Towards High Resolution MS in Regulated Bioanalysis Benno Ingelse MSD 3 rd EB focus meeting June 12 th 13 th Brussels, Belgium
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
utline Quads vs HRMS HRMS: Quantitative applicability Quan Qual workflows uture considerations
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
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
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. 118. cps. 118 115 11 15 1.93 Haloperidol 1 nm in plasma 95 9 85 8 75 7 65 6 H 55 5 45 4 35 Cl 3 25 2 15 1 5.1.2.3.4.5.6.7.8.9 1. 1.1 1.2 1.3 1.4 1.5 Time, min +MRM (9 pairs):.928 min from Sample 7 (std.1um) of Test_mix_rat_plasma_23eb21_EL.wiff (Turbo Spray) Max. 118. cps. 118 115 11 15 1 95 9 85 8 75 7 65 6 55 5 45 4 35 3 25 2 15 1 5 329.3/161.7 39./25. 296.1/215. 345.1/277. 388.2/19.2 477.3/266.2 386.2/122.1 376.2/165.1 455.4/165.1 Q1/Q3 Masses, amu
Why Mass Accuracy and Stability Matters: arrow Window XIC from Rat Plasma t=15 min 1e+6 8e+5 3 mda L-873724 5e+5 4e+5 1 mda L-873724 6e+5 3e+5 4e+5 2e+5 2e+5 1e+5 5e+5 1 mda L-873724 4e+5 3e+5 2e+5 1e+5 5e+5 5 mda L-873724 4e+5 3e+5 2e+5 1e+5 5e+5 4e+5 5 mda L-873724 5e+5 4e+5 1 mda L-873724 3e+5 3e+5 2e+5 2e+5 1e+5 1e+5 1 2 3 4 5 Time (min) 1 2 3 4 5 Time (min)
ull Scan Based Quantitation Standard, 6 compound Mix,.1 24feb21_PKTest_1 1 % H 1.42 1: T MS ES+ 376.146.Da Haloperidol 1.1e3 1 nm in plasma XIC 3 mda window Synapt G2 1.33 1.63 1.93 1.98.25.5.75 1. 1.25 1.5 1.75 2. Standard, 6 compound Mix,.1 24feb21_PKTest_1 149 (1.416) 1 371.1472 Cl Time 1: T MS ES+ 4.56e4 % 372.1511 551.3395595.3644 265.189 31.741 463.2855 77.3527 874.79 m/z 2 3 4 5 6 7 8 9 1
Plasma is a Dirty Matrix Standard, 6 compound Mix,.1 24feb21_PKTest_1 15 (1.425) 371.1468 1 Standard, 6 compound Mix,.1 24feb21_PKTest_1 15 (1.425) 376.1473 1-1.9 ppm 1: T MS ES+ 3.99e4 1: T MS ES+ 884 %! 375.7216! 376.2263 377.1489 % 375.8494!;375.8943! 376.256 376.532! 377.85 376 377! 377.2366 m/z 31.749 265.1913 372.156 57.3187 551.349 595.3692 874.4479 64.3943 853.4789 875.273 m/z 2 3 4 5 6 7 8 9 1
Why Resolution Matters: Rat Plasma t=15 min Experimental Data 482.1712 R=22-1.53 ppm 481.5 482. 482.5 483. 483.5 M/ΔM = 2 At a resolution <2, WHM the compound will be over quantified due to un-resolved contaminants. 481.5 482. 482.5 483. 483.5 M/ΔM = 1 Both improved mass resolution and chromatographic resolution are needed for accurate quantification. 481.5 482. 482.5 483. 483.5 M/ΔM = 5 481.5 482. 482.5 483. 483.5 m/z
utline Quads vs HRMS HRMS: Quantitative applicability Quan Qual workflows uture considerations
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)
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.
