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Bioanalysis lecturesChapter 1 & 2
Bioanalyis is the analysis of pharmaceuticals and their metabolites as well as biomarkers and
therapeutic proteins at low concentrations in complex biological samples. To do so, it is necessary to
use advance analytical techniques with high sensitivity and selectivity and to optimize the
methodology based on a profound knowledge of the physico-chemical properties of the analyte as
well as a good understanding about possibly interfering matrix components.
The workflow of bioanalysis is the sample preparation -> separation of the sample -> detection of the
sample based on the properties of the molecule -> data analysis from the sample in terms of
reliability and interpretation of the results.
Applications for bioanalysis are
- Doping
- Environmental and occupational safety analyses
- Clinical and forensic toxicology
- Drug metabolism and pharmacokinetics
- Laboratory medicine
- Therapeutic drug monitoring
- Analysis of therapeutic proteins
Bioanalysis is challenging because there are millions of different proteins with many forms. Among
these forms there is difference in frequency, therefore there are many different proteins in the
sample within your sample. Thus it is like finding a needle in the hay.
Definitions:
Qualitative bioanalysis is substance identification.
Quantitative bioanalysis is substance concentration or amount determination.
Matrix is the biological material in which a substance must be determined, so the sample.
Blank matrix is the biological material without the analyte.
Calibrator is a blank matrix containing a known concentration of the analyte.
Calibration line relates the measured signal to the analyte concentration.
Internal standard is the substance that resembles the analyte and corrects for method variability.
Sample preparation is the link between the sample and the analytical procedure. Sample preparation
serves multiple purposes, concentration adjustment so enrich it or dilute it. Also, removal of
interfering compounds, stabilization of analytes and adjustment of analyte properties.
Separation will separate molecules that give the same or a similar response, this will increase the
depth of the analysis, improve sensitivity and selectivity. This will allow the sample to be compatible
with the detection systems.
Data analysis and biostatistics are the identification and quantification. This is done to ensure the
compound is what you expect it to be and the concentration is quantified and the precision is
determined. In case of biomarkers you need discrimination of cases from controls.
To select a bioanalytical method you should have a goal of the analysis. The psycho-chemical
properties of the analyte should be known. The biological matrix should be looked at, the
concentration range should be determined to obtain a certain sensitivity. It is important to look at
,the required throughput, so how many analyses should be performed. Admissible costs should be
considered and look at the available instrumentation and trained personnel.
To measure an exogenous compound is easier than an endogenous compound. This is easy because
the target analyte is different from any molecule in the human body. It can be added to analyte-free
authentic biological matrix to generate calibrators. It can likely be synthesized in stable-isotope-
labelled form and used as internal standard for mass spectrometry. Reference material is generally
available.
To measure an endogenous compound the target analyte is already in the human body. An analyte-
free matrix is not available. It is difficult to obtain a stable-isotope-labelled analogue to serve as
internal standard. Reference material is often not available or is not identical to the endogenous
analyte, which may in itself be heterogenous.
Sample preparation
Liquid-liquid extraction is the distribution of molecules in an aqueous phase and an organic phase. It
is characterised by the distribution coefficient which is P = Corg/Caq. A high concentration in the
organic phase is wanted and a low concentration in the
aqueous phase is wanted. The phase ratio can be
adjusted by yourself, this is the volume used for the
organic and aqueous phase. This can be used to favour
the organic phase. The fraction in the organic phase
and the aqueous phase is always one. To optimize the
extraction yield the choice of organic solvent is of
importance, adjust the pH (lower for acids, higher for
bases) of the aqueous phase, salting out and ion-pair
extraction (introducing a ion-pair and thus make a pair
n
that is in the organic phase). f org=1−( f aq )
Advantages of liquid-liquid extraction is that it is easily performed and that the mechanism is easily
understandable. The disadvantages are however that there is limited choice of selectivity, large
volumes of organic solvents, potential loss during solvent evaporation, emulsions, analyte adsorption
and difficult to automate.
Solid phase extraction (SPE) is reminiscent of chromatography. There is a cartridge with all beads, the
sample is added and the matrix is rinsed out of the cartridge and then the analyte is eluted.
Reversed-phase SPE there are a lot of phases available. These materials have to be conditioned, this
means that the molecules on the side chains have to be available for binding, this is called the
conditioning step. Normal-phase SPE is when the stationary phase is hydrophilic and the mobile
phase is hydrophobic. Ion-exchange SPE, weak ion-exchangers are only charged in a certain pH,
strong ion-exchangers are always charged, no matter the pH. Hydrophobic interactions mostly
replace water. Van Der Waals interaction form attraction between molecules. Electrostatic
interaction is due to a charge on a molecule.
Chapter 3
Ligand binding assays are based on equilibrium binding. The ligand binding assay is easy to perform,
in each wells on a microtiter plate an ligand binding assay can be assessed. To detect whether there
is a virus infection in the blood you detect virus derived proteins. The virus sample will bind on the
surface and the unbound material is washed away. Then a protein-rich solution is added to block any
plastic surface. In the next step you add an antibody which contains an enzyme. This antibody will
, bind to the virus sample if it is present. Then the excess is washed away and a substrate is added. The
enzyme will turn over the substrate and there is a signal that can be detected.
A ligand binding
assay is a saturation
binding assay. The
amount of complex
that is formed is
maximalised by one
of the two products.
A Scatchard analysis
has an equilibrium.
This Scatchard plot
will have the intersection with the X axis as the B max.
Immunoassays are the most widely used ligand binding assays. Immunoassays require affinity
binders. Drug-protein conjugates must be prepared to render low-molecular-weight pharmaceuticals
immunogenic. Antibodies recognize only a small part of the antigen. An antibody is a quite big
molecule consisting of a light and a heavy chain.
Monoclonal antibodies are made by immunisation of a mouse, then the B cells are isolated from the
spleen. Then cultivation of myeloma cells and the fusion of myeloma and B cells happens to ensure
that the B cells survive. Separation of cell lines happens to make them monoclonal and grow these
and then screen them for suitability. In vitro or in vivo multiplication and then harvesting of the
antibody.
Receptor assays are ligand binding assays that measure pharmacologically active molecules based on
receptor binding. Production of receptors by recombinant DNA technology. Due to difficulty of
purifying active receptors, they are often used in semi-purified form.
The two large classes of receptors are the soluble receptors and membrane bound receptors. The
soluble receptors are considered to be the easy receptors because they bind small ligands and are
intracellular. The membrane bound receptors have a integrate structure, they transfer a signal across
the membrane.
Detection principles are radioactivity, colorimetric, enzyme multiplied immunoassay technique,
ChemoLuminescence, fluorescence polarization and fluorescence resonance energy transfer.
For radioactivity you need radioisotopes, these radioisotopes can be detected by liquid scintillation
counting, liquid chromatography with radioactivity detection or by autoradiography. Quantification is
1 Bq = 1 disintegration/sec.
For colorimetric detection is used. The technique used is ELISA which uses
antibodies that can bind to the proteins. To these proteins a second antibody can
be added to the second epitope. The specificity of this method is increased a lot
because of the antibody binding to a specific substrate. Then the unbound
antibodies are washed away and another antibody is bound to the antibody on
top that will convert a molecule into another molecule that for example emits
fluorescence.
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