Determination of Urinary Catecholamines and Metanephrines in a single run Analysis by offline SPE and LC-MS/MS for Clinical Research Linda Côté Senior Clinical Application Specialist Agilent Technologies
Objectives In this presentation, we will be discussing: A four minute method for quantifying catecholamines and their metabolites: epinephrine, norepinephrine, dopamine, metanephrine normetanephrine and 3-methoxytyramine The use of offline solid phase extraction (SPE) for simultaneous extraction of all six analytes from urine The chromatographic separation of all six analytes with conditions compatible with LC-MS/MS Typical method performance results Tips and tricks
Pathways of catecholamine synthesis and O-methylation 5 by Endocrine Society Eisenhofer G et al. JCEM 5;9:68-75 3
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Which ones and in which matrix urine or plasma? Free catecholamines and metanephrines in urine Total catecholamines and metanephrines in urine Free catecholamines in plasma Free metanephrines in plasma VMA in urine HVA in urine 5-HIAA in urine 6
Which ones and in which matrix urine or plasma? Free catecholamines and metanephrines in urine Total catecholamines and metanephrines in urine Free catecholamines in plasma (in progress) Free metanephrines in plasma (in progress) VMA in urine (in progress) HVA in urine (in progress) 5-HIAA in urine (in progress) 7
Compound structures HO HO OH HO OH HO NH HO NH HO HN CH 3 Dopamine (D) C8HNO Neutral Mass: 53.8 Norepinephrine (NE) C8HNO3 Neutral Mass: 69.7 Epinephrine (E) C9H3NO3 Neutral Mass: 83.9 CH 3 CH 3 OH CH 3 OH O O O HO NH HO NH HO HN CH 3 3-Methoxytyramine (3-MT) C9H3NO Neutral Mass: 67.9 Normetanephrine (NMN) C9H3NO3 Neutral Mass: 83.9 Metanephrine (MN) CH5NO3 Neutral Mass: 97. 8
Product Ion Scans (MS/MS) D 3-MT-HO NE NMN-HO E MN-HO 9
Product Ion Scans (MS/MS) Must separate E and NMN by chromatography D 3-MT-HO NE NMN-HO E MN-HO
Product Ion Scans (MS/MS) Must separate 3-MT and MN by chromatography D 3-MT-HO NE NMN-HO E MN-HO
Final Method Gradient to Include 3-MT Need to keep resolution between E and NMN Also between MN and 3-MT
Internal Standards Analyte Dopamine Norepinephrine Epinephrine 3-Methoxytyramine Normetanephrine Metanephrine Internal Standard Dopamine-D4 Norepinephrine-D6 Epinephrine-D6 3-Methoxytyramine-D4 Normetanephrine-D3 Metanephrine-D3 3
Sample preparation Calibrators are prepared with clean urine matrix from Golden West Biologicals Isotopically labelled Internal standards 4 hours collection of urine Native urine for free catecholamines and metabolites (typically for catecholamines) Acid-hydrolysed urine for total (typically for metanephrines) Solid phase extraction (SPE) is used to cleanup urine 4
Solid Phase Extraction (SPE) 5
Diphenyl boronate-catecholamine complex The diphenyl boronate forms a stable negatively charged complex (Fig. ) with cishydroxyl groups of catecholamines, which is strongly retained on a C8 extraction sorbent when operating in alkali media. This allows for column washing with methanol-buffer solutions to remove interfering compounds without the loss of the catecholamines which are eluted by disrupting the complex under acid conditions. [] Talwar et al., Journal of Chromatography B, 769 () 34 349 6
