Lecture 1: Introduction and Applications Flashcards
What is meant by the term ‘diagnostics’?
Measuring things that are endogenous within us (e.g. proteins, metabolites, cell structures, etc.) and looking for changes.
What is meant by the term ‘therapeutics’?
Supplement/add/change the system for the benefit of somebody’s health e.g. adding a protein, or supplementing a hormone.
What are precision nanomedicines?
The application of nanotechnology to medicines and healthcare.
What is nanotechnology?
Nanotechnology is the science, engineering, and technology conducted at the nanoscale (1-100 nanometers).
Describe the chemistry a therapy may have in terms of its structure and why
Therapies would normally have a lipophilic section to allow transfer into the cell and a polar (lone pair of electrons) section that allows receptor recognition (because you must trigger a response when giving a therapy).
What is the aim of precision medicines to move medicine forward?
Precision medicines now are about improving the specificity and durability of therapy (to give less and reduce side effects without reducing the positive response).
Describe micelles and liposomes as nanomedicine types
Micelles and liposomes are naturally abundant within us (how we package up any garbage to be removed) and they allow us to partition the therapeutic within the core or membrane of the particle to deliver it to the source.
Generally, how can nanomedicines aid therapeutics?
Can attach the therapeutic to various cells to be able to monitor and image the therapy to see where it is going. Also, to facilitate the transfer of the therapeutic.
What is a problem with the use of nanomedicines in therapeutics?
You can’t measure the therapeutic particle when it is inside a liposome, which is bad considering the high amount of therapeutics relying on liposomes. So, the analytical protocol (how you measure the analyte) has to be able to separate the target analyte and has to go through an additional step to release the drug from the particle itself.
What is the reality of developing measurement methods?
Target/biomarker present in samples (serum, blood, urine, etc) at trace levels with other more abundant species (salts, albumin, etc). (The target analyte is in a much smaller quantity than everything else in the sample).
What is the consequence of the target/biomarker present in samples being in trace amounts?
The target analyte may be ‘masked’ by other components & little detected (poor sensitivity).
What is the solution for the target/biomarker present in samples being in trace amounts?
Separate sample so we can detect (& quantify with confidence) individual
components, using preparation (extraction/purification/separation) techniques.
(Samples are mainly aqueous-based so this does help define what you do)
What should you consider when developing measurement methods?
Analyte (and interference) molecular size
Analyte (and interference) solubility
Analyte (and interference) chemical functionalities
Explain why you should consider the analyte (and interference) molecular size when developing measurement methods
The chemistry of the analyte and the rest of the sample (the more selective we are about pulling out our target analyte, the cleaner the data)
Explain what you should consider about the analyte (and interference) solubility when developing measurement methods
Polar – soluble in water
Lipophilic – soluble in organic solvents
Explain what you should consider about the analyte (and interference) chemical functionalities when measurement methods
i.e. weakly basic or acidic and pH control
What are the 2 interactions used in partition and adsorption?
Polarity and Ion exchange
What does partition mean?
Dissolving
What does adsorption mean?
Sticking stuff to a surface
Explain polarity interactions used in partition and adsorption
Like dissolves with like.
Like goes with like.
Explain ion exchange interactions used in partition and adsorption
Opposites attract
Example: pKa as a measure of dissociation
What would a qualitative experiment consist of doing?
Using structural information of analyte to develop a method and identify the target.
What would a quantitative experiment consist of doing?
Create a calibration standard line (should have R2>0.99) over a concentration
range.
QCs to test the quantitative performance of the calibration line.
For MS methods, add an internal standard (e.g. d2, 2/1 H ) to standards & measured sample to normalize against variations in instrument performance
What type of questions would you expect a qualitative experiment to answer?
What is present and what does it look like?
What type of questions would you expect a quantitative experiment to answer?
How much is present?
What are the critical stages to consider for quantification?
- Sample collection
- Quantification method needs calibration and quality control (QC) samples
- Sample preparation and extraction
- Analytic method
- Data processing and reporting
Explain the sample collection stage of quantification
You need to have a sample that is representative and homogenous for analysis.
You should consider sample containers and:
- Protection from UV light?
- Temperature?
- Leaching of sodium/plastics/lubricants from non-stick surfaces/adhesives?
- Storage time?
Explain the calibration and QC sampling stage of quantification
Ideally ‘matrix-matched’ to replicate interferences observed with unknown samples.
QC samples to test and characterise method – i.e. accuracy, precision, recovery (e.g. sample preparation stage), matric effects, stability….
Acceptance of data usually depends on QC samples being successfully quantified within predefined limits.
Map a response/concentration graph to check quantification of a drug (work out the best concentration for a drug) – gives a detector response.
Explain the sample preparation and extraction stage of quantification
Need to isolate target analyte from interference and concentre if at trace levels
Broad range of sample preparation techniques – more specific for the target analyte the better for quantitation!
E.g. pK, Kow…
Explain the analytical method stage of quantification
Needs to be fit-for-purpose… elective, sensitive… VAM!
Method development – points to ask/consider:
- Is the approach/equipment capable of what it to do?
- Do you have sufficient measurement confidence – separation and selectivity? Is it reliable?
- Have you tuned the instrument to best detect the target analyte?
- Are you operating under the best conditions for sensitive quantitation?
- Do you have sufficient data points for a quantitative measurement?
Explain the data processing and reporting stage of quantification
Be clear, transparent and consistent with all data processing! (Another analyst should be able to repeat what you have done.)
Typically use automated data processing rather than manual approaches for
consistency – still needs sense check by analyst!
Data should be inspected to ensure quality.
Predefined acceptance criteria should be used for data processing & determining key figures of merit/performance metrics such as
repeatability, calibration linearity…
As quantitation is typically carried out in regulated laboratories (e.g. Good Laboratory Practice (GLP)) data processing & reporting typically has audit & sample tracking functionalities recording what you have done & when.
