Bioanalysis
Encyclopedia
Bioanalysis is a sub-discipline of analytical chemistry
covering the quantitative
measurement of xenobiotics (drugs and their metabolite
s, and biological molecules
in unnatural locations or concentrations) and biotics (macromolecule
s, protein
s, DNA
, large molecule drugs, metabolites) in biological systems.
, toxicokinetics
, bioequivalence
and exposure–response (pharmacokinetics/pharmacodynamics studies). Bioanalysis also applies to drugs used for illicit purposes, forensic investigations, anti-doping testing in sports, and environmental concerns.
Bioanalysis was traditionally thought of in terms of measuring small molecule drugs. However, the past twenty years has seen an increase in biopharmaceuticals (e.g. proteins and peptides), which have been developed to address many of the same diseases as small molecules. These larger biomolecules have presented their own unique challenges to quantification.
.
Initially, nonspecific assays were applied to measuring drugs in biological fluids. These were unable to discriminate between the drug and its metabolites; for example, aspirin
(circa 1900) and sulfonamides (developed in the 1930s) were quantified by the use of colorimetric
assays. Antibiotic
s were quantified by their ability to inhibit bacterial growth. The 1930s also saw the rise of pharmacokinetics, and as such the desire for more specific assays. Modern drugs are more potent, which has required more sensitive bioanalytical assays to accurately and reliably determine these drugs at lower concentrations. This has driven improvements in technology and analytical methods.
The most frequently used techniques are: liquid chromatography coupled with tandem mass spectrometry (LC–MS/MS
) for 'small' molecules and enzyme-linked immunosorbent assay (ELISA
) for macromolecules
Bioanalytical laboratories often deal with large numbers of samples, for example resulting from clinical trials. As such, automated sample preparation methods and liquid-handling robots are commonly employed to increase efficiency and reduce costs.
(EBF), on the other hand, is independent of any larger society or association.
Analytical chemistry
Analytical chemistry is the study of the separation, identification, and quantification of the chemical components of natural and artificial materials. Qualitative analysis gives an indication of the identity of the chemical species in the sample and quantitative analysis determines the amount of...
covering the quantitative
Quantitative analysis (chemistry)
In chemistry, quantitative analysis is the determination of the absolute or relative abundance of one, several or all particular substance present in a sample....
measurement of xenobiotics (drugs and their metabolite
Metabolite
Metabolites are the intermediates and products of metabolism. The term metabolite is usually restricted to small molecules. A primary metabolite is directly involved in normal growth, development, and reproduction. Alcohol is an example of a primary metabolite produced in large-scale by industrial...
s, and biological molecules
Biomolecule
A biomolecule is any molecule that is produced by a living organism, including large polymeric molecules such as proteins, polysaccharides, lipids, and nucleic acids as well as small molecules such as primary metabolites, secondary metabolites, and natural products...
in unnatural locations or concentrations) and biotics (macromolecule
Macromolecule
A macromolecule is a very large molecule commonly created by some form of polymerization. In biochemistry, the term is applied to the four conventional biopolymers , as well as non-polymeric molecules with large molecular mass such as macrocycles...
s, protein
Protein
Proteins are biochemical compounds consisting of one or more polypeptides typically folded into a globular or fibrous form, facilitating a biological function. A polypeptide is a single linear polymer chain of amino acids bonded together by peptide bonds between the carboxyl and amino groups of...
s, DNA
DNA
Deoxyribonucleic acid is a nucleic acid that contains the genetic instructions used in the development and functioning of all known living organisms . The DNA segments that carry this genetic information are called genes, but other DNA sequences have structural purposes, or are involved in...
, large molecule drugs, metabolites) in biological systems.
Modern bioanalytical chemistry
Many scientific endeavors are dependent upon accurate quantification of drugs and endogenous substances in biological samples; the focus of bioanalysis in the pharmaceutical industry is to provide a quantitative measure of the active drug and/or its metabolite(s) for the purpose of pharmacokineticsPharmacokinetics
Pharmacokinetics, sometimes abbreviated as PK, is a branch of pharmacology dedicated to the determination of the fate of substances administered externally to a living organism...
