EAS Sirocco 2003 - Waters · PDF file2Pall Life Sciences, Ann Arbor, MI ... © 2003 Waters Corporation Drawing of Plate ... Mass Spectrom. 2003; in press

© 2003 Waters Corporation

Kimvan Tran1, Diane M. Diehl1, Tim Schultz2, Wilf Wixwat2, Brian P. Murphy1, Jeff Kane2, Emily Berlin2, Kevin Seeley2, Ziling Lu1

1Waters Corporation, Milford, MA2Pall Life Sciences, Ann Arbor, MI

High-Throughput, Simplified Protein Precipitation: A Novel 96-Well Format for Precipitation and Filtration

EAS 2003

Pall Life Sciences

©2003 Waters Corporation


Outline

• Introduction

• Product Development

• Sirocco™ Protein Precipitation Plate– Design and Attributes

• Performance

• Summary



Introduction

– Protein precipitation (PPT) is a quick and dirty method for sample preparation in the pharmaceutical industry

– Various methods are utilized– Commercial PPT plates exist, but many are

difficult to use and suffer from poor performance

– To facilitate faster and better PPT, Waters has co-developed an new PPT plate with Pall Life Sciences – a valued technical partner



Market Research

• Surveyed customers doing protein precipitation

• They talked about:– Labor intensive sample prep (to feed the LC/MS)– Equipment intensive steps– Looking for relief in sample prep – real time savings.

• “Eliminate steps that would save real time, I would pay a reasonable premium.”

So why aren’t more scientists using current PPT plate technologies?



Problems with Existing Plates

• Tend to leak before customer was ready to vortex

• Precipitate in the filtrate

• Plates suffer from plugging

• Cross contamination

• Some manufacturers recommend crashing the protein in tubes and transfer to the filter plate. This creates extra steps and transfers (i.e. time).

• Binding on glass fiber filters

• Extractables/leachables that interfere with analyses, especially in LC/MSn



Development Criteria

• No cross contamination

• Minimize the levels of:

– Leaking – customer controls flow– Extractables/leachables

– Ion suppression/enhancement

• Additional Concerns• Smaller volumes of plasma – need greater recovery

of supernatant (without drawing precipitate)

• Precipitate fouling HPLC column



New Sirocco™ Protein Precipitation Plate

• 96-well, 1 mL/well filtration plate

• Contains a valve mat on the tips –which open and allow flow under vacuum*

• Stack of membranes, smallest is 0.45 µm (nylon-based)

• Vented cap mat

• Product – 5 pack– 5 filter plates– 5 vented cap mats in separate bag

*patent pending

Pall Life Sciences



Drawing of Plate – Partial Valve Mat

Shows 8 rows with valve mat – 4 rows of tip only



Single Valve

There is a slit at the bottom of the valvethat opens under 3-4” Hg vacuum



All Materials Extensively Tested for Ion Suppression/Enhancement and Extractables

• Ion suppression or enhancement is NOT observed with any of the materials, including the final plate configuration

• Levels of extractable materials are negligible and orders of magnitude cleaner than the competitive plates– Examined both by UV and MS

Let’s take a look at some data.



Ion Suppression/Enhancement Protocol*

• Two MeOH/water mobile phase solutions were prepared– One with 8 basic analytes, m/z 260-609, ES+– One with 8 acidic analytes, m/z 114-503, ES-

• HPLC flow rate was 0.2 mL/min of above mobile phases into a single quadrupole mass spectrometer

• Candidate materials were soaked in ACN and ACN:acid

• ACN and ACN:acid were flowed through prototype plates

• With HPLC pump on, these material solutions were infused into the MS source via a syringe pump

• Monitored the m/z of the mobile phase analytes and compared responses to blank solutions for degree of ion suppression or enhancement

*Mallet, C. R.; Lu, Z.; Mazzeo, J. R. Rapid Commun. Mass Spectrom. 2003; in press.



