Pathology And Laboratory Medicine Clinical And Forensic Applications Of Capillary Electrophoresis

PATHOLOGY AND LABORATORY MEDICINE

CLINICAL

AND FORENSIC

APPLICATIONS

OF CAPILLARY

ELECTROPHORESIS

EDITED BY

JOHN R. PETERSEN

AMIN A. MOHAMMAD

HUMANA PRESS

Clinical and Forensic Applications

of Capillary Electrophoresis

Clinical and Forensic Applications of Capillary Electrophoresis

edited by John R. Petersen and Amin A. Mohammad, 2001

Cardiac Markers

edited by Alan H. B. Wu, 1998

Clinical Pathology of Pancreatic Disorders

edited by John A. Lott, 1997

Molecular Diagnostics: For the Clinical Laboratorian

edited by William B. Coleman and Gregory J. Tsongalis, 1997

PATHOLOGY AND LABORATORY MEDICINE

Series Editors: Stewart Sell and Alan Wu

Edited by

John R. Petersen

and

Amin A. Mohammad

University of Texas Medical Branch, Galveston, TX

Humana Press Totowa, New Jersey

Clinical and Forensic

Applications

of Capillary

Electrophoresis

PATHOLOGY AND LABORATORY MEDICINE

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Clinical and forensic applications of capillary electrophoresis / edited by John R.

Petersen and Amin A. Mohammad.

p. ; cm.

Includes bibliographical references and index.

ISBN 0-89603-645-6 (alk. paper)

1. Capillary electrophoresis. 2. Clinical chemistry. 3. Chemistry, Forensic. I. Petersen,

John R. II. Mohammad, Amin A.

[DNLM: 1. Clinical Laboratory Techniques. 2. Electrophoresis, Capillary--methods. 3.

Amino Acids--blood. 4. Blood Proteins--analysis. 5. Forensic Medicine--methods. 6.

Immunoassay--methods. QU 25 C6408 2001]

RB43.8.C36 C54 2001

616.07'56--dc21

2001016677

Preface

v

Since the advent of the clinical laboratory in the 20th century, the need to

report more accurate results, faster, and at a lower cost has driven technol￾ogy. One area that has lagged behind the rest of the laboratory is electro￾phoretic separations of analytes that are clinically relevant. Because of this,

electrophoresis has been relegated to the very specialized sections of the

laboratory, limiting its use in patient care.

Electrophoresis, as we use it today, was first described by Tiselius in his

PhD thesis in 1937. In pioneering experiments that have led to the methods

used today, he used a U-shaped quartz tube to show the zonal separation of

serum in free solution using Schlieren optics to monitor the migration of the

protein bands. Driven by the desire to make electrophoresis easier, a number

of matrixes—such as paper, cellulose acetate, agarose, starch gel, and poly￾acrylamide—were investigated and, in one form or another, are still used

today. From the basic method described by Tiselius a number of innovative

electrophoretic methods have now been developed, including immunoelec￾trophoresis, isoelectric focusing (IEF), isotachophoresis (ITP), and size

separation by gradient electrophoresis.

Tiselius’s basic concept of using a tube for electrophoretic separation

received little notice until the late 1960s when Hjerten described the first

capillary electrophoresis (CE) apparatus. In spite of the pioneering work by

Hjerten, CE remained relatively unknown until 1981 when Jorgenson and

Lukacs described the separation and fluorescent detection of amino acids,

peptides, and urine proteins by capillary zone electrophoresis. Since then,

all of the classical separation techniques—IEF, ITP, zone electrophoresis,

and micellar electrokinetic chromatography (MEKC)—have allowed CE to

rival the versatility of high pressure liquid chromatography (HPLC). MEKC,

which in its simplest form is the addition of detergent to the buffer, has

enabled CE to be used in an area once thought impossible for electrophore￾sis techniques, the separation of small, electrically neutral molecules.

CE has come a long way since it was first described. Current methods

are capable of being automated, and, because it is a microtechnique, the

method conserves precious samples and minimizes the use of hazardous

organic chemicals. Although CE has not made inroads into the clinical

vi Preface

laboratory that many anticipated, we expect that, in the future, it will find its

“proper” place. Because this “proper” place may surprise everyone involved

in the clinical applications of CE, this book is not meant to give an in-depth

methodological description of the use of CE in the clinical laboratory, but to

give an overview of its current use.

