critical evaluation of common sample preparation …...2.1mm id 1mm id each point represents an...

©2012 Waters Corporation 1

Critical evaluation of common sample preparation techniques for bioanalysis on

microfluidic LC/MS performance

Paul Rainville

Waters Corporation


Capillary/nano LC/MS in DMPK


Why ? – Need for sensitivity – Limited sample volumes – Multiple injections/ same

sample – Single model PK data – “Green” approach

Why not ? – Plenty of sensitivity – Plenty sample available – Speed on analysis – Proteomics not DMPK

platform – Chromatographic

performance* – Robustness*

Capillary/nano-scale LC/MS in DMPK


Signal Intensity Improvement in comparison to a 2.1mm format

75µm ID

150µm ID

300µm ID 2.1mm ID

1mm ID

Each point represents an average response of a variety of sm mols Note: Constant load 1uL injected at all scales


Separation Comparison

Parent Drug (Glyburide)

Hydroxylation products

2.1 mm i.d. 150 µm i.d.


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


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


Protein precipitation (PPT)

– 1000 1 µL injections of 2:1 crashed human plasma



– a standard mixture of

dextromethorphan and propranolol (critical pair) was injected every 50 plasma injections

Robustness Evaluation Test Case 1

Add Precipitation Solvent

Place supernatant into separate vial

Dilute depending on analyte and % organic in solution

A typical Protein Precipitation workflow

Vortex and Centrifuge

http://www.google.com/url?sa=i&rct=j&q=&esrc=s&frm=1&source=images&cd=&cad=rja&docid=NNE2k5tJiWMRaM&tbnid=6NUvubOU6y2e1M:&ved=0CAUQjRw&url=http://www.3tailer.com/venous-blood-collection-tube-bd-vacutainer-plasma-tube-3-ml-13-x-75-mm-light-green-top-bx-100&ei=4ALfUY2ANdi14AOZ9oHQCw&bvm=bv.48705608,d.dmg&psig=AFQjCNGx7NXZTezDLEKIMmfrmszi2_oB5Q&ust=1373656125262816


Time3.20 3.25 3.30 3.35 3.40

%

0

120312_WAA176_CD_1228 2: MRM of 1 Channel ES+ 272.201 > 171 (Dextromethorphan)

6.60e5

First injection

Time3.20 3.25 3.30 3.35 3.40

%

0


9.90e5

Time3.20 3.25 3.30 3.35 3.40

%

1


8.16e5

After 1000 injections of crashed plasma

Time3.20 3.25 3.30 3.35 3.40

%

0

100121712_WAA176_CD_1171 2: MRM of 1 Channel ES+

272.201 > 171 (Dextromethorphan)6.54e5

Equivalent to 200uL

injection on 2.1mm scale!

Equivalent to 1mL

injection on 2.1mm scale!

1 uL

injection

5 uL

injection

Robustness Testing (PPT data)


Time1.00 2.00 3.00 4.00 5.00 6.00

psi

0.000

1000.000

2000.000

3000.000

4000.000

5000.000

6000.000

7000.000121712_WAA176_CD_01 nBSM System Pressure

Range: 2426

Last injection

First injection

No of plasma injections

Peak Area RSD: 5.5%

Robustness Testing (PPT data)


Peptide Test (PPT data)

Injection #3 Injection #302 Injection #576


Different devices Different plasma sources (PPT data)

Average of all Peptides, all Tiles

Peak Width RSD

4.16%

Retention Time RSD

0.57%

Area RSD 15.5%

Peptide P00924

Retention Time 6.64 min

SD of Retention Time

0.09

Average Peak Width at 10%

2.64 s



Add Extraction Solvent

Transfer to new vessel

Evaporate to dryness

Reconstitute in aqueous to match RP starting conditions

A typical LLE workflow

Vortex then Centrifuge

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



Robustness Testing (LLE data)

5 uL injection

Equivalent to 1mL injection on

2.1mm scale!

First injection

After 1000 injections of LLE/dry

down/recon



A typical Protein BioA workflow

Affinity isolation

Trypsin digestion

Reduction, alkylation

Spiking 13C15N-peptide IS

Acidify sample, inject

https://www.google.com/search?q=mAb&source=lnms&tbm=isch&sa=X&ei=gwN5UsSWK_jfsASZg4DwBA&ved=0CAcQ_AUoAQ&biw=1024&bih=577


http://www.google.com/url?sa=i&rct=j&q=&esrc=s&frm=1&source=images&cd=&cad=rja&docid=yZKhGPUQWYLFGM&tbnid=uPu3GPLBhyp7CM:&ved=0CAUQjRw&url=http://www.automation-drive.com/igd-antibody&ei=Vwh5UtCmO_ihsAT6_4CgBQ&psig=AFQjCNHNnNMEbia3D_puU9k5qJ-gQmLJsg&ust=1383749000892672


Robustness Testing (mAb digest data)

First injection

First injection

First injection

1000 injection

1000 injection

1000 injection


Separations Device

Incoming flow

Analytical Column

ESI tip assembly


Ceramic Microfluidic Device


Peer-reviewed Beta collaborators

M Lassman et al.

http://www.google.com/url?sa=i&rct=j&q=&esrc=s&frm=1&source=images&cd=&cad=rja&docid=SZtDt0RZifnGjM&tbnid=gEEHuDhz4UNfyM:&ved=0CAUQjRw&url=http://www.bioanalysis-zone.com/news/bioanalysis-324-19th-international-reid-bioanalytical-forum/&ei=UId6Upa8MomfkQfx94DoCw&bvm=bv.55980276,d.cWc&psig=AFQjCNEVAXczYKtvx67NfeJydDfGFAOPJw&ust=1383848111633142


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


Acknowledgements

Jim Murphy Jay Johnson Mark Wrona Catalin Doneanu Erin Chambers

critical evaluation of common sample preparation …...2.1mm id 1mm id each point represents an...

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