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

Chapter 1 High-Throughput Sample Preparation Techniques and Their Application to Bioanalytical Protocols and Purification of Combinatorial Libraries ...1 Michael G.. Preparation Techniq

H igH -T HrougHpuT A nAlysis in

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

Edited by

Perry G Wang

CRC Press

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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]

Chapter 1 High-Throughput Sample Preparation Techniques and Their Application to

Bioanalytical Protocols and Purification of Combinatorial Libraries 1

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

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

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

Pharma-ceutical 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 cer-tain automation Conventional analytical approaches can no longer keep pace with the new break-throughs 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 spectrom-etry (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 (HPLC-MS) and the use of tandem mass spectrometry (HPLC/MS-MS) have proven to be the most impor-tant 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

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 neering His expertise focuses on high-throughput analysis of drugs and their metabolites in biologi-cal matrices with LC/MS/MS

engi-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 ence 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

Confer-He earned a B.S in chemistry from Shandong University and an M.S and Ph.D in environmental engineering from Oregon State University

Min Shuan Chang

Abbott Laboratories

Abbott Park, Illinois, USA

Kojo S J Elenitoba-Johnson

Department of Pathology

University of Michigan Medical School

Ann Arbor, Michigan, USA

Brehm Research Laboratory

Wright State University

Fairborn, Ohio, USA

Exploratory Drug Metabolism

Schering-Plough Research Institute

Kenilworth, New Jersey, USA

Pharmaceutical Development Center

MD Anderson Cancer CenterThe University of TexasHouston, Texas, USA

Douglas E McIntyre

Agilent TechnologiesSanta Clara, California, USA

Patrick J Rudewicz

GenentechSouth San Francisco, California, USA

Young Shin

GenentechSouth San Francisco, California, USA

Katty X Wan

Abbott LaboratoriesAbbott Park, Illinois, USA

Dong Wei

Biogen Idec, Inc

Cambridge, Massachusetts, USA

Quanyun A Xu

Pharmaceutical Development Center

MD Anderson Cancer CenterThe University of TexasHouston, Texas, USA

Richard Xu

Micro-Tech ScientificVista, California, USA

Contributors

Biogen Idec, Inc.

Cambridge, Massachusetts, USA

Qin Yue

GenentechSouth San Francisco, California, USA

Wanlong Zhou

Brehm Research LaboratoryWright State UniversityFairborn, Ohio, USA

Preparation Techniques

and Their Application to

Bioanalytical Protocols

and Purification of

Combinatorial Libraries

Krishna Kallury

Contents

1.1 Need for High-Throughput Sample Purification and Clean-Up in Drug Discovery 2

1.2 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

. 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 prod-ucts 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 high-throughput screening, computational chemistry, and automation of laboratory procedures, led to a significantly accelerated drug discovery process compared to the traditional one-compound-at-a- time approach During the high-throughput biological screening of combinatorial compounds, ini-tial 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 tis-sue 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 chromato-graphic 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 puri-fication 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 metabo-lites in biological matrices, it is suggested that the readers refer to the latest literature available

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

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

. rapid puriFiCation teCHniques For drugs

and metabolites in biologiCal matriCes

1.2.1 State - of -a rt S ample p reparation p rotocolS

niques 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 fun-

of the principles of these methods are presented For isolating drugs and metabolites from biological matrices, several approaches have been reported, which consist of:

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 desiredDetect 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

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 chro-matographic column would result in the precipitation or absorption of proteins on the column pack-ing 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/

lytical techniques are shown in Table 1.1 Major classes encountered in plasma consist of inorganic

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

interferences identified in Human plasma

Concentration (mg/l) reference

Potassium [K + ] Calcium [Ca 2 + ] Magnesium [Mg 2 + ] 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 × 10 3 to 3.4 × 10 3 148.6 to 199.4 92.2 to 112.2 19.5 to 31.6 3.5 × 10 3 to 3.8 × 10 3 1.5 × 10 3 to 2.1 × 10 3 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

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

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

of dozens of samples per hour One important factor affecting the performance of a mass detec-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,

table . (Continued)

interferences identified in Human plasma

Concentration (mg/l) reference 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

NA NA NA NA

The literature clearly reviews how plasma constituents and endogenous materials adversely

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 S olid p haSe e xtraction (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) condi-tioning 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 func-tionalized neutral and ion exchange polymeric SPE sorbents, respectively, along with structure and

table .

