Bioanalysis of Pharmaceuticals E-Book
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- Herausgeber: John Wiley & Sons
- Kategorie: Wissenschaft und neue Technologien
- Sprache: Englisch
Bioanalysis of Pharmaceuticals: Sample Preparation, Separation Techniques and Mass Spectrometry is the first student textbook on the separation science and mass spectrometry of pharmaceuticals present in biological fluids with an educational presentation of the principles, concepts and applications. It discusses the chemical structures and properties of low- and high-molecular drug substances; the different types of biological samples and fluids that are used; how to prepare the samples by extraction, and how to perform the appropriate analytical measurements by chromatographic and mass spectrometric methods.
Bioanalysis of Pharmaceuticals: Sample Preparation, Separation Techniques and Mass Spectrometry:
- Is an introductory student textbook discussing the different principles and concepts clearly and comprehensively, with many relevant and educational examples
- Focuses on substances that are administered as human drugs, including low-molecular drug substances, peptides, and proteins
- Presents both the basic principles that are regularly taught in universities, along with the practical use of bioanalysis as carried out by researchers in the pharmaceutical industry and in hospital laboratories
- Is aimed at undergraduate students, scientists, technicians and researchers in industry working in the areas of pharmaceutical analyses, biopharmaceutical analyses, biological and life sciences
The book includes multiple examples to illustrate the theory and application, with many practical aspects including calculations, thus helping the student to learn how to convert the data recorded by instruments into the real concentration of the drug substances within the biological sample.
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Veröffentlichungsjahr: 2015
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Table of Contents
Cover
Title Page
Copyright
Contributing Authors
Preface
Chapter 1: Introduction
1.1 What Is Bioanalysis?
1.2 What Is the Purpose of Bioanalysis, and Where Is It Conducted?
1.3 Bioanalysis Is Challenging
1.4 The Different Sections of This Textbook
Chapter 2: Physicochemical Properties of Drug Substances
2.1 Bioanalysis in General
2.2 Protolytic Properties of Analytes
2.3 Partitioning of Substances
2.4 Stereochemistry
2.5 Peptides and Proteins
Chapter 3: Biological Samples: Their Composition and Properties, and Their Collection and Storage
3.1 Introduction
3.2 Blood, or Whole Blood
3.3 Plasma and Serum
3.4 Urine
3.5 Feces
3.6 Saliva
3.7 Cerebrospinal Fluid
3.8 Synovial Fluid
3.9 Hair and Nails
3.10 Tissue (Biopsies)
Chapter 4: General Chromatographic Theory and Principles
4.1 General Introduction
4.2 General Chromatographic Theory
4.3 Theory of Partition
4.4 Retention
4.5 Separation Efficiency
4.6 Resolution
4.7 Selectivity
4.8 The Separation Process
4.9 Chromatographic Principles
4.10 Reversed Phase Chromatography
4.11 Size Exclusion Chromatography (SEC)
4.12 Ion Exchange Chromatography
4.13 Chiral Separations
Chapter 5: Quantitative and Qualitative Chromatographic Analysis
5.1 Collection of Chromatographic Data
5.2 Quantitative Measurements
5.3 Calibration Methods
5.4 Validation
5.5 Qualitative Analysis
Chapter 6: Sample Preparation
6.1 Why Is Sample Preparation Required?
6.2 What Are the Main Strategies?
6.3 Protein Precipitation
6.4 Liquid–Liquid Extraction
6.5 Solid-Phase Extraction
6.6 Dilute and Shoot
6.7 What Are the Alternative Strategies?
Chapter 7: High-Performance Liquid Chromatography (HPLC) and High-Performance Liquid Chromatography–Mass Spectrometry (LC-MS)
7.1 Introduction
7.2 The Solvent Delivery System
7.3 Degassing and Filtering of Mobile Phases
7.4 Injection of Samples
7.5 Temperature Control
7.6 Mobile Phases
7.7 Stationary Phases and Columns
7.8 Detectors
7.9 Mass Spectrometric Detection
Chapter 8: Gas Chromatography (GC)
8.1 Basic Principles of GC
8.2 GC Instrumentation
8.3 Carrier Gas
8.4 Stationary Phases
8.5 Separation Selectivity in GC
8.6 Columns
8.7 Injection Systems
8.8 Detectors
8.9 Derivatization
8.10 Gas Chromatography–Mass Spectrometry (GC-MS)
Chapter 9: Analysis of Small-Molecule Drugs in Biological Fluids
9.1 Plasma and Serum Samples
9.2 Whole Blood Samples
9.3 Dried Blood Spots
9.4 Urine Samples
9.5 Saliva
References
Chapter 10: Analysis of Peptide and Protein Drugs in Biological Fluids
References
Chapter 11: Regulated Bioanalysis and Guidelines
11.1 Introduction
11.2 The Evolution of Regulated Bioanalysis
11.3 Bioanalytical Method Validation
11.4 Pre-study Validation
11.5 In-Study Validation
11.6 Documentation
11.7 Regulatory Requirements to Bioanalysis
11.8 Quality Systems in Regulated Bioanalysis
Index
End User License Agreement
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Guide
Cover
Table of Contents
Preface
Begin Reading
List of Illustrations
Chapter 1: Introduction
Figure 1.1 Effects of pharmacokinetics and pharmacodynamics on the dose–response relationship
Chapter 2: Physicochemical Properties of Drug Substances
Figure 2.1 Ionization of acids and bases as a function of pH
Figure 2.2 Distribution of an analyte A between an upper organic phase and a lower aqueous phase
Figure 2.3 Chemical structures of ibuprofen and salicylic acid with log P and pKa values
Figure 2.4 Classification of isomers
Figure 2.5 The chemical structure of cis-clopenthixol and trans-resveratrol
Figure 2.6 Enantiomeric drug compounds
Figure 2.7 A drug substance with several chiral centers
Figure 2.8 General structure of amino acid, including its chiral C-atom and the charge–pH dependency of amino acids
Figure 2.9 Structure of gonadorelin including three-letter abbreviation
Figure 2.10 Overview of the different protein structure levels
Figure 2.11 Acetylation and amidation of terminal amino acids in a polypeptide chain
Chapter 3: Biological Samples: Their Composition and Properties, and Their Collection and Storage
Figure 3.1 Whole blood after centrifugation
Figure 3.2 Whole blood sample in its collection vial
Figure 3.3 Sampling for dried blood spot
Figure 3.4 Containers for urine collection
Figure 3.5 Collection of saliva using a cotton bud
Chapter 4: General Chromatographic Theory and Principles
Figure 4.1 Schematic presentation of a chromatographic separation
Figure 4.2 A schematic chromatogram showing relevant parameters
Figure 4.3 A Gaussian peak with relevant parameters assigned
Figure 4.4 Peak-to-valley ratio between two not fully resolved peaks