We arranged Clinical and Forensic Applications of Capillary Electro￾phoresis into six main sections. Section I covers the history and some of the

potential applications of CE. This section also covers the principles neces￾sary for the clinical laboratory scientist to understand the basics of CE. Sec￾tion II covers the separation of proteins, probably the first use of CE in the

clinical laboratory. The section describes the potential problems and solu￾tions when using CE to separate proteins, along with outlining how CE has

been used to separate serum and CSF proteins, detect serum and urine

paraproteins, and separate lipoproteins and hemoglobin variants. Section III

covers metabolic diseases, which are usually detected by abnormalities in

small molecules, such as amino acids, organic acids, or steroids. Section IV

covers the use of CE in immunoassay, where CE is used as a separation

method. Although this may seem trivial at first glance, it opens up the possi￾bility of simple, yet highly sensitive, analysis at the point of care. Section V

describes what may be the future of CE in the clinical laboratory, the use of

CE in molecular diagnostics, both for the detection of diseases and

quantiation of viral loads and its use in the forensic DNA identification labo￾ratory. Finally, Section VI describes how CE can be used in conjunction

with mass spectrometry, its potential use in detection of heavy metal poi￾soning, therapeutic drug monitoring, and clinical and forensic toxicology.

John R. Petersen

Amin A. Mohammad

Contents

Preface ................................................................................................. v

List of Contributors .......................................................................... ix

SECTION I. INTRODUCTION ............................................................................. 1

1 Capillary Electrophoresis: General Overview and Applications

in the Clinical Laboratory

Manjiri Lele, Subodh M. Lele, John R. Petersen,

and Amin A. Mohammad ............................................................ 3

2 Basic Principles and Modes of Capillary Electrophoresis

Harry Whatley ................................................................................. 21

SECTION II. PROTEIN ELECTROPHORESIS ...................................................... 59

3 Capillary Coatings for Protein Analysis

Kannan Srinivasan, Chris Pohl, and Nebojsa Avdalovic ....... 61

4 Clinical Serum Protein Capillary Zone Electrophoresis

Carl R. Jolliff.................................................................................... 79

5 Serum and Urine Paraprotein Capillary Electrophoresis

Carl R. Jolliff.................................................................................... 93

6 Cerebrospinal Fluid Protein Electrophoresis

John R. Petersen and Amin A. Mohammad.............................. 105

7 Lipoprotein Analysis

Rainer Lehmann ............................................................................ 113

8 Clinical Analysis of Structural Hemoglobin Variants

and Hb A1c by Capillary Isoelectric Focusing

James M. Hempe, Alfonso Vargas,

and Randall D. Craver............................................................. 145

SECTION III. METABOLIC DISEASES ............................................................ 165

9 Amino Acid Analysis

Mary Kathryn Linde ..................................................................... 167

vii

10 Organic Acids

Kern L. Nuttall and Norberto A. Guzman................................ 193

11 Steroids

Cecilla Youh, Amin A. Mohammad, and John R. Petersen ... 209

SECTION IV. IMMUNOASSAY ...................................................................... 221

12 Capillary Electrophoresis Based Immunoassay

Bode Adesoji, Amin A. Mohammad, and John R. Petersen .....223

SECTION V. MOLECULAR DIAGNOSTICS ..................................................... 235

13 Quantitation of Viral Load

Jill M. Kolesar................................................................................ 237

14 The Application of Capillary Electrophoresis in the Analysis

of PCR Products Used in Forensic DNA Typing

Bruce R. McCord and John M. Butler........................................ 261

15 Combining Capillary Electrophoresis with Electrospray Ionization

Mass Spectrometry

Samir Cherkaoui............................................................................ 285

SECTION VI. MASS SPECTROMETRY, THERAPEUTIC DRUG MONITORING,

AND TOXICOLOGY .......................................................................... 315

16 Capillary Electrophoresis-Mass Spectrometry

of Biologically Active Peptides and Proteins

Stephen Naylor and Andy J. Tomlinson ................................... 317

17 Serum Drug Monitoring by Capillary Electrophoresis

Zack K. Shihabi ............................................................................. 355

