasma extract C8 C6 C5 C4 C3 C Time (min) 8.84E F: FT + p ESI Full ms [.-7.] 12418_ E F: FT + p ESI Full ms [.-7.] 12418_ E F: FT + p ESI Full ms [.-7.] 12418_ E F: FT + p ESI Full ms [.-7.] 12418_ E F: FT + p ESI Full ms [.-7.] 12418_ E F: FT + p ESI Full ms [.-7.] 12418_ E F: FT + p ESI Full ms Alanine Hilic-Exactive plus [.-7.] 12418_ Passing & Bablok Fit Alanine Biochrom LC-Fluorescence Identity Passing & Bablok (I) fit ( x)

15 Quantitative amino acid & acylcarnitine analysis in plasma using HILIC coupled to an Exactive Plus high resolution mass spectrometer Selected amino acids from LC-HR chromatograms from plasma extract Relative Abundance min min min Relative Abundance b-ala Relative Abundance RT: SM: 5B E Tau _ E6 Phe _ E6 Leu Ile _ E6 Tyr _ E4 Met _11 1.8E6 Val _ E5 Thr _ Time (min) RT: SM: 5B E5 Ser _ E5 Ala Sar _ E Hyp _ E Pro _ E Gly _ E Cit _ E Glu _ Time (min) RT: SM: 5B 1 5.1E6 Arg _ E Orn 1242_ E Pip _ E Lys _ E Car _ E Ans _ Time (min) RT: SM: 5B E F: C FT + p ESI Full ms [.-7.] 12418_15 Relative Abundance Labelled ISTD acylcarnitines spiked in a plasma extract C1 C8 C6 C5 C4 C3 C Time (min) 8.84E F: FT + p ESI Full ms [.-7.] 12418_ E F: FT + p ESI Full ms [.-7.] 12418_ E F: FT + p ESI Full ms [.-7.] 12418_ E F: FT + p ESI Full ms [.-7.] 12418_ E F: FT + p ESI Full ms [.-7.] 12418_ E F: FT + p ESI Full ms [.-7.] 12418_ E F: FT + p ESI Full ms [.-7.] 12418_15 Thursday Poster Number 423

16 Criteria for evaluation 1. detection selectivity in HR full scan 2. accuracy and precision of mass determination 3. dynamic range and linearity of Calibration curves 4. level accuracy of UNK samples in comparison to the quantification performed with our TQ- analyses 5. ease of use and bottleneck in clinical research environment

17 Mass distribution at the LLOQ (plain) and ULOQ (dashed) in plasma (dynamic range of 1,) with the Exactive Plus 1 2 ppm Relative Abundance 1 ng/ml 1, ng/ml + 9 hours m/z

18 [ F] Temperature variability in our research labs AC Room Temp. at the location 79 [ C] min = 3-5 [ C] = 5-9 [ F] Temperature at the position depends on the Air-Conditioning flow strength and Additional room ventilation system Time [days]

19 Mass Accuracy on the Exactive HR- and temperature fluctuation Room Temp. [ C] Room temp. Exactive temp Time [hours] External Calibration: Mass Accuracy of caffein [ppm] Exactive can cope with temperature shifts of a few C in 1 hour and keep accurate mass determinations

20 Mass Accuracy on the Exactive Plus HR-: how we have worked? Mass accuracy [ppm] Exactive plustemp [ C] Room Temp [ C] 3. 5 calibration Check the mass accuracy with mobile phase «contaminants» Si(CH 3 ) 2 O)) 6, m/z = C 24 H 38 O 4, m/z = Re-calibrate if mass accuracy is > 4 or 5 ppm. 3. Possibly, use lock mass with these «contaminants» Time [days] calibration

21 Criteria for evaluation 1. detection selectivity in HR full scan 2. accuracy and precision of mass determination 3. dynamic range and linearity of Calibration curves 4. level accuracy of UNK samples in comparison to the quantification performed with our TQ- analyses 5. eas4 of use and bottleneck in a clinical research environment

22 Calibration Curves of LC- Exactive- and LC-TQ- LC-ESI TQ discovery - LC-ESI Exactive - Ciclosporine A Dasatinib Fluconazole µg/l Sirolimus Area Ratio Area Ratio Area Ratio µg/l Imatinib µg/l Area Ratio Area Ratio Posaconazole Area Ratio µg/l µg/l µg/l Henry H et al., RC, 212

23 Calibration Curves in plasma extract analysed by LC Exactive Plus Imatinib : 1 1, ng/ml 25-OH-Vitamin D3 : 1 17 nm Area Ratio 15 1 Area Ratio , 4, 6, 8, 1, ng/ml. 1 1 nm

24 Criteria for evaluation 1. detection selectivity in HR full scan 2. accuracy and precision of mass determination 3. dynamic range and linearity of Calibration curves 4. level accuracy of UNK samples in comparison to the quantification performed with our TQ- analyses 5. ease of use and bottleneck in a clinical research environment

