Waters Ostro Sample Preparation Products User Manual

[ Care and Use ManUal ]

ostro sample preparation plate

Contents

I. INTRODUCTION

II. PROCEDURE

III. ORDERING INFORMATION

I. INTRODUCTION

Ostro™ sample preparation plates provide a novel approach for the

removal of phospholipids from biological samples. The Ostro 96-well

plate design uses an in-well protein precipitation in combination

with a single, rapid, pass-through method that provides consistent,

high extraction recoveries for acids, bases, and neutrals. Designed

specifically to meet the needs of the bioanalytical scientist, Ostro

enhances a laboratory’s performance by providing greater repro-

ducibility, improved recovery, and increased phospholipid

removal when compared with competitive techniques.

Step 2: Plasma Loading

Add between 50 and 200 μL of plasma sample into the wells. The

Ostro plate is designed to prevent plasma samples from leaking

through the plate.

Step 3: Precipitation

Solvent Loading

Forcefully/rapidly add 1% HCOOH (formic acid) in acetonitrile to the

wells in a ratio of 3:1 solvent to plasma.

A straightforward 6-step procedure was designed to facilitate in-well

protein precipitation and produce filtrates free of proteins and other

endogenous components. These filtrates are then suitable for high-

throughput LC/MS/MS analysis.

II. PROCEDURE

Step 1: Preparation

Place the Ostro plate onto a 2 mL collection plate for processing.

Ostro Plate

96-Well 2 mL Collection Plate

Note: The forceful/rapid addition of the solvent aids in mixing the

plasma with this solvent.

Note: Take care to ensure that all of the pipette tips are properly

positioned in the samples well to prevent any loss of mixing of samples.

1Ostro Sample Preparation Plate

[ Care and Use ManUal ]

Step 4: Mixing

Aspiration (Preferred)

Aspirate the samples up and down 3X by a manual pipette, or an

automated sample handler may be used to ensure complete mixing.

Aspiration is the preferred mixing mode which will provide complete

mixing and a clear filtrate. Alternatively, an automated sample han-

dler may be used.

Step 5: Sample Filtration

Positive Pressure (Preferred)

After mixing, place the Ostro plate/collection plate on a positive

pressure processor and set the flow for 60 psi for 5 minutes. A higher

pressure can be applied if flow does not occur.

Note: The Ostro plate’s proprietary design prevents liquid flow

through the device until force has been applied.

Vacuum

Vortexing

If vortexing is chosen for mixing, it is critically important to vortex

vigorously and thoroughly to observe the best product performance.

Apply a cap mat and securely seal (e.g. use a cap roller). This will

prevent leakage and cross talk during mixing. Shake (mix) the loaded

Ostro plate/collection plate assembly on a vortexer.

Note: There is no need to remove the cap mat from the Ostro plate

before applying vacuum. If positive pressure is to be used, remove the

cap mat, carefully.

Note: Excessive mixing at too high a setting (e.g., setting 8 on a

1-10 scale) could cause cross talk. Under-mixing (e.g., setting 2 on

a 1-10 scale) may cause clogging or a cloudy filtrate.

After mixing, place the Ostro plate/collection plate assembly on a

vacuum manifold (e.g. Waters Vacuum Manifold P/N 186001831),

and apply 15” Hg of vacuum for 5-10 minutes. A higher vacuum set-

ting can be use if flow does not occur at 15” Hg.

Step 6: Analysis

Discard the Ostro plate and remove the collection plate containing

clear filtrate from the vacuum device or positive pressure manifold.

Seal the collection plate and transfer it to the LC/MS/MS autosampler

or store for later analysis. Extracts may be directly injected, diluted

prior to injection, or evaporated and reconstituted with an appropriate

solvent.

2Ostro Enhanced Protein Precipitation Plate