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Summary Bioanalysis
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BioanalysisChapter 1 introduc4on
Bioanalysis = the analysis of pharmaceu4cals and their metabolites as well as biomarkers and
therapeu4c proteins at low concentra4ons in complex biological samples. Since the
concentra4on of the complex biological samples is very large, there are advanced analy4cal
techniques necessary. Therefore, there is needed to use advanced analy4cal techniques with
high sensi4vity and selec4vity to op4mize the methodology based on a profound knowledge
of the physico-chemical proper4es of the analyte, as well as a good understanding about
possibly interfering matrix components (albumin).
Applica4ons:
- Doping, relies on high performance analy4cal methods to detect drugs/substances of
abuse that may have performance enhancing effects
- Clinical and forensic toxicology, to detect molecules, measure biomarkers and relate
them to toxic effects or to the development of a disease
- New pharmaceu4cals: pharmacokine4cs and metabolism
- Biomarker discovery and analysis, they are molecules used and analyzed in circula4on
to diagnose or prognos4cate a disease or to look at the effect of therapeu4cs
- Laboratory medicine (diagnosis/prognosis)
- Therapeu4c Drug Monitoring (TDM), related to drugs that have a narrow therapeu4c
window because these need to be measured accurately and precisely preven4ng an
overdose or an under-dose
- Analysis of therapeu4c proteins
- Environmental and occupa4onal safety analyses
Blood is highly complex including a lot of proteins with different dynamic concentra4on
ranges. So, when you want do detect a certain protein you have to make sure that the
concentra4on of the sample prepara4on is in the right range.
Why is it so challenging?
⇨ Let’s assume that we need to measure a tumor biomarker in blood to follow to
efficacy of a given therapy to assess the risk to develop cancer: blood contains
millions of different proteins (also called “proteoforms”), some of which are present
at much higher concentra4on than a puta4ve cancer biomarker and can also
originate from different organs.
⇨ Let’s assume that the cancer biomarker circulates at 0.6 ng/mL (realis4c): blood
(serum or plasma) has an overall protein concentra4on of about 60 mg/mL. that
means that each ‘biomarker molecule’ there will be 108 molecules of other proteins
in blood (assuming the molecular weight).
Defini4ons
Qualita4ve bioanalysis = substance iden4fica4on, to really be sure that the correct molecule
is measured.
1
,Quan4ta4ve bioanalysis = substance concentra4on or amount of a given substance in a
complex biological material.
Analyte = the substance that is being measured. It can either be foreign to the organism, in
which case it is called exogenous or xenobio4c analyte, or occur naturally and be called an
endogenous or bio4c analyte. Most tradi4onal drugs are xenobio4cs, while modern
biopharmaceu4cals may resemble endogenous molecules. Biomarkers are always
endogenous.
Matrix = biological material in which the analyte must be determined, also provides
interac4on
- Blood: serum is obtained a\er coagula4on of blood a\er which the cellular frac4on
and the blood clot are removed by centrifuga4on or filtra4on. If blood is collected in
a tube containing an an4-coagulant (such as heparin or EDTA) and subsequently
centrifuged, plasma is obtained. Plasma contains a lot of proteins. The composi4on of
plasma have to be taken into account because they may affect the analy4cal method.
- Urine, easily obtainable but very variable in its biological composi4on and needs to
be collected very carefully in a sense that it is not contaminated and is not too much
affected.
- Cerebrospinal fluid, collected in the case of neurological disorders. It is a quite
invasive procedure and can not be done on the rou4ne basis
- Bronchoalveolar lavage, collected from the lung during bronchoscopy which is also a
very invasive procedure requiring anesthesia and not useful for rou4ne diagnos4cs.
- Hair and 4ssue (biopsy)
Blank matrix = an aliquot of the matrix, which does not contain the analyte of interest. For
the determina4on of xenobio4cs, blank matrix can be obtained from individuals that have
not been dosed. For endogenous analytes, blank matrix typically does not exist. Or the
molecules can be so widely distributed and used that this is not easy to find. As a subs4tute,
synthe4c solu4ons, matrix from which the analyte has been removed or matrix from another
species may be used as the blank. It is important to have a blank matrix to develop a
quan4ta4ve analy4cal method.
Calibrator = an aliquot of blank matrix to which a known concentra4on of the analyte has
been added. Does my analy4cal method accurately determine this concentra4on? Need:
Calibra4on line/plot/curve = a plot of the instrumental response as a func4on of the
concentra4on for a set of calibrators, to which the response of unknown samples is
compared to establish their concentra4ons.
