(Critical reviews in combinatorial chemistry) perry g wang high throughput analysis in the pharmaceutical industry CRC press (2008)

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CRITICAL REVIEWS IN COMBINATORIAL CHEMISTRY
Series Editors
BING YAN
School of Pharmaceutical Sciences
Shandong University, China

ANTHONY W. CZARNIK
Department of Chemistry
University of Nevada–Reno, U.S.A.

A series of monographs in molecular diversity and combinatorial chemistry,
high-throughput discovery, and associated technologies.
Combinatorial and High-Throughput Discovery and Optimization of Catalysts and Materials
Edited by Radislav A. Potyrailo and Wilhelm F. Maier
Combinatorial Synthesis of Natural Product-Based Libraries
Edited by Armen M. Boldi
High-Throughput Lead Optimization in Drug Discovery
Edited by Tushar Kshirsagar
High-Throughput Analysis in the Pharmaceutical Industry
Edited by Perry G. Wang

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Edited by

Perry G. Wang


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CRC Press
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© 2009 by Taylor & Francis Group, LLC
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Library of Congress Cataloging‑in‑Publication Data
High‑throughput analysis in the pharmaceutical industry / edited by Perry G. Wang.
p. ; cm. ‑‑ (Critical reviews in combinatorial chemistry)
Includes bibliographical references and index.
ISBN‑13: 978‑1‑4200‑5953‑3 (hardcover : alk. paper)
ISBN‑10: 1‑4200‑5953‑X (hardcover : alk. paper)
1. High throughput screening (Drug development) 2. Combinatorial chemistry. I. Wang, Perry G. II.
Title. III. Series.
[DNLM: 1. Combinatorial Chemistry Techniques‑‑methods. 2. Drug Design. 3. Pharmaceutical
Preparations‑‑analysis. 4. Technology, Pharmaceutical‑‑methods. QV 744 H6366 2009]
RS419.5.H536 2009
615’.19‑‑dc22
Visit the Taylor & Francis Web site at

and the CRC Press Web site at


2008003895


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Contents
Chapter 1

High-Throughput Sample Preparation Techniques and Their Application to
Bioanalytical Protocols and Purification of Combinatorial Libraries........................1
Krishna Kallury

Chapter 2

Online Sample Extraction Coupled with Multiplexing Strategy to Improve
Throughput............................................................................................................... 73
Katty X. Wan

Chapter 3

Optimizing LC/MS Equipment to Increase Throughput
in Pharmaceutical Analysis...................................................................................... 93
Michael G. Frank and Douglas E. McIntyre

Chapter 4

Throughput Improvement of Bioanalytical LC/MS/MS by Sharing Detector
between HPLC Systems.......................................................................................... 119
Min Shuan Chang and Tawakol El-Shourbagy

Chapter 5

High-Throughput Strategies for Metabolite Identification in Drug Discovery...... 141
Patrick J. Rudewicz, Qin Yue, and Young Shin

Chapter 6

Utilizing Microparallel Liquid Chromatography for High-Throughput
Analyses in the Pharmaceutical Industry............................................................... 155
Sergio A. Guazzotti

Chapter 7

Strategies and Techniques for Higher Throughput ADME/PK Assays.................205
Walter Korfmacher

Chapter 8

High-Throughput Analysis in Drug Metabolism during
Early Drug Discovery............................................................................................. 233
Yau Yi Lau

Chapter 9

High-Throughput Analysis in Support of Process Chemistry and Formulation
Research and Development in the Pharmaceutical Industry.................................. 247
Zhong Li


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Chapter 10 Online Solid Phase Extraction LC/MS/MS for High-Throughput
Bioanalytical Analysis............................................................................................ 279
Dong Wei and Liyu Yang
Chapter 11 Applications of High-Throughput Analysis in Therapeutic Drug Monitoring....... 299

Quanyun A. Xu and Timothy L. Madden
Chapter 12 High-Throughput Quantitative Pharmaceutical Analysis in Drug Metabolism
and Pharmacokinetics Using Liquid Chromatography–Mass Spectrometry......... 319
Xiaohui Xu
Chapter 13 Designing High-Throughput HPLC Assays for Small and Biological
Molecules................................................................................................................ 339
Roger K. Gilpin and Wanlong Zhou
Chapter 14 Advances in Capillary and Nano HPLC Technology for Drug Discovery
and Development.................................................................................................... 355
Frank J. Yang and Richard Xu
Chapter 15 High-Throughput Analysis of Complex Protein Mixtures by Mass
Spectrometry........................................................................................................... 377
Kojo S. J. Elenitoba-Johnson
Index............................................................................................................................................... 393


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Preface
I had the pleasure of developing and exploiting the high-throughput techniques used for drug
analysis in the pharmaceutical industry at Abbott Laboratories. My major duties as project leader
involved bioanalytical method development and validation by liquid chromatography with tandem
mass spectrometry (LC/MS/MS). While organizing a symposium titled “High-Throughput Analyses
of Drugs and Metabolites in Biological Matrices Using Mass Spectrometry” for the 2006 Pittsburgh
Conference, it became my dream to edit a book called High-Throughput Analysis in the Pharmaceutical Industry.
It is well known that high-throughput, selective and sensitive analytical methods are essential
for reducing timelines in the course of drug discovery and development in the pharmaceutical
industry. Traditionally, an experienced organic chemist could synthesize and finalize approximately
50 compounds each year. However, since the introduction of combinatorial chemistry technology to
the pharmaceutical industry, more than 2000 compounds can be easily generated yearly with certain automation. Conventional analytical approaches can no longer keep pace with the new breakthroughs and they now constitute bottlenecks to drug discovery. In order to break the bottlenecks,