Functionalized neutral polymeric sorbents

examples from literature (plasma samples only)

Rosuvastatin (71); NSAIDs (72); fexofenadine (73); catechins (74);

valproic acid (75) Phenomenex (see

2006 Catalog,

SPE products)

Strata-X Polar functionalized

styrene-divinylbenzene polymer

Reversed phase with weakly acidic, hydrogen bond donor, acceptor, and dipolar interactions

Cetirizine (76); pyridoxine (77); omeprazole (78); mycophenolic acid (79); 25-hydroxy-vitamin D 3 (80) Varian (see

Catalog, SPE

products)

Focus Polar functionalized

styrene-divinylbenzene polymer

Reversed phase with strong hydrogen bond donor, acceptor, and dipolar character

Fluoxetine, verapamil, olanzapine, tramadol, loratidine, and sumatriptane (81); verdanafil (82) Varian (see

Catalog, SPE

products)

Bond Elut Plexa

Highly cross-linked polymer with hydroxylated surface

Hydrophobic retention of small molecules and hydrophilic exclusion of proteins

See catalog

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) contrib-utes to the ionic mechanism and the backbone polymer to hydrophobic retention This is exemplified

table .

Functionalized ion exchange polymeric sorbents

examples from literature (plasma samples only)

Waters Oasis

MCX

N-vinylpyrrolidone

Sulfonated divinylbenzene-Mixed mode with strong cation exchange and reversed phase activities

Alkaloids (83); illicit drugs (84); general screening of therapeutic and toxicological drugs (85) Oasis

MAX

Quarternary amine functionalized divinylbenzene- N-vinylpyrrolidone

Mixed mode with strong anion exchange and reversed phase activities

NSAIDs (86); glutathione (87)

Oasis

WCX

Carboxy functionalized divinylbenzene- N-vinylpyrrolidone

Mixed mode with weak cation exchange and reversed phase activities

Basic drugs (88)

Oasis

WAX

Cyclic secondary/tertiary amine functionalized divinylbenzene- N-vinylpyrrolidone

Mixed mode with weak anion exchange and reversed phase activities

NSAIDs (86)

Phenomenex Strata-

X-C

Sulfonated styrene- divinylbenzene polymer with polar surface modification

Mixed mode with both strong cation exchange and reversed phase interactions

Stanazolol (89);

antidepressant drugs (90); sulfonamides (91); acrylamide (92) Strata-

X-CW

divinylbenzene polymer

Carboxylated styrene-Mixed mode with weak cation exchange, hydrogen bond donor and acceptor, and reversed phase activities

Phenothiazine drugs (93); basic drugs (94)

Strata-

X-AW

functionalized styrene- divinylbenzene polymer

Primary and secondary amine-Weak anion exchange and reversed phase interactions

Nucleotide phosphates (95)

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

THC-COOH (main metabolite)

1 2

3 4 4a 5 6

7 8 9

8a

5a 11a

O N O

O (–)

H H

O H

OH

H H

Zwitter ionic form of tetracyclines Fully enolized form of tetracyclines

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

during the extraction of benzodiaz-epine 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 meth-anol 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 contain-ing 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 temaze-pam 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 .

results of spe of benzodiazepines from plasma

sorbent

main mode of interaction benzodiazepine

% recovery with methanol

% recovery with methanol/% ammonia

strata-C18-E (silica

based)