18 Applications of Capillary Zone Electrophoresis

in the Analysis of Metal Ions of Clinical Significance

Lokinendi V. Rao, John R. Petersen,

Amin A. Mohammad, and Anthony O. Okorodudu ........... 385

19 Clinical and Forensic Drug Toxicology: Analysis of Illicit

and Abused Drugs in Urine by Capillary Electrophoresis

Wolfgang Thormann and Jitka Caslavska............................... 397

20 Screening Biological Specimens for Drugs of Forensic

Significance

John C. Hudson, Murray J. Malcolm, and Mauro Golin ..........423

Index ................................................................................................ 437

viii Contents

Contributors

BODE ADESOJI • Department of Pathology, University of Texas Medical

Branch, Galveston, TX

NEBOJSA AVDALOVIC • Dionex Corporation, Sunnyvale, CA

JOHN M. BUTLER • GeneTrace Systems Inc., Alameda, CA

JITKA CASLAVSKA • Department of Clinical Pharmacology, University

of Bern, Bern, Switzerland

SAMIR CHERKAOUI • Laboratory of Pharmaceutical Analytical

Chemistry, University of Geneva, Switzerland

RANDALL D. CRAVER • Department of Pediatrics, Louisiana State

University School of Medicine, New Orleans, LA

MAURO GOLIN • Forensic Laboratory, Royal Canadian Mounted Police,

Regina, Saskatchewan, Canada

NORBERTO A. GUZMAN • The R.W. Johnson Pharmaceutical Research

Institute, Raritan, NJ

JAMES M. HEMPE • Department of Pediatrics, Louisiana State

University School of Medicine, New Orleans, LA

JOHN C. HUDSON • Forensic Laboratory, Royal Canadian Mounted

Police, Regina, Saskatchewan, Canada

CARL R. JOLLIFF • Physician’s Laboratory Services Inc., Lincoln, NE

JILL M. KOLESAR • Department of Pharmacy (CHS), University

of Wisconsin-Madison, Madison, WI

RAINER LEHMANN • Department of Internal Medicine IV, Division

of Endocrinology, Metabolism and Pathobiochemistry, Medical

Center Hospital, University of Tübingen, Tübingen, Germany

MANJIRI LELE • Department of Pathology, University of Texas Medical

Branch, Galveston, TX

SUBODH M. LELE • Department of Pathology, University of Texas

Medical Branch, Galveston, TX

MARY KATHRYN LINDE • College of Professional Studies, Shawnee State

University, Portsmouth, OH

MURRAY J. MALCOLM • Forensic Laboratory, Royal Canadian Mounted

Police, Regina, Saskatchewan, Canada

ix

x Contributors

BRUCE R. MCCORD • Deparment of Chemistry, Clippinger Laboratories,

Ohio University, Athens, OH

AMIN A. MOHAMMAD • Department of Pathology, University of Texas

Medical Branch, Galveston, TX

STEPHEN NAYLOR • Department of Biochemistry and Molecular Biology,

Department of Pharmacology, Mayo Foundation, Rochester, MN

KERN L. NUTTALL • Department of Pathology, University of Utah

School of Medicine, Salt Lake City, UT

ANTHONY O. OKORODUDU • Department of Pathology, University

of Texas Medical Branch, Galveston, TX

JOHN R. PETERSEN • Department of Pathology, University of Texas

Medical Branch, Galveston, TX

CHRIS POHL • Dionex Corporation, Sunnyvale, CA

LOKINENDI V. RAO • University of Texas Medical Branch/Texas

Department of Criminal Justice Laboratory Services, Huntsville, TX

ZACK K. SHIHABI • Department of Pathology, Bowman Gray School

of Medicine, Wake Forest University Baptist Medical Center,

Winston-Salem, NC

KANNAN SRINIVASAN • Dionex Corporation, Sunnyvale, CA

WOLFGANG THORMANN • Department of Clinical Pharmacology,

University of Bern, Bern, Switzerland

ANDY J. TOMLINSON • Department of Biochemistry and Molecular

Biology, Department of Pharmacology, Mayo Foundation, Rochester,

MN

ALFONSO VARGAS • Department of Pediatrics, Louisiana State

University School of Medicine, New Orleans, LA

HARRY WHATLEY • Beckman Coulter, Fullerton, CA

CECILLA YOUH • Department of Pathology, University of Texas Medical

Branch, Galveston, TX

Capillary Electrophoresis 1

I

Introduction

2 Lele et al.

Capillary Electrophoresis 3

3

From: Clinical and Forensic Applications of Capillary Electrophoresis

Edited by: J. R. Petersen and A. A. Mohammad © Humana Press Inc., Totowa, NJ

1

Capillary Electrophoresis

General Overview and Applications in the Clinical Laboratory

Manjiri Lele, Subodh M. Lele, John R. Petersen, and Amin Mohammad

1. INTRODUCTION

Electrophoresis was first described by Arne Tiselius (1) in 1930, for

which he received a Nobel Prize in 1948. In this pioneering experiment, he

used a U-shaped quartz tube to show the separation of different proteins in