a revolutionary improvement of conventional methodology is needed. Therefore, new tools and
approaches for analysis combined with the technologies such as combinatorial chemistry, genomics,
and biomolecular screening must be developed. Fortunately, liquid chromatography/mass spectrometry (LC/MS)-based techniques provide unique capabilities for the pharmaceutical industry. These
techniques have become very widely accepted at every stage from drug discovery to development.
This book discusses the most recent and significant advances of high-throughput analysis in the
pharmaceutical industry. It mainly focuses on automated sample preparation and high-throughput
analysis by high-performance liquid chromatography (HPLC) and mass spectrometry (MS). The
application of high-performance liquid chromatography combined with mass spectrometry (HPLCMS) and the use of tandem mass spectrometry (HPLC/MS-MS) have proven to be the most important analytical techniques for both drug discovery and development. The strategies for optimizing
the application of these techniques for high-throughput analysis are also discussed. Microparallel
liquid chromatography, ADME/PK high-throughput assays, MS-based proteomics, and advances in
capillary and nano-HPLC technology are also introduced in this book.
I sincerely hope that readers—ranging from college students to professionals and academics in
the fields of pharmaceutics and biotechnology—will find the chapters in this book to be helpful and
valuable resources for their current projects and recommend this volume to their colleagues.
I would like to note my appreciation to all the contributors who found time in their busy
schedules to provide the chapters herein. Many thanks to my previous colleagues, Shimin Wei,
Min S. Chang, and Tawakol El-Shourbagy for their friendship and support. I would like to take this
opportunity to acknowledge and thank the late Dr. Raymond Wieboldt for his priceless mentoring,
without which I could not have been so successful in establishing my career in the pharmaceutical
industry. I would also like to thank Bing Yan, Lindsey Hofmeister, Pat Roberson, Marsha Hecht,
and Hilary Rowe for their much valued assistance throughout the preparation of this book. My
thanks and gratitude go also to my family, whose support and encouragement greatly assisted me
in editing this book.
Perry G. Wang
Wyomissing, Pennsylvania


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Editor
Dr. Perry G. Wang is currently a principal scientist at Teleflex Medical. His interests include
analytical method development and validation, medicated device products, and environmental engineering. His expertise focuses on high-throughput analysis of drugs and their metabolites in biological matrices with LC/MS/MS.
Dr. Wang received a President’s Award for Extraordinary Performance and Commitment in
2005 for his dedication in leading the Kaletra® reformulation project at Abbott Laboratories. He
was presented with a President’s Award for Excellence while he worked in the U.S. Environmental
Protection Agency’s research laboratories.
Dr. Wang is an author of more than 20 scientific papers and presentations. He organized and
presided over symposia for the Pittsburgh Conference in 2006 and 2008, respectively. He has been
an invited speaker and presided over several international meetings including the Pittsburgh Conference and the Federation of Analytical Chemistry and Spectroscopy Societies (FACSS). His current
research focuses on developing new medicated-device products applied to critical care medicine
and testing drug release kinetics and impurities released from drug-device combination products.
He earned a B.S. in chemistry from Shandong University and an M.S. and Ph.D. in environmental
engineering from Oregon State University.


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Contributors
Min Shuan Chang
Abbott Laboratories
Abbott Park, Illinois, USA

Zhong Li
Merck Research Laboratories

West Point, Pennsylvania, USA

Kojo S. J. Elenitoba-Johnson
Department of Pathology
University of Michigan Medical School
Ann Arbor, Michigan, USA

Timothy L. Madden
Pharmaceutical Development Center
MD Anderson Cancer Center
The University of Texas
Houston, Texas, USA

Tawakol El-Shourbagy
Abbott Laboratories
Abbott Park, Illinois, USA
Michael G. Frank
Agilent Technologies
Waldbronn, Germany
Roger K. Gilpin
Brehm Research Laboratory
Wright State University
Fairborn, Ohio, USA
Sergio A. Guazzotti
Engineering Division
Nanostream, Inc.
Pasadena, California, USA
Krishna Kallury
Phenomenex
Torrance, California, USA

Douglas E. McIntyre
Agilent Technologies
Santa Clara, California, USA
Patrick J. Rudewicz
Genentech
South San Francisco, California, USA
Young Shin
Genentech
South San Francisco, California, USA
Katty X. Wan
Abbott Laboratories
Abbott Park, Illinois, USA
Dong Wei
Biogen Idec, Inc.
Cambridge, Massachusetts, USA

Walter Korfmacher
Exploratory Drug Metabolism
Schering-Plough Research Institute
Kenilworth, New Jersey, USA

Quanyun A. Xu
Pharmaceutical Development Center
MD Anderson Cancer Center
The University of Texas
Houston, Texas, USA

Yau Yi Lau
Abbott Laboratories

Abbott Park, Illinois, USA

Richard Xu
Micro-Tech Scientific
Vista, California, USA


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Xiaohui Xu
Pharmaceutical Research Institute
Bristol-Myers Squibb
Princeton, New Jersey, USA

Liyu Yang
Biogen Idec, Inc.
Cambridge, Massachusetts, USA

Frank J. Yang
Micro-Tech Scientific
Vista, California, USA

Qin Yue
Genentech
South San Francisco, California, USA
Wanlong Zhou
Brehm Research Laboratory
Wright State University
Fairborn, Ohio, USA

