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.
2
, 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.
3
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.
2
, 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.
3