Catecholamines and metanephrines in a single run - One SPE cartridge is used to recover both catecholamines and metanephrines - SPE Bond Elut Plexa was chosen for best recoveries - Simple acid elution for direct injection into LC-MS/MS - ph control for stabilization of catecholamines - Metanephrines are also retained under the same conditions, even though they are methylated and do not contain the cis-diol moiety for the covalent linkage binding mechanism - Studies have shown that metanephrines do have some affinity for this sorbent [] Ann Clin Biochem 9; 46: 9 36. DOI:.58/acb.8.88 7
Bond Elut Plexa A Unique Polar Enhanced Polymeric Sorbent Going thru the Plexa cartridge Unique chemistry with Polarity gradient: Hydrophilic OH ligands on the exterior Frequency of OH is reduced as getting inside Hydrophobic deep inside the pore Plexa particle Frequency of OH oligomers is reduced 8
Bond Elut Plexa How does it works Apply Sample Washing Elution Water Rich Hydrophilic component allows excellent phase transfer of analytes into the polymer core Analytes remain tightly bound in the hydrophobic core. Specially engineered pore structure allows excellent mass transfer out of polymer Analyte of interest Pores Hydrophobic core Large endogenous proteins do not bind to the surface of the polymer and cannot access pore structure. Interferences ( lipids and proteins) washed away without into the analytes of interest Clean extract with high recovery
Sample Preparation Solid Phase Extraction (SPE) Prepare complexed samples:.5 ml urine*, calibrators, QCs* Add 4 µl of internal standards mix Add.8 ml of complexing agent Verify ph, must be between 7.5-9.5. If necessary adjust to ph 8.5 with NH4OH Step : Step : Step 3: Step 4: Condition SPE cartridge (Bond Elut Plexa, 3 mg, 3 ml) with: ml of MeOH ml of wash buffer. M NH4Cl-NH4OH Add complexed samples Wash with ml of 5% MeOH wash buffer. M NH4Cl-NH4OH Dry at full vacuum for 5 minutes Elute with ml of 5% formic acid in water. Apply vacuum 5 Hg for 3 seconds Transfer to autosampler vial * Native for free catecholamines, hydrolyzed for total metanephrines (add 5 µl HCl 6N, incubate at 9 deg. C for 5 min., cool at RT) [] Ann Clin Biochem 9; 46: 9 36. DOI:.58/acb.8.88 [] Talwar et al., Journal of Chromatography B, 769 () 34 349
Four Minute Method LC Conditions Agilent 9 LC Analytical Column: Pursuit 3 PFP, x 5 mm, 3 µm Guard Column: Pursuit 3 PFP MetaGuard mm Columns Temp: 4 C Injection volume: µl Needle Wash: ::: MeOH:ACN:IPA:H O +.% formic acid ( sec) Injector Temp: 4 C Mobile Phase: Flow rate: 9 Pump Gradient: Stop time: Re-equilibration time: A:.% Formic Acid in Water B: Methanol.3 ml/min. Time (min) %B..5.5 6 4. 6 4 min. 3 min. (use automated column regeneration for increased throughput)
Automated Column Regeneration (ACR) For increased throughput Position (Port > ) Position (Port > ) Pump Column A Pump Column A ALS Waste ALS Waste 9 3 9 3 8 4 8 4 Pump 7 6 5 Pump 7 6 5 Column B Column B Mass Spec Mass Spec
QQQ MS/MS Method Conditions Agilent 646 QQQ Mass Spectrometer Agilent 646 QQQ MS/MS Ion Source: Agilent Jet Stream (AJS) Ion Mode: Positive Ionization Ion source conditions Drying gas temperature: 35 C Drying gas flow: 5 L/min Nebulizer pressure: 35 psi Sheath gas temperature: 375 C Sheath gas flow: L/min Capillary voltage: 3 V Nozzle voltage: V Q/Q3 resolution:.7 unit EMV V 3