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Sample
1 High-Throughput
Preparation Techniques
and Their Application to
Bioanalytical Protocols
and Purification of
Combinatorial Libraries
Krishna Kallury
Contents
1.1
1.2

Need for High-Throughput Sample Purification and Clean-Up in Drug Discovery...............2
Rapid Purification Techniques for Drugs and Metabolites in Biological Matrices.................3
1.2.1 State-of-Art Sample Preparation Protocols.................................................................3
1.2.2 Matrix Components and Endogenous Materials in Biological Matrices....................3
1.2.3 Solid Phase Extraction (SPE)......................................................................................6
1.2.3.1 Interactions of Sorbent and Analyte in SPE and Selective Extractions
Based on Sorbent Chemistry.........................................................................7
1.2.3.2 Elimination of Proteinaceous and Endogenous Contaminants from
Biological Matrices to Minimize Ion Suppression during SPE:
Comparison of Ion Exchange and Mixed Mode Sorbents........................... 14
1.2.3.3 Formats for Rapid and/or High-Throughput Solid Phase Extraction
of Drugs in Biological Matrices.................................................................. 15
1.2.3.4 Online Solid Phase Extraction as Tool for High-Throughput
Applications.................................................................................................24
1.2.3.5 Utility of 384-Well Plates for High-Throughput Applications

and In-Process Monitoring of Cross Contamination...................................26
1.2.3.6 Utility of Multisorbent Extraction for SPE High-Throughput
Method Development................................................................................... 27
1.2.4 Recent Developments in Liquid–Liquid Extraction (LLE) for Clean-Up
of Biological Matrices: Miniaturization and High-Throughput Options..................28
1.2.4.1 Automated Liquid–Liquid Extraction without Solid Support..................... 31
1.2.4.2 Solid-Supported Liquid–Liquid Extraction................................................. 33
1.2.4.3 Liquid Phase Microextraction (LPME)....................................................... 35
1.2.5 Protein Precipitation Techniques and Instrumentation
for High-Throughput Screening................................................................................44
1.2.5.1 Use of Protein Precipitation in Tandem with Other Sample
Preparation Techniques............................................................................... 50



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High-Throughput Analysis in the Pharmaceutical Industry

1.3

Other Sample Preparation Technologies: Latest Trends........................................................ 53
1.3.1 Solid Phase Microextraction (SPME) as Sample Preparation Technique................. 53
1.3.2 Sample Clean-Up through Affinity Purification Employing Molecularly
Imprinted Polymers................................................................................................... 56
1.4 Purification of Synthetic Combinatorial Libraries................................................................60
1.4.1 HPLC-Based High-Throughput Separation and Purification
of Combinatorial Libraries........................................................................................ 61
1.4.2 Scavenger-Based Purification of Combinatorial Libraries Generated

by Solution Phase Synthesis......................................................................................64
1.5 Concluding Remarks.............................................................................................................. 68
1.6 Additional Reading................................................................................................................ 68
References......................................................................................................................................... 68

1.1 Need For High-Throughput Sample Purification
and Clean-Up in Drug Discovery
The drug discovery process took a revolutionary turn in the early 1990s through the adaptation of
combinatorial chemistry for generating large volumes of small organic molecules (generally having
molecular weights below 750 Daltons) so that the products of all possible combinations of a given
set of starting materials (building blocks) can be obtained at once. The collection of these end products is called a combinatorial library.
Production of such libraries can be achieved through either solid phase synthesis or solution
chemistry. This newly acquired capability of synthetic chemists to produce a large number of
compounds with a wide range of structural diversity in a short time, when combined with highthroughput screening, computational chemistry, and automation of laboratory procedures, led to a
significantly accelerated drug discovery process compared to the traditional one-compound-at-atime approach. During the high-throughput biological screening of combinatorial compounds, initial sample purification to remove assay-interfering components is required to ensure “true hits” and
prevent false positive responses. This created needs for rapid purification of combinatorial synthesis
products along with rapid evaluation of the purities of these large numbers of synthetic products.
In addition, screening biological activities of combinatorial libraries at the preclinical and clinical
(phases I through III) trial stages generates drug and metabolite samples in blood, plasma, and tissue matrices. Because these biological matrices carry many other constituents (proteins, peptides,
charged inorganic and organic species) that can interfere with the quantitation of the analytes and
also damage the analytical instrumentation (especially mass spectrometers and liquid chromatographic columns), rapid clean-up methods are required to render the samples amenable for analysis
by fast instrumental techniques. This chapter addresses the progress made during the past decade
in the areas of rapid purification of combinatorial libraries and sample preparation and clean-up for
high-throughput HPLC and/or LC/MS/MS analysis.
In addition to the large volume synthesis of small molecules, combinatorial approaches are also
used to generate catalysts, oligonucleotides, peptides, and oligosaccharides. High-throughput purification has also found applicability for the isolation and clean-up of natural products investigated
for biological activity. Several reviews and monographs are available on various topics related to
the synthetic and biological screening aspects of the drug discovery process. Since the focus of this
chapter is on the purification of combinatorial libraries and clean-up of drugs and their metabolites in biological matrices, it is suggested that the readers refer to the latest literature available
on solid phase1–10 and solution phase11–17 combinatorial synthesis, ADME studies,18–26 rapid instrumental analysis techniques,27–33 and high-throughput methods in natural products chemistry34–40 for

more detailed insights into these areas of relevance to combinatorial synthesis and high-throughput
screening.


