Agilent Technologies. Technological Innovation and Growth of Liquid Chromatography Mass Spectrometry for Research in the Clinical Laboratory

Transcription

1 Agilent Technologies Technological Innovation and Growth of Liquid Chromatography Mass Spectrometry for Research in the Clinical Laboratory AACC 2013 Mass Spectrometry in the Clinical Laboratory: Best Practices and Current Applications Sept 17-18, St. Louis, MO 1

2 Outline Summary Triple Quadrupole Mass Spectrometry LC/MS/MS use has seen incredible success and growth in the last decade for a wide range of small molecules clinical research. New and innovative technologies/methodologies spanning the entire sample analysis workflow -- improving sensitivity, specificity, and throughput -- will always be introduced. The promise of biomarker research is still unfulfilled but successes in targeted proteomics demonstrate that the next clinical frontier is here now and will continue to grow with LC/MS/MS. 2

3 Introduction Tools for Design and Development Customer Examples Large Molecule Research Summary

4 Method HW/SW Certified LC/MS Research in a Clinical Lab Chemistries Finance Support

5 Cost Certification TAT Expertise Capability

6 Why Do Laboratories Adopt LC/MS? High Specificity High Sensitivity Wide dynamic range Can measure multiple analytes in one analysis Easier method development Solid economical and financial benefit

7 Introduction Tools for Design and Development Customer Examples Large Molecule Research Summary

8 LCMS Laboratory Workflow Design & Development Sample Prep Analysis Reporting Target Selection Define Performance Goals Operational & Regulatory Requirements Transfer Research Component

9 Analytical Methods in the Library REFERENCES FOR VARIOUS ANALYTES OF INTEREST Acylycarnitines Aldosterone Alpha-OH trizolam Alprazolam Amino Acids Amphetamine Androstenedione Clonazepam Codeine Corticosterone Cortisol Cyclosporine 11-deoxycortisol DHEA Diazepam DHEAS Dihydrocodeine EDDP Estradiol Estriol Estrone Everolimus Fentanyl Flunitrazepam Flurazepam Hydrocodone Hydromorphone Lorazepam Methamphetamine Methylmalonic Acid Midazolam Mycophenolic Acid Oxycodone 17-OH progesterone Propoxyphene Sirolimus Succinylacetone Tacrolimus Temazepam Testosterone Trizolam 25-OH Vitamin D2 and D3

10 Analytical Methods: Multiplex Application for Steroid Research Comprehensive and Robust LC/MS/MS Analysis Confident analysis of 13 steroids in less than 8 minutes Range of quantitation from pg/ml to ng/ml MS/MS specificity = No Cross Reactivity Confident analysis of over 65 drugs in less than 6 minutes Range of quantitation from 100 pg/ml to over 500 ng/ml in urine Short cycle times allow quick turnaround for confirmatory testing Amenable to automation for higher throughput Cost effective

11 Improving Throughput: Parallel LC injections Improving MS productivity Stream 2 Injection 2 Injection 4 Stream 1 Injection 1 MS idle time MS MS idle idle time time Injection 3 Injection 5 Injection MS Analysis Up to twice the number of analyses in the same time! 11

12 Introduction Tools for Design and Development Customer Examples Large Molecule Research Summary

13 High Productivity LC/MS Analyses for Clinical Research Laboratories Cory Bystrom

14 Data quality 4 ng/ml patient sample Injection 38 2 ng/ml patient sample Injection 1284

15 Improving Throughput: Ultra-Fast SPE-MS MS analysis in Seconds LC/MS RF/MS Run Assay Run Assay 1-5 min/sample Data Acquisition Data Acquisition Data Analysis 7-13 sec/sample Data Analysis Faster speed to results Increased analysis capacity 15

16 RapidFire Methods Step Lamotrigine Levetiracetam OMHC Felbamate Voriconazole Inject 600 ms 600 ms 600 ms 600 ms 600 ms Load 3500 ms 3500 ms 3500 ms 3500 ms 3500 ms Wash 0 ms 0 ms 0 ms 0 ms 0 ms Elute 3500 ms 4000 ms 4000 ms 4000 ms 3500 ms Equil 500 ms 1000 ms 1000 ms 500 ms 1000 ms Total (sec): (x2) = MFMER slide-16

