Paul D. Rainville Ph.D. Health Sciences Group Waters Corporation Waters Corporation 1

Transcription

1 A new level of sensitivity and standardization: The promise of integrated micro fluidic devices coupled with mass spectrometry for the analysis of biofluids Paul D. Rainville Ph.D. Health Sciences Group Waters Corporation 2015 Waters Corporation 1

2 Current Challenges Personalized and Translational Medicine Biomarker Discovery Multiple Molecule Classes Existing biomarkers that present analytical challenges Biomolecules Low systemic exposure Multiple analysis from a single sample Costs and Changing Environment Investment in innovation Instrument Versatility Operating costs (reagents) 2015 Waters Corporation 2

3 Why micro fluidics coupled with MS? Micro fluidics allows for: Improved Sampling Efficiency by the Mass Spectrometer Improved Ionization Efficiency Reduced Ion Suppression 2015 Waters Corporation 3

4 Improved Sampling Efficiency by the Mass Spectrometer Fine ESI plume Broad diverse ESI plume 0.200µm droplets Nanospray Microspray Electrospray IonSpray 25µm droplets 2015 Waters Corporation 4

5 Enhancement in comparison to a 2.1mm format 75µm ID 150µm ID 1mm ID 300µm ID 2.1mm ID 2015 Waters Corporation 5

6 Perception and Challenges of sub 2.1 mm ID chromatography 2015 Waters Corporation 6

7 Integrated Microfluidics 1.7 µm particles 35 µm ID 2015 Waters Corporation 7

8 Ion Source 2015 Waters Corporation 8

9 Ceramic Microfluidic Fabrication Cut Ceramic Material Fired Wafer Final Diced Part 2015 Waters Corporation 9

10 Ratio of Signal Response, Area (ikey/2.1mm) Signal Response on ionkey/ms compared to a 2.1mm, Peptides AEFVEVTK VLGIDGGEGKEELFR LVNELTEFAK NVNDVIAPAFVK VLYPNDNFFEGK ikey Flow Rate (µl/min) 2015 Waters Corporation 10

11 % % Comparison of Original Analytical Scale and ionkey/ms Method pg/ml teriparatide extracted from human plasma MRM of 4 Channels ES+ 687 > (Teriparatide 6+) 5.00e4 ionkey/ms method S/N:RMS= µl plasma 15 µl injection 45:1 S:N MRM of 4 Channels ES > (Teriparatide 6+) 5.00e original 2.1 mm method S/N:RMS= µl plasma 30 µl injection 11:1 S:N 0 Time Waters Corporation 11

12 Representative QC Statistics Teriparatide QC Concentration (pg/ml) Mean (N=5) Calculated Concentration (pg/ml) SD %CV Mean Accuracy Can I Successfully Adapt a Highly Optimized Analytical Scale Method? Yes! 2015 Waters Corporation 12

13 % % % % Robustness Testing (PPT) First injection After 1000 injections of crashed plasma _WAA176_CD_01 2: MRM of 1 Channel ES > 171 (Dextromethorphan) 9.90e5 1 ul injection Equivalent to 200uL injection on 2.1mm scale! _WAA176_CD_1228 2: MRM of 1 Channel ES > 171 (Dextromethorphan) 6.60e Time Time _WAA176_CD_01 2: MRM of 1 Channel ES > 171 (Dextromethorphan) 8.16e5 5 ul injection _WAA176_CD_ : MRM of 1 Channel ES > 171 (Dextromethorphan) 6.54e5 Equivalent to 1mL injection on 2.1mm scale! Time Time 2015 Waters Corporation 13

14 psi Robustness Testing (PPT) _WAA176_CD_ Last injection nbsm System Pressure Range: First injection Peak Area RSD: 5.5% Time No of plasma injections 2015 Waters Corporation 14

15 Reproducibility of ikey ikey Peptide 1 Peptide 2 Peptide 3 Peptide 4 Peptide AVG RT ST DEV % RSD RT Waters Corporation 15

16 Ion Suppression Infusion Pump SM Column Tee ESI Source TQ-MS LC 2015 Waters Corporation 16

17 Matrix Suppression: Peptide Example (LVNELTEFAK) x -224x 0% Time % Time 150 um ID 2.1 mm ID 2015 Waters Corporation 17

18 Matrix Suppression: Small Molecule (Dextromethorphan) x -24.5x 0% Time % Time um ID 2.1 mm ID 2015 Waters Corporation 18

19 Applications 2015 Waters Corporation 19

20 Oral Fluid for Forensic Assays Many advantages to the use of oral fluid samples compared with other matrices Convenience, immediacy of sample collection o Reduced sample collection overheads e.g., no requirement for specifically-trained staff (c.f. blood) Observed collection - reduced opportunity for specimen adulteration o No requirement for gender-specific staff for collection (c.f. urine) Relatively clean matrix however collection devices used will influence the specimen for analysis For some drugs, concentrations in oral fluid parallel those in blood: o o Can provide better indication of current impairment (c.f urine) Shorter detection window Can better indicate recent consumption (c.f. urine) 2015 Waters Corporation 20

21 2015 Waters Corporation 21

22 Experimental Conditions Mobile phase A: Water, 0.1% Formic Acid Mobile phase B: Acetonitrile, 0.1% Formic Acid Analytical Flow rate: 3 µl/min Trapping Flow rate: 15 µl/min Trapping Time: 1.0 min Analytical Gradient: 5% B to 45% B in 6 min., 45% B to 85 %B in 2 min, equilibrate at 5% for 3 min. Trapping Gradient: 0.5% B Weak needle wash: Water, 0.1% Formic Acid Strong needle wash: ACN, 0.1% Formic Acid Column Temp: 30⁰C, Trap Temp: 25⁰C 5 ul Full Loop Column Chemistry: HSS T3, 130Å, 1.7 µm Trap Chemistry: HSS T3, 130Å, 5.0 µm Column Length:10 cm, Trapping Column Length: 5 cm 2015 Waters Corporation 22