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High-Throughput Sample Preparation Techniques



1.2 Rapid Purification Techniques for Drugs
and Metabolites in Biological Matrices
1.2.1 State-of-Art Sample Preparation Protocols
A number of advances have been made during the past decade to convert sample preparation techniques used for about 30 years for the clean-up of drugs in biological matrices into formats that are
amenable for high volume processing with or without automation. Detailed accounts about the fundamentals of these techniques can be found in the literature.41–50 Therefore, only brief descriptions
of the principles of these methods are presented. For isolating drugs and metabolites from biological
matrices, several approaches have been reported, which consist of:
•
•
•
•
•
•
•
•

Solid phase extraction (SPE)
Liquid—liquid extraction (LLE)
Protein precipitation (PPT)
Affinity separations (MIP)
Membrane separations

Preparative high performance liquid chromatography (HPLC)
Solid phase microextraction (SPME)
Ultrafiltration and microdialysis

SPE, LLE, and PPT are the most commonly used sample preparation techniques and hence most of
the discussion will be devoted to them. The others will be dealt with briefly. All of these methods
have certain ultimate goals:
• Concentrate analyte(s) to improve limits of detection and/or quantitation
• Exchange analyte from a non-compatible environment into one that is compatible with
chromatography and mass spectrometric detection
• Remove unwanted matrix components that may interfere with the analysis of the desired
compound
• Perform selective separation of individual components from complex mixtures, if desired
• Detect toxins in human system or in environment (air, drinking water, soil)
• Identify stereochemical effects in drug activity and/or potency
• Follow drug binding to proteins
• Determine stability and/or absorption of drugs and follow their metabolism in human
body

1.2.2Matrix Components and Endogenous Materials in Biological Matrices
Biological matrices include plasma, serum, cerebrospinal fluid, bile, urine, tissue homogenates,
saliva, seminal fluid, and frequently whole blood. Quantitative analysis of drugs and metabolites
containing abundant amounts of proteins and large numbers of endogenous compounds within these
matrices is very complicated. Direct injection of a drug sample in a biological matrix into a chromatographic column would result in the precipitation or absorption of proteins on the column packing material, resulting in an immediate loss of column performance (changes in retention times,
losses of efficiency and capacity). Similar damage can occur to different components of the ESI/MS/
MS system commonly utilized for analyzing drugs. Matrix components identified by different analytical techniques are shown in Table 1.1. Major classes encountered in plasma consist of inorganic
ions, proteins and/or macromolecules, small organic molecules, and endogenous materials.51–56
Mass spectrometry is the most preferred technique employed during high-throughput screening.
It provides specificity based on its capability to monitor selected mass ions, sensitivity because it
affords enhanced signal-to-noise ratio, and speed due to very short analysis times that allow analysis

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High-Throughput Analysis in the Pharmaceutical Industry

Table 1.1
Interferences Identified in Human Plasma
Classification

Components

Concentration
(mg/L)

Reference

Inorganic ions

Sodium [Na+]
Potassium [K+]
Calcium [Ca2+]
Magnesium [Mg2+]
Chloride [Cl-]
Hydrogencarbonate [HCO3-]
Inorganic phosphorus [P], total
Iron [Fe] in men
Iron [Fe] in women
Iodine [I], total

Copper [Cu] in men
Copper [Cu] in women

3.2 ×103 to 3.4 × 103
148.6 to 199.4
92.2 to 112.2
19.5 to 31.6
3.5 × 103 to 3.8 × 103
1.5 × 103 to 2.1 × 103
21.7 to 41.6
1.0 to 1.4
0.9 to 1.2
34.9 × 10-3 to 79.9 × 10-3
0.7 to 1.4
0.9 to 1.5

51

Proteins/Macromolecules
(g/L)

Prealbumin
Albumin
Acid-a1-glycoprotein
Apolipoproteins (globulins)
Haptoglobin (a2-globulin)
Hemopexin (b1-globulin)
Transferin (b2-globulin)
Ceruloplasmin (a2-globulin)
Transcortin (a1-globulin)

Transcobalamin
a2-Macroglobulin
a1-Antitrypsin
Protein-binding metal (a1-globulin)
Antithrombin III (a2-globulin)
Fibrinogen
Immunoglobulins (g-globulins)

0.1 to 0.4
42.0
0.2 to 0.4
4.0 to 9.0
1.0
0.7
2.9
0.4
0.04
94.0 × 10-8
2.5
2.5
0.06
0.2
4.0
15.0 to 16.0

51

Endogenous components
(small organic molecules)

Amino Acids
Alanine
Valine
Leucine
Serine
Threonine
Methionine
Aspartate
Glutamate
Phenyl alanine
Glycine
Lysine
Tyrosine
Proline
Cystine
Tryptophan

NA
NA
NA
NA
NA
28.8 mM
NA
43.5 mM
55.8 mM
NA
127.3 mM
NA
289.1 mM

NA
55.7 mM

52


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High-Throughput Sample Preparation Techniques

Table 1.1 (Continued)
Interferences Identified in Human Plasma
Classification

Endogenous phospholipids

Prostaglandins
Hormones
Polysaccharides
Unseen endogenous matrix
components (dosing excipients)

Concentration
(mg/L)