, toxicokinetics
Toxicokinetics
Toxicokinetics is the description of what rate a chemical will enter the body and what happens to it once it is in the body. It is an application of pharmacokinetics to determine the relationship between the systemic exposure of a compound in experimental animals and its toxicity...
, bioequivalence
Bioequivalence
Bioequivalence is a term in pharmacokinetics used to assess the expected in vivo biological equivalence of two proprietary preparations of a drug...
and exposure–response (pharmacokinetics/pharmacodynamics studies). Bioanalysis also applies to drugs used for illicit purposes, forensic investigations, anti-doping testing in sports, and environmental concerns.
Bioanalysis was traditionally thought of in terms of measuring small molecule drugs. However, the past twenty years has seen an increase in biopharmaceuticals (e.g. proteins and peptides), which have been developed to address many of the same diseases as small molecules. These larger biomolecules have presented their own unique challenges to quantification.
History
The first studies measuring drugs in biological fluids were carried out to determine possible overdosing as part of the new science of forensic medicine/toxicologyToxicology
Toxicology is a branch of biology, chemistry, and medicine concerned with the study of the adverse effects of chemicals on living organisms...
.
Initially, nonspecific assays were applied to measuring drugs in biological fluids. These were unable to discriminate between the drug and its metabolites; for example, aspirin
Aspirin
Aspirin , also known as acetylsalicylic acid , is a salicylate drug, often used as an analgesic to relieve minor aches and pains, as an antipyretic to reduce fever, and as an anti-inflammatory medication. It was discovered by Arthur Eichengrun, a chemist with the German company Bayer...
(circa 1900) and sulfonamides (developed in the 1930s) were quantified by the use of colorimetric
Colorimetry (chemical method)
In physical and analytical chemistry, colorimetry or colourimetry is a technique "used to determine the concentration of colored compounds in solution."...
assays. Antibiotic
Antibiotic
An antibacterial is a compound or substance that kills or slows down the growth of bacteria.The term is often used synonymously with the term antibiotic; today, however, with increased knowledge of the causative agents of various infectious diseases, antibiotic has come to denote a broader range of...
s were quantified by their ability to inhibit bacterial growth. The 1930s also saw the rise of pharmacokinetics, and as such the desire for more specific assays. Modern drugs are more potent, which has required more sensitive bioanalytical assays to accurately and reliably determine these drugs at lower concentrations. This has driven improvements in technology and analytical methods.
Bioanalytical techniques
Some techniques commonly used in bioanalytical studies include:- Hyphenated techniques
- LC–MSLiquid chromatography-mass spectrometryLiquid chromatography–mass spectrometry is an analytical chemistry technique that combines the physical separation capabilities of liquid chromatography with the mass analysis capabilities of mass spectrometry. LC-MS is a powerful technique used for many applications which has very high...
(liquid chromatography–mass spectrometry) - GC–MSGas chromatography-mass spectrometryGas chromatography–mass spectrometry is a method that combines the features of gas-liquid chromatography and mass spectrometry to identify different substances within a test sample. Applications of GC-MS include drug detection, fire investigation, environmental analysis, explosives investigation,...
(gas chromatography–mass spectrometry) - LC–DAD (liquid chromatography–diode array detection)
- CE–MSCapillary electrophoresis-mass spectrometryCapillary electrophoresis–mass spectrometry is an analytical chemistry technique formed by the combination of the liquid separation process of capillary electrophoresis with mass spectrometry. Ions are typically formed by electrospray ionization, but they can also be formed by matrix-assisted...
(capillary electrophoresis–mass spectrometry)
- LC–MS
- Chromatographic methods
- HPLCHigh-performance liquid chromatographyHigh-performance liquid chromatography , HPLC, is a chromatographic technique that can separate a mixture of compounds and is used in biochemistry and analytical chemistry to identify, quantify and purify the individual components of the mixture.HPLC typically utilizes different types of stationary...
(high performance liquid chromatography) - GC (gas chromatography)
- UPLC (ultra performance liquid chromatography)
- Supercritical fluid chromatography
- HPLC
- ElectrophoresisElectrophoresisElectrophoresis, also called cataphoresis, is the motion of dispersed particles relative to a fluid under the influence of a spatially uniform electric field. This electrokinetic phenomenon was observed for the first time in 1807 by Reuss , who noticed that the application of a constant electric...