Lower limit of quan4ta4on (LLOQ) = the lowest concentra4on in a calibra4on curve
Upper limit of quan4fica4on (ULOQ) = the highest concentra4on in a calibra4on curve
Internal standard = a substance with proper4es very similar to that of the analyte, but which
gives an instrumental response that can be dis4nguished from that of the analyte. It is
typically added to the sample and its response is used to correct for the experimental
variability of the bioanaly4cal procedure.
Selec4vity = the ability of a method to reliably determine an analyte in the presence of
(many) other compounds in the matrix
Specificity = the ability of a method to generate an instrument response only for the analyte
of interest
Sensi4vity = the ability of a method to determine low concentra4ons of an analyte
2
,Small molecules are compounds with a low molecular mass that can usually be generated by
chemical synthesis. They can be taken orally because they survive condi4ons in the GI and
rapidly diffuse. The majority (90%) of drugs belongs to the class of small molecules. Large
molecules (biologics/biopharmaceu4cals) with a higher molecular mass (10.000 Da to
150.000 Da). They are produced by biotechnological processed in micro-organisms or in
gene4cally engineered mammalian cell lines. They are typically dosed by injec4on or infusion
because they would be digested in the GI. There are also drugs with intermediate size
1.000-10.000 Da.
Sample prepara4on
- Concentra4on adjustment to fit the sensi4vity of the analy4cal equipment
o Enrichment: interested in low abundance, low concentra4on molecules in
complex biological samples. It invokes also the removal of interfering
compounds as well as capture of the molecule of interest
o Dilu4on: to measure high abundance compounds that may saturate the
equipment or detector
- Removal of interfering compounds
- Stabiliza4on of analytes, use the right temperature and pH
- Adjustment of analyte proper4es, modify the analyte
Separa4on: separate the molecules from each other that give the same or a similar response
(R/S-thalidomide, isomers with different effects), increase the depth of the analysis (e.g., in
biomarker discovery and in clinical chemistry), improve the sensi4vity (e.g., fluorescence),
improve the selec4vity (e.g., ion-pair chromatography) and make the sample compa4ble
with detec4on systems (e.g., mass spectrometry).
Detec4on: good detec4on methods must be sensi4ve and specific for the molecule in
ques4on. This is achieved by proximity assays = a signal is only generated when two
molecules are close together (ligand-binding assay).
Data analysis and biosta4s4cs: iden4fica4on and quan4fica4on of substances. Any analy4cal
method result in raw data which need to be analyzed and o\en compared to sta4s4cal
methods. In the case of biomarkers, it includes the discrimina4on of the cases from the
controls and the diagnosis/prognosis.
Guide for Selec4ng a Bioanaly4cal Method
1. Goal of the analysis (e.g., qualita4ve or quan4ta4ve)
2. Physico-chemical proper4es of the analyte, what is the structure?
3. Biological matrix, highly complex or diluted?
4. Concentra4on range (required sensi4vity), what is the expected concentra4on of the
molecule to be biologically ac4ve or relevant?
5. Required throughput, is it measured in one or more samples and in what 4me?
6. Admissible cost, how much may it cost?
7. Available instrumenta4on and trained personnel
3
, Measuring an exogenous (xenobio4c) compound: the target analyte is different from any
molecule in the human body (e.g., a synthe4c drug molecule). It can be added to analyte-
free authen4c biological matrix to generate calibrators and it can likely be synthesized in
stable-isotope-labelled form and used as internal standard for mass spectrometry. Reference
material is generally available.
Measuring an endogenous compound (e.g., a biomarker, much more challenging): the target
analyte is already in the human body (e.g., a protein biomarker). Analyte-free authen4c
biological matrix is not available (resort to a surrogate matrix to generate calibrators or use
the standard addi4on method, which is tedious). It may be difficult to obtain a stable-
isotope-labelled analogue to serve as internal standard. Reference material is o\en not
available or is not iden4cal to the endogenous analyte, which may in itself be heterogenous.
Summary
- Bioanalysis seeks to detect and to quan4fy analytes in complex biological samples
such as blood plasma.
- Analytes may be foreign to the organism (e.g., drug molecules) or endogenous
compounds (e.g., protein biomarkers).
- Bioanaly4cal methods build on a sequence of ‘unit opera4ons’ from sample
prepara4on to data analysis.
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