17 Days Turn Around Time Comparison: Average TAT Before Average TAT After Lamotrigine Levetiracetam OMHC Felbamate Voriconizole 2013 MFMER slide-17

18 Introduction Tools for Design and Development Customer Examples Large Molecule Research Summary

19 What about protein and peptides as biomarkers? Similar challenges to small molecules Sensitivity Specificity Throughput Reproducibility Sample Preparation Page 19

20 SISCAPA: Enrich Target Peptides and Decrease Sample Complexity 20

21 L/H (fwd) or H/L (rev) Area Ratio L/H (fwd) or H/L (rev) Area Ratio Comparison of Standard Flow Ion Funnel QQQ-MS to Nanoflow QQQ-MS Mesothelin forward and reverse curves (log/log) 100 Agilent 6490 (400ul/min) + Bravo 100 AB 4000 Qtrap (300nl/min) + Kingfisher 10 Bravo1-Forward-Meso Bravo1-Reverse-Meso 10 Meso:Xlink:Reverse Meso:Xlink:Forward 1 Endogenous level: 3fmol/10ul = 16ng/ml 1 Endogenous level: 3fmol/10ul = 16ng/ml fmol Spiked Varying Peptide Standard flow ion funnel QQQ-MS fmol Spiked Varying Peptide Nanoflow QQQ-MS Forward curve: IS=heavy, standard addition above endogenous level with light Reverse curve: IS=light, heavy peptide spiked in at known levels (no endogenous of heavy) AB 4000 Qtrap data courtesy of Dr. Leigh Anderson Edit: added disclaimer that AB data is from Dr Leigh Anderson 21

22 Reproducibility for 110 Injections (10 fmol SIS Peptides and 2.5 µg Plasma Digest On-column) Protein Adiponectin: IFYNQQNHYDGSTGK Antithrombin-III : DDLYVSDAFHK Apolipoprotein A-II precursor: SPELQAEAK Apolipoprotein C-III: GWVTDGFSSLK Ceruloplasmin : EYTDASFTNR Heparin cofactor II: TLEAQLTPR Histidine-rich glycoprotein: DGYLFQLLR Kininogen-1: TVGSDTFYSFK L-selectin: AEIEYLEK Plasminogen: LFLEPTR Vitamin D-binding protein: THLPEVFLSK von Willebrand Factor: ILAGPAGDSNVVK Response %RSD Ret. Time %RSD Plasminogen LFLEPTR 7.9% RSD, n=4 12.3% RSD, n=4 2.2% RSD n= % RSD, n=4 The samples were provided by Derek Smith and Christoph H. Borchers from the UVic-Genome BC Proteomics Centre 22 February 12, 2012

23 Increasing Throughput for SISCAPA With Faster Chromatography 20 min 3 min 9 min 23 February 12, 2012

24 Introduction Tools for Design and Development Customer Examples Large Molecule Research Summary

25 Summary Triple Quadrupole Mass Spectrometry LC/MS/MS has seen incredible success and growth in the last decade or so for a wide range of small molecules clinical research. New and innovative technologies/methodologies spanning the entire sample analysis workflow -- improving sensitivity, specificity, and throughput -- will always be introduced. The promise of biomarker research is still unfulfilled but successes in targeted proteomics demonstrate that the next clinical frontier is here now and will continue to grow with LC/MS/MS. 25

26 Thank you! Chemistry and Toxicology Clinical & Translational Research 1200 Infinity LC 7700 ICP-MS 6400 Triple Quadrupole LC/MS Multiple Affinity Removal Kits Bravo RapidFire Mass Spec HT Front End 5977 Single Quadrupole GCMS Molecular Research HPLC Columns HPLC-Chips 7000 Triple Quadrupole GCMS Agilent Technologies: Your Partner in Clinical Research 6500 QTOF LCMS Anatomic & Molecular Pathology Halo and SureSelect NGS Target Enrichment & SureCall Software SurePrint CGH + SNP Microarrays & Cytogenomics Software Dako Omnis Antibodies qpcr FISH probes Hybridizer Kits and Reagents