Figure 4.5 Peak broadening due to eddy diffusion
Figure 4.6 Illustration of mass transfer between the mobile and stationary phases
Figure 4.7 Illustration of mass transfer in the mobile and stationary phases
Figure 4.8 A schematic presentation of the van Deemter plot showing the optimum efficiency (at minimum H) and the optimum flow rate, u
Figure 4.9 Silanol groups: (1) free (isolated) silanol, (2) germinal silanols, and (3) associated silanols on the surface of silica
Figure 4.10 Chromatogram of hydroxyatrazin before (showing strong tailing) and after addition of a carboxylic acid to the mobile phase. HPLC system: column silica 120 mm × 4.6 mm, 5 µm, with dichloromethane + methanol (95:5 v/v) as mobile phase without and with propionic acid added
Figure 4.11 Retention factor of opiates obtained on unmodified silica versus the polarity of the mobile phase. Opiates: +, codeine; ○, morphine; v, normorphine; □, noscapine; Δ, papaverine; and •, thebaine
Figure 4.12 Derivatization of silica with a chlorosilane reagent
Figure 4.13 C18 column packing material (A) before and (B) after end capping with trimethylchlorosilane
Figure 4.14 Polystyrene–divinylbenzene copolymer
Figure 4.15 Hydrophobic interactions between the hydrocarbon chain of C18 material and the hydrophobic parts of naproxen
Figure 4.16 Chromatogram of two analytes with different sizes of side chain. The separation was obtained using reversed phase chromatography
Figure 4.17 Example of solvent selectivity. Separation of 7 test solutes on a C18 reversed phase HPLC column using (a) methanol or (b) acetonitrile as organic modifier. (1) benzylalcohol; (2) acetophenone; (3) phenylethanol; (4) propiophenone; (5) anisole; (6) toluene; and (7) p-cresol
Figure 4.18 Viscosity of mixtures of water and organic solvents. EtOH, ethanol; THF, tetrahydrofuran; MeOH, methanol; MeCN, acetonitrile
Figure 4.19 Variation of the retention factor, k, for a weak acid and a weak base as a function of pH in the mobile phase
Figure 4.20 Separation selectivity at high and low pH. Separation is achieved using gradient elution on an ACE UltraCore 2.5 µm Super C18, 50 mm × 2.1 mm column. (a) At pH 3.0, an acetonitrile gradient containing ammonium formate pH 3.0 was used. (b) At pH 10.7, an acetonitrile gradient containing 18 mM ammonia pH 10.7 was used. Sample: (1) atenolol; (2) methylphenylsulfoxide; (3) eserine; (4) prilocaine; (5) bupivacaine; (6) tetracaine; (7) 1,2,3,4-tetrahydro-1-naphthol; (8) carvedilol; (9) nitrobenzene; (10) methdilazine; (11) amitriptyline; and (12) valerophenone. Reproduced with permission of Advanced Chromatography Technologies Ltd, UK ([email protected]/www.ace-hplc.com)
Figure 4.21 Structures of the ions octanesulfonate, heptafluorobutyrate, and tetrabutylammonium
Figure 4.22 Liquid chromatography (LC) versus size exclusion chromatography (SEC) and the calibration curve for SEC
Chapter 5: Quantitative and Qualitative Chromatographic Analysis
Figure 5.1 A chromatographic peak at the limit of quantification
Figure 5.2 An example of a quantitative determination calibration curve
Figure 5.3 Chromatograms showing the use of internal standards (IS). (a) An example where the internal standard is separated from the analyte. (b) An example where the internal standard is an isotopically labeled analyte and the two compounds are measured by mass spectrometry (MS) at to different mass-to-charge ratios (m/z) values
Figure 5.4 The standard addition calibration curve
Figure 5.5 Parameters to be validated in a bioanalytical method
Figure 5.6 Chromatograms of bromohexine and its metabolites in urine using (a) ultraviolet and (b) radiochemical detection
Figure 5.7 High-performance liquid chromatography–ultraviolet–mass spectrometry–nuclear magnetic resonance (HPLC-UV-MS-NMR) system for metabolite identification
Chapter 6: Sample Preparation
Figure 6.1 Principle of protein precipitation
Figure 6.2 96-well protein precipitation
Figure 6.3 Principle of liquid–liquid extraction
Figure 6.4 Distribution ratio (log D) for ibuprofen (acidic drug substance) between 1-octanol and aqueous solution as function of pH
Figure 6.5 Distribution ratio (log D) for amitriptyline (basic drug substance) between 1-octanol and aqueous solution as function of pH
Figure 6.6 Overview of molecular interactions in liquid–liquid extraction
Figure 6.7 Principle of supported liquid extraction
Figure 6.8 Principle of solid-phase extraction
Figure 6.9 (a) Photo and (b) illustration of a solid-phase extraction (SPE) column. A broad range of SPE columns are commercially available, with different masses and chemistries of the stationary phase
Figure 6.10 Conditioning and solvation of a solid-phase extraction stationary phase
Figure 6.11 Solid-phase extraction columns and vacuum manifold
Figure 6.12 Different stationary phases for reversed-phase solid-phase extraction
Figure 6.13 Example of polymeric solid-phase extraction stationary phase
Figure 6.14 Secondary interactions in solid-phase extraction
Figure 6.15 Overview of some stationary phases for ion exchange solid-phase extraction
Figure 6.16 Retention of amphetamine on a strong cation exchange solid-phase extraction column
Figure 6.17 Retention of amphetamine on a mixed-mode solid-phase extraction column
Figure 6.18 Polymeric-based stationary phase for mixed-mode solid-phase extraction
Figure 6.19 Overview of some stationary phases for normal-phase solid-phase extraction
Figure 6.20 Photo of a solid-phase extraction 96-well plate
Figure 6.21 Dilute and shoot
Figure 6.22 Principle of dialysis
Figure 6.23 Schematic drawing of a microdialysis probe
Figure 6.24 Schematic drawing of an immunosorbent
Figure 6.25 Schematic overview of molecularly imprinted polymer production
Figure 6.26 Schematic drawing of an oligosorbent
Figure 6.27 Illustration of a fiber-solid-phase microextraction device
Figure 6.28 Schematic illustration of a supported liquid membrane extraction process exemplified by a basic analyte
Chapter 7: High-Performance Liquid Chromatography (HPLC) and High-Performance Liquid Chromatography–Mass Spectrometry (LC-MS)
Figure 7.1 Main structure of a liquid chromatography system
Figure 7.2 The main parts in a piston pump
Figure 7.3 Isocratic and gradient elution of a sample containing analytes with large differences in retention
Figure 7.4 A two-position, six-port injection valve in the load position (a) and in the inject position (b)
Figure 7.5 Octadecylsilylsilica (ODS column packing material) for reversed phase chromatography
Figure 7.6 The development in column packing materials
