Critical evaluation of common sample preparation techniques for bioanalysis on microfluidic LC/MS performance Paul Rainville Waters Corporation ©2012 Waters Corporation 1 Capillary/nano LC/MS in DMPK ©2012 Waters Corporation 2 Capillary/nano-scale LC/MS in DMPK  Why ?  Why not ? – Need for sensitivity – Plenty of sensitivity – Limited sample volumes – Plenty sample available – Multiple injections/ same sample – Speed on analysis – Single model PK data – “Green” approach – Proteomics not DMPK platform – Chromatographic performance* – Robustness* ©2012 Waters Corporation 3 Signal Intensity Improvement in comparison to a 2.1mm format 75µm ID Each point represents an average response of a variety of sm mols Note: Constant load 1uL injected at all scales 150µm ID 1mm ID 300µm ID ©2012 Waters Corporation 2.1mm ID 4 Separation Comparison Hydroxylation products 2.1 mm i.d. ©2012 Waters Corporation Parent Drug (Glyburide) 150 µm i.d. 5 Sample Preparation Schemes  Place into format acceptable for injection onto LC/MS system.  Various techniques depending on goal of analysis: – Protein precipitation* – Liquid-liquid extraction* – Solid-phase extraction – Solid-liquid extraction – Digestion* – Affinity – Filter – Centrifugation – Dilution ©2012 Waters Corporation 6 Methods  Various biological matrices tested with small and large molecules: – Plasma* – Urine – Bile – Microsomes  Criteria: 1000 injections (approx. 5 days) – Chromatographic peak shape – System pressure  Chromatographic conditions: – 150 µm X 50 mm prototype microfluidic device 1.7 µm BEH C18, , temperature controlled, 3-4 µL/ min, gradient elution, formic acid/ MeCN , various injection volumes ©2012 Waters Corporation 7 Robustness Evaluation Test Case 1 A typical Protein Precipitation workflow  Protein precipitation (PPT) – 1000 1 µL injections of 2:1 crashed human plasma Add Precipitation Solvent Vortex and Centrifuge Place supernatant into separate vial Dilute depending on analyte and % organic in solution ©2012 Waters Corporation – 1000 3 µL injections of 2:1 crashed human plasma – 1000 5 µL injections of 2:1 crashed human plasma – a standard mixture of dextromethorphan and propranolol (critical pair) was injected every 50 plasma injections 8 Robustness Testing (PPT data) First injection 120312_WAA176_CD_01 After 1000 injections of crashed plasma 2: MRM of 1 Channel ES+ 272.201 > 171 (Dextromethorphan) 9.90e5 120312_WAA176_CD_1228 % 0 2: MRM of 1 Channel ES+ 272.201 > 171 (Dextromethorphan) 6.60e5 % 1 uL injection Equivalent to 200uL injection on 2.1mm scale! 3.20 3.25 3.30 3.35 121712_WAA176_CD_01 3.40 0 Time 3.20 3.25 3.30 3.35 121712_WAA176_CD_1171 100 2: MRM of 1 Channel ES+ 272.201 > 171 (Dextromethorphan) 8.16e5 3.40 Time 2: MRM of 1 Channel ES+ 272.201 > 171 (Dextromethorphan) 6.54e5 % % 5 uL injection Equivalent to 1mL injection on 2.1mm scale! 1 3.20 ©2012 Waters Corporation 3.25 3.30 3.35 3.40 Time 0 3.20 3.25 3.30 3.35 3.40 Time 9 Robustness Testing (PPT data) 121712_WAA176_CD_01 7000.000 nBSM System Pressure Range: 2426 Last injection 6000.000 5000.000 psi 4000.000 Peak Area RSD: 5.5% First injection 3000.000 2000.000 1000.000 0.000 1.00 2.00 3.00 4.00 5.00 6.00 Time No of plasma injections ©2012 Waters Corporation 10 Peptide Test (PPT data) Injection #3 ©2012 Waters Corporation Injection #302 Injection #576 11 Different devices Different plasma sources (PPT data) Peptide P00924 Average of all Peptides, all Tiles Retention Time 6.64 min Peak Width RSD 4.16% SD of Retention Time 0.09 Retention Time RSD 0.57% Average Peak Width at 10% 2.64 s Area RSD 15.5% ©2012 Waters Corporation 12 Robustness Evaluation Test Case 2 A typical LLE workflow Add Extraction Solvent Vortex then Centrifuge Transfer to new vessel Solvents Tested Hexane Ethyl Acetate Methyl tert Butyl Ether 1:10 ratio plasma:solvent Dry down Recon in initial gradient conditions X 5 enrichment 5 µL injection volume Evaporate to dryness Reconstitute in aqueous to match RP starting conditions ©2012 Waters Corporation 13 Robustness Testing (LLE data) After 1000 injections of LLE/dry down/recon First injection 5 uL injection Equivalent to 1mL injection on 2.1mm scale! ©2012 Waters Corporation 14 Robustness Evaluation Test Case 3 A typical Protein BioA workflow Affinity isolation Reduction, alkylation Spiking 13C15N-peptide IS Trypsin digestion https://www.google.com/search?q=mAb&source=lnms&tbm=isch&sa=X&e i=gwN5UsSWK_jfsASZg4DwBA&ved=0CAcQ_AUoAQ&biw=1024&bih=577 Acidify sample, inject ©2012 Waters Corporation 15 Robustness Testing (mAb digest data) First injection 1000 injection First injection 1000 injection First injection 1000 injection ©2012 Waters Corporation 16 Separations Device ESI tip assembly Incoming flow Analytical Column ©2012 Waters Corporation 17 Ceramic Microfluidic Device ©2012 Waters Corporation 18 Peer-reviewed Beta collaborators M Lassman et al. ©2012 Waters Corporation 19 Summary  A 150 µm i.d. ceramic micro-fluidics prototype device was successfully implemented in the analysis of biofluid samples with good chromatographic performance.  Common sample preparation techniques already utilized in BioA workflows can be readily implemented when scaling down chromatography to 150 µm i.d. – PPT – LLE – IA/Digestion ©2012 Waters Corporation 20 Acknowledgements  Jim Murphy  Jay Johnson  Mark Wrona  Catalin Doneanu  Erin Chambers ©2012 Waters Corporation 21