High resolution mass spectrometry for bioanalysis at Janssen. Current experiences and future perspectives

Transcription

1 High resolution mass spectrometry for bioanalysis at Janssen. Current experiences and future perspectives Lieve Dillen Drug Safety Sciences Analytical Sciences, Non-regulated Bioanalysis

2 Presentation outline Rationale for Quan on HR in non-regulated BAN (only Q-TOF) for in vivo samples Mass spec workflow considerations Three examples Conclusions 2

3 Rationale for HR MS quantification PRO Sensitivity of HR instruments considerably improved Mass spec simplified Optimisation minimal Quan performed on extracted ion chromatogram Added value: post-acquisition evaluation of data in Quan/Qual mode Information on metabolites Information on biomarkers CON Expensive equipment Often shared between groups Mass spec simplified? Selections to be considered Large datasets Time consuming data mining and processing 3

4 Sensitivity of HR instruments Sensitivity factor relative versus the response on API4000 Q-TOF1 Q-TOF2 Q-TOF MS/MS cpd acetaminophen galantamine 1 2 loperamide midazolam norethindrone omeprazole prednisone 1 1 risperidone cpd cpd cpd cpd cpd tolbutamide '-OH mephenytoin

5 Mass spec workflows can be simplified? No optimisation of SRM But following considerations Resolution mode Include fragmentation (Ms E, Ms all ) Scan time Mass range Centroid or profile data Extraction window 5

6 Extraction window, profile vs centroid Exact (M+H + ) mass: mda MEW (+ 10) 5 mda MEW (+ 2.5) FWHM=M/R MEW= 2 FWHM 6

7 mass accuracy and centroid data Rs Extraction around measured mass Extraction around theoretical mass 7

8 Impact of resolution on sensitivity Chromatography constant Scan time constant (200 ms) Extraction window 20mDa (+/- 10 mda) cpd 1 cpd 2 cpd 3 ng/ml Rs % Rs % ng/ml Rs % Rs % cpd cpd cpd

9 MS E MS E for structural info -> Qual Do we compromise Quan with MS E sensitivity # datapoints per peak Ms E 3s PW = 7 DP 3s PW = 15 DP cpd 1 cpd 2 cpd 3 cpd 4 cpd 5 cpd 6 ng/ml % PA MS E vs PA Rs

10 Example 1: first study Q-TOF vs QQQ Study design: bile excretion study 3 rats dosed PO 10 mg/kg Plasma time profile (0.5, 1, 2, 4, 7, 24h) Bile and urine collection (0-1h, 1-4h, 4-7h, 7-24h) Protein precipitation 1000 PO E1 E2 E3 Mean P O Concentration (ng/ml) time (h) 10

11 Past and Current Approaches Predefined separate Quan and Qual approaches Quan -> fast SRM method (< 3 min run) - Bioanalysis Qual -> pool samples per time point 10 min LC run - Biotransformation Integrated Quan-Qual approach Bioanalist analyses on HR-MS all samples available for Qual but no compromise in LC run? Data handover to biotransformation for evaluation 11

12 Example 1:evaluate Quan/Qual Quan with SRM, fast LC Quan with HR-MS, fast LC including MS E Comparison Quan performance Qual evaluation with fast LC Qual evaluation following re-analysis of samples with extended LC run Comparison Qual performance 12

13 SRM-quantitative results Equipment: API 4000 Triple Quadrupole LC-MS/MS + UPLC Column: Acquity UPLC BEH C18 50 x 2.1 mm 1.7 µm Gradient profile Time (min) Flow ml/min % Formic Acid 0.1 % % CH3CN Injection volume (µl) LLOQ (ng/ml) linearity (ng/ml) Batch +QC Bile ok Plasma ok urine 0.1* ok * Low IV because of high concentrations 13

