BIOANALYSIS
Lecture 1 – Principles of bioanalysis
Bioanalysis is the analysis of pharmaceuticals, their metabolites, biomarkers and
therapeutic proteins in complex biological samples.
Why is it so challenging? There is 60 mg proteins per mL blood. A tumor marker is
0.6 ng per mL blood.
60 mg = 6 · 107 ng proteins
0.6 ng tumor marker
Thus for 1 tumor, there are 1 · 108 (100 million) other proteins!
Qualitative VS quantitative –
These are both analytical procedures, which provide information on the substance.
Often these procedures are combined.
Matrix: biological material in which a substance must be determined.
This includes biofluids.
Blank matrix: biological material without the analyte.
Blank solvent: only contains the used solvent.
The matrix can have a considerable effect on the way the analysis is
conducted, and the quality of the results are obtained. Such effects are
called matrix effects.
Calibration is the general method for determining the concentration of a substance in
an unknown sample by comparing the unknown to a set of standard samples of
known concentration. The calibrator is the blank matrix that contains a known
concentration of the analyte. The calibration line relates the instrumental response
(analytical signal) to the analyte concentration. This is also known as standard
curve.
1
,The dynamic range refers to the range
of concentrations over which an
analytical method can provide accurate
and reliable results.
‘Ratio of the largest up to the smallest
measurable signal that is equal to the
noise – ideally 3-5 orders of magnitude.’
When the concentration of analyte
exceeds the upper limit of the dynamic range, the instrument can become saturated.
In a saturated state, the instrument's response no longer increases proportionally
with increases in analyte concentration. Instead, it levels off, reaching a plateau.
The linear range refers to the portion of the dynamic range where the relationship
between analyte concentration and the instrument's response is linear.
Only fit a line through the linear dynamic range!
• ULOQ: upper limit of quantification - highest concentration of substance that
can be accurately measured by the analytical method.
• LLOQ: lower limit of quantification – lowest concentration of substance that
can be accurately measured by the analytical method.
• LOD: limit of detection - lowest concentration of substance that can be
detected.
The slope of the line defines the
sensitivity, which is the ability to determine
low concentrations of an analyte.
𝐂𝐡𝐚𝐧𝐠𝐞 𝐢𝐧 𝐝𝐞𝐭𝐞𝐜𝐭𝐨𝐫 𝐬𝐢𝐠𝐧𝐚𝐥
Sensitivity = 𝐂𝐡𝐚𝐧𝐠𝐞 𝐢𝐧 𝐚𝐧𝐚𝐥𝐲𝐭𝐞 𝐜𝐨𝐧𝐜𝐞𝐧𝐭𝐫𝐚𝐭𝐢𝐨𝐧
Example: (40; 1.226) & (20; 0.6)
1.226−0.6 0.626
Sensitivity = = = 0.03
40−20 20
Matrix effect –
A matrix effect describes the changes observed in the detection or quantification of
an analyte when other substances are present in the sample.
It can be seen that the graph with water as
matrix and the graph with plasma as matrix
differ. The response is higher for the same
concentration when water is used as matrix.
Because of matrix effects, calibration lines
can only be used for quantification when the
same matrix is used.
2
,Internal standard: substance that resembles the analyte and
corrects for method variability.
In chemical analysis, the internal standard method involves
adding the same amount of a chemical substance to each
sample and calibration solution. The internal standard
responds proportionally to changes in the analyte and
provides a similar, but not identical, measurement signal.
It is useful when the quantity of sample or instrument varies from run-to-run or when
sample loss occurs during sample preparation.
𝐀𝐫𝐞𝐚 𝐨𝐟 𝐚𝐧𝐚𝐥𝐲𝐭𝐞 𝐬𝐢𝐠𝐧𝐚𝐥 𝐀𝐫𝐞𝐚 𝐨𝐟 𝐢𝐧𝐭𝐞𝐫𝐧𝐚𝐥 𝐬𝐭𝐚𝐧𝐝𝐚𝐫𝐝 𝐬𝐢𝐠𝐧𝐚𝐥
= F ( 𝐂𝐨𝐧𝐜𝐞𝐧𝐭𝐫𝐚𝐭𝐢𝐨𝐧 𝐨𝐟 𝐢𝐢𝐧𝐭𝐞𝐫𝐧𝐚𝐥 𝐬𝐭𝐚𝐧𝐝𝐚𝐫𝐝)
𝐂𝐨𝐧𝐜𝐞𝐧𝐭𝐫𝐚𝐭𝐢𝐨𝐧 𝐨𝐟 𝐚𝐧𝐚𝐥𝐲𝐭𝐞
Example:
1. Concentrations of analyte (A) and internal standard (IS)
2. Define detector signal
3. Define F
A: 20 mM
IS: 15 mM
Detector signal A: 1000
Detector signal IS: 800
1000 800 50
=F( ) → 50 = F (53.33) → F = 53.33 → F = 0.94
20 15
Specificity: the ability to generate only an instrument response for one single
analyte in the presence of other components (e.g. matrix, impurities, etc).