free solution as contiguous bands. His work was published in 1937 (1) but

received little notice until the late 1960s, when Hjerten (2) described the

first capillary electrophoresis (CE) apparatus. Hjerten’s apparatus consisted

of three units: 1) a high voltage power supply; 2) a detector; and 3) a unit

holding a 1–3 mm ID quartz capillary tube, which was immersed in a cool￾ing bath (2). He used this apparatus to prove numerous theoretical concepts

in CE and was able to separate inorganic ions, proteins, nucleic acid, and

microorganisms by capillary zone electrophoresis (CZE) or capillary iso￾electric focusing (CIEF). In spite of the pioneering work by Hjerten, CE was

still relatively unknown until Jorgenson and Lukacs (3–5) published a series

of papers in 1980. The availability of polyiimide-coated fused silica capil￾laries with a 75–100-µm internal diameter, in addition to sensitive absor￾bance detectors developed for micro-bore high-performance liquid

chromatography (HPLC), were instrumental in the development of com￾mercial CE applications. The smaller internal diameter eliminated band

broadening caused by convection, whereas the plug flow characteristics of

the electroosmotic flow (EOF) allowed efficiencies reaching hundreds of

thousand of theoretical plates. Since the landmark publication in 1980 by

Jorgenson and Lukacs research dealing with the applications of CE has

grown exponentially. Consistent with the theme of this book, this chapter

will try to provide a general overview of current and future applications of

CE in clinical chemistry. It is not meant to be a comprehensive review of

4 Lele et al.

general literature, but instead an attempt to give a reader a flavor of its

potential power in solving some of the challenging problems that arise in a

clinical laboratory.

2. INTERGRATION OF CE INTO THE CLINICAL LABORATORY

Clinical laboratories had evolved considerably since the early 1980s and

will continue to do so during the next millennium. Reduced turnaround time,

convenience, patient satisfaction (quick diagnosis), and physician satisfac￾tion (improved real-time clinical decision-making) are some of the benefits

gained from having more clinical testing to be done in the hospital labora￾tory. At the same time economies of scale, reduced cost, regionalization,

and continuity of care are responsible for outsourcing laboratory testing to

reference laboratories. These two opposing forces have placed tremendous

pressure on clinical chemists to develop assays that are inexpensive, fast,

amenable to laboratory automation, while still being accurate and precise.

Thus the million dollar question is: “Can capillary electrophoresis fulfill

some or any of these conditions?” This is a key question that every labora￾tory director, laboratory manager, or administrative director must answer

before introducing a new technology for patient testing. Let us now examine

applications of CE from this perspective.

2.1. Cost

Because CE is a separation technique, it will compete with more traditional

chromatographic techniques such as gas chromatography (GC) and HPLC.

However, unlike the classical chromatographic techniques, CE can also com￾pete with classical electrophoresis, such as agarose gel and isoelectric focus￾ing. Table 1 gives a typical cost comparison for the procedures that CE could

replace in a clinical laboratory. As shown in this table, in certain instances CE

can be less expensive. The main cost savings comes from the use of fused

silica capillaries, which are less expensive than HPLC or GC columns. For

instance, the cost of an HPLC column used for clinical testing can range from

$250 to 300, whereas a 10-m piece of CE fused silica capillary will cost about

$80. Assuming that a CE method will use a 57-cm piece of fused silica capil￾lary and that the capillary will last ~200 injections, a very conservative esti￾mate, one can expect at least 1800 injections from 10 m of fused silica capillary

instead of 800 injections for an HPLC column. Even neutral-coated capillar￾ies, which are appreciably more expensive than uncoated, fused silica capil￾laries, are less expensive than reverse-phase (RP) or ion exchange HPLC

columns. It should be noted that in this analysis we did not account for the

labor component because in our experience, the hands-on labor for CE is com￾parable to traditional chromatographic and electrophoretic techniques.