Towards High Resolution MS in Regulated Bioanalysis

Transcription

1 Towards High Resolution MS in Regulated Bioanalysis Benno Ingelse MSD 3 rd EB focus meeting June 12 th 13 th Brussels, Belgium

2 Contributors Gary Adamson Ken Anderson Kevin Bateman Cynthia Chavez-Eng Inhou Chu Raj Desai Bart Emary Jason Hoar Mike Holkenborg Benno Ingelse Harrie Peters Enzo Pucci Amy Wang Yang Xu Bella Yao Sean Yu Rena Zhang

3 utline Quads vs HRMS HRMS: Quantitative applicability Quan Qual workflows uture considerations

4 Triple Quadrupole Mass Spectrometry Selective Based on two stages of mass selection, parent m/z and fragment ion m/z Sensitive Beam instrument with high duty cycle Robust Calibration stable over long period Compact Data Sets MRM data files are very small and easy to process 25+ years of constant development for quantitative applications

5 High Resolution Mass Spectrometry Selective? Based on resolving power Sensitive? Pulsed (T) or Indirect Detection (T traps) based instruments Robust? Calibration stable over long period (using internal calibration protocols) Large Data Sets ull scan data files are large and time consuming to process ~3-5 years of recent development for quantitative applications

6 Selectivity: SRM Based Quantitation XIC of +MRM (9 pairs): 376.2/165.1 amu from Sample 7 (std.1um) of Test_mix_rat_plasma_23eb21_EL.wiff (Turbo Spray... Max cps Haloperidol 1 nm in plasma H Cl Time, min +MRM (9 pairs):.928 min from Sample 7 (std.1um) of Test_mix_rat_plasma_23eb21_EL.wiff (Turbo Spray) Max cps / / / / / / / / /165.1 Q1/Q3 Masses, amu

7 Why Mass Accuracy and Stability Matters: arrow Window XIC from Rat Plasma t=15 min 1e+6 8e+5 3 mda L e+5 4e+5 1 mda L e+5 3e+5 4e+5 2e+5 2e+5 1e+5 5e+5 1 mda L e+5 3e+5 2e+5 1e+5 5e+5 5 mda L e+5 3e+5 2e+5 1e+5 5e+5 4e+5 5 mda L e+5 4e+5 1 mda L e+5 3e+5 2e+5 2e+5 1e+5 1e Time (min) Time (min)

8 ull Scan Based Quantitation Standard, 6 compound Mix,.1 24feb21_PKTest_1 1 % H : T MS ES Da Haloperidol 1.1e3 1 nm in plasma XIC 3 mda window Synapt G Standard, 6 compound Mix,.1 24feb21_PKTest_1 149 (1.416) Cl Time 1: T MS ES+ 4.56e4 % m/z

9 Plasma is a Dirty Matrix Standard, 6 compound Mix,.1 24feb21_PKTest_1 15 (1.425) Standard, 6 compound Mix,.1 24feb21_PKTest_1 15 (1.425) ppm 1: T MS ES+ 3.99e4 1: T MS ES+ 884 %! ! % !; ! ! ! m/z m/z

10 Why Resolution Matters: Rat Plasma t=15 min Experimental Data R= ppm M/ΔM = 2 At a resolution <2, WHM the compound will be over quantified due to un-resolved contaminants M/ΔM = 1 Both improved mass resolution and chromatographic resolution are needed for accurate quantification M/ΔM = m/z

11 utline Quads vs HRMS HRMS: Quantitative applicability Quan Qual workflows uture considerations

12 Challenges in Analytical Workflows 1. Perform Quantitative analysis. 2. Increase Method Selectivity. 3. Reduce Method development time. 4. Increase laboratory throughput. Single Reaction Monitoring (ew Generation Triple Quads) Resolving Challenges in Regulated Bioanalysis High Resolution Mass Spectrometer (ew Generation of High Resolution detector)

13 Challenges in Analytical Workflows 1. Quantitative analysis: v Able to reach level of sensitivity given by the SRM. v Good Linearity. 2. Method selectivity: v Can resolve chromatographic interference. v Decrease baseline noise (á signal to noise). 3. Method development: v Reduce method development working days. v Methods (extraction and chromatographic ) simplified. 4. Laboratory throughput: v Decrease total injection runtime. v Decrease total extraction time.