14 Equipment : Synapt G2S + UPLC (identical conditions) Mass range : (plasma), (bile, urine) Analyser : resolution mode Scan time : 0.1 s QUAN: 20 mda MEW Q-TOF-based quantitative results Injection volume (µl) LLOQ (ng/ml) linearity (ng/ml) Batch +QC s Bile ok Plasma Not ok * urine ok * range of curve too small to cover the range of the 3 QC s 14

15 Comparison Q-ToF vs API-4000 data 15

16 Qualitative outcomes from Q-ToF data Combined Metabolite Peaks (All Found and Unexpected Peaks) [Analyte] 2.96e6 100 Parent Plasma % Gluc Combined Metabolite Peaks (All Found and Unexpected Peaks) [Analyte] Time 8.83e6 100 Parent Urine % O 270 +Gluc Time Combined Metabolite Peaks (All Found and Unexpected Peaks) [Analyte] 100 Glucuronide conjugation e6 Bile % O-2H Time

17 Are QUAL results inferior due to LC compromise? Samples re-analysed with 10 min LC gradient Acquisition kept identical (except for scan time) Treshold settings identical for met ID More metabolites measured in Qual mode (different scan time) But XIC in Quan results also revealed metabolite Differences in response btw Quan and Qual (matrix impact) 17

18 Conclusions example 1 Analytical performance comparable between QQQ and Q-TOF PK Profiles comparable Chromatography very compressed for MetID Metabolites largely co-eluting Difficult to make good use of MS E data (IMS?) Some optimization of retention time for the parent feasible Qualitative outcomes still valuable Identified major excretion pathways and circulating metabolite Increased LC peak capacity will improve data quality 18

19 Example 2: Bile excretion study Decision upfront for longer LC run (Rt UD 3.5 min) Quan for parent compound plasma conc ng/ml PO 10 mg/kg study 1 SRM study 2 HRMS Time (h) after administration study 1 SRM study 2 HR MS Time (h) ng/ml ng/ml ± ± ± ± ± ± ± ± ± ± BQL BQL LLOQ LLOQ 2.00 Bile Rat 1 Rat 2 ng/ml % of dose ng/ml % of dose 0-7h h Total Rat 1 Rat 2 Urine ng/ml % of dose ng/ml % of dose 0-7h h Total

20 Example 2: Bile excretion study Plasma 0.5h: combined metabolites M+O parent Met Id Rt (min) PPM Area Abs Met Met M+O+glucuronide parent M+O M+O

21 Example 2: Bile excretion study Urine 0-7h: combined metabolites M+O Met Id Rt (min) PPM Area Abs Met Unassigned Met parent M+O M+O

22 Example 2: Bile excretion study Bile 7-24h: combined metabolites Met Id Rt (min) PPM Area Abs gluc gluc M+O+gluc Parent M+O

23 Conclusion : example 2 Major drug related material is not UD Bioanalytical method can be optimized Separation UD and M+O Consider stab testing of N-oxide early on in project PK/PD correlation difficult Evaluate activity of metabolite Monitor metabolite in PD experiment 23

24 Example 3: metabolite + biomarker evaluation Lead project: nephrotox due to metabolite Bu project: evaluate in vivo metabolites early on Include biomarker Evaluate creatinine in plasma Chromatography adapted for creatinine analysis Acquity UPLC HSS T3 2.1x100mm 1.8µ Solvent A: Ammonium Acetate 0.01M Solvent B: Acetonitrile LC 0.5 ml/min Time (min) % A % B

25 Creatinine Metabolite 2 Metabolite 1 Compound X 25

26 Conclusions Select studies for Quan/Qual Workflows not as simple as promised Always compromise between Quan efficiency and Qual data quality Untargeted Quan/Qual for endogenous markers, sample prep and LC matters Q needed? -> LC-TOF or LC-exactive 26

27 Acknowledgements Stijn Van Asten Willy Lorreyne Willy Cools Liesbeth Vereyken Pascale Proost Russell Mortishire-Smith Filip Cuyckens Drug Safety Sciences