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 6 6. 12 8.7 1 4.2 11 11.7 12 n/a n/a 2.5 6 of 6 2.1 96 3. 99 2.1 99 11. 97 15.8 99 5 6 of 6 2.1 97 2.8 12 1.1 99 6.4 96 8.2 1 1 6 of 6 1.9 99 1.6 11 2.2 11 5. 97 4.3 12 1 6 of 6 1.7 1.8 13 1.2 12 2.9 11 6.5 16 5 6 of 6 1.3 13.9 12.6 14 5. 13 3.8 15 1 6 of 6 1.4 11.8 99.7 1 2.7 11 5.3 99 175 6 of 6 1.4 1 1.2 98 1.9 97 4.1 12 2.7 94 2 6 of 6 1.7 1.7 97 1.4 97 1.6 11 2.7 93 Range of Values 1.3-6. 96-13.7-8.7 97-12.6-4.2 97-12 1.6-11.7 96-13 2.7-15.8 93-16 a - AB 56 T quant with 4 mda width b - AB 56 MRM quant with 4 mda width
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 4 8 6 2 3 8. x 1 5 3.8x1 4-4.8x1 4 API 5 2 85 3 28 7.5 x 1 5 7.5x1 4-1.2x1 5 AB 55 2 125 5 25 2.3 x 1 5 1.3x1 5-1.6x1 5 AB 56 a 8 45 1 5 8. x 1 5 5.x1 4-7.x1 4 AB 56 b 8 42 4 11 7. x 1 3 2.x1 4-3.x1 4 a - AB 56 T quant with 4 mdalton width b - AB 56 MRM quant with 4 mdalton width
Progesterone Quantitation (2.5 nm) API 4 vs Xevo G1.37nM" Sample ID: "" ile: "cal line RG 232.wiff" mu" 58 2.79 56 54 52 API 4 2 µl 5 48 46 44 42 2.2 4 4.34 38 36 34 2.6 32 2.9 3 1.86 3.63 28 1.99 26 24 2.11 2.57 2.68 22 1.93 2.26 2.61 2 18 16 14 Xevo Q-T.32 1 µl 2.55 2.15 2.33 2.36 2.51 1.75 2.44 1.77 2.2 2.72 2.84 3.69 2.89 3.58 3.52 4.6 3.12 3.48 3.75 4.17 4.22 12 1 8 1.4 3.32 4.1 1.47 3.15 3.4 1.6 2.943.4 3.82 3.89 4.38 1.69 3.96 1.35 3.26 1.6 1.41 1.21 1.58 1.65 6.9.39 1.12.6.22.24.97 1.17 4.54.6.72.94 2 1.33.2.4.6.8 1. 1.2 1.4 1.6 1.8 2. 2.2 2.4 2.6 2.8 3. 3.2 3.4 3.6 3.8 4. 4.2 4.4 Time, min
utline Quads vs HRMS HRMS: Quantitative applicability Quan Qual workflows uture considerations
Correlation of Data (3 cpds) from Qtof and QqQ 1 % Parent remaining (data from API) 9 8 7 6 5 4 3 2 ± 2% range 1 2 4 6 8 1 % Parent remaining (data from Xevo)
Guiding L CH 3 R1 Chemistry optimisation CH 3 R1 R2 H R2 Extensive -demethylation dramatically increased clearance Extension side-chain minimizes -demethylation
Guiding L Monitoring undesired dealkylation 14 12 1 concentration 8 6 R RH 4 2 ER-ant ER-ago 5 1 15 2 25 3 35 time (minutes)
In vivo Metabolites (4 hr rat plasma time point) RT:. - 5. 1 8 6 4 2 1 8 6 4 2 1 8 6 4 2 1 8 6 4 2 1 8 6 4 2 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: 482.1715 RT: 2.64 BP: 482.181 RT: 3.7 BP: 482.1764 RT: 2.8 BP: 498.1663 RT: 2.82 BP: 46.1393 RT: 2.82 BP: 442.1287 RT: 2.99 BP: 442.1289 RT: 3.7 BP: 444.1449 RT: 3.71 BP: 498.1568 RT: 3.78 BP: 444.1363..2.4.6.8 1. 1.2 1.4 1.6 1.8 2. 2.2 2.4 2.6 2.8 3. 3.2 3.4 3.6 3.8 4. 4.2 4.4 4.6 4.8 5. Time (min) L: 8.3E5 m/z= 482.164-482.1846 : TMS + p ESI ull ms [12.-1.] MS ICIS 8416_KB_724_rat2_T4 _1 L: 1.2E6 m/z= 498.1544-498.1794 : TMS + p ESI ull ms [12.-1.] MS ICIS 8416_KB_724_rat2_T4 _1 L: 9.58E5 m/z= 46.1285-46.1515 : TMS + p ESI ull ms [12.-1.] MS ICIS 8416_KB_724_rat2_T4 _1 L: 2.8E5 m/z= 442.1183-442.145 : TMS + p ESI ull ms [12.-1.] MS ICIS 8416_KB_724_rat2_T4 _1 L: 2.49E5 m/z= 444.134-444.1562 : TMS + p ESI ull ms [12.-1.] MS ICIS 8416_KB_724_rat2_T4 _1
Metabolite Profiles in Plasma 3.5 3. L-873,724 M1 M2 M3 M4 2.5 Peak Area Ratio 2. 1.5 1..5. 4 8 12 16 2 24 Time (hr)
Multiplexed Analysis Drug Concentration (nm) 1 1 1 1 1 4 8 12 16 2 24 Time (hr) 1.5 mg 5 mg 12.5 mg 25 mg 5-mg 1-mg 2-mg Biomarker Conc. (nm) 1 9 8 7 6 5 4 3 2 1 4 8 12 16 2 24 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
utline Quads vs HRMS HRMS: Quantitative applicability Quan Qual workflows uture considerations
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
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
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
Tamoxifen 2mDa XIC, MS nly, IMS.25 ng/ml.5 ng/ml 1 ng/ml
Tamoxifen 2mDa XIC, IMS full scan DT 1-2 (no filter).25 ng/ml.5 ng/ml 1 ng/ml
Tamoxifen 2mDa XIC + 92-12 DT filter.25 ng/ml.5 ng/ml 1 ng/ml
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?
EB HRMS