Figure 7.7 Height equivalent to a theoretical plate as a function of the flow rate of the mobile phase for 1.5, 3.5, 5, and 10 μm particles
Figure 7.8 Effect of the reduction in particle size and column length and the analysis time and chromatographic separation
Figure 7.9 Effect of particle diameter (μm) and internal column diameter (Ø) on peak signal with identical injection volume
Figure 7.10 The electromagnetic spectrum, showing the connection between frequency and wavelength
Figure 7.11 Diagram illustrating light excitation of electron
Figure 7.12 Diagram illustrating electronic states in fluorescence
Figure 7.13 Schematic diagram of a single wavelength UV detector (left) and a diode array detector (right)
Figure 7.14 Sketch of flow cell in a UV detector
Figure 7.15 Schematic diagram of a fluorescence detector
Figure 7.16 Excitation and emission spectra of quinine
Figure 7.17 Oxidation of catecholamines release electrons that are detected by the electrochemical detector
Figure 7.18 Typical setup of a liquid chromatography–mass spectrometer (LC-MS) system
Figure 7.19 Choosing the right interface depends on the analyte size and the polarity
Figure 7.20 Schematic representation of the electrospray and the formation of ions
Figure 7.21 (a) Protonated haloperidol (thus positively charged) having ; and (b) de-protonated acetylsalicylic acid (thus negatively charged) having
Figure 7.22 Angiotensin II consists of eight amino acids, and three of them can be positively charged (gray boxes). The mass of angiotensin II is 1045.53; adding three protons makes up a mass of 1048.53 and will be detected like . Adding two protons makes up a mass of 1046.53 and will be detected like
Figure 7.23 Atmospheric pressure chemical ionization with gas molecular reactions
Figure 7.24 Protonated epitestosterone gives rise to m/z 289 (= [M + H]
+
) in the mass spectrum (m/z 290 and 291 are isotopes)
Figure 7.25 Atmospheric pressure photo-ionization with the three types of ionization
Figure 7.26 The high velocity of electrons (initiated by a spark) caused by a frequency of 30–40 MHz of the coil causes argon gas to ionize. In this way, a plasma at high temperature is created
Figure 7.27 Three mass spectra with increasing mass resolution
Figure 7.28 (a) Ions move from the source toward the detector in the z-direction. Only the stable oscillating ions will reach the detector (black line). Unstable ions will collide with one of the quadrupoles (gray line). (b) x/y view of the quadrupole. The electrical field applied is composed of a DC component (U) and an RF component (Vcos ϖt)
Figure 7.29 Schematic representation of a triple quadrupole mass spectrometer
Figure 7.30 The ion trap consists of a ring electrode and two end-cap electrodes (a). On the right (b), from top to bottom: ions enter the ion trap and are cooled down by He (white dots). After the ions are trapped, they are scanned out of the ion trap. Light ions leave the trap before the heavier ones
Figure 7.31 The linear ion trap consists of three quadrupole-like electrodes where the first and last quadrupoles are coupled to a direct voltage with the same polarity. An ion trap field is created within the second quadrupole
Figure 7.32 (a) Linear time-of-flight mass analyzer. Heavy ions (largest circles) travel slower than lighter ions (smallest circles). The velocity of the ions traveling through the field-free flight tube thus correlates with the m/z value. (b) In a reflectron ToF mass analyzer, all ions are deflected by a reflectron to reach higher resolution and mass accuracy by a longer flight path
Figure 7.33 Schematic representation of ion mobility spectrometry. The drift tube consists of several focusing rings, creating an electric field that is opposite in direction to the drift gas flow
Figure 7.34 (a) Electrical signal measured in ion cyclotron resonance or an orbitrap mass analyzer. (b) Signal is decomposed in all the frequencies. (c) Separate frequencies of three masses, where
Figure 7.35 Detectors used in mass spectrometry: (a) electron multiplier, (b) microchannel plate, and (c) Faraday cup
Figure 7.36
Figure 7.37 General principle of tandem mass spectrometry. Step 1: mass selection; step 2: collision-induced dissociation; and step 3: fragment measurement
Figure 7.38
Figure 7.39 A chromatogram is a collection of points, and each point represents a mass spectrum. In this example, only four mass spectra are shown
Figure 7.40 Centroid scan (a) and profile scan (b) of protonated lidocaine
Figure 7.41
Figure 7.42 (a) Extracted ion chromatogram of metoprolol obtained with a low-resolution mass spectrometer; and (b) extracted ion chromatogram of metoprolol obtained with a high-resolution mass spectrometer
Figure 7.43 Base peak chromatogram showing four peaks of four different substances with their respective mass spectra and signal intensities
Figure 7.44 Full scan and selected ion monitoring from same-drug analysis
Figure 7.45 Postextraction addition approach to estimate the effect of the matrix on the analysis
Figure 7.46 Postcolumn infusion approach to estimate the effect of the matrix on the analysis. The chromatogram shows: (a) the continuous signal of drug X infused by the syringe pump; (b) the continuous signal of drug X infused by the syringe pump after injecting a blank extracted biological sample in the HPLC system; and (c) the signal of drug X injected in the HPLC system. The gray areas indicate the time ranges with signal disturbances
Figure 7.47 Chromatograms of time-resolved analyte and two deuterated internal standards
Chapter 8: Gas Chromatography (GC)
Figure 8.1 Schematic illustration of a gas chromatograph
Figure 8.2 Photograph of a gas chromatograph
Figure 8.3 Illustration of an isothermal and temperature programmed gas chromatography
Figure 8.4 Isothermal versus temperature programming in gas chromatography analysis of n-alkanes
Figure 8.5 van Deemter plots with different carrier gases
Figure 8.6 The basic skeleton of polydimethylsiloxane, polyphenylmethylsiloxane, and polycyanopropylmethylsiloxane. The numbers of repeating units (
n
,
p
, and
x
) can vary significantly form one stationary phase to another
Figure 8.7 The basic skeleton of polyethylene glycol
Figure 8.8 Split or splitless injector for a capillary gas chromatograph
Figure 8.9 Schematic illustration of a flame ionization detector
Figure 8.10 Schematic illustration of a nitrogen–phosphorous selective detector
Figure 8.11 Schematic illustration of an electron capture detector
Figure 8.12 Silylation of a hydroxyl group
Figure 8.13 Derivatization of an α-amino acid with trifluoroacetic acid anhydride and methanol