Selectivity: the ability to reliably determine several analytes in the presence of
(many) compounds in the matrix but exhibits a degree of preference for the
substance of interest.
Many analytical methods are not truly specific. Due to different proteoforms of a
protein, achieving specificity becomes hard. It's essential to ensure that the method
can specifically detect and quantify the desired proteoform without interference from
other similar molecules. However, this is difficult. Therefore, prefer selectivity over
specificity.
Bioanalysis workflow –
The analyte concentration might be influenced
during the pre-analytical phase. This should be
avoided to obtain a reliable result that reflects
the original concentration.
3
, These errors give high risk for the patient.
Sample preparation method –
• Analyte
• Biofluid
• Analysis
Measuring an endogenous compound (e.g. biomarker):
• Target analyte is already in the human body
• Analyte-free authentic biological matrix is not
available. Resort to a surrogate matrix to generate
calibrators or use the standard addition method.
• 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.
The surrogate matrix is a similar biological fluid that doesn't contain the target
analyte.
Stable-isotope-labeled analogs of the target analyte serve as internal standards.
When added to the sample, they mimic the behavior of the analyte. They are the
most trustworthy compounds. However, synthesizing stable-isotope-labeled analogs
for endogenous compounds can be difficult.
Measuring an exogenous (xenobiotic) compound:
• Target analyte is different from any molecule in the human body (e.g. a
synthetic drug molecule)
• 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
Small VS large analytes –
Paracetamol is a small analyte, whereas
Alemtuzumab is a large analyte.
Small molecules can penetrate through
the cells, where large molecules have to
bind external cellular proteins to pass cell
membranes.
Large analytes pose challenges in characterization due to their complex structures.
Small analytes are relatively easier to characterize.
4
Lecture 1 – Principles of bioanalysis
Bioanalysis is the analysis of pharmaceuticals, their metabolites, biomarkers and
therapeutic proteins in complex biological samples.
Why is it so challenging? There is 60 mg proteins per mL blood. A tumor marker is
0.6 ng per mL blood.
60 mg = 6 · 107 ng proteins
0.6 ng tumor marker
Thus for 1 tumor, there are 1 · 108 (100 million) other proteins!
Qualitative VS quantitative –
These are both analytical procedures, which provide information on the substance.
Often these procedures are combined.
Matrix: biological material in which a substance must be determined.
This includes biofluids.
Blank matrix: biological material without the analyte.
Blank solvent: only contains the used solvent.
The matrix can have a considerable effect on the way the analysis is
conducted, and the quality of the results are obtained. Such effects are
called matrix effects.
Calibration is the general method for determining the concentration of a substance in
an unknown sample by comparing the unknown to a set of standard samples of
known concentration. The calibrator is the blank matrix that contains a known
concentration of the analyte. The calibration line relates the instrumental response
(analytical signal) to the analyte concentration. This is also known as standard
curve.
1
,The dynamic range refers to the range
of concentrations over which an
analytical method can provide accurate
and reliable results.
‘Ratio of the largest up to the smallest
measurable signal that is equal to the
noise – ideally 3-5 orders of magnitude.’
When the concentration of analyte
exceeds the upper limit of the dynamic range, the instrument can become saturated.
In a saturated state, the instrument's response no longer increases proportionally
with increases in analyte concentration. Instead, it levels off, reaching a plateau.
The linear range refers to the portion of the dynamic range where the relationship
between analyte concentration and the instrument's response is linear.
Only fit a line through the linear dynamic range!
• ULOQ: upper limit of quantification - highest concentration of substance that
can be accurately measured by the analytical method.
• LLOQ: lower limit of quantification – lowest concentration of substance that
can be accurately measured by the analytical method.
• LOD: limit of detection - lowest concentration of substance that can be
detected.
The slope of the line defines the
sensitivity, which is the ability to determine
low concentrations of an analyte.