Reversed phase Nordiazepam

Diazepam Oxazepam Lorazepam Temazepam

104 101 97 95 95

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

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

20 mg of plant mate-rial (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 tridge, 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

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

Flavonoid aglycones (isolated by

acid hydrolysis of the corresponding

H3C

R2 R1

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) selec-tively 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

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 sam-ples 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),

ous 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)

maX % nH  oH

aq meoH

% nH  oH in meoH

% HCooH in meoH Collected Fractions Washing elution Washing elution Washing elution Washing elution

Although Certify is a mixed mode sorbent with C8 and sulfonic acid moieties, the authors ratio-of certain drugs during methanol wash The weak WCX ion exchanger was also excluded for similar reasons Both the mixed mode strata-X-C and the ion exchange sorbent SCX were found to be most amenable for the derivatization-based GC/MS analysis and both yielded pure extracts However, the yields were consistently lower with strata-XC than SCX and the authors hypothesized that this was due to the inability of the 5% ammonia/methanol eluent to completely disrupt the hydrophobic and dipolar interactions between the analytes and XC

investigated These drugs were divided into two groups—one consisting of desmethylmirtazapine, O-desmethylvenlafaxine, desmethylcitalopram, didesmethylcitaloporam, reboxetine, paroxetine, maprotiline, fluoxetine, norfluoxetine, and m-chlorophenylpiperazine The other group included mirtazapine, viloxazine, desmethylmianserin, citalopram, mianserin, fluvoxamine, desmethylser-traline, sertraline, melitraen, venlafaxine, and trazodone They tested protein precipitation by four methods: dilution with (1) pH 2.5 or pH 6.5 phosphate buffer, (2) glycine hydrochloride, (3) 2% phosphoric acid, and (4) organic solvents (methanol and acetonitrile) Since the sorbents used for SPE were cation exchangers, Willie’s group did not investigate inorganic salts

Terbutaline 226/152 Propranol 260/116

Supernatant

EtOH/ZnSO 4 aq 1:1 (v/v)

HLB

30% MeOH in 2% NH4OH aq

Figure . Comparison of ion suppression data for propranolol and terbutaline after solid phase extraction

with HLB, MAX, and MCX 109 (Reproduced with permission from Elsevier.)

a-1-acid glycoprotein of the plasma (isoelec-tric point 3.0) at pH 2.5 for both reagents On the other hand, the lower recoveries for acetonitrile (62%) and methanol (78%) were interpreted as arising from the hydrophobic binding of the drugs to albumin and lower solubility of the drugs in acetonitrile Phosphoric acid gave 73% recovery The importance of load pH and disruption of hydrophobic interactions while using ion exchange and mixed mode sorbents is thus emphasized

Of particular interest is the comparison of the performance of cation exchange and mixed mode sorbents for their efficacy in cleaning up endogenous phospholipids Unlike the protein-related materials that are eluted in the very early stages of HPLC, these phospholipids elute in the hydro-phobic region and interfere with drug peaks which also elute around the same time

compared SPEC SCX disks with SPEC MP1 disks and Oasis MCX SPEC-SCX is a phenylsulfonic acid, while MP1 is a mixed mode C8/sulfonic acid and MCX is a polymeric sulfonic acid on a divinylbenzene–vinylpyrrolidone polymer backbone The sorbents were conditioned with methanol and then with 2% formic acid The sample was loaded in 2% formic acid solution and washing was done with 2% formic acid, followed by acetonitrile:methanol (70:30) Analytes were eluted with two aliquots of methanol:acetonitrile:water:ammonia (45:45:10:4% v/v/v/v) The eluent was dried under nitrogen and the residue reconstituted in the mobile phase (80% 10mM ammonium formate containing 0.2% formic acid and 20% 10mM ammonium formate in methanol with 0.2% formic acid) Their data on desloratadine and its 3-hydroxy analog (see Figure 1.7), along with data on phosphatidyl-choline indicates that MCX retains about seven times as much phospholipids as SCX does and MP1 retains around 60 times more than SCX (see Figure 1.8) Post-column infusion experiments with blank plasma extracts showed ion suppression in the hydrophobic region for MP1 and MCX, but not for SCX (see Figures 1.9 through 1.11) The observations were rationalized through hydrophobic retention of the phospholipids by the mixed mode sorbents; SCX did not exhibit such retention mechanisms