Figure 8.14 Electron ionization (upper reaction) and one major fragmentation (lower reaction) during gas chromatography–mass spectrometry (GC-MS) of chlorambucil
Figure 8.15 EI mass spectrum of chlorambucil
Figure 8.16 Mass spectrum (electron ionization) of (a) amphetamine and (b) lysergide (lysergic acid diethylamide, or LSD)
Figure 8.17 Magnified mass spectrum for chlorambucil in the mass range of 250–310
Figure 8.18 Mass spectrum (electron ionization) of morphine (an analgesic drug). The signal at mass 286 corresponds to molecular ions with one
13
C-atom (M+1)
+
, and 285 corresponds to the actual molecular mass of the compound (M)
+
Figure 8.19 Isotope patterns of chlorine (one and two atoms) and bromine (one and two atoms). Two mass units between each peak
Figure 8.20 Mass spectrum (electron ionization) of p-amino-benzoic acid
Figure 8.21 Mass spectrum (electron ionization) of bromazepam
Figure 8.22 Mass spectrum (electron ionization) of clonazepam
Figure 8.23 Mass spectra (electron ionization and chemical ionization) of amphetamine
Figure 8.24 Total ion current chromatogram (TIC) and mass spectrum for component 3 in a mixture of four components
Figure 8.25 Selected ion chromatogram (mass 286) for gas chromatography–mass spectrometry (GC-MS) analysis of human plasma. N-desmethylclobazam eluted into the mass spectrometer at 9.5 minutes (retention time) and formed ions with mass 286, resulting in the peak observed in the selected ion chromatogram. Plasma samples contain a large number of other substances, but these do not appear because the ionization or fragmentation does not form ions with mass 286
Figure 8.26 Calibration curve for the quantitative determination of N-desmethylclobazam in plasma based on six standard solutions with different concentrations of N-desmethylclobazam
Chapter 9: Analysis of Small-Molecule Drugs in Biological Fluids
Figure 9.1
Figure 9.2
Figure 9.3 Photo of dried blood spots (DBS) on specially manufactured absorbent filter paper (a DBS card).
Chapter 10: Analysis of Peptide and Protein Drugs in Biological Fluids
Figure 10.1
Figure 10.2
Figure 10.3
Figure 10.4
Figure 10.5
Figure 10.6 Chromatogram of a digested enfuvirtide standard. Peak A, C, and E represent the measured product peptides A, C, and E described in this chapter.
Figure 10.7
Figure 10.8
Figure 10.9
Figure 10.10
Figure 10.11
Chapter 11: Regulated Bioanalysis and Guidelines
Figure 11.1 Level of method characterization during the process of development of a new drug. The level of method characterization increases dramatically after the drug candidate has been selected for development and remains hereafter constant from the first regulated toxicological studies in the early preclinical development phase throughout the clinical development phase. CS = candidate selection, TKs = toxicokinetics, and FIM = first in man
List of Tables
Chapter 1: Introduction
Table 1.1 Therapeutic range of common drugs subjected to therapeutic drug monitoring
Chapter 2: Physicochemical Properties of Drug Substances
Table 2.1
Table 2.2 Energy in bonds or of intermolecular forces
Table 2.3 Amino acid abbreviations and key properties
Chapter 4: General Chromatographic Theory and Principles
Table 4.1 The relationship between α and the number of
N
needed to obtain a resolution of 1.5
Table 4.2 Relationship between the retention factor,
k
, and the efficiency,
N
Table 4.3 Mobile phases with similar eluting strength
Table 4.4 pKa values (25 °C) of buffer compounds frequently used in HPLC
Table 4.5 Derivatization of an enantiomeric analyte with a chiral reagent
Chapter 6: Sample Preparation
Table 6.1 Different approaches to protein precipitation
Table 6.2 Frequently used liquid–liquid extraction solvents and their physiochemical properties
Table 6.3 Kamlet and Taft solvatochromic parameters (α, β, and π*) for selected solvents
Chapter 7: High-Performance Liquid Chromatography (HPLC) and High-Performance Liquid Chromatography–Mass Spectrometry (LC-MS)
Table 7.1 UV cut-off for common solvents (1 cm path length)
Table 7.2 Effect of column length and particle size on column efficiency (
N
)
Table 7.3 Some typical high-performance liquid chromatography columns and the corresponding eluent consumption
Table 7.4 Some commercial available liquid chromatography detectors commonly used in bioanalysis and their typical performance
Chapter 8: Gas Chromatography (GC)
Table 8.1 Masses and occurrence of sTable isotopes
Table 8.2 Typical fragmentations from the molecular ion
Chapter 11: Regulated Bioanalysis and Guidelines
Table 11.1 Elements of a Method Validation
Bioanalysis of Pharmaceuticals
Sample Preparation, Separation Techniques, and Mass Spectrometry
Editors
STEEN HONORÉ HANSEN
School of Pharmaceutical Sciences, University of Copenhagen, Denmark
STIG PEDERSEN-BJERGAARD
School of Pharmacy, University of Oslo, Norway School of Pharmaceutical Sciences, University of Copenhagen, Denmark
This edition first published 2015
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Contributing Authors
Leon Reubsaet
School of Pharmacy, University of Oslo, Norway
Chapters 2
,
7
, and
10
Trine Grønhaug Halvorsen
School of Pharmacy, University of Oslo, Norway
Chapters 6
and
10
Astrid Gjelstad
School of Pharmacy, University of Oslo, Norway
Chapter 6
Martin Jørgensen
Drug ADME Research, H. Lundbeck AS, Denmark
Chapter 11
Morten A. Kall
Department of Bioanalysis, H. Lundbeck AS, Denmark
Chapter 11
Preface
The field of bioanalysis is very broad, complex, and challenging, and therefore writing an introductory textbook in this field is a difficult task. From our point of view, a good introductory student textbook is limited in the number of pages, discusses the different principles and concepts clearly and comprehensively, and contains many relevant and educational examples. Given these criteria, we have narrowed our focus on bioanalysis. First, we have limited our discussion to the chemical analysis of pharmaceuticals that are present in biological fluids. The focus is directed toward substances that are administered as human drugs, including low-molecular drug substances, peptides, and proteins. Endogenous substances are not discussed. Second, the discussion of different analytical methods has been limited to those based on chromatography and mass spectrometry. Certainly, different immunological methods are also used, but teaching all the principles and applications of chromatographic, mass spectrometric, and immunological methods was too ambitious to meet our criteria for a good introductory student textbook.