𝐂𝐡𝐚𝐧𝐠𝐞 𝐢𝐧 𝐝𝐞𝐭𝐞𝐜𝐭𝐨𝐫 𝐬𝐢𝐠𝐧𝐚𝐥
Sensitivity = 𝐂𝐡𝐚𝐧𝐠𝐞 𝐢𝐧 𝐚𝐧𝐚𝐥𝐲𝐭𝐞 𝐜𝐨𝐧𝐜𝐞𝐧𝐭𝐫𝐚𝐭𝐢𝐨𝐧
Example: (40; 1.226) & (20; 0.6)
1.226−0.6 0.626
Sensitivity = = = 0.03
40−20 20
Matrix effect –
A matrix effect describes the changes observed in the detection or quantification of
an analyte when other substances are present in the sample.
It can be seen that the graph with water as
matrix and the graph with plasma as matrix
differ. The response is higher for the same
concentration when water is used as matrix.
Because of matrix effects, calibration lines
can only be used for quantification when the
same matrix is used.
2
,Internal standard: substance that resembles the analyte and
corrects for method variability.
In chemical analysis, the internal standard method involves
adding the same amount of a chemical substance to each
sample and calibration solution. The internal standard
responds proportionally to changes in the analyte and
provides a similar, but not identical, measurement signal.
It is useful when the quantity of sample or instrument varies from run-to-run or when
sample loss occurs during sample preparation.
𝐀𝐫𝐞𝐚 𝐨𝐟 𝐚𝐧𝐚𝐥𝐲𝐭𝐞 𝐬𝐢𝐠𝐧𝐚𝐥 𝐀𝐫𝐞𝐚 𝐨𝐟 𝐢𝐧𝐭𝐞𝐫𝐧𝐚𝐥 𝐬𝐭𝐚𝐧𝐝𝐚𝐫𝐝 𝐬𝐢𝐠𝐧𝐚𝐥
= F ( 𝐂𝐨𝐧𝐜𝐞𝐧𝐭𝐫𝐚𝐭𝐢𝐨𝐧 𝐨𝐟 𝐢𝐢𝐧𝐭𝐞𝐫𝐧𝐚𝐥 𝐬𝐭𝐚𝐧𝐝𝐚𝐫𝐝)
𝐂𝐨𝐧𝐜𝐞𝐧𝐭𝐫𝐚𝐭𝐢𝐨𝐧 𝐨𝐟 𝐚𝐧𝐚𝐥𝐲𝐭𝐞
Example:
1. Concentrations of analyte (A) and internal standard (IS)
2. Define detector signal
3. Define F
A: 20 mM
IS: 15 mM
Detector signal A: 1000
Detector signal IS: 800
1000 800 50
=F( ) → 50 = F (53.33) → F = 53.33 → F = 0.94
20 15
Specificity: the ability to generate only an instrument response for one single
analyte in the presence of other components (e.g. matrix, impurities, etc).
Selectivity: the ability to reliably determine several analytes in the presence of
(many) compounds in the matrix but exhibits a degree of preference for the
substance of interest.
Many analytical methods are not truly specific. Due to different proteoforms of a
protein, achieving specificity becomes hard. It's essential to ensure that the method
can specifically detect and quantify the desired proteoform without interference from
other similar molecules. However, this is difficult. Therefore, prefer selectivity over
specificity.
Bioanalysis workflow –
The analyte concentration might be influenced
during the pre-analytical phase. This should be
avoided to obtain a reliable result that reflects
the original concentration.
3
, These errors give high risk for the patient.
Sample preparation method –
• Analyte
• Biofluid
• Analysis
Measuring an endogenous compound (e.g. biomarker):
• Target analyte is already in the human body
• Analyte-free authentic biological matrix is not
available. Resort to a surrogate matrix to generate
calibrators or use the standard addition method.
• 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.
The surrogate matrix is a similar biological fluid that doesn't contain the target
analyte.
Stable-isotope-labeled analogs of the target analyte serve as internal standards.
When added to the sample, they mimic the behavior of the analyte. They are the
most trustworthy compounds. However, synthesizing stable-isotope-labeled analogs
for endogenous compounds can be difficult.
Measuring an exogenous (xenobiotic) compound:
• Target analyte is different from any molecule in the human body (e.g. a
synthetic drug molecule)
• 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
Small VS large analytes –
Paracetamol is a small analyte, whereas
Alemtuzumab is a large analyte.
Small molecules can penetrate through
the cells, where large molecules have to
bind external cellular proteins to pass cell
membranes.
Large analytes pose challenges in characterization due to their complex structures.
Small analytes are relatively easier to characterize.
4