... Formats for rapid and/or High-throughput solid phase

extraction of drugs in biological matrices

erence around 1995 to cater to the high-throughput sample preparation needs of bioanalysis The

be detailed here In this well format, the sorbent is packed at the bottom of the plate with popular bed mass sizes ranging from 10 to 500 mg Further refinements of this 96-well flow-through system include miniaturization of the plate and well geometry to accommodate as little as 2 mg of sorbent

Cl N

N H

Elsevier.)

Liquid handling systems such as the Tomtec Quadra Model 320 or Packard Multiprobe II EX (HT) are used to automate the solid phase extraction process The former carries 96 pipette tips for simultaneous delivery of liquid into all 96 wells, while the latter is designed with 8 tips Process-ing of a well plate using the Tomtec takes 10 min Multiprobe processing requires 30 to 60 min to complete SPE on a 96-well plate This compares favorably in terms of time and labor to manual SPE of a 96-well plate that requires more than 5 hr for completion of one extraction Other popu-lar liquid handling systems include the Sciclone Advanced Liquid Handler Workstation (Zymark), Cyberlab (Gilson, Inc.), Multimek (Beckman Coulter), and Personal Pipettor (Apricot Designs), all

contains detailed accounts of these automated liquid stations; they are not discussed here due to spatial considerations

A few examples from the latest literature will be presented to illustrate the use of the 96- and higher well formats and pipette tip formats for high-throughput sample preparation An interest-ing example of orthogonal extraction chromatography and ultra-pressure liquid chromatography (UPLC) of plasma samples of desloratadine and 3-hydroxy-desloratadine (see Figure 1.7 for struc-

SCX

MCX

MP1

2.0e+7 0.0

1.5e5 1.4e5 1.2e5 1.0e5 8.0e4 6.0e4 4.0e4 2.0e4 0.0 1.11e5 1.00e5 8.00e4 6.00e4 4.00e4 2.00e4 0.00 1.4e4 1.2e4 1.0e4 80000 60000 40000 20000 0.0

150

necessary to achieve significantly better signal-to-noise ratio compared to the Shimadzu experiment representing an LLOQ of 0.478 pg for desloratadine and 0.525 pg for the 3-hydroxy metabolite

In a control experiment, the peak widths with UPLC were found at 0.15 min and at 0.16 min for desloratadine and its 3-hydroxy analog, respectively The corresponding values from the Shimadzu experiment were 0.37 min and 0.32 min, respectively Nevertheless, only a marginal improvement

in sensitivity (peak height) was found under UPLC conditions The accuracy and precision values for the two drugs under the two sets of LC conditions were very similar

over 10 min Subsequently, low vacuum (0.5 scfh) was applied and washing was done with 2% formic acid followed by 5% methanol in water After application of low vacuum, the drug was eluted with

the plate under low vacuum The eluate was evaporated under nitrogen (15 min) and reconstituted in acetonitrile The addition of TFA during sample dilution was aimed at keeping the carboxylic groups

of cetirizine in the protonated form and the piperazine ring nitrogens protonated The reconstituted

Scientific) using a mobile phase of acetonitrile:water:TFA:acetic acid (93:7.0:0.025:1 v/v/v/v) under isocratic conditions and MRM detection on an API 3000 or 4000 mass spectrometer using a run time

standard A minimum detection limit of 1.0 ng/mL was achieved Matrix lot-to-lot reproducibility tests revealed an RSD of 5%; the RSD for precision and accuracy was less than 3% For LLOQ

