Lecture 1: Introduction and Applications

Question 1

What is meant by the term ‘diagnostics’?

Answer 1

Measuring things that are endogenous within us (e.g. proteins, metabolites, cell structures, etc.) and looking for changes.

Question 2

What is meant by the term ‘therapeutics’?

Answer 2

Supplement/add/change the system for the benefit of somebody’s health e.g. adding a protein, or supplementing a hormone.

Question 3

What are precision nanomedicines?

Answer 3

The application of nanotechnology to medicines and healthcare.

Question 4

What is nanotechnology?

Answer 4

Nanotechnology is the science, engineering, and technology conducted at the nanoscale (1-100 nanometers).

Question 5

Describe the chemistry a therapy may have in terms of its structure and why

Answer 5

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).

Question 6

What is the aim of precision medicines to move medicine forward?

Answer 6

Precision medicines now are about improving the specificity and durability of therapy (to give less and reduce side effects without reducing the positive response).

Question 7

Describe micelles and liposomes as nanomedicine types

Answer 7

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.

Question 8

Generally, how can nanomedicines aid therapeutics?

Answer 8

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.

Question 9

What is a problem with the use of nanomedicines in therapeutics?

Answer 9

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.

Question 10

What is the reality of developing measurement methods?

Answer 10

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).

Question 11

What is the consequence of the target/biomarker present in samples being in trace amounts?

Answer 11

The target analyte may be ‘masked’ by other components & little detected (poor sensitivity).

Question 12

What is the solution for the target/biomarker present in samples being in trace amounts?

Answer 12

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)

Question 13

What should you consider when developing measurement methods?

Answer 13

Analyte (and interference) molecular size

Analyte (and interference) solubility

Analyte (and interference) chemical functionalities

Question 14

Explain why you should consider the analyte (and interference) molecular size when developing measurement methods

Answer 14

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)

Question 15

Explain what you should consider about the analyte (and interference) solubility when developing measurement methods

Answer 15

Polar – soluble in water

Lipophilic – soluble in organic solvents

Question 16

Explain what you should consider about the analyte (and interference) chemical functionalities when measurement methods

Answer 16

i.e. weakly basic or acidic and pH control

Question 17

What are the 2 interactions used in partition and adsorption?

Answer 17

Polarity and Ion exchange

Question 18

What does partition mean?

Answer 18

Dissolving

Question 19

What does adsorption mean?

Answer 19

Sticking stuff to a surface

Question 20

Explain polarity interactions used in partition and adsorption

Answer 20

Like dissolves with like.
Like goes with like.

Question 21

Explain ion exchange interactions used in partition and adsorption

Answer 21

Opposites attract

Example: pKa as a measure of dissociation

Question 22

What would a qualitative experiment consist of doing?

Answer 22

Using structural information of analyte to develop a method and identify the target.

Question 23

What would a quantitative experiment consist of doing?

Answer 23

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

Question 24

What type of questions would you expect a qualitative experiment to answer?

Answer 24

What is present and what does it look like?

Question 25

What type of questions would you expect a quantitative experiment to answer?

Answer 25

How much is present?

Question 26

What are the critical stages to consider for quantification?

Answer 26

1. Sample collection 2. Quantification method needs calibration and quality control (QC) samples 3. Sample preparation and extraction 4. Analytic method 5. Data processing and reporting

Question 27

Explain the sample collection stage of quantification

Answer 27

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?

Question 28

Explain the calibration and QC sampling stage of quantification

Answer 28

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.

Question 29

Explain the sample preparation and extraction stage of quantification

Answer 29

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…

Question 30

Explain the analytical method stage of quantification

Answer 30

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?

Question 31

Explain the data processing and reporting stage of quantification

Answer 31

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.