23 % % Opioid Separations in Oral Fluid 1:Oxymorphone-3b-D-glucuronide 2:Morphine-3b-D glucuronide 3: Morphine-6b-D-glucuronide 4: Hydromorphone-3b-Dglucuronide 5: Morphine 6: Codeine-6b-D-glucuronide 7: Dihydrocodeine 8: Oxymorphone 9: Hydrocodone 10: Oxycodone 11: 6-Acetylmorphone 12: O-desmethyl Tramadol 13: Codeine 14: Norbuprenorphine-glucuronide 15: Norfentanyl 16: Tramadol 17: Normeperedine 18: Meperidine 19: buprenorphine-glucuronide 20: Norbuprenorphine 21: Fentanyl 22: Buprenorphine 23: EDDP+ 24: Propoxyphene 25: Methadone Opioid Pre-spike, 5to45%Bin10mins@3uLmin, 30C, Opioid Pre-spike, 5to45%Bin10mins@3uLmin, 30C, 5ul loop FL, 3.5 kv, 150 x 100 HSS, HSS T3 Trap Time Waters Corporation Time pg on-column

24 % Morphine Sensitivity Comparison Morphine Opioid Pre-spike, 30C, 5ul loop FL, 3.5 kv, 150 x 100 HSS, HSS T _2pt1_Opioids Pre Spike_1 7: MRM of 2 Channels ES+ 100 TIC (Morphine) 1.12e8 ionkey/ms 2.1 mm ACQUITY LOQ: 81.6 pg/ml 10.2 X ionkey/ms LOQ: 8.0 pg/ml 2.1 mm ACQUITY 0 Time Waters Corporation 24

25 Response Morphine spiked into oral fluid Morphine- Oral Fluid Matrix Calibration Curve 100 pg/ml Morphine Oral Fluid Matrix MRM Compound name: Morphine Correlation coefficient: r = , r^2 = Calibration curve: * x Response type: External Std, Area Curve type: Linear, Origin: Exclude, Weighting: 1/x, Axis trans: None _Trapping_ Morphine_Calibration Curve_ Morphine * F7:MRM of 2 channels,es > , 286.1> e LOQ: 13 pg/ml R 2 = 0.99 % S/N: 78.2 Peak-Peak Ignoring Zeros No Extra Processing Morphine * Conc min 2015 Waters Corporation 25

26 Alzheimer Disease A substantial effort in the characterization of Alzheimer Disease (AD) biomarkers for clinical diagnosis and for the development of new drugs. Work has been directed towards two biomarkers: Plaque forming A Beta peptide Microtubule binding Tau peptide CRsQSrtIGYBQ&ved=0CAcQ_AUoAg#imgrc=y0l8FYbCy6f7OM%253A%3Bcz2Ck8LW6O3alM%3Bhttp%253A%252F%252Fwww.natur e.com%252fnature%252fjournal%252fv461%252fn7266%252fimages%252f461895af1.2.jpg%3bhttp%253a%252f%252fwww.nature.com%252fnature%252fjournal%252fv461%252fn7266%252ffig_tab%252f a_F1.html%3B800%3B573 FOR RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC PROCEDURES 2015 Waters Corporation 26

27 Tau Quantification by microflow LC/MS Work flow FOR RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC PROCEDURES 2015 Waters Corporation 27

28 Data from Study FOR RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC PROCEDURES 2015 Waters Corporation 28

29 Diabetes The American Centers for Disease Control and Prevention (CDC) has just declared that we are facing a diabetes epidemic. More than 285 million people across the world suffer from diabetes, 90% of whom have Type 2 Diabetes. It is estimated that by the year million people will be inflicted with diabetes worldwide. In the United States, CDC projects that by 2050 one in three individuals will be diagnosed with diabetes. In Europe, there are 55 million diabetics, and by 2030 this number is expected to grow to 66 million. Russia and Germany lead in European diabetes prevalence with 9.6 million and 7.5 million diabetics respectively. Diabetes is also becoming a major health concern in Asia with over 90 million diabetics in China and 50 million in India. Available at: FOR RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC PROCEDURES 2015 Waters Corporation 29

30 GLP 1 Quantification by immunoaffinity and microflow LC/MS GLP 1 is a hormone peptide derived from the proglucagon gene and is translated into several different peptides. Measurement of GLP-1 is critical for the development of antidiabeteic compounds as the the ability to extend the length of their biological effect is an indication of the effect of DPP-IV inhibitors a class of antihyperglycemic compounds for Type 2 diabetes. FOR RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC PROCEDURES 2015 Waters Corporation 30

31 Data from Study FOR RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC PROCEDURES 2015 Waters Corporation 31

32 Summary Ceramic-based microfluidics provides a reproducible and robust platform for the analysis of targeted and untargeted analytes prepared from biological fluids. The application of ceramic-based microfluidics coupled with mass spectrometry had been applied successfully to a number of analyte classes in various biofluids. The lower operating flow rate of microfluidics may reduce ion suppression originating from co-eluting interferences Waters Corporation 32

33 Thank You Waters Corporation Greg Roman Michelle Wood Rob Lee Jim Murphy Jay Johnson JD Loughlin Erin Chambers Mary Lame Kendon Graham All of our ionkey/ms collaborators and customers And the audience for their time 2015 Waters Corporation 33