Components
Fatty Acid Derivatives
2-Hydroxybutyrate
3-Hydroxybutyrate

3-Methyl-2-hydroxybutyrate
Palmitate
Oleate
Stearate
Laurate
Linoleate

NA
NA
NA
125.8 mM
NA
NA
NA
NA

Other Small Organics
Urea
Glycerate
Creatinine
Glycerol phosphate isomer
Citrate
Ascorbic acid

NA
NA
106.5 mM
NA
318.6 mM
NA

Carbohydrate Derivatives
Glucose
Myoinositol
Inositol phosphates

NA
24.5 mM
NA

Purine Derivatives
Urate
Nucleosides

331.5 mM
NA

Steroids
Cholesterol

2109.7 mM

Phosphatidylcholine
Lysophosphatidylcholines (18:2,
16:0 and 18:0)
Prostaglandin D2 and F2
Melatonin
Glycosaminoglycans
Hydroxypropyl-b-cyclodextrin
Polyethyleneglycol 400

Propyleneglycol
Tween 80

Reference

NA

53

NA
NA
NA
NA
NA
NA
NA

54
55
56

of dozens of samples per hour. One important factor affecting the performance of a mass detector is ion suppression, with the sample matrix, coeluting compounds, and cross talk contributing
to this effect. Operating conditions and parameters also play a role in inducing matrix effects that
result in suppression of the signal, although enhancement is also observed occasionally. The main
cause is a change in the spray droplet solution properties caused by the presence of nonvolatile or
less volatile solutes. These nonvolatile materials (salts, ion-pairing agents, endogenous compounds,


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High-Throughput Analysis in the Pharmaceutical Industry

drugs, metabolites) change the efficiency of droplet formation or droplet evaporation, affecting the
concentrations of charged ions in the gas phase reaching the detector.
The literature clearly reviews how plasma constituents and endogenous materials adversely
affect the quantitation of drugs and their metabolites in these matrices.57–70 It follows that when drugs
or metabolites in biological matrices are analyzed, a thorough purification step must be invoked to
eliminate (or at least minimize) these adverse effects. In the context of high-throughput screening of
ADME (or DMPK) samples, the following discussion elaborates on protocols popularly employed
for the high-throughput clean-up of biological matrix components and/or endogenous materials.

1.2.3 Solid Phase Extraction (SPE)
Application of SPE to sample clean-up started in 1977 with the introduction of disposable cartridges
packed with silica-based bonded phase sorbents. The solid phase extraction term was devised in
1982. The most commonly cited advantages of SPE over liquid–liquid extraction (LLE) as practiced
on a macroscale include the reduced time and labor requirements, use of much lower volumes of
solvents, minimal risk of emulsion formation, selectivity achievable when desired, wide choices of
sorbents, and amenability to automation. The principle of operation consists of four steps: (1) conditioning of the sorbent with a solvent and water or buffer, (2) loading of the sample in an aqueous or
aqueous low organic medium, (3) washing away unwanted components with a suitable combination
of solvents, and (4) elution of the desired compound with an appropriate organic solvent.
With increasing popularity of the SPE technique in the 1980s and early 1990s, polymeric
sorbents started to appear to offset the two major disadvantages of silica based sorbents, i.e., smaller
surface area resulting in lower capacities and instability to strongly acidic or basic media. Around
the mid-1990s, functionalized polymers were introduced to overcome the shortcomings of the first
generation polymers such as lower retention of polar compounds and loss of performance when
the solvent wetting them accidentally dried. Tables 1.2 and 1.3 list some of the popular polar functionalized neutral and ion exchange polymeric SPE sorbents, respectively, along with structure and

Table 1.2
Functionalized Neutral Polymeric Sorbents

Source

Sorbent

Chemistry

Mode of Interaction

Waters (see
2006–2007
Catalog, SPE
products)
Phenomenex (see
2006 Catalog,
SPE products)

Oasis HLB

Divinylbenzene-N-vinylpyrrolidone copolymer

Reversed phase with some
hydrogen bond acceptor
and dipolar reactivity

Strata-X

Polar functionalized
styrene-divinylbenzene
polymer

Varian (see
Catalog, SPE
products)

Focus

Polar functionalized
styrene-divinylbenzene
polymer

Varian (see
Catalog, SPE
products)

Bond Elut
Plexa

Highly cross-linked
polymer with
hydroxylated surface

Reversed phase with weakly
acidic, hydrogen bond
donor, acceptor, and
dipolar interactions
Reversed phase with strong
hydrogen bond donor,
acceptor, and dipolar
character
Hydrophobic retention of

small molecules and
hydrophilic exclusion of
proteins

Examples from Literature
(Plasma Samples Only)
Rosuvastatin (71); NSAIDs
(72); fexofenadine (73);
catechins (74);
valproic acid (75)
Cetirizine (76); pyridoxine
(77); omeprazole (78);
mycophenolic acid (79);
25-hydroxy-vitamin D3 (80)
Fluoxetine, verapamil,
olanzapine, tramadol,
loratidine, and sumatriptane
(81); verdanafil (82)
See catalog


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High-Throughput Sample Preparation Techniques

Table 1.3
Functionalized Ion Exchange Polymeric Sorbents
Source

Sorbent

Waters

Oasis
MCX

Sulfonated divinylbenzeneN-vinylpyrrolidone

Mixed mode with strong
cation exchange and reversed
phase activities

Oasis
MAX

Quarternary amine
functionalized divinylbenzene-
N-vinylpyrrolidone
Carboxy functionalized
divinylbenzene-
N-vinylpyrrolidone
Cyclic secondary/tertiary amine
functionalized divinylbenzene-
N-vinylpyrrolidone