The present book is the first introductory student textbook on chromatography and mass spectrometry of pharmaceuticals present in biological fluids, highlighting an educational presentation of the principles, concepts, and applications. We discuss the chemical structures and properties of low- and high-molecular pharmaceuticals, the different types of biological samples and fluids that are used, how to prepare the samples by extraction, and how to perform the final analytical measurement by use of chromatography and mass spectrometry. Many examples illustrate the theory and applications, and the examples discuss all practical aspects, including the calculations. Thus, in this textbook, you will even learn how to convert the numbers recorded by the instrument to the concentration of the actual drug substances in the biological sample.
Bioanalysis is an applied scientific discipline, and this represents another challenge in terms of writing an introductory student textbook. University professors are well trained in teaching the basic principles. However, bioanalysis is mainly performed outside the university by researchers in the pharmaceutical industry, in contract laboratories, and in hospital laboratories. Thus, the researchers outside the university have the best overview of the most important applications and techniques in practical use. To address this, both university professors and researchers from the pharmaceutical industry have authored this textbook. Hopefully, this has resulted in a textbook that reflects bioanalysis in the year 2015. The authors have been in close contact with colleagues for advice, and we would especially like to thank Elisabeth Leere Øiestad for fruitful discussions.
The present textbook is intended for the fourth- or fifth-year university pharmacy or chemistry student. Reading the textbook requires basic knowledge in organic chemistry and biochemistry, as well as in analytical chemistry. With respect to the latter, we have given priority to discuss the analytical techniques in a fundamental and educational frame, and detailed knowledge on instrumental analytical methods is not required prior to reading this textbook.
Good luck with the reading!
Oslo and Copenhagen, June 2014Steen Honoré Hansen, Stig Pedersen-Bjergaard, Leon Reubsaet, Astrid Gjelstad,Trine Grønhaug Halvorsen, Martin Jørgensen, and Morten A. Kall
Readers can access PowerPoint slides of all figures at
http://booksupport.wiley.com
Chapter 1Introduction
Stig Pedersen-Bjergaard
School of Pharmacy, University of Oslo, Norway School of Pharmaceutical Sciences, University of Copenhagen, Denmark
Welcome to the field of bioanalysis! Through reading of this textbook, we hope you get fascinated by the world of bioanalysis, and also we hope that you learn to understand that bioanalysis is a highly important scientific discipline. In this chapter, five fundamental questions are raised and briefly discussed as an introduction to the textbook: (i) What is bioanalysis? (ii) What is the purpose of bioanalysis? (iii) Where is bioanalysis conducted? (iv) Why do you need theoretical understanding and skills in bioanalysis? And (v) how do you gain the understanding and the skills from reading this textbook?
1.1 What Is Bioanalysis?
In this textbook, we define bioanalysis as the chemical analysis of pharmaceutical substances in biological samples. The purpose of the chemical analysis is normally both to identify (identification) and to quantify (quantification) the pharmaceutical substance of interest in a given biological sample. This is performed by a bioanalytical chemist (scientist) using a bioanalytical method. The pharmaceutical substance of interest is often termed the analyte, and this term will be used throughout the textbook. Identification of the analyte implies that the exact chemical identity of the analyte is established unequivocally. Quantification of the analyte implies that the concentration of the analyte in the biological sample is measured. It is important to emphasize that quantification is associated with small inaccuracies, and the result is prone to errors. Thus, the quantitative data should be considered as an estimate of the true concentration. Based on theoretical and practical skills, and based on careful optimization and testing of the bioanalytical methods, the bioanalytical chemist tries to reduce the error level, providing concentration estimates that are very close to the true values.
Bioanalytical data are highly important in many aspects. As an example, a patient serum sample is analyzed for the antibiotic drug substance gentamicin, and gentamicin is measured in the sample at a concentration of 5 µg/ml. First, the identification of gentamicin in the blood serum sample confirms that the patient has taken the drug. This is important information because not all patients actually comply with the prescribed medication. Second, the exact concentration of gentamicin measured in the blood serum sample confirms that the amount of gentamicin taken is appropriate, as the recommended concentration level should be in the range of 4–10 µg/ml. For aminoglycoside antibiotics such as gentamicin, it is recommended to monitor the concentration in blood if the treatment is expected to continue for more than 72 hours as these antibiotics have the potential to cause severe adverse reactions, such as nephrotoxicity and ototoxicity.
As will be discussed in much more detail in this book, not only blood serum samples are used for bioanalysis. Bioanalysis can be performed on raw blood samples (whole blood) or on blood samples from which the blood cells have been removed (serum or plasma). Alternatively, bioanalysis can be performed from urine or saliva as examples, depending on the purpose of the bioanalysis. Bioanalysis is performed both on human samples and on samples from animal experiments.
1.2 What Is the Purpose of Bioanalysis, and Where Is It Conducted?
Bioanalysis is conducted in the pharmaceutical industry, in contract laboratories associated with the pharmaceutical industry, in hospital laboratories, in forensic toxicology laboratories, and in doping control laboratories. In the pharmaceutical industry and in the associated contract laboratories, bioanalysis is basically conducted to support the development of new drugs and new drug formulations. In hospital laboratories, bioanalysis is used to monitor existing drugs in patient samples, to check that individual patients take their drugs correctly. In forensic toxicology laboratories and doping laboratories, bioanalysis is used to check for abuse of drugs and drug-related substances.
1.2.1 Bioanalysis in the Pharmaceutical Industry
Bioanalytical laboratories are highly important in the development of new drugs and new drug formulations in the pharmaceutical industry. Thus, identification and quantification of drug substances and metabolites in biological samples like blood plasma, urine, and tissue play a very important role during drug development. Drug development begins with the identification of a medical need and hypotheses on how therapy can be improved. Drug discovery is the identification of new drug candidates based on combinatorial chemistry, high-throughput screening, genomics, and ADME (absorption, distribution, metabolism, and elimination). By combinatorial chemistry, a great number of new drug candidates are synthesized, and these are tested for pharmacological activity and potency in high-throughput screening (HTS) systems. The HTS systems simulate the interaction of the drug candidates with a specific biological receptor or target. Once a lead compound is found, a narrow range of similar drug candidates is synthesized and screened to improve the activity toward the specific target. Other studies investigate the ADME profile of drug candidates by analyzing samples collected at different time points from dosed laboratory animals (in vivo testing) and tissue cultures (in vitro testing).
