Sample Preparation and

Liquid Chromatography Solutions for Quantitative

BIOANALYSIS

BIOANALYSIS CONSUMABLE SOLUTIONS

Waters solutions for bioanalysis, which include sample preparation products, columns,

analytical standards and reagents, LC systems, mass spectrometers, and software, offer

the most sensitive, robust, and reproducible results. Whether you are working in the area

of discovery or development, non-regulated or regulated environments, Waters system

solutions comprehensively meet the converging needs of scientists, analysts, laboratory

managers, and quality assurance (QA) and informational technology (IT) professionals.

For the ultimate in bioanalytical data quality, laboratory productivity, and compliance,

Waters chemistries and consumables provide a comprehensive starting point for

your workflow.

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BIOANALYSIS CONSUMABLE SOLUTIONS

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■ Unique design eliminates time-consuming steps for increased throughput

■ Precipitate-free filtrate for maximum system uptime vs. classical methods

■ Ideally suited for cleanliness, MS compatibility, and analyte recovery

Key Benefits:

SAMPLE PREPARATION SOLUTIONS

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PROCESSING TIME COMPARISON

With Sirocco Plates, samples can be processed in significantly less time than with classical PPT, thus increasing laboratory throughput.

RECOVERY

The Sirocco Plate was tested for analyte recovery using seven different analytes spiked into rat plasma. The results show excellent recoveries across all analytes listed including bases (b), strong acid (sa), acids (a), neutrals (n) and quarternary amine (qa).

SIMPLY CLEAR

The Sirocco™ Protein Precipitation (PPT) Plate eliminates additional, time-consuming sample-handling steps traditionally done with classical PPT. Unique membrane and valve-tip technology enable the analyst to carry out in-well protein precipitation followed by the final filtration step. This approach leads to efficient sample processing, resulting in clear filtrates from small sample volumes. Sirocco Plates can be automated to process large numbers of samples efficiently.

Classical PPT Sirocco PPT

Load Sample

Mix

Vacuum

45 minutes 15 minutes

Load Sample

Mix

Centrifuge

Transfer Supernatant

Direct Inject

Direct Inject

0

20

40

60

80

100

120

Sirocco Supelco Varian

% R

ecov

ery

N-acety

lproca

inamide

(b)

Propra

nolol

(b)

Pyren

esulfo

nic Acid

(sa)

T6692 (q

a)

Ketopr

ofen (

a)

Niflumic A

cid (z

)

Ethiny

l Estr

adiol

(n)

SIROCCO PROTEIN PRECIPITATION PLATE

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PHOSPHOLIPID REMOVAL

The level of 5 phospholipids remaining after sample preparation using the Ostro Plate is less than with both liquid-liquid extraction (LLE) and solid-supported liquid extraction (SSLE).

REPRODUCIBILITY

Comparative % removal of total phospholipids from 6 different lots of plasma using the Ostro Plate (0.19% RSD), phospholipid removal plate from competitor A (24.5% RSD), and phospholipid removal plate from competitor B (40.9% RSD).

THE FASTER WAY TO CLEANER

Ostro™ Sample Preparation Products provide a novel solution for the cleanup of phospholipids in plasma and serum. Requiring minimal to no method development and using a simple protocol, this technology can be quickly implemented to optimize your laboratory’s workflow. Providing cleaner, more reproducible extracts than competitive phospholipid removal devices or techniques, the Ostro 96-well Plate allows for more sensitive analyses, increased sample throughput, and reduced instrument downtime.

Sum of 5 abundant phosphatidylcholine containing phospholipids with m/z 496, 524, 704, 758, and 806.