Mixed mode with strong anion
exchange and reversed phase
activities
Mixed mode with weak cation

exchange and reversed phase
activities
Mixed mode with weak anion
exchange and reversed phase
activities

Strata-
X-C

Sulfonated styrene-
divinylbenzene polymer
with polar surface modification

Mixed mode with both strong
cation exchange and reversed
phase interactions

Strata-
X-CW

Carboxylated styrenedivinylbenzene polymer

Strata-
X-AW

Primary and secondary aminefunctionalized styrenedivinylbenzene polymer

Mixed mode with weak cation
exchange, hydrogen bond
donor and acceptor, and

reversed phase activities
Weak anion exchange and
reversed phase interactions

Oasis
WCX
Oasis
WAX
Phenomenex

Chemistry

Mode of Interaction

Examples from Literature
(Plasma Samples Only)
Alkaloids (83); illicit drugs
(84); general screening of
therapeutic and
toxicological drugs (85)
NSAIDs (86); glutathione
(87)
Basic drugs (88)

NSAIDs (86)

Stanazolol (89);
antidepressant drugs (90);
sulfonamides (91);
acrylamide (92)

Phenothiazine drugs (93);
basic drugs (94)

Nucleotide phosphates (95)

manufacturer information, modes of interaction, and references on representative applications in sample preparation. Other known hydrophilic polymeric materials are summarized in a recent review.96
1.2.3.1 Interactions of Sorbent and Analyte in SPE and Selective
Extractions Based on Sorbent Chemistry
The interactions of a sorbent and an analyte fall into three classes: hydrophobic (also called dispersive or van der Waals interactions with associated energy of 1 to 5 kJ/mol), polar, and ionic. Polar
interactions are further divided into dipole-induced dipole (2 to 7 kJ/mol), dipole–dipole (5 to 10
kJ/mol), hydrogen bonding (10 to 25 kJ/mol), and ion–dipole (10 to 50 kJ/mol). Ionic interactions
are electrostatic with the highest associated energy levels of 50 to 500 kJ/mol. These energy values
reflect the fact that when analytes interact with neutral sorbents only through hydrophobic interactions, a thorough organic wash (with 100% solvent) cannot be carried out and hence extracts may
contain some contaminants or interference. On the other hand, sorbents possessing ion exchange
functionalities can retain ionizable analytes via ionic mechanisms and are amenable to 100% organic
solvent washes, thereby furnishing much cleaner extracts.58
Ion exchange resins based on poly(styrene-divinylbenzene) backbones display mixed mode
retention mechanisms. The ion exchange functionality (sulfonic acid or carboxylic acid for cation
exchangers and quarternary or primary, secondary, or tertiary amines for anion exchangers) contributes to the ionic mechanism and the backbone polymer to hydrophobic retention. This is exemplified


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High-Throughput Analysis in the Pharmaceutical Industry
COOH
OH
CH3

OH

CH3

C5H11

O

CH3

O

CH3

Tetrahydrocannabinol (THC)

C5H11

THC-COOH (main metabolite)

Figure 1.1  Structures of THC and THC-COOH, its main metabolite from urine.

by a recent report demonstrating the retention of a hydrophobic molecule like THC carboxylic acid (a
metabolite of THC, the major constituent of marijuana; see Figure 1.1) on a strong cation exchanger
like strata-X-C even when subjected to a 30 to 40% acetonitrile wash without breakthrough.97
The mechanisms of retention of apparently basic analytes on either strong or weak cation
exchanger resins depend upon the structures of these analytes and the intra-molecular interactions
of the functional groups on these analytes. Thus, tetracycline and its analogs are not eluted from the
sulfonic acid-functionalized strata-X-C resin with methanol containing 5% ammonium hydroxide or
with acetonitrile containing 0.1M oxalic acid. However, these antibiotics are eluted from strata-X-C
with acetonitrile containing 1.0M oxalic acid. On the other hand, they could be easily eluted from the
carboxy functionalized weak cation exchanger strata-X-CW with methanol containing formic acid.

The differences in the elution patterns for the two sorbents have been explained98 by invoking
the zwitterionic structures for the antibiotics under the basic pH conditions employed for strata-X-C

R2 HO
7

8

8a

6

H

5a

5

11a

10a

9

H3C

CH3
R1

11

10

4a
12a

CH3

N

4

1

OH

3

O

2

C

12

OH
O
HO
O

OH
Tetracycline (TC): R1 = R2 = H
Oxytetracycline (OTC): R1 = OH, R2 = H
Chlorotetracycline (CTC): R1 = H, R2 = Cl
H
H3C
CH3
H

HO

H

N+

O

HO

O

H3C

CH3
HO

H

CH3
H

H

N

CH3
H
O

O

(–)

OH

NH2

OH

O
N

H
H
Zwitter ionic form of tetracyclines

OH

O

HO

O

N

H
Fully enolized form of tetracyclines

Figure 1.2  Neutral, zwitterionic, and fully enolic forms of tetracyclines.