25 Passing & Bablok Fit of immunosupressive drugs in serum: SRM versus HR-FS acquisition (N=1) HR- (HR-FS) Ciclosporine A Identity Passing & Bablok (I) fit x TQ- (SRM) HR- (HR-FS) Everolimus 5.5 Identity 5 Passing & Bablok (I) fit x TQ- (SRM) 16 Sirolimus 14 Tacrolimus HR- (HR-FS) 12 1 Henry H et al., RC, Identity 4 Passing & Bablok (I) fit x HR- (HR-FS) 4 Identity Passing & Bablok (I) fit x TQ- (SRM) TQ- (SRM) 1 8 6

26 Passing & Bablok Fit of 25-OH-vitamine D3 in plasma : SRM versus HR-FS acquisition (N=1) 25-OH-vitamine D3 [nm] Determined by LC-Triple Quadrupole Identity Passing & Bablok (I) fit ( x) OH-vitamin D3 [nm] Determined by LC-Exactive plus

27 Passing & Bablok Fit of imatinib in plasma: SRM versus HR-FS acquisition (N=17) 3, 2, TQ- [ng/ml] 2, 1, 1, Identity Passing & Bablok (I) fit 1.851x R2 =.988 1, 1, 2, 2, 3, HR- [ng/ml] Exactive plus Exactive

28 Criteria for evaluation 1. detection selectivity in HR full scan 2. accuracy and precision of mass determination 3. dynamic range and linearity of Calibration curves 4. level accuracy of Cs, QCs and UNK samples in comparison to the quantification performed with our TQ- analyses 5. ease of use and bottleneck in a clinical research environment

29 Ease of use in a clinical research environment User friendly for lab techs Easy follow up of mass accuracy status by mass deviation of contaminant ions Si(CH 3 ) 2 O)) 6 at m/z = C 24 H 38 O at 4 m/z = Work with fast PCs Handle Go of data: processing and storage Are there bottlenecks for a shift of technology in Quan analyses? cost difference between TQ- and HR- no need of Qual analyses no HR specific guidelines by Authorities conservatism

30 The Goal of our work Is Exactive Plus HR- able to perform robust quantitative analyses in a busy and productive clinical research lab? Quan analyses by HR-FS : better «picture» of the samples : troubleshooting, adducts, charge states... time saving Qual analyses by HR-FS : in comparison to TQ- identification of unknowns fate of the analyte in vivo : e.g. drug metabolite profiles in samples semi-quantitative metabolite phenotyping = metabotyping

31 Parent Drug : imatinib M1 hydroxy-deacetyl-imatinib M2 demethyl-imatinib M3 demethyl-ketone-imatinib M4 demethyl-hydroxy/n-oxide-imatinib M5 hydroxy-desaturated-imatinib M6 hydroxy/n-oxide-imatinib M7 demethyl-carboxilic-imatinib M8 demethyl-glucuronide-imatinib M9 glucuronide-imatinib M1 hydroxy-glucuronide-imatinib Patient #1 4 Patient # Profiling imatinib metabolites in parallel to imatinib quantification in donor plasma Relative metabolite amounts A: 2x A: 1x A: 2x A: 5x A: 8x A: 4x A: 4x A: 4x A: 2x A: 3x A: 6x Time [min] metabolite ID#

32 Semiquantitative Determination of Metabotypes using HR- : a Fundamental Step Forward in Clinical Research Analyses Quan Qual Targeted quantitative analysis therapeutic drug research biomarker research Validated metabotyping (*) Relative quantification of lipid profiles drug metabolite profiles etc Quan or Quan/Qual analyses using LC-HR-FS acquisition interpretation Metabolomics Metabotype (*) Validation Identification of unknowns; biochemical explanation, etc. Metabotype (*) Discovery Fundamental + metabolomics sample stratification Personalized medicine research (*) metabolite phenotyping = metabotyping

33 Conclusion and Summary The LC- analyses that we performed in HR-FS acquisition mode with the Exactive and Exactive Plus, showed that : HR analysis is as specific as SRM analysis (R 25, at all analyte m/z) HR analysis is compatible with usual plasma extraction (PP, SPE and SLE) Exactive Plus is compatible with a clinical research labs needs HR-FS acquisition mode allows simultaneous Qual analyses that should have a strong impact in clinical research such as the semi-quantitative determination of metabotypes

34 Acknowledgements Maciej Bromirski, Olaf Scheibner, Sara Robinson, Bénédicte Duretz and Subodh Nimkar Thermo Fisher Scientific, Bremen, Paris, San Jose Emmanuel Kottelat, René Nellen and Hamid Sobhi Quantitative Mass Spectrometry Facility, CHUV Hugues Henry, Steve Bruce and Alexandre Béguin Clinical Chemistry, CHUV