Drug candidates passing the discovery phase are subjected to toxicity testing and further metabolism and pharmacological studies in the preclinical development phase. Both in vivo and in vitro tests are conducted, and various animal species are used to prove the pharmacokinetic profile of the candidate. The detailed information about the candidate forms the basis for further pharmaceutical research on the synthesis of raw materials, the development of dosage forms, quality control, and stability testing.
The clinical development phase can begin when a regulatory body has judged a drug candidate to be effective and to appear safe in healthy volunteers. In phase I, the goal is to establish a safe and efficient dosage regimen and to assess pharmacokinetics. Blood samples are collected and analyzed from a small group of healthy volunteers (20–80 persons). The data obtained form the basis for developing controlled phase II studies. The goal of phase II studies is to demonstrate a positive benefit–risk balance in a larger group of patients (200–800) and to further study pharmacokinetics. Monitoring of efficacy and monitoring of possible side effects are essential. Phase II studies can take up to two years to fulfill. At the end of phase II, a report is submitted to the regulatory body, and conditions for phase III studies are discussed. Additional information supporting the claims for a new drug is provided. Phase III begins when evidence for the efficacy of the drug candidate and supporting data have demonstrated a favorable outcome to the regulatory body. The phase III studies are large-scale efficacy studies with focus on the effectiveness and safety of the drug candidate in a large group of patients. In most cases, the drug candidate is compared with another drug already in use for treatment of the same condition. Phase III studies can last two to three years or more, and 3000–5000 patients can be involved. Carcinogenetic tests, toxicology tests, and metabolic studies in laboratory animals are conducted in parallel. The cumulative data form the basis for filing a new drug application to the regulatory body and for future plans for manufacturing and marketing. The regulatory body thoroughly evaluates the documentation that is provided before a market approval can be authorized and the drug product can be legally marketed. The time required from drug discovery to product launch is up to 12 years. Phase IV studies are studies that are conducted after product launch to demonstrate long-term effects and new claims, expand on approved claims, examine possible drug–drug interactions, and further assess pharmacokinetics. Several thousand patients participate in phase IV studies.
Bioanalytical measurements are conducted during drug discovery, preclinical development, and clinical development, and they are intended to (among other things) generate the experimental data to establish the pharmacokinetics, the toxicokinetics, and the exposure–response relationships for a new drug. The pharmacokinetics of a certain drug substance describes how the body affects the drug after administration (ADME): how the drug is absorbed (A) and distributed (D) in the body, and how the drug is metabolized (M) by metabolic enzymes and chemically changed to different types of metabolites, which in turn are excreted (E) from the body. Bioanalysis is used extensively in pharmacokinetic studies, among others, to establish blood concentration–time profiles, and to measure the rate of drug metabolism and excretion. This involves a large number of both animal and human samples.
Toxicokinetics studies, in contrast, are intended to investigate the relationship between the exposure of a new drug candidate in experimental animals and its toxicity. This type of information is used to establish a relationship between the possible toxic properties of a drug in animals and those in humans. Toxicokinetic studies involve bioanalysis in both animal and human samples.
Exposure–response studies investigate the link between pharmacokinetics and pharmacodynamics. Pharmacodynamics is the study of the biochemical and physiological effects of a drug substance on the body. Exposure–response studies thus establish the link between the dose, the blood concentration, and the effect. Also for exposure–response studies, bioanalysis of a large number of blood samples has to be conducted. Frequently, such bioanalytical measurements are outsourced by the pharmaceutical company to a contract laboratory that is highly specialized in bioanalysis. High quality of the bioanalytical data is mandatory, because these data from drug discovery, preclinical, and clinical studies are used to support the regulatory filings.
1.2.2 Bioanalysis in Hospital Laboratories
Bioanalysis is also very important in many hospital laboratories. Here, the focus is on measuring the drug concentration in blood samples of patients to check that they are properly medicated. This is called therapeutic drug monitoring (TDM), and it refers to the individualization of dosage by maintaining serum or plasma drug concentrations within a target range to optimize efficacy and to reduce the risk of adverse side effects. The target range of a drug is also called the therapeutic range or the therapeutic window; it is the concentration range between the lowest drug concentration that has a positive effect and the concentration that gives more adverse effects than positive effects. Variability in the dose–response relationship between individual patients is due to pharmacokinetic variability and pharmacodynamic variability, as shown in Figure 1.1.
Figure 1.1 Effects of pharmacokinetics and pharmacodynamics on the dose–response relationship
Pharmacodynamic variability arises from variations in drug concentrations at the receptor and from variations in the drug–receptor interaction. Pharmacokinetic variability is due to variations in the dose to plasma concentration relationship. Major sources of pharmacokinetic variability are age, physiology, disease, compliance, and genetic polymorphism of drug metabolism. Indications for including a drug in a therapeutic drug-monitoring program are:
There is an experimentally determined relationship between the plasma drug concentration and the pharmacological effect.
There is a narrow therapeutic window.
The toxicity or lack of effectiveness of the drug puts the patient at risk.
There are potential patient compliance problems.
The dose cannot be optimized by clinical observations alone.
Many drugs do not meet the criteria to be included in a TDM program. They are safely taken without determining drug concentrations in plasma because the therapeutic effect can be evaluated by other means. For example, the coagulation time effectively measures the efficacy of an anticoagulant drug, and the blood pressure indicates the efficacy of a drug used in the treatment of hypertension. In these situations, it is preferred to adjust the dosage on the basis of medical response.
The two major situations when TDM is advised are (1) for drugs used prophylactically to maintain the absence of a condition (e.g., depressive or manic episodes, seizures, cardiac arrhythmias, organ rejection, and asthma relapses) and (2) to avoid serious toxicity for drugs with a narrow therapeutic window (e.g., antiepileptic drugs, antidepressant drugs, digoxin, phenytoin, theophylline, cyclosporine and HIV protease inhibitors, and aminoglycoside antibiotics). The therapeutic range of some drugs subjected to TDM is shown in Table 1.1.
Table 1.1 Therapeutic range of common drugs subjected to therapeutic drug monitoring
Drug
Therapeutic range
Drug
Therapeutic range
Amitriptyline
120–150 ng/ml
Nortriptyline
50–150 ng/ml
Carbamazepine
4–12 µg/ml
Phenobarbital
10–40 µg/ml
Desipramine
150–300 ng/ml
Phenytoin
10–20 µg/ml
Digoxine
0.8–2.0 ng/ml
Primidone
5–12 µg/ml
Disopyramide
2–5 µg/ml
Theophylline
10–20 µg/ml
Ethosuximide
40–100 µg/ml
Valproic acid
50–100 µg/ml
Lithium
4–8 µg/ml
1.2.3 Bioanalysis in Forensic Toxicology Laboratories
Bioanalysis is a core discipline also in forensic toxicology laboratories, where a large number of blood, urine, and saliva samples are analyzed to identify abuse of drugs and narcotics. The focus is on drugs and their metabolites, narcotics, and other substances that are toxicologically relevant. Serious cases that are of criminal relevance may include:
Analysis of pharmaceuticals and addictive drugs that may impair human behavior.