Ostro Competitor A Competitor B

1 2 3 4 5 60

10

20

30

40

50

60

70

80

90

100

% P

hosp

holip

id R

emov

al

0

50000

100000

150000

200000

250000

300000

350000

400000

Sum

of A

rea

Coun

ts o

f 5 P

hosp

holip

ids

Ostro LLE SSLE

SAMPLE PREPARATION SOLUTIONS

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■ Removes significantly more phospholipids than competitive products or techniques for cleaner extracts

■ Improves reproducibility for more consistent, robust methods

■ Increases throughput with easy-to-implement protocol

Key Benefits:

OSTRO SAMPLE PREPARATION PLATE

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■ Highest sensitivity for traditional pharmaceutical compounds and therapeutic peptides

■ Minimal matrix effects for LC/MS/MS analyses

■ Unmatched product quality, performance, and reproducibility

Key Benefits:

SAMPLE PREPARATION SOLUTIONS

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SENSITIVITY

QUALITY

Waters careful process design and stringent quality controls have set a new standard in batch-to batch and lot-to-lot reproducibility for SPE sorbents. This enables scientists to develop robust, accurate, and precise bioanalytical methods.

MATRIX EFFECTS

Following extraction with an Oasis mixed-mode sorbent, the matrix effects for a panel of 5 antidepressant drugs were evaluated in multiple lots of urine. The RSDs of the matrix factors were determined to be between 5.5 and 8.4%, which are well within the 15% CV limit recommended by regulatory agencies.

SENSITIVITY IN ITS PUREST FORM

Sensitivity is one of the key requirements for a successful bioanalytical assay. Achieving optimum sensitivity requires the isolation of compounds of interest from interferences present in biological samples. The patented Oasis® Solid-Phase Extraction (SPE) Products are available in a wide array of formats and sorbents, allowing scientists to develop methodologies that are selective for the compounds of interest. Oasis Products enable scientists to create highly accurate and reproducible SPE methods that are unsurpassed in sensitivity and selectivity.

The Oasis mixed-mode sorbents, all built upon the unique water-wettable Oasis HLB copolymer, provide dual modes of retention, enabling greater cleanup selectivity and sensitivity for both acidic and/or basic compounds—even if the sorbent in the wells runs dry. Achieve unsurpassed sensitivity with the Oasis μElution Plate which can concentrate a sample up to 15x without requiring evaporation or reconstitution.

RSDs of recovery for: Procanamide: 1.90% Ranitidine: 1.68% Acetominophen: 1.65%

105

95

850 5 10 15 years

Reco

very

%

No Batch Reservations Needed: Consistently high recoveries over more than 15 years of production.

Analyte % RSD of Matrix Factors

Amitriptyline 6.03

Nortriptyline 5.53

Imipramine 8.36

Desipramine 7.15

Doxepin 5.96

OASIS SAMPLE EXTRACTION PRODUCTS

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1.19

1.18

1.28

A. Spiked sample

B. Matrix blank

A. Spiked sample

B. Matrix blank%

0

100

%

0

100

1.54 1.58 1.62 1.66 1.70 1.74 1.78 1.10 1.20 1.30

1.10 1.20 1.30 min1.54 1.58 1.62 1.66 1.70 1.74 1.78 min

%

0

100

%

0

100

1.61

1.521.55

1.66

1.73

LOQ of benzodiapine alprazolam 1 pg/mL from 375 μL of Plasma LOQ of Fluticasone Propionate 5 pg/mL from 375 μL of Plasma

EXTREME FLEXIBILITY AND EXCEPTIONAL SELECTIVITY

Due to the increased sensitivity, improved throughput, and exceptional assay reliability provided by UPLC® Technology, this system is now the platform of choice for bioanalytical assays. With four different column technologies and different particle substrates and over twenty sub-2-µm column chemistries available for small molecules and biotherapeutic applications, Waters has a wide range of column solutions to meet your application needs.

As a starting point, we recommend Waters BEH C18, CSH™ C18, and BEH HILIC columns as they offer the flexibility to address the bioanalytical needs for the widest range of compounds. For HPLC-MS systems, we suggest the same column chemistries in our eXtended Performance [XP] 2.5 µm Columns format for maximum resolution and throughput that can also provide a bridge from HPLC to UPLC technologies.

With the introduction of CORTECS™ Columns, based on a new silica-based solid-core particle, we add a new dimension to ultra performance chromatographic separations, with high efficiency, high resolution, and reduced analysis time separations. CORTECS columns are available in C18, C18+, and HILIC chemistries.

LIQUID CHROMATOGRAPHY COLUMNS

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UPLC separation of Teriparatide (MW4117) and internal standard from a 125 pg/mL extracted human plasma sample, using an ACQUITY UPLC CSH 2.1 x 50 mm Column.

