26 microsampling tim schlabach - agilent
- 1. Microsampling/DBS Latest Innovations from Agilent Technologies CE Pre Extraction Image Capillary Sheath N2 Liquid Gas CE-MS Chip-MS DBS-MS Agilent Restricted 1
- 2. New Polaris-Chip for highest resolution Stationary Phase Inert polyimide Polaris C18-A, polar embedded phase, 3 袖m, 180 , 200 m2/g 150 mm x 75 袖m ID Enrichment column 1 袖g sample loading Analytical column Sprayer-tip December 12, 2012
- 3. Loading capacity for new Polaris Chip Shown: E.coli TIC and EICs of 500 ng injection superimposed Determined using 9 representative peptides across elution range P1 P2 P3 P4 P5 P6 P7 P8 P9 December 12, 2012
- 4. Agilent CE-ESI-MS Setup MassHunter SW control: - CE instrument - MS instrument - LC makeup flows
- 5. Small anionic molecules in Metabolomics by CE-ToF-MS CE-MS is ideally suited to handle ionic or polar metabolites in complex mixtures reproducibly Page 5
- 6. Dried Blood Spots (DBS) Analysis 袖Lssample mLs sample Stable on card at ambient temp Must store frozen Easy to ship Difficult to ship Non-biohazardous Biohazardous CPSA Shanghai 6 12/12/2012
- 7. An Automated Online Card Extraction LC/MS System for Dried Blood Spots Analysis Robotic Gripper ISTD Dilutor DBS CardRacks Camera Switching Clamp Module Valves CPSA Shanghai 7 12/12/2012
- 8. Agilent Automated Card Extraction (AACE) LC/MS System Flow Schematic Master Control Prolab Card Extration Analytical Column 6460 QQQ Instrument Trap 1 Column 10-Port 6-Port Valve Valve ISTD Loop Trap 2 Column ISTD A B A A B Clamped Waste DBS Card Dilution Pump 1200 Isocratic LC Extraction Pump (1) Analytical Pump (2) 1260 Binary LC 1260 Binary LC CPSA Shanghai 8 12/12/2012
- 9. Reproducible and sensitive DBS/MS system LOQs for all compound 0.5 ng/mL in blood Clozapine Norclozapine Clozapine-N-oxide MRM chromatograms of clozapine, noreclozapine, and clozapine-N-oxide Agilent Restricted 9
- 10. Summary: AACE System vs. Offline DBS Comparable Quantitation Performance Parameters AACE LC/MS System LOQ Range Linearity Accuracy Reproducibility Precision Compound (ng/mL in blood) (ng/mL) (R2) (%) (%) (%) Clozapine 0.5 0.5 10000 0.9996 92.1 106.8 0.61 5.61 4.82 Norclozapine 0.5 0.5 5000 0.9988 85.2 109.9 0.84 6.05 8.94 Clozapine N-oxide 0.5 0.5 - 5000 0.9981 93.2 104.1 0.68 6.15 3.82 Analysis Time ~1 hr Offline Extraction DBS LOQ Range Linearity Accuracy Reproducibility Precision Compound (ng/mL in blood) (ng/mL) (R2) (%) (%) (%) Clozapine 0.5 0.5 10000 0.9997 88.0 103.2 0.61 8.92 4.89 Norclozapine 0.5 0.5 10000 0.9991 89.3 107.1 0.08 8.98 8.36 Clozapine N-oxide 0.5 0.5 - 10000 0.9991 91.8 108.4 0.25 8.66 6.64 Analysis Time ~5 hrs CPSA Shanghai 10 12/12/2012
- 11. DBS Summary fast and fit for purpose Agilent Automated Card Extraction (AACE) Automated flow-through DBS analysis Fully integrated with MassHunter LC/MS Data Processing and Reporting Pre- and post- sampling DBS card images Processed quantitative data Chain of custody Clozapine study Sensitivity: 0.5 ng/mL LLOQ Linear assay over 4 orders of magnitude Comparable results with offline method Time savingss, 1 hour versus 5 hours CPSA Shanghai 11 12/12/2012
Editor's Notes
- #3: Collecting high quality MS/MS produces more peptide matchesKey elements:Efficient recognition of expected isotope patternAccumulate transients long enough to produce a good MS/MSSelect precursor likely to produce good MS/MSImproved chromatography results in sharper peaks which improves sensitivity and reduces the complexity of the DDA as peptides are better resolved. The Polaris C18-A material (from Varian) has proven to be an excellent material and we hope to have this chip available in December.Finally, increasing the sensitivity of the mass spec will always improve protein id on complex proteomics samples. With the 6550, we have increased sensitivity with iFunnel technology and increased our acquisition rate capabilities.
- #4: 03/08/2011Chip DE51G02065 BSA gradient starting at 100% Water or 2.7% water - any difference?Mobile Phase:A: Water (0.1%FA)B: ACN (0.1%FA)Gradient:t 1-%B 2-%B 0.00 2.7 0 0.10 4.5 4.5 2.00 9.0 9.0 30.00 26.8 26.8 35.00 80.0 80 36.00 80.0 80 38.00 2.7 0 Flow0.450 uL/minInjection volume2 ul (IFV 3)Sample: Sample: 250 fmol BSAPressure (init, enrichment mode):CapP (0%B, 3ul/min) = 103 bar NanoP (0%B, 0.26ul/min) = 140 bar Data:D:\\MassHunter\\data\\Polaris\\110804Data evaluation: Skyline to produce list of theoretical trypsin fragments (allowing 1 missed cleavage) = 296 peptides (C:\\8540p 110730\\Steph R&D\\Proteomics\\BSA - Michrom_1missedcleavage_0001_sequence1.xls) >>> EICs either multiple masses in combined in one chromatogram or for exact evaluation in the area up to 7.5 min = RT single EICs
- #9: The example is based on a SCAP DBS System with a binary high pressure gradient pumpfor the extraction, an isocratic pump for the dilution and a binary high pressure gradient pumpfor the chromatographic separation.The SCAP DBS System functions as the master of the analytical setup and samples areanalyzed according to the SCAP DBS Sample List. The SCAP DBS method starts theextraction pump program, the dilution pump program and the analytical LC-MS/MS system.The restart for the next DBS extraction is performed from the extraction pump program whilethe analytical gradient is still running. Thus, the extraction overlaps the analytical run andthereby shortens the cycle time (time duration from one injection until next injection).Two traps increased selectivity from complex matricsremoval of lipids from blood.