LC for rapid analysis based on their analyses of several drugs and analyses by other laboratories

An automated solid phase extraction method for human biomonitoring of urinary polycyclic aromatic hydrocarbon (PAH) metabolites using the RapidTrace SPE workstation was recently

wood, and tobacco Exposure is primarily through inhaling polluted air or tobacco smoke, and by ingestion of contaminated and processed food and water Dermal exposure may also be a major path-way Following absorption in the human body, PAHs are rapidly biotransformed into hydroxylated metabolites by cytochrome P450 mono-oxygenases and these are further converted into glucuro-nide or sulfate conjugates to enhance their polarity and consequently aid in their urinary excretion

added and the metabolites were derivatized with MSTFA The silyl derivatives were analyzed by GC/MS using a DB-5 column Appropriate C13 labeled metabolites were used as standards and the molecular ions and their [M-15] fragment ions were monitored Variations were within stipulated

exchange SPE sorbent (MP1) in this tip format is also available from Varian

When these tips are used for extraction, a sample solution is aspirated and then dispensed using

an automated liquid handler like Tomtec Quadra and circulates across the solid phase media The use of a monolithic glass fiber results in a design that has less sorbent density than that used in a traditional plate format and enables free flow of a liquid across the media without assistance from

of which is shown in Figure 1.13 SCH 56984, a closely related compound, was used as an internal standard The extraction procedure was the same as in a typical SPE procedure: conditioning, appli-cation of the sample solution, wash, and elution The wash and elution solutions were pre-aliquoted into individual wells of a 96-well block before placement on the Tomtec Prior to aspiration, a 50- to 150-mL air gap was drawn into the m-SPE tips followed by an aliquot of the sample solution and

air from the system air compressor was blown into the tips to dislodge remaining liquid No vacuum application or manual operator intervention was needed

phase B was acetonitrile:methanol:formic acid (90:10:0.1 v/v/v) with a gradient from 10% A at 0.3 min to 75% in 1.3 min, held until 2.5 min, then to 100% B at 2.6 min and back to 10% at 3.6 min and equilibrated until 4 min Posaconazole and the internal standard had retention times of 2.0 and 2.1 min, respectively

Wash–elution and aspiration–dispensing cycle optimization experimental results are shown in Figures 1.14 and 1.15, respectively A comparison of recovery yields between the tip experiment and

a 96-well plate containing 15 mg of Varian SPEC C18 under the same extraction conditions gave a value of 70% for the latter, a figure obtained from three aspiration–dispensing cycles for the former For intra-run accuracy of calibration standards, a %CV range from –3.6% to 3.5% was recorded,

respectively Run precisions were 1.1 to 9.2% and 5.1 to 5.7%, respectively, for calibration and QC samples An LLOQ of 10 ng/mL was established

An analogous pipette tip-based solid phase extraction of ten antihistamine drugs from human

pipette tip volume, C18-bonded monolithic silica gel with a diameter of 2.8 mm and thickness of

1 mm) was utilized The monolithic silica with a continuous mesoporous (pore size ~20 nm) silica

pipette tip and chemically modified with the C18 phase The advantages of this sorbent include ease of extraction coupled with rapidity compared to conventional SPE cartridges The small bed volume and the sorbent mass within the MonoTip C18 permit use of a small volume of solvent, smaller elution volumes, and reduced evaporation times, leading to higher throughput A plasma

Posaconazole

SCH 56984 Formula Weight: 700.8

Formula Weight: 686.8

7 6 5 4

4 OH 2% HCOOH 2

1 0

# of Aspirate and Dispense

40 20 0

(Reproduced with permission from Elsevier.)