- Ligand binding assays
- Dual polarisation interferometryDual Polarisation InterferometryDual polarization interferometry is an analytical technique that can probe molecular scale layers adsorbed to the surface of a waveguide by using the evanescent wave of a laser beam confined to the waveguide...
- ELISAELISAEnzyme-linked immunosorbent assay , is a popular format of a "wet-lab" type analytic biochemistry assay that uses one sub-type of heterogeneous, solid-phase enzyme immunoassay to detect the presence of a substance in a liquid sample."Wet lab" analytic biochemistry assays involves detection of an...
(Enzyme-linked immunosorbent assay) - MIAMagnetic immunoassayMagnetic immunoassay is a novel type of diagnostic immunoassay using magnetic beads as labels in lieu of conventional enzymes , radioisotopes or fluorescent moieties . This assay involves the specific binding of an antibody to its antigen, where a magnetic label is conjugated to one element of...
(magnetic immunoassay) - RIARadioimmunoassayRadioimmunoassay is a very sensitive in vitro assay technique used to measure concentrations of antigens by use of antibodies...
(radioimmunoassay)
- Dual polarisation interferometry
- Mass spectrometryMass spectrometryMass spectrometry is an analytical technique that measures the mass-to-charge ratio of charged particles.It is used for determining masses of particles, for determining the elemental composition of a sample or molecule, and for elucidating the chemical structures of molecules, such as peptides and...
- Nuclear magnetic resonanceNuclear magnetic resonanceNuclear magnetic resonance is a physical phenomenon in which magnetic nuclei in a magnetic field absorb and re-emit electromagnetic radiation...
The most frequently used techniques are: liquid chromatography coupled with tandem mass spectrometry (LC–MS/MS
Liquid chromatography-mass spectrometry
Liquid chromatography–mass spectrometry is an analytical chemistry technique that combines the physical separation capabilities of liquid chromatography with the mass analysis capabilities of mass spectrometry. LC-MS is a powerful technique used for many applications which has very high...
) for 'small' molecules and enzyme-linked immunosorbent assay (ELISA
ELISA
Enzyme-linked immunosorbent assay , is a popular format of a "wet-lab" type analytic biochemistry assay that uses one sub-type of heterogeneous, solid-phase enzyme immunoassay to detect the presence of a substance in a liquid sample."Wet lab" analytic biochemistry assays involves detection of an...
) for macromolecules
Sample preparation and extraction
The bioanalyst deals with complex biological samples containing the analyte alongside a diverse range of chemicals that can have an adverse impact on the accurate and precise quantification of the analyte. As such, a wide range of techniques are applied to extract the analyte from its matrix. These include:- Protein precipitationProtein precipitationPrecipitation is widely used in downstream processing of biological products, such as proteins. This unit operation serves to concentrate and fractionate the target product from various contaminants. For example, in the biotechnology industry protein precipitation is used to eliminate contaminants...
- Liquid–liquid extraction
- Solid phase extractionSolid phase extractionSolid-phase extraction is a separation process by which compounds that are dissolved or suspended in a liquid mixture are separated from other compounds in the mixture according to their physical and chemical properties. Analytical laboratories use solid phase extraction to concentrate and purify...
Bioanalytical laboratories often deal with large numbers of samples, for example resulting from clinical trials. As such, automated sample preparation methods and liquid-handling robots are commonly employed to increase efficiency and reduce costs.
Bioanalytical organisations
There are several national and international bioanalytical organisations active throughout the world. Often they are part of a bigger organisation, e.g. Bioanalytical Focus Group and Ligand Binding Assay Bioanalytical Focus Group, which are both within the American Association of Pharmaceutical Scientists (AAPS) and FABIAN, a working group of the Analytical Chemistry Section of the Royal Netherlands Chemical Society. The European Bioanalysis ForumEuropean Bioanalysis Forum
The European Bioanalysis Forum is an organisation comprising bioanalytical scientists working within the pharmaceutical industry R&D. Scope of the EBF is on bioanalysis of small and large molecules with bioanalysis being defined as:...
(EBF), on the other hand, is independent of any larger society or association.