ES+ Data for Sirocco™ Plate

Ion Supression - ES+ - Sirocco PPT Plate

-50.0%

-40.0%

-30.0%

-20.0%

-10.0%

0.0%

10.0%

20.0%

30.0%

40.0%

50.0%

260 291 354 411 473 485 531 609

Molecule

Supp

ress

ion

or E

nhan

cem

ent (

%)

ACNAcid/ACN



ES- Data for Sirocco™ Plate

Ion Supression - ES- - Sirocco PPT Plate

-50.0%

-40.0%

-30.0%

-20.0%

-10.0%

0.0%

10.0%

20.0%

30.0%

40.0%

50.0%

114 132 204 242 281 357 407 503

Molecule

Supp

ress

ion

or E

nhan

cem

ent (

%)

ACN



UV Data - Sirocco™AU

0.00

0.05

0.10

0.15

0.20

Minutes0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00

AU

0.00

0.05

0.10

0.15

0.20

Minutes0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00

ACN Blank - 254 nm

ACN Blank - 210 nm

AU

0.00

0.05

0.10

0.15

0.20

Minutes0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00

Sirocco™ – 210 nm

AU

0.00

0.05

0.10

0.15

0.20

Minutes0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00

Sirocco™ – 254 nm



AU

0.00

0.05

0.10

0.15

0.20

Minutes0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00

UV Data – Competitive PlatesAU

0.00

0.05

0.10

0.15

0.20

Minutes0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00

Plate B254 nm

Plate B210 nm

AU

0.00

0.05

0.10

0.15

0.20

Minutes0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00

AU

0.00

0.05

0.10

0.15

0.20

Minutes0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00

Plate A254 nm

Plate A210 nm



100 200 300 400 500 600 700 800 900 1000m/z0

100

%

0

100

%

Scan ES+ 1.00e7152.020810146816

156.95185620992203.98874474368

245.05362617344263.99571384896368.6862750016 842.0283384448

415.5776337088Scan ES+ 1.00e7

ACN Blank

Plate BACN

Extractables Data

100 200 300 400 500 600 700 800 900 1000m/z0

100

%

0

100

%

Scan ES+ 1.00e7124.0560163135488130.54458299520

155.00534958720172.97723284224

245.05361022912368.8159530624Scan ES+ 1.00e7

ACN Blank

Plate AACN

100 200 300 400 500 600 700 800 900 1000m/z0

100

%

0

100

%

Scan ES+ 1.00e7172.912322340608174.92367422208

245.05364084480246.99972278656

260.10351579776368.5564434208Scan ES+ 1.00e7

ACN Blank

Sirocco™ACN



Eliminates Cloudy Filtrates

Plate A

Plate B

Sirocco™

Precipitate in the filtrate

Consistently clean filtrates, no precipitate in the tips.

Precipitate in the tips



Protocol

• Set the Sirocco™ plate on a collection plate. This keeps the valve tips suspended in wells of collection plate during processing.

1. Add crash solvent (typically acetonitrile with ISTD)2. Rapidly inject plasma into wells3. Apply vented cap mat (use a roller for good seal)4. Vortex PPT plate/collection plate stack at a medium

speed – ensures complete mixing and precipitation –for 1 minute.

5. Filter on vacuum manifold at 8-10 ”Hg.Note: Never remove cap mat or valve tips – this eliminates cross talk

between wells. The entire assembly is disposed after filtration.



Plasma Flow Times

Sirocco Plate Flow Results

0.00

0.50

1.00

1.50

2.00

2.50

3.00

3.50

0 50 100 150 200 250

Plasma (µL)

Tim

e (m

in)

Rat PlasmaPorcine Plasma



Recovery Data

• 300 µL ACN with internal standard added to plate (N = 6)

• Rat plasma (100 µL) spiked with 10 pq/µL of 8 analytes

• Vortexed and filtered for 3 minutes

% Recovery RSD (%)Propranolol 97.9 4.7Labetalol 103.4 3.8Atenolol 98.5 4.5Nadolol 98.4 4.1Metoprolol 102.9 4.3Alprenolol 102.1 8.7Pindolol 85.5 8Oxprenolol 103.5 5.8



Summary

• Novel design of the Sirocco™ Protein Precipitation Plate enables faster and more reliable protein precipitation– Simple protocol– Leak-free– No cross contamination– Faster processing– No ion suppression or enhancement– Minimal levels of extractables/leachables– Clean filtrates

Documents

EAS Sirocco 2003 - Waters · PDF file2Pall Life Sciences, Ann Arbor, MI ... © 2003 Waters Corporation Drawing of Plate ... Mass Spectrom. 2003; in press