... online solid phase extraction as tool for High-throughput applications

Features that make online SPE more attractive compared to off-line SPE consist of:

Direct elution of analyte from extraction cartridge into LC system

Elimination of time-consuming evaporation, reconstitution, and preparation for injectionAchievement of maximum sensitivity for detection because the entire volume of eluate

up such as dual and multiple columns, turbulent flow chromatography, restricted access media,

ations and the ability of the cartridges to withstand high pressures and pH extremes

the most promising and viable mode is use of online SPE cartridges based on economic consider-Generally, an online SPE LC/MS/MS system consists of three major hardware components: an online SPE module, a separation (LC) module, and a detection (MS) module A multicomponent LC pumping assembly with two individual HPLC pumping units connected by one or two switching valves (six- or ten-port type) is used in most of the online applications reported in the literature One pump is used for plasma sample loading and washing of the SPE cartridge; the other is used for ana-lytical separation of compounds eluted from the SPE cartridge after removal of plasma proteins

for verapamil, indiplon, and six investigative drug compounds, using a strata-X online extraction

the analytical column This combination permits exploitation of the speed of the monolithic columns and provides the advantages of polymeric online SPE that also include the ability to utilize hydro-phobic and hydrophilic interactions simultaneously in addition to the favorable features cited above The flow rate for achieving optimal removal of proteins was established initially, by comparing 2, 3, and 4 mL/min flow rates using 90:10 water:acetonitrile with 0.1% formic acid as the mobile phase;

4 mL/min was determined to yield the cleanest profile

A single six-port switching valve was used in two settings In position A, the autosampler (HTC Pal, LEAP Technologies) loads the plasma sample onto the strata-X, followed by a 30-sec wash-ing using the same mobile phase as above at 4 mL/min; in position B, the drugs are back-eluted off strata-X (after the 30-sec wash, the six-port valve switches to connect the monolithic column) into the monolithic column that effectively provides baseline separations for all eight drug compounds The autosampler syringe depth was adjusted such that the syringe needle only slightly penetrated the top layer of the diluted plasma solution in the autosampler vial This avoided clogging from the diluted plasma sample The linear range was validated from 1.95 to 1000 ng/mL of each drug and greater than 0.997 correlation coefficient values were obtained The set-up enabled the analysis of

sure, chromatographic retention time, baseline noise level, or peak shape for each analyte The method proved to be rugged and comparison of off-line LLE data with results from this online method for pharmacokinetic screening samples for 0.25 to 12 hr time periods showed that the online SPE method was as efficient as the LLE method

autosampler as a device to measure and introduce both sample (analyte) and IS, two off-line (manual) sample preparation steps (measuring fixed amounts of samples and spiking with fixed amount of IS) can be eliminated The applicability of this method for propranolol and diclofenac using ketoconazole and ibuprofen as ISs, respectively, was demonstrated on the Symbiosis system

introduction of sample and IS are illustrated in Figure 1.16 The IS may be injected from a vial via autosampler or directly into the injection loop (using one of the injection modes of the Symbiosis autosampler), the latter avoiding cross contamination possible with the former The variation (RSD)

in IS peak areas of samples spiked with IS off-line were 10.1% for ketoconazole and 2.1% for ibuprofen For online introduction, the values were 6.8 and 3.1% for ketoconazole and ibuprofen,

(IS) Stock Bottle

(IS) Stock Bottle

(IS) Stock Bottle

(IS Reservoir)

(IS Reservoir)

(IS Reservoir) 96-well plate

(IS Reservoir)

Injection Valve

Injection Valve

Injection

Valve

Injection Valve

Injection Valve Injection Loop

Injection Loop

Injection Loop

Injection Loop Needle

Tubing Injection Needle

Needle Tubing Injection Needle

Needle Tubing Injection Needle

Needle Tubing Injection Needle

Needle Tubing Injection Needle

Needle Tubing Injection Needle

Mobile Phase Internal Standard Sample

Mobile Phase Internal Standard Sample

Mobile Phase Internal Standard Sample

Mobile Phase Internal Standard Sample

Mobile Phase Internal Standard Sample

Mobile Phase Internal Standard Sample

Syringe Syringe

Figure .