MRM Transitions Table Compound Prec Ion Prod Ion Dwell Frag (V) CE (V) CAV (V) Dopamine* 54. 37. 75 8 3 Dopamine 54. 9. 75 8 3 Dopamine-D4 58. 4. 75 8 3 Norepinephrine* 7. 5. 65 4 5 Norepinephrine 7. 7 65 5 Norepinephrine-D6 76. 58. 65 4 5 Epinephrine* 84. 66. 7 8 5 Epinephrine 84. 7. 7 4 5 Epinephrine-D6 9. 7. 7 8 5 3-Methoxytyramine* 5. 9. 35 3 3-Methoxytyramine 5. 9 35 3 3-Methoxytyramine-D4 55. 95. 35 4 3 Normetanephrine* 66. 34 5 6 3 Normetanephrine 66. 6. 5 3 Normetanephrine-D3 69. 37. 5 6 3 Metanephrine* 8. 65. 6 5 Metanephrine 8. 48. 6 5 Metanephrine-D3 83. 68. 6 5 * = Quantification transition 4
Example chromatogram 6. Norepinephrine. Epinephrine 3. Normetanephrine 4. Dopamine 5. Metanephrine 6. 3-Methoxytyramine 3 4 5 5
Example chromatogram E/NMN and MN/3-MT Resolution is Critical 6. Norepinephrine. Epinephrine 3. Normetanephrine 4. Dopamine 5. Metanephrine 6. 3-Methoxytyramine 3 4 5 6
Calibration curves 7
Calibration curves in log scales Relative Responses. Dopamine y =.3 * x - 7.6E-4 Relative Responses. Norepinephrine y =. * x - 9.63E-5 Relative Responses. Epinephrine y =.3 * x - 3.78E-4 Concentration (ng/ml) Concentration (ng/ml) Concentration (ng/ml) x 3-Methoxytyramine-HO y =.5 * x +.4 x Normetanephrine-HO y =.4 * x - 4.74E-4 x Metanephrine-HO y =.3 * x -. Relative Responses. Relative Responses. Relative Responses.. Concentration (ng/ml) Concentration (ng/ml) Concentration (ng/ml) 8
Results Summary of Analyte Performance Compound R Concentration Concentration Accuracy (%) Intraday CV (%) Interday CV (%) (ng/ml) (nmol/l) n = 3 n = 3 n = 5 Dopamine.9997.56. 7.5..7 6.5 48. 99..7. 658.3.3..3 Norepinephrine.9999.56 9..9.9 5.4 6.5 369.4. 3.5 4. 59.9..6.6 Epinephrine.9998.56 8.5.6 4.3.7 6.5 34..9.5. 5458.4.3.4.3 Note: Signal to noise ratios and CVs indicate that LLOQs are lower than measured here for all analytes 9
Results Summary of Analyte Performance Compound R Concentration Concentration Accuracy (%) Intraday CV (%) Interday CV (%) (ng/ml) (nmol/l) n = 3 n = 3 n = 5 3-Methoxytyramine.9999 4.69 8 95.7. 3.6 87.5.4.9.9. 3 794....3 Normetanephrine.9999 4.69 5.6..5 3. 87.5 3.45...5 3 6375..7.. Metanephrine.9999 4.69 3.8.5.3.8 87.5 95.7..5. 3 5.6.8.. Note: Signal to noise ratios and CVs indicate that LLOQs are lower than measured here for all analytes 3
Results Inter-run Over 3 Days for Commercial QC (BioRad Lyphocheck) Level Level Compound Free/Total Range (HPLC) Measured CV (%) Range (HPLC) Measured CV (%) Dopamine Free 44.4 75. 6.4 3.4 377 69 59.8 Norepinephrine Free 3.3 5.6 38.4 5.8 56 39 9 4.8 Epinephrine Free 9.6 9. 4.3 5.3 67.8 4 86.7 3 Methoxytyramine Total 8.6 48.7 44.7 3.8 38 57 557.7. Normetanephrine Total 366 3.7.4 84 63 379..8 Metanephrine Total 69. 6 9. 434 655 6.5 All measurements are in ng/ml Bio-Rad QC material was used. Ranges provided were for free catecholamines and total metanephrines 3
