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Summary of all courses - Bioanalysis (WBFA032-05)

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Summary of all Bioanalysis courses given at the University of Groningen

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  • April 22, 2024
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  • 2022/2023
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Chapter 1: Principles of bioanalysis
Klont

Bioanalysis: the analysis of pharmaceuticals and their metabolites as well as biomarkers and
therapeutic proteins at low concentrations in complex biological samples. Use:
- Advanced analytical techniques with high sensitivity and selectivity.
- Profound knowledge of the psycho-chemical properties of the analyte to optimize the
methodology.
- Good understanding about possible interfering matrix components.

Workflow: sample preparation  separation  detection  data analysis.

Applications: doping, environmental and occupational safety analysis, clinical and forensic
toxicology, drug metabolism and PK, laboratory medicine, TDM, analysis of therapeutic proteins.

The human plasma proteome (all proteins) is very big  makes bioanalysis hard because proteins
can be in very low concentration and there can be millions of different proteins (proteoforms) in a
sample, which can be in much higher concentrations.

Definitions
- Qualitative bioanalysis: substance identification.
- Quantitative bioanalysis: substance concentration or amount.
- Matrix: biological material in which a substance must be determined.
- Blank matrix: biological material without the analyte.
- Calibrator: blank matrix containing a known concentration of the analyte.
- Calibration line: relates the measured signal to the analyte concentration.
- Internal standard: substance that resembles the analyte and corrects for method variability.

Sample preparation
- Concentration adjustment (enrichment or dilution, mostly enrichment).
- Removal of interfering compounds.
- Stabilization of analytes.
- Adjustment of analyte properties.

Separation
(Mostly done by chromatography)
- Separate molecules that give the same or a similar response.
- Increase the depth of analysis.
- Improve sensitivity / selectivity.
- Make sample compatible with detection systems.

Data analysis & biostatistics  identification and quantification. Method validation with
biostatistics. In the case of biomarkers there is discrimination between cases and controls  used for
prognosis and diagnosis. The highest concentration of controls is the same as the lowest
concentration of patients.

Selecting a bioanalytical method
- Goal of analysis (quantitative of qualitative).
- Physico-chemical properties of analyte.
- Biological matrix.
- Concentration range.
- Required throughput.
1

, - Admissible costs.
- Available instrumentation and trained personnel.

Examples of biological matrices  blood, urine, breath, meconium, cerebrospinal fluid,
bronchoalveolar lavage, hair, tissue.

Measuring an exogenous (xenobiotic) compound
- Target analyte different from any molecule in the human body.
- Can be added to analyte-free authentic biological matrix to generate calibrators.
- Can be synthesized in stable-isotope-labelled form and used as internal standard dor mass
spectrometry.
- Reference material is generally stable

Measuring an endogenous compound (e.g. biomarker)
- Target analyte is already in the human body.
- Analyte-free authentic biological matrix is not available.
- It may be 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.

Exogenous compound  easier.

EDTA is an anti-coagulation  addition to blood before centrifugation prevents the formation of
plasma after centrifuging.




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, Chapter 2: Sample preparation
Klont

Liquid-Liquid Extraction (LLE)

Distributes molecules between and aqueous phase and an organic phase. Depending on the density
the phases are separated on the top and on the bottom. Goal  get as much of the molecule in the
org phase, and as many of the interfering substances in the aq phase.
Org phase: hydrophobic. Aq phase: hydrophilic.
- Advantages  easily performed (few steps and mechanis is simple).
- Disadvantages  limited choice of selectivity, large volumes of org solvent, potential loss
during solvent evaporation, emulsions, analyte adsorption and difficult to automate.

Distribution coefficient: P = Corg / Caq (high P is more in the org phase)
Phase ratio: V = Vorg / Vaq
Fractions: forg + faq = 1

Optimization of extraction yield
- Choice of organic solvent  should not be able to mix with water.
- Adjust the pH of the aqueous phase  to make compounds more hydrophobic.
- Salting out (add salt to the aqueous phase)  to make aq phase more hydrophilic.
- Ion-pair extraction  neutralize permanent charged molecules by forming ion pairs that are
hydrophobic, go to org phase.

Influence of the pH
In




case of acid  low pH (more H+)  neutral molecule = high forg.
In case of base  high pH (less H+)  neutral molecule = high forg.

pH of acid needs to be below the pKa of compound  neutral charge.
pH of base needs to be above the pKa of compound  neutral charge.
(Buffer of a pH 2 units above (basic) or below (acidic) pKa is added to the solution.)

pKa  pH at which the molecule is 50% charged and 50% neutral.

Extraction of a basic drug molecule with pKa = 11 and P = 100.
P = 100 = 102  Log(P) = 2.
11 – 2 = 9  at pH 9, 50% is in the org and 50% is in the aq.




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