Configuration and operational details of online introduction of internal standard for quantita-tive analysis of drugs from biological matrices 118 (Reproduced with permission from the American Chemical Society and the authors.)

... utility of -Well plates for High-throughput applications

and in-process monitoring of Cross Contamination

An application involving the use of monoclonal antibody fragments for selective extraction of the d-enantiomer of an experimental drug belonging to the diarylalkyl triazole system was reported

Progress with the 384-well plate solid phase extraction has been slow since the first examples

increased cross contamination, lack of appropriate supplies and tools, lack of demand and interest, presence of other upstream and downstream bottlenecks, and sample volume and sensitivity limits

ritonavir, the active ingredients of the Kaletra anti-HIV drug Samples in individual vials were

Sample Flow-throughs (1-6) Matrix metal, copper or cobalt antibody: ENA5His

or ENA5His Y96V

Sample application (1 – 10) × 50 µL incubation time, 3 or 5 min

suspension 1, 3, 5 times, Washes 2, 3, 4 times 7,2–14.4 bed volumes PBS,

a significant impact on the overall throughput unless shorter LC/MS methods such as UPLC, high temperature LC, multiparallel micro-HPLC, and nanoelectrospray infusion are used For example,

a run time of 2.2 min will allow handling of 570 samples in 21 hours, while a 1.5-min run time will facilitate running of 840 samples in the same 21-hour period With respect to availability of appa-ratus and disposables, the authors note that SPE using a centrifuge minimizes cross-contamination, but the technique is difficult to automate On the other hand, one must be careful about cross con-tamination while using a vacuum Centrifugation minimizes this contamination Suitable disposable pipette tips for mixing samples in a deep-well 384-formatted microtiter plate are difficult to locate; only recently was this problem addressed

The concept of rectangular experimental designs for multiunit platforms (RED-MUPs) as a part of statistical experimental design (also known as design of experiments or DOE) was explored in a recent

or combinations of drugs and/or drug metabolites, impurities, and degradation products from aque-The complexity of the method in terms of number of steps and solvents needed depends on the sorbent chemistry The development in a simplified scenario involves running an analyte in several concentrations in multiple replicates and assaying for recovery and performance This procedure

sorbents are to be evaluated, the process becomes time-consuming if multiple 96-well plates (each with one sorbent packed in all the wells) must be screened separately This process may take a week

or more and consume an analyst’s precious time as well The most plausible solution is to pack different sorbents in the same well plate and use a universal procedure that applies to all of them

An example of such a multisorbent method development plate is the four-sorbent plate recently

and SPE conditions

Four polymeric sorbents with different chemistries and interaction mechanisms are packed in a 96-well plate in a configuration wherein three vertical columns are dedicated to each sorbent (total

24 wells; see Figure 1.18) These sorbents consist of the strata-X neutral polar/non-polar balanced functionalized styrene–divinylbenzene polymer, the strong strata-X-C cation exchanger with sulfonic acid moieties located on the phenyl rings of the same base polymeric skeleton, the weak strata-X-CW cation exchanger with a carboxyl-functionalized PSDVB, and a weak strata-X-AW anion exchanger with primary and secondary amine groups on the PSDVB skeleton The four sorbents cover all possi-ble types of interactions any analyte can exhibit The strata-X displayed strong hydrophobic and π–π interactions, coupled with moderate hydrogen bonding and weakly acidic properties The strata-X-C yielded strong cation exchange and hydrophobic interactions, along with weak hydrogen bonding and moderate π–π interactions At the same time, strata-X-CW showed weak cation exchange and strong hydrogen bonding properties with much lower hydrophobicity; strata-X-AW exhibited strong anion exchange activity along with moderate hydrophobicity and weak hydrogen bonding