H


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High-Throughput Sample Preparation Techniques

(see Figure 1.2). At acidic pH, the antibiotics exist in their non-ionized enol forms and can be eluted
from the weaker carboxylic resin with formic acid, which is stronger than the surface carboxylic
acid moieties. However, the sulfonic acid is a stronger acid than either formic or even 0.1M oxalic
acid (pH 2.0) and hence the basic dimethylamino moieties on the antibiotics preferentially stay with
the sulfonic acid; these antibiotics can be eluted with 1.0M oxalic acid (pH 0.8) from these strong
ion exchange resins. Neutral polar functionalized polymers like Oasis HLB or strata-X do not retain
the tetracyclines even when a 5% methanol wash is used.
Another interesting selectivity difference was observed99 during the extraction of benzodiazepine drugs from plasma employing different sorbents. With silica-based strata-C18E, the neutral
polymeric strata-X sorbent, or the strata-X-CW weak cation exchanger, diazepam, nordiazepam,
oxazepam, lorazepam, and temazepam could all be eluted in excellent yields (Table 1.4) with methanol. On the other hand, with the strong strata-Screen C (silica-based sulfonic acid) and strata-X-C
cation exchangers, methanol eluted oxazepam, lorazepam, and temazepam, while methanol containing 5% ammonia was needed to elute diazepam and nordiazepam.

The differential elution with strong cation exchangers does not stem from differences in pH
(see Figure 1.3 for structures and pH values). On the contrary, oxazepam, lorazepam, and temazepam possess a hydroxyl at the C-3 position of the diazepine ring system that can stabilize their enolic
forms while simultaneously promoting hydrogen bonding with the basic N-4 nitrogen, resulting in the

Table 1.4
Results of SPE of Benzodiazepines from Plasma
Sorbent

Main Mode of
Interaction

Benzodiazepine

% Recovery with
Methanol

% Recovery with
Methanol/5% Ammonia

strata-C18-E (silica
based)

Reversed phase

Nordiazepam
Diazepam
Oxazepam
Lorazepam
Temazepam

104
101
97
95
95

Not applicable

strata-Screen C

Cation exchange

Nordiazepam
Diazepam
Oxazepam
Lorazepam
Temazepam

9
24
63
104
87

90
93

strata-X

Reversed phase

Nordiazepam
Diazepam
Oxazepam
Lorazepam
Temazepam

103
98
94
95
92

Not applicable

strata-X-CW

Weak cation
exchanger

Nordiazepam
Diazepam
Oxazepam
Lorazepam
Temazepam

94
97
96
100

98

Not applicable

strata-X-C

Strong cation
exchanger

Nordiazepam
Diazepam
Oxazepam
Lorazepam
Temazepam

14
18
65
88
87

96
95


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10

High-Throughput Analysis in the Pharmaceutical Industry
H 3C

N
Cl

H

O

O

N

O

H
N

OH
Cl

N

Cl

N

N
R

Oxazepam (R = H, pKa 1.7)

Diazepam (pKa 3.3)

Nordiazepam (pKa 1.7) Lorazepam (R = Cl, pKa 1.3)

H 3C

O

N

H
N
OH

Cl

N

OH
OH

Cl

N
Cl

Temazepam (pKa 1.6)

Lorazepam (enol form)

Figure 1.3  Structures and pKa values of benzodiazepines.

failure of this nitrogen to interact with the sulfonic acid moieties of strata-X-C and the silica-based
strata-Screen C through ionic mechanism. Consequently, these drugs are eluted off in methanol.
Selective extraction of the flavonoid components from the ginkgolide and bilabalide terpenoids
(see Figure 1.4) of health supplement extracts from Ginkgo biloba leaves has been demonstrated100
by solid phase extraction with the weak strata-X-CW cation exchanger. While the terpenoids could
be eluted with 60:40 methanol:water, the flavonoids required a strong organic (methanol:acetonitrile:
water, 40:40:20 or acetonitrile:dichloromethane, 50:50) for elution. In comparison, the silica-based
strata-C18E and the neutral strata-X polymer did not exhibit this kind of selectivity (see Table 1.5
for recovery data), the former eluting all components with 60:40 methanol:water, while the latter
eluted the terpenoid partially in this solvent and partially with the stronger organic.
This protocol was modified to enable automation. In a later publication,107 20 mg of plant material (Arabidopsis thaliana) was extracted with 1 mL of methanol, water, and formic acid. The extract
was transferred to glass tubes in an Aspec XL4 robot. After an initial clean-up with a C18 cartridge, the extract was evaporated and the residue reconstituted in formic acid and transferred to the
robot. SPE purification was carried out with Oasis MCX. After buffering and methanol wash, the
cytokinins were eluted with methanol and aqueous ammonium hydroxide (see Figure 1.5). After
evaporation, the residue was derivatized with either propionic anhydride or benzoic anhydride. The
derivatives were analyzed by LC/MS/MS using a 10 × 1 mm BetaMax Neutral Guard cartridge
as the LC column. Lower detection limits in the femtomole to attomole range were obtained. The
protocol was also successfully applied to non-cytokinin compounds such as adenosine mono-,
di-, and tri-phosphates, adenosine, uridinophosphoglucose, and flavin mononucleotide with the same
limits of detection. The ESI sensitivity of the derivatives was found to be far superior compared to
underivatized cytokinins and nucleotides. The procedure can be applied to strongly hydrophilic
molecules from any biological matrix and serves as an example of high-throughput automated solid
phase extraction.
The propensity of mixed mode cation exchange resins to retain highly water-soluble compounds
like gamma-aminobutyric acid (GABA) was demonstrated in a recent publication.108 Animal tissue

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11

High-Throughput Sample Preparation Techniques
R

O

OH
O

HO

HO

HO
O

R2
H3C

O

O

O

Flavonoid aglycones (isolated by
acid hydrolysis of the corresponding
flavonoid glycosides)

Kaempferol (R = H)
Quercetin (R = OH)
Isorhamnetin (R = OCH3)

R1

C(CH3)3

O
R3

O

O

Ginkgolides A (R1 = OH; R2 = R3 = H);
B (R1 = R2 = OH; R3 = OH); C
(R1 = R2 = R3 = OH)

Bilabalide

O
O

HO

C(CH3)3
OH

O

O O
O
O

Figure 1.4  Structures of flavonoids and terpenoids from Gingko biloba.