Detection of poisons and evaluation of their relevance in determining causes of death.
In forensic toxicology, the analyte is essentially unknown. Therefore, samples are first screened for the presence of drugs or drugs of abuse. In case of a positive sample, the drug or the drug of abuse is confirmed with a second bioanalytical method. Due to the serious legal consequences of forensic cases, particular emphasis is placed on the quality and reliability of bioanalytical results. The work always involves the application of at least two different analytical methods (screening and confirmation) based on different physical or chemical principles.
1.2.4 Bioanalysis in Doping Control Laboratories
Bioanalysis also is very important in doping control laboratories, where blood and urine samples are tested for doping agents. Only laboratories accredited by the World Anti-Doping Agency (WADA) take part in the testing. WADA was established in 1999 as an international agency to promote, coordinate, and monitor the fight against doping in sport. One of WADA's most significant achievements was the acceptance and implementation of the World Anti-Doping Code (the Code). The Code is the core document that provides the framework for antidoping policies, rules, and regulations within sport organizations and among public authorities. The Code works in conjunction with five international standards aimed at bringing harmonization among antidoping organization in various areas. The standards are:
List of Prohibited Substances and Methods
International Standard for Testing
International Standard for Laboratories
International Standard for Therapeutic Use Exemptions
International Standard for the Protection of Privacy and Personal Information.
The prohibited list is the standard that defines substances and methods that are prohibited to athletes at all times (both in competition and out of competition), substances prohibited in competition, and substances prohibited in particular sports. The prohibited list is updated annually.
The purpose of the International Standard for Testing is to plan for effective in-competition and out-of-competition testing and to maintain the integrity and identity of the samples collected. The International Standard for Therapeutic Use Exemptions and the International Standard for the Protection of Privacy and Personal Information ensure that the process of granting an athlete therapeutic-use exemptions is harmonized and that all relevant parties adhere to the same set of privacy protections.
The purpose of the International Standard for Laboratories is to ensure that laboratories produce valid test results. The standard further ensures that uniform and harmonized results are reported from all accredited laboratories. In addition, the document specifies the criteria that must be fulfilled by antidoping laboratories to achieve and maintain their WADA accreditation.
1.3 Bioanalysis Is Challenging
Bioanalysis is highly challenging because most target pharmaceutical substances are present in blood, urine, and saliva samples at very low concentrations. Typically, the concentration level is at the low ng/ml level, but in some cases, target pharmaceuticals have to be detected even down to the pg/ml level. This relies on very sensitive instrumentation and high operator skills. In addition, the target pharmaceuticals coexist with a broad range of endogenous compounds that are naturally present in biological samples. There can be thousands of different components, and many of them can be present at high concentration levels. Therefore, in most cases, a successful bioanalysis procedure requires the isolation of target pharmaceuticals from the biological matrix, before the final measurement with a sensitive instrument. Thus, experience and skills on how to prepare samples are extremely important in bioanalysis. The intention of the current textbook is to provide the reader with the required theoretical understanding and skills related to the understanding, development, and application of bioanalytical methods and procedures.
1.4 The Different Sections of This Textbook
The first part of this book is focused on the chemical properties of drug substances (Chapter 2) and the properties of the different biological fluids in use (Chapter 2). Careful reading of Chapter 2 is important for readers who are not familiar with pharmaceutical substances or the chemical properties of these substances. Understanding the chemical properties of the target pharmaceuticals is highly important in order to understand the bioanalytical procedures. Chapter 3 discusses the properties of different biological fluids, and if you are unfamiliar with biological fluids, you should read this chapter carefully. Understanding the properties of the biological fluids is mandatory in order to understand bioanalytical procedures.
Chapters 4–8 teach the different techniques and their principles, with foci on sample preparation, separation, and detection. These are chapters that are similar to the content of general textbooks in analytical chemistry. So, if you have been through general courses in analytical chemistry, this part of the textbook will be repetition. The discussion about sample preparation gives the reader an understanding of how to isolate target pharmaceuticals from the bulk biological matrix. The discussion about separation is focused on chromatographic separation, in which target pharmaceuticals are separated from any other substances in the sample, and the discussion about detection is focused on the final measurement of the substance, in most cases by mass spectrometry.
Chapters 9 and 10 are a collection of examples of bioanalytical procedures that are typically not found in general textbooks in analytical chemistry. In this part of the textbook, we make use of all previous knowledge and try to give you the full understanding. In these chapters, we discuss practical examples, and all discussions are related to the theory presented in Chapters 4–8. Remember when you read Chapters 9 and 10 that you should understand rather than remember all the details. Hopefully, the combination of Chapters 4–10 should give the reader a very good understanding of bioanalytical procedures. Finally, regulatory aspects related to bioanalytical procedures are discussed in Chapter 11. This is important to make sure that we can rely on the data generated from the application of bioanalytical methods.
Good luck with your journey into the world of bioanalysis!
Chapter 2Physicochemical Properties of Drug Substances
Steen Honoré Hansen1 and Leon Reubsaet2
1School of Pharmaceutical Sciences, University of Copenhagen, Denmark
2School of Pharmacy, University of Oslo, Norway
The development of both sample preparation strategies and chromatographic methods is based on the physicochemical properties of the substances to be analyzed as well as the principles of the analytical technique used. Why is it that in some cases mass spectrometric detection is needed to determine a substance, whereas in other cases UV detection is sufficient? Can chromatographic behavior be predicted from simply looking at the chemical structure of the analyte? In this chapter, the most important physicochemical properties of small-molecule drug substances as well as those of peptide and protein biopharmaceuticals are discussed. The discussions are short and comprehensive, as most of this information should already be known from learning general chemistry. The properties discussed here will be used in other chapters in this book in relation to sample preparation and subsequent analysis.
2.1 Bioanalysis in General
In bioanalysis, the task often is to perform qualitative or quantitative measurements of analytes in complex matrices consisting of thousands of other chemical entities. Therefore, a high degree of selectivity is needed to be able to “pick the needle out of the haystack” and in this way increase the reliability of the data obtained. In many bioanalytical methods, the selectivity is incorporated at several stages: in the sample preparation, in the following chromatographic separation, and in the detection step. To be able to optimize the selectivity, a basic knowledge of some fundamental chemical and physicochemical properties is needed.