Results Inter-run Over 3 Days for Commercial QC (BioRad Lyphocheck) Level Level Compound Free/Total Range (HPLC) Measured CV (%) Range (HPLC) Measured CV (%) Dopamine Free 9 49 4 3.4 465 45 333.8 Norepinephrine Free 85 35 7 5.8 9 4 35 4.8 Epinephrine Free 5.5 4 78 5.3 37 57 473 3 Methoxytyramine Total 7 9 67 3.8 8 34 3335. Normetanephrine Total 64.4 59 89 758.8 Metanephrine Total 35 59 46 33 33.5 All measurements are in nmol/l Bio-Rad QC material was used. Ranges provided were for free catecholamines and total metanephrines 3
Example chromatogram Dopamine and Dopamine-D4 Calibration 6.5 ng/ml QC Level QC Level Target Quant x 4 8 7 6 5 4 3 + MRM (54. -> 37.) 5_Cal5_S.96 x 4 5 4 3 + MRM (54. -> 37.) 44_QC_S.96 x 5 4.5 4 3.5 3.5.5.5 + MRM (54. -> 37.) 45_QC_S.96 x 5 4.5 4 + MRM (58. -> 4.) 5_Cal5_S.947 x 5 3 + MRM (58. -> 4.) 44_QC_S.947 x 5 3.5 3 + MRM (58. -> 4.) 45_QC_S.947 ISTD 3.5 3.5.5.5.5.5.5.5.5.5.5.5.5 3 3.5 Counts vs. Acquisition Time (min).5.5.5 3 3.5 Counts vs. Acquisition Time (min).5.5.5 3 3.5 Counts vs. Acquisition Time (min) 33
Example chromatogram Epinephrine and Epinephrine-D6 Separation from Normetanephrine and other interference is critical Calibration 6.5 ng/ml QC Level QC Level x 4 7 + MRM (84. -> 66.) 5_Cal5_S.75 x 4.6 + MRM (84. -> 66.) 44_QC_S x 4 8 + MRM (84. -> 66.) 45_QC_S 6.4 7 5. 6 Target Quant 4 3.8.6.4. *.57 5 4 3 *.63 ISTD x 5 3.5 3.5.5 + MRM (9. -> 7.) 5_Cal5_S.38 x 5.6.4..8.6 + MRM (9. -> 7.) 44_QC_S.44 x 5.4..8.6 + MRM (9. -> 7.) 45_QC_S.44.4.4.5...5.5.5 3 3.5 Counts vs. Acquisition Time (min).5.5.5 3 3.5 Counts vs. Acquisition Time (min).5.5.5 3 3.5 Counts vs. Acquisition Time (min) 34
Example chromatogram Norepinephrine and Norepinephrine-D6 Separation from interferences is critical Calibration 6.5 ng/ml QC Level QC Level Target Quant ISTD x 3 8 7 6 5 4 3 x 4 6 5 4 3 + MRM (7. -> 5.) 5_Cal5_.768 + MRM (76. -> 58.) 5_Cal5_.767.5.5.5 3 3.5 Counts vs. Acquisition Time (min) x 3..8.6.4..8.6.4. x 4..8.6.4. + MRM (7. -> 5.) 44_QC_ *.87 + MRM (76. -> 58.) 44_QC_ *.8.5.5.5 3 3.5 Counts vs. Acquisition Time (min) x 3 4 3.5 3.5.5.5 x 4.9.8.7.6.5.4.3.. + MRM (7. -> 5.) 45_QC_ *.83 + MRM (76. -> 58.) 45_QC_ *.86.5.5.5 3 3.5 Counts vs. Acquisition Time (min) 35
Example chromatogram 3-Methoxytyramine and 3-Methoxytyramine-D4 Target Quant x 5 Separation from Metanephrine is critical Calibration 87.5 ng/ml QC Level QC Level.5.5.5 + MRM (5. -> 9.) 5_Cal5_SP 3.38 x 4..8.6.4. + MRM (5. -> 9.) 44_QC_SP 3.38 x 5 3.5 3.5.5.5 + MRM (5. -> 9.) 45_QC_SP 3.3 ISTD x 5.5.5.5 + MRM (55. -> 95.) 5_Cal5_SP 3.3 x 5.6.4..8.6.4. + MRM (55. -> 95.) 44_QC_SP 3.3 x 5..8.6.4. + MRM (55. -> 95.) 45_QC_SP 3.3.5.5.5 3 3.5 Counts vs. Acquisition Time (min).5.5.5 3 3.5 Counts vs. Acquisition Time (min).5.5.5 3 3.5 Counts vs. Acquisition Time (min) 36
Example chromatogram Normetanephrine and Normetanephrine-D3 Calibration 87.5 ng/ml QC Level QC Level Target Quant x 4 5 4.5 4 3.5 3.5.5.5 + MRM (66. -> 34.) 5_Cal5_S.68 x 4 4 3.5 3.5.5.5 + MRM (66. -> 34.) 44_QC_S.634 x 5.8.6.4..8.6.4. + MRM (66. -> 34.) 45_QC_S.68 x 4 7 + MRM (69. -> 37.) 5_Cal5_S.65 x 4 + MRM (69. -> 37.) 44_QC_S.6 x 4 4 + MRM (69. -> 37.) 45_QC_S.65 6 3.5 3.5 ISTD 5 4 3 3.5.5 3.5.5.5.5.5.5.5 3 3.5 Counts vs. Acquisition Time (min).5.5.5 3 3.5 Counts vs. Acquisition Time (min).5.5.5 3 3.5 Counts vs. Acquisition Time (min) 37