14 Small Molecule - MK-A Clinical method- monoisotope 821, Human plasma, Isocratic, 2x5 mm UPLC, L/L cleanup Expected Conc. o. f Values API 4 API 5 AB 55 AB 56 a % % % % % C.V. Accuracy % C.V. Accuracy % C.V. Accuracy % C.V. Accuracy AB 56 b % % C.V. Accuracy (ng/ml) Used 1 6 of n/a n/a of of of of of of of of Range of Values a - AB 56 T quant with 4 mda width b - AB 56 MRM quant with 4 mda width

15 Small Molecule - MK-A Sensitivity of Detection: MS Vol (ul) Injection Intensity at LLQ Average oise S/ Intensity at ULQ Range of IS Response API x x x1 4 API x x x1 5 AB x x x1 5 AB 56 a x x1 4-7.x1 4 AB 56 b x x1 4-3.x1 4 a - AB 56 T quant with 4 mdalton width b - AB 56 MRM quant with 4 mdalton width

16 Progesterone Quantitation (2.5 nm) API 4 vs Xevo G1.37nM" Sample ID: "" ile: "cal line RG 232.wiff" mu" API 4 2 µl Xevo Q-T.32 1 µl Time, min

17 utline Quads vs HRMS HRMS: Quantitative applicability Quan Qual workflows uture considerations

18 Correlation of Data (3 cpds) from Qtof and QqQ 1 % Parent remaining (data from API) ± 2% range % Parent remaining (data from Xevo)

19 Guiding L CH 3 R1 Chemistry optimisation CH 3 R1 R2 H R2 Extensive -demethylation dramatically increased clearance Extension side-chain minimizes -demethylation

20 Guiding L Monitoring undesired dealkylation concentration 8 6 R RH 4 2 ER-ant ER-ago time (minutes)

21 In vivo Metabolites (4 hr rat plasma time point) RT: Parent M1 M2 M3 M4 S S S S S H H H H H H H H H H H C C RT: 3.1 BP: RT: 2.64 BP: RT: 3.7 BP: RT: 2.8 BP: RT: 2.82 BP: RT: 2.82 BP: RT: 2.99 BP: RT: 3.7 BP: RT: 3.71 BP: RT: 3.78 BP: Time (min) L: 8.3E5 m/z= : TMS + p ESI ull ms [12.-1.] MS ICIS 8416_KB_724_rat2_T4 _1 L: 1.2E6 m/z= : TMS + p ESI ull ms [12.-1.] MS ICIS 8416_KB_724_rat2_T4 _1 L: 9.58E5 m/z= : TMS + p ESI ull ms [12.-1.] MS ICIS 8416_KB_724_rat2_T4 _1 L: 2.8E5 m/z= : TMS + p ESI ull ms [12.-1.] MS ICIS 8416_KB_724_rat2_T4 _1 L: 2.49E5 m/z= : TMS + p ESI ull ms [12.-1.] MS ICIS 8416_KB_724_rat2_T4 _1

22 Metabolite Profiles in Plasma L-873,724 M1 M2 M3 M4 2.5 Peak Area Ratio Time (hr)

23 Multiplexed Analysis Drug Concentration (nm) Time (hr) 1.5 mg 5 mg 12.5 mg 25 mg 5-mg 1-mg 2-mg Biomarker Conc. (nm) Time (hr) 1.5 mg 5 mg 12.5 mg 25 mg 5-mg 1-mg 2-mg Drug Measurements Biomarker Measurements H S H H Metabolite Identification S H H

24 utline Quads vs HRMS HRMS: Quantitative applicability Quan Qual workflows uture considerations

25 uture Drug Development Workflows When compared to a SRM, will HRMS instruments: Resolve chromatographic problems? Can have improved selectivity with HRMS, but will not fix ionization issues Perform bioanalytical assays? Yes Simplify method development time? Yes Be robust and reliable? Yes Generate manageable data files??, yet to be done significant hurdle Enable data processing throughput??, need suitable software

26 uture Challenges HRMS hardware is ready for routine assay support Sensitivity, Linearity, Selectivity are acceptable and improving Software continues to lag hardware How to handle large datasets? Validate data processing steps? Centroid vs. Profile data? Data reduction acceptable or not? Cost vs. Benefit of ownership QQQ s are a commodity with large user base HRMS are more complex with fewer users uture developments in hardware and software will narrow the gap between QQQ and HRMS

27 Ion Mobility with HRMS Waters G2-S using Ion Mobility Additional level of selectivity beyond mass resolving power by using ion mobility Early data looks promising, but much more work is needed Data file size and data processing software will need to be addressed

28 Tamoxifen 2mDa XIC, MS nly, IMS.25 ng/ml.5 ng/ml 1 ng/ml

29 Tamoxifen 2mDa XIC, IMS full scan DT 1-2 (no filter).25 ng/ml.5 ng/ml 1 ng/ml

30 Tamoxifen 2mDa XIC DT filter.25 ng/ml.5 ng/ml 1 ng/ml

31 What if HRMS was developed instead of QQQ technology? ull scan based data acquisition with sensitivity and selectivity equal to today s modern QQQ with fast data processing used routinely in today s bioanalytical labs. What would the discussion be like if QQQ s just came along now? I have to figure out how the compound fragments to analyze it? I need a different MS method for every compound? I have to know what I am looking for before I run my samples? ominal mass? Really? You must be joking? What about all the other things in my samples?

32 EB HRMS