Table 1.5
Selective Elution of Flavonoids from Terpenoids in Ginkgo biloba Leaf Extracts
Strata C18-E
Analyte (MW)

Elut 1

Elut 2

Quercetin (302)
Kaempferol (286)
Isorhamnetin (316)
Bilobalide (326)
Ginkgolide A (408)

83
84
90
113
82

8
13
13

0
0

NH-R1

Strata-X
Elut 1

Strata-X-CW
Elut 2

0
0
0
9
51

Elut 1

89
85
80
102
45

25
0
0
86
78

CH3

CH2COOH

N

N
N

N

N
H
Auxin (Indole-3-acetic acid)

O

R2
Cytokinins and nucleosides
R1 = -CH2CH = CHCH2OH
CH2CH = CHCH2O Ribofuranoside (or glucopyranoside)
-CH2CH2CH(CH3)CH2OH
-CH2Phenyl
Cytokinin nucleotides
R2 = H or beta-D-ribofuranosyl-5'-monophosphate

OH
CH3

Abscisic Acid

Figure 1.5  Structures of auxin and abscisic acid derivatives of cytokinin.

COO–

Elut 2
76
90
103
2
0


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12

High-Throughput Analysis in the Pharmaceutical Industry

was extracted with simultaneous protein precipitation using 2% sodium dodecylsulfate in 0.1M
potassium dihydrogen phosphate buffer (pH 2). After centrifugation, the supernatant was loaded
onto an Oasis MCX cartridge. Washing with methanol and formic acid in acetonitrile (10:90) selectively eluted gamma-hydroxybutyric acid and 1,4-butanediol. GABA was then eluted with water:
methanol:ammonia (94.5:5:05 v/v). All the analytes were derivatized with N-(t-butyldimethylsilyl)N-methyl trifluoroacetamide (MTBSTFA) and analyzed by GC/MS. This procedure is potentially
suitable for evaluating PMI (postmortem interval) in humans because the amount of GABA in blood
increases after death and the increase may be correlated to time of death.
Relative extraction efficiencies of polar polymeric neutral, cation, and anion exchange sorbents
(HLB, MCX, and MAX) for 11 beta antagonists and 6 beta agonists in human whole blood were
probed.109 Initial characterization of MCX and MAX for acidic and basic load conditions, respectively,
showed that both the agonists and antagonists were well retained on MCX, while they were recovered
from MAX in the wash with either methanol or 2% ammonia in methanol (see Table 1.6). Blood samples were treated with ethanol containing 10% zinc sulfate to precipitate proteins and the supernatants

loaded in 2% aqueous ammonium hydroxide onto the sorbents. After a 30% methanol and 2% aqueous
ammonia wash, the analytes were eluted with methanol (HLB), 2% ammonia in methanol (MCX),
or 2% formic acid in methanol (MAX). The best recoveries were observed with MCX under aqueous conditions or blood supernatant (after protein precipitation) spiked sample load conditions (see
Table 1.7). Ion suppression studies by post-column infusion showed no suppression for propranolol
and terbutaline with MCX, while HLB and MAX exhibited suppression (see Figure 1.6).

Table 1.6
Comparison of MCX and MAX for SPE of b-Agonists and Antagonists
SPE Column
Equilibration
and Loading
Washing Elution

MCX 2% HCOOH
aq MeOH
2% NH4OH in MeOH

Collected Fractions

Washing

Elution

b-Antagonists:
Atenolol
Sotalol
Carteolol
Pindolol
Timolol
Metoprolol

Bisoprolol
Labetalol
Betaxolol
Propranolol
Carvediol

0
0
0
Trace
Trace
Trace
0
Trace
Trace
Trace
1.5

100
93
96
58
90
92
93
85
90
84
83

0
0
0
0
0
0
0
0
0
0
0

b-Agonists:
Salbutamol
Terbutaline
Fenoterol
Formoterola
Clenbuterol
Bambuterol

0
0
0
Trace
Trace
0

98
101
104

53
94
97

0
Trace
0
0
Trace
0

2% HCOOH in
MeOH
Washing

MAX 2% NH4 OH
aq MeOH
2% NH4OH in MeOH

2% HCOOH in
MeOH

Elution

Washing

Elution

Washing

102
94
97
76
85
88
90
83
93
91
82

99
0
91
86
99
91
90
0
86
73
65

5
113
4
15
4
17

18
108
23
28
23

98
0
91
87
97
91
86
0
86
70
63

4
111
2
16
3
16
18
114
22
29
22

99
95
54
32
93
96

27
0
0
26
84
91

53
80
94
41
6
3

38
0
0
18
77
92

47
77

87
42
5
4

Source: From M. Joseffson and A. Sabanovic, J. Chromatogr. A, 2006, 1120, 1. With permission from Elsevier.

Elution