Applications of Chromatography Flashcards
what is an analyte?
Something we measure e.g drug, hormone, drug, electrolyte
Do immunoassays and other assays need to be sensitive?
yes
what are common assays?
Immunoassay, spectrophotometry, mass spectrometry
why would you use mass spectometry? - advantages
Used instead of immunoassay when selectivity of the antibody is not good.
It is selective, specific and sensitive
Disadvantages of mass spec?
slow
Expensive
Requires more training
Used in specilised and less accessible
Why would you use mass spec?
Identify a substance, modification, a metabolite
Quantify’s concentration and the rate of formation
What is precision?
How repeatable is this
What is accuracy?
How close to the correct answer is this
How would you compare assays?
Bland altman plot
Bland Altman Plot - how do you do this?
You plot the average of method 1 and 2 along the x-axis and the y-axis is the difference between the two assays - you want it to be 0 (you compare something