CSH C18

■ Alternative selectivity for method development and screening

■ Superior peak shape and loading capacity for basic compounds

■ Exceptional stability at low and high pH

Teriparatide[rhPTH (1–34)]

rhPTH (1–38), ISTD

-780

-79:

MRM of 4 Channels ES+4.20e5

min

BEH C18

■ Universal starting point for method development and screening for small and large molecules

■ Maximum flexibility and a wide pH range (1–12)

■ Long column lifetimes

min0.2 0.4 0.6 0.80 1.0 1.2 1.4 1.6 1.8 2.0 2.2 2.4 2.6 2.8

%

0

100

0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 2.2 2.4 2.6 2.8

%

0

100MRM of 5 Channels ES+ TIC

MRM of 5 Channels ES+ TIC

Low pH (pH 3)

High pH (pH 11)1. Desipramine2. Nortriptyline3. Doxepin4. Imipramine5. Amitriptyline

3

1,4,2,5

1

2

4

3

5

Comparison data between low and high pH.

■ Maximize laboratory productivity by reducing analysis time while improving data quality

■ Reach lower limits of detection by enabling higher MS response with narrow chromatographic peaks

■ Improve sample characterization by reducing MS suppression due to residual matrix components

Key Benefits:

BEH, CSH, AND SOLID-CORE PARTICLE COLUMN TECHNOLOGIES

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BEH HILIC

■ Ideal column for highly polar analytes and other difficult separations including lipids

■ Complimentary selectivity

■ Exceptional chemical stability and peak shape compared to silica columns

Cer(d18:1/17:0)0.59

PG(14:0/14:0)2.27

PE(14:0/14:0)4.63

PC(14:0/14:0)6.08

SM(d18:1/12:0)7.78

LPC(20:0)8.20

min

HILIC UPLC chromatogram of lipid standards.

SOLID-CORE PARTICLE TECHNOLOGY

CORTECS C18

■ Silica-based solid core particle technology column

■ Maximum efficiency and maximum resolution

■ Ultimate peak capacity with the ACQUITY UPLC® System

5.52

4.29

5.26

4.303.60

5.26 5.46

4.28

3.60

5.81

4.13

5.285.73

4.27

4.04 5.78

4.23

3.21 5.19min

2.0 4.0 6.0 8.0

%

0

100

2.0 4.0 6.0 8.0

%

0

100

2.0 4.0 6.0 8.0

%

0

100

min2.0 4.0 6.0 8.0

%

0

100

2.0 4. 6.0 8.0

%

0

100

2.0 4.00 6.0 8.0

%

0

100

2.0 4.0 6.0 8.0

%

0

100 Insulin glulisine RT 4.28 min

Insulin aspart RT 4.27 min

Insulin glargineRT 4.13 min

Bovine insulin (IS)RT 4.23 min

Insulin detemir RT 5.52 min

Insulin glulisine RT 4.29 min

Human insulinRT 4.30 min

UPLC-MS/MS chromatogram for human insulin, five insulin analogs, and bovine insulin (IS).

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PRECISE FORMULATION AND ABSOLUTE TRACEABILITY

Waters understands the importance of high quality analytical standards and reagents for ensuring continuous analytical instrumentation performance and workflow success. As a result, Waters now offers standards and reagents that are pure, precisely formulated, reproducible, and traceable to exact specifications.

From system performance standards to application specific standards, Waters provides the right product for optimal results.

ANALYTICAL STANDARDS AND REAGENTS

■ Reduce variability in the laboratory

■ Streamline the laboratory process

■ Increase confidence in analytical results and reproducibility

■ Provide absolute standard traceability

Key Benefits:

Analytical Standards and Reagents Catalog, Literature Code: 720004231EN

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CERTIFIED FOR CONFIDENCE

Waters understands the importance of auto sampler vials and plates for the performance of analytical instrumentation. Quality vials are essential for reliable analytical results. All Waters Certified Vials are tested by LC or LC-MS to assure quality and cleanliness. The test results confirm the vials, cap, and septa have been properly manufactured, handled, and packaged, and are free from any potential contaminants which could result in chromatographic ghost peaks.