Example chromatogram Metanephrine and Metanephrine-D3 Separation from interference is critical Calibration 87.5 ng/ml QC Level QC Level Target Quant x 5.9.8.7.6.5.4.3.. + MRM (8. -> 65.) 5_Cal5_S 3.4 x 4.5.5.5 + MRM (8. -> 65.) 44_QC_S 3.4 x 5.8.6.4..8.6.4. + MRM (8. -> 65.) 45_QC_S 3.4 ISTD x 5.6.4..8.6.4. + MRM (83. -> 68.) 5_Cal5_S 3.4.5.5.5 3 3.5 Counts vs. Acquisition Time (min) x 5..8.6.4. + MRM (83. -> 68.) 44_QC_S 3.4.5.5.5 3 3.5 Counts vs. Acquisition Time (min) x 5.9.8.7.6.5.4.3.. + MRM (83. -> 68.) 45_QC_S 3.4.5.5.5 3 3.5 Counts vs. Acquisition Time (min) 38
Results Recoveries Observed Using SPE Procedure Compound Absolute Recoveries %* (n = 9) Relative recoveries % With ISTDs corrections** (n = 9) Average SD Range Average SD Dopamine 73.5.4 95.-3.4..5 Norepinephrine.5 4.9 99.-.4.. Epinephrine 9.3 3.6 94.5-4.4..9 3-Methoxytyramine 53. 3.6 94.4-.5. 3. Normetanephrine 88.7 7.5 97.3-... Metanephrine 93.9 3.6 97.-3.9.. * ISTDs peak areas spiked in formic acid subjected to SPE compared with spiked formic acid without SPE ** Calculated concentrations with ISTDs peak area ratios corrections (with SPE) versus theoretical concentrations 39
Results Matrix Effects Observed Using SPE Procedure Compound Matrix effects %* (n = 9) Accuracies % With ISTDs corrections** (n = 9) Average SD Range Average SD Dopamine. 3.9 95.4-.5..3 Norepinephrine 3. 5. 94.7-4.4. 3.6 Epinephrine 7. 3. 95.-4.6. 3.7 3-Methoxytyramine 88. 8.5 95.4-4.. 3. Normetanephrine 93.8 3. 96.4-.8..8 Metanephrine 3..3 95.3-.3..8 * ISTDs peak areas spiked in urine subjected to SPE compared with spiked formic acid subjected to SPE ** Calculated concentrations with ISTD corrections (urine with SPE) versus theoretical concentrations 4
Stability of extracted spiked urine 8 hours in Autosampler at 6⁰C Calibration Levels Stability in Autosampler 8 hours at 6 ºC % Difference D NE E 3-MT NMN MN.7-4. -. -. 4.7.4.8 -.8.7.5.4.5 3. -.8. -.4.3.4 4 -. -4.6.9 -. -.8.3 5 -.7 -.9.7 -... 6 -.8.6..6.9 -. 7 -.4 -... -.5 -. 8. -.5.9. -.5 -.6 9 -. -.3.7 -.7 -.6 -.8 4
Stability of Extracted Spiked Urine Urine Extracts Instability Urine extracts kept at 4⁰C x 4 8 7 6 5 4 3 x 4 6 5 4 Cpd 5: Norepinephrine-D6.758: + MRM (76. -> 58.) 7_Cal5_SPE7_Urine_Gr-6_AJS_Cleaned-col.d.758.9 Norepinephrine-D6 extracted on Oc. th and injected on Oc. 5 th Cpd 5: Norepinephrine-D6.885: + MRM (76. -> 58.) 7_Cal5_SPE7_Urine_Gr-6_AJS_Cleaned-col_MG.d.885.55 Norepinephrine-D6 extracted on Oc. th and injected on Oc. 3 th 3..4.6.8..4.6.8..4.6.8 3 3. 3.4 3.6 3.8 Counts vs. Acquisition Time (min) 4
Tips and Tricks Mobile phase quality; grade and freshness Inline filter between needle seat and injection valve If sensitivity decreases, check cleanness of spray chamber and run Scan method to monitor background ions levels Use needle wash to reduce carryover Perform a wash method with % methanol at the end of a batch Use guard column and change when necessary Minimize extra column volume Delay volume adjustment 43
Conclusions A four minute method has been developed for quantifying catecholamines and their metabolites: epinephrine, norepinephrine, dopamine, metanephrine normetanephrine and 3-methoxytyramine for research Offline solid phase extraction (SPE) for simultaneous extraction of all six analytes from urine is shown with excellent recoveries Chromatographic separation of all six analytes with conditions compatible with LC-MS/MS have been developed Typical method performance results are well within acceptable criteria 44