2.2 Protolytic Properties of Analytes
Many small molecules show protolytic properties, which cause them to be present in an ionic state as well as a neutral form. The degree of ionization controlled by the surrounding aqueous solvent very much influences the properties of the molecules and thus their behavior in each step in the bioanalytical method.
pH is defined in dilute aqueous solution and is an expression for the acidity or alkalinity of an aqueous solution. The pH concept is extremely important and has great influence on living organisms as well as in analytical chemistry. Water can react with itself to form a hydronium ion and a hydroxide ion:
This is called autoprotolysis, as the water in this case acts as an acid and a base. The autoprotolysis constant is:
and it indicates that only a very small amount of water is ionized. The concentration of the two ions, H3O+ and OH−, in pure water is therefore 10−7 M of each ion.
pH is defined as the negative logarithm to the activity, aH+, or the concentration of the hydrogen ion, [H+] (being equivalent to the hydronium ions):
Strong acids and strong bases are fully ionized in dilute aqueous solution, and the activity and concentration of [H+] therefore can be considered to be identical.
Weak acids and weak bases are not completely ionized in aqueous solution and are therefore in equilibrium with the unionized acid or base. When we ignore the weak autoprotolysis of water, we get the following general equation for a weak acid:
When an acid (H+) is added to such a system, the H+ will partly be removed by association with A− to form HA, and if a base (OH−) is added it will be partly neutralized by H+ and more HA will dissociate. pH will thus be maintained in the solution. A system like this is called a buffer system, and the purpose of a buffer is to maintain the pH in the solution. pKa is defined as the negative logarithm to Ka:
and it is obvious that the highest buffer capacity is achieved at a pH value equivalent to the pKa value of the buffer substance. Combining Equations 2.4 and 2.5 results in a most useful equation called the Henderson–Hasselbalch equation:
At pH = pKa, equal concentrations of the acid and corresponding base are present. If the ratio between HA and A− becomes 9/1 (only 10% base) pH will decrease one unit, and if the ratio becomes 99/1 (1% base) the pH value will decrease by two units. This is illustrated in Figure 2.1. Equivalent estimations can be performed when increasing the base content. One example of a buffer system is arterial plasma, and this is featured in Box 2.1.
Figure 2.1 Ionization of acids and bases as a function of pH
Box 2.1 Arterial Plasma Is a Buffer System
pH in arterial plasma is buffered by a special bicarbonate system. When acid is added CO2 is formed, which is actively controlled by the lungs, and if base is added the increase in bicarbonate is actively controlled by the kidneys. In this way it is possible to maintain a pH of 7.4, although the pKa value of the bicarbonate is 6.1. Plasma collected for bioanalysis no longer has the contact to the lungs and the kidneys, but the plasma still has some buffer capacity, mainly due to the content of about 8% of proteins.
It is convenient to have general knowledge of the pKa values of a number of functional groups, as presented in Table 2.1.
Table 2.1 Typical pKa values of important functional groups
Functional group
pK
a
Comments (depending on chemical structure)
R-COOH, carboxylic acid
4–5
Can be lower (more acidic)
a
R-NH
2
; R1, R2, NH; and R1, R2, R3, N, aliphatic amines
8–11
Can be lower (less basic)
a
Aromatic amines
About 5
—
Quaternary ammonium ions
—
Ions with no protolytic properties; are always positively charged
Ar-OH, phenols
8–10
Can be lower (more acidic)
a
R-OH, alcohols
>12
Can for practical purposes be considered as neutral substances
R-SO
2
OH, sulfonic acid
About 1
Are for all practical purposes always negatively charged
R-CO—NH—CO—R and R—SO
2
NH—R
7–11
Weak to very weak acids
a This depends on other chemical groups in the molecule.
The pKa value for bases refers to the protonated form of the bases. However, the basicity of bases may also be expressed equivalent to the pKa of acids. In that case, the term pKb is used and
2.3 Partitioning of Substances
A prerequisite in chromatography as well as in many sample preparation techniques is the partitioning of molecules between more or less immiscible phases (gas–liquid, gas–solid, liquid–liquid, or liquid–solid). When molecules are in solution, they will be exposed to a number of intermolecular interactions. These include, among other things, diffusion, collisions, dipole–dipole interactions, hydrogen bonding, and electrostatic interactions, as illustrated in Table 2.2. The nature of the interactions taking place is dependent on the physical and chemical nature of the analytes, and these interactions will determine how the molecules are distributed between different phases.
Table 2.2 Energy in bonds or of intermolecular forces
Type of bond or intermolecular force
Example of interacting molecules
Energy in kJ/mol (kcal/mol)
Covalent
RH
2
C–CH
2
R
400–1200 (100–300)
Ionic
R
4
N
+
• • •
−
OOC-R
200–800 (50–200)
Hydrogen bond
H
3
CO
H
• • • HO
H
20–50 (5–12)
Dipole–dipole
H
3
CC≡N • • • C
6
H
5
Cl
12–40 (3–10)
Dipole-induced dipole
H
3
CC≡N • • • C
6
H
6
10–25 (2–6)
Dispersion or van der Waals
C
6
H
6
• • • C
6
H
14
5–20 (1–5)
Ionic interactions can be as strong as a covalent bond but are often limited to one interaction per molecule. In contrast, van der Waal interactions are relatively weak but have many interactions per molecule and therefore are also very important.
The partition or distribution between phases (see Figure 2.2) is also influenced by pH, and thus a thorough knowledge of the pH concept, including pKa, as well as of distribution constants will ease the development of bioanalytical methods (e.g., the chromatographic separation). The distribution is dependent on the nature of the two phases as well as the temperature. If we want to alter the partition between the two phases, we must change one of these variables. The equilibrium distribution for a substance A is given by the partition ratio, which is also called the distribution constant:
where [A]org is the concentration of compound A in the organic phase and [A]aq is the concentration of compound A in the water phase.
Figure 2.2 Distribution of an analyte A between an upper organic phase and a lower aqueous phase
The distribution constant is a constant relating to a specific molecular species, but often the molecules of a compound can be present as different species, for example by dissociation in the aqueous phase:
or by dimerization in the organic phase:
These equilibria are normally very fast, and it is therefore appropriate to look at the total distribution of all the species of a compound between the two phases:
The concentration distribution ratio, DC, between the two phases can also be converted to the mass distribution ratio, Dm, by multiplying the concentrations with the matching phase volumes:
where Vorg and Vaq