SAMPLE VIALS AND PLATES

■ Compatible with all autosamplers

■ Specially designed vials and plates for limited sample volumes

■ Complete selection of vial sizes and materials

Key Benefits:

Waters TruView LCMS Certified Vials White Paper, Literature Code: 720004097EN Waters LCMS Certified Vials White Paper, Literature Code: 720001517EN

1.0 1.1 1.2 1.3 1.4 1.5 1.6 1.7 1.8 1.9 2.0 2.1 2.2 2.3 2.4 2.5 2.6 2.7 2.8 2.9 3.0 min

%

0

1002.00

1.0 1.1 1.2 1.3 1.4 1.5 1.6 1.7 1.8 1.9 2.0 2.1 2.2 2.3 2.4 2.5 2.6 2.7 2.8 2.9 3.0 min

%

0

100 1.98

1.0 1.2 1.4 1.6 1.8 2.0 2.2 2.4 2.6 2.8 3.0 min

%

0

100

1.94

T initial

T initial

T initial

T 4hr

T 4hr

T 4hr

Competitor A

Competitor B

TruView LCMSCertified

Comparison of loss of sample between Waters TruView™ LCMS Certified Vials and competitive vials. TruView LCMS Certified Vials are recommended for ng/mL concentrations and lower.

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Tomorrow’s pharmaceuticals will need to be more targeted, effective, safer, and get approved and to market faster than ever. To succeed in that world, you’ll need technology that is designed to support continuous improvement and a technology partner who truly understands your methods and goals. Only one company offers both: Waters.

For regulated laboratories, Waters offers the Regulated Bioanalysis Platform Solution, a fully integrated platform for quantitative bioanalysis. It delivers robust assays with the highest sensitivity. The platform brings together industry-leading technology for every stage of analysis with a fully networked, scalable informatics system that gives you a tremendous advantage in data management and reporting for compliance and validation. These are technologies that Waters continually innovates, upgrades, and improves. They’re the best today. They’ll be even better tomorrow.

SOLUTIONS FOR THE WAY YOU’LL WORK TOMORROW

BIOANALYSIS SOLUTIONS

Blood

Plasma

Serum

Urine

Tissue

Biological Fluids

BEH Technology™ (ACQUITY UPLC and XBridge® Columns)

CSH Technology (ACQUITY UPLC and XSelect® Columns)

Solid-core Particle Technology (CORTECS Columns)

Sirocco Protein Precipiration Plate

Ostro Sample Preparation Plate

Oasis Sample Extraction Cartridges and Plates

ACQUITY UPLC I-Class

ACQUITY UPLC H-Class

SamplePreparation

Columns LiquidChromatography System

MassSpectrometer

DataManagement

Sample

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Key Benefits:■ Advanced, analytical software for

increased MS sensitivity

■ Adaptive workflows to help you function as a unified team

■ Sample preparation tools for purity, consistency, and quality

■ Better tools for ever-increasing regulatory demands

STANDARDS, VIALS, SAMPLE PREPARATION, COLUMNS, INSTRUMENTS, AND SOFTWARE

UNIFI® Software

MassLynx® Software

Xevo® TQD

Xevo TQ-S

SamplePreparation

Columns LiquidChromatography System

MassSpectrometer

DataManagement

Sample

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Waters Corporation 34 Maple Street Milford, MA 01757 U.S.A. T: 508 478 2000 F: 508 872 1990 www.waters.com

Waters, UPLC, ACQUITY UPLC, Xevo, XBridge, XSelect, UNIFI, MassLynx, The Science of What’s Possible, and Oasis are registered trademarks of Waters Corporation. Ostro, BEH Technology, CORTECS, Sirocco, TruView, and CSH are trademarks of Waters Corporation.

©2014 Waters Corporation. Printed in the U.S.A. January 2014 720003931EN AO-IGS

www.waters.com/BA

References

1. E. Chambers, D. M. Wagrowski-Diehl, Z. Lu, J. R. Mazzeo J., Systematic and comprehensive strategy for reducing matrix effects in LC/MS/MS analyses. Chromatogr. B 852 (2007) 22–34.