References [] Simultaneous measurement of urinary metanephrines and catecholamines by liquid chromatography with tandem mass spectrometric detection M J Whiting, Clinical Biochemistry and Pharmacology Laboratory, SA Pathology, Flinders Medical Centre, Bedford Park 54, South Australia Ann Clin Biochem 9; 46: 9 36. DOI:.58/acb.8.88 [] Extraction and separation of urinary catecholamines as their diphenyl boronate complexes using C solid-phase extraction 8 sorbent and high-performance liquid chromatography Dinesh Talwar*, Cathie Williamson, Allison McLaughlin, Alan Gill, Denis St.J. O Reilly Department of Clinical Biochemistry, Macewen Building, Royal Infirmary, Glasgow G4 OSF, UK Journal of Chromatography B, 769 () 34 349 [3] Extraction of Catecholamines from Urine AN 7A, Argonaut, Dr Wéber Consulting KFT, www.weber.hu/pdfs/spe/an7s_catecholaminesurine.pdf 45
Solutions preparations Solutions preparations:. % Formic acid in water: ml of formic acid in L water 5 % Formic acid in water: 5 ml of formic acid in L water HCl.N in water: 83 ul of HCl 6N in 5 ml water M NH4Cl-NH4OH buffer: Dissolve 7g of NH4Cl in L water and adjust ph to 8.5 with NH4OH (about 8 ml of NH4OH 5%). Store at 4 deg.c Diphenyl-boronate complexing agent: Wash buffer. M NH4Cl-NH4OH: 5% Methanol in wash buffer. M NH4Cl-NH4OH: To ml of M NH4Cl-NH4OH buffer, add 4 mg of diphenylboronic acid ethanolamine ester and g of disodium EDTA. Diphenylboronic acid does not dissolve easily and may require to mixing slowly overnight to dissolve completely. Adjust ph to 8.5 with NH4OH. Store at 4 deg.c and check ph before use Add 5 ml of M NH4Cl-NH4OH buffer to 45 ml of water. Add 5 mg EDTA. Adjust ph to 8.5 with NH4OH (about 3 ml of NH4OH 5%). Store at 4 deg.c and check ph before use Add 5 ml of methanol to 475 ml of wash buffer. M NH4Cl-NH4OH. Adjust ph to 8.5 with NH4OH. Store at 4 deg.c and check ph before use 46
Supplies Supplier: Part number: Description: Agilent 933 SPE cartridges Bond Elut Plexa, 3 mg, 3 ml, 5/pk Agilent 34 Vac Elut SPS 4 (SPE manifold) Sigma 57-U SPE vacuum trap VWR 5498-37 Vacuum Pressure Pump, Gast Agilent 58-868 Screwcap vials with septa, 5/pk Agilent A355X HPLC column Pursuit 3 PFP, x 5 mm, 3 µm Agilent A35MG Meta Guard column Pursuit 3 PFP, mm, pk 3 Agilent 567-4638 9 Infinity Inline filter,.3 µm 47
Chemicals Supplier: Part Description: number: VWR LC3-.5 Burdick & Jackson Methanol LCMS grade Sigma-Aldrich 9438 Formic acid Golden West MSG5 DC Mass Spect Gold urine Biologics Sigma-Aldrich D9754-5G -Aminoethyl diphenylborinate Biorad 376 Lyphochek Quantitative Urine control, Level Normal Biorad 377 Lyphochek Quantitative Urine control, Level Abnormal VWR RC3753 Hydrochloric acid 6. N VWR CAJT66- Ammonium Chloride 48
Standards Cambridge Isotopes: PN: DLM739-.5g, Description: 3-methoxytyramine-d4 HCl Medical Isotopes: PN: 5376, Description: 3-methoxytyramine HCl 49
Questions Thank You! Acknowledgements Kevin McCann Agilent Technologies Clinical Applications Specialist Rory Doyle Agilent Technologies Clinical Applications Specialist Christophe Deckers Agilent Technologies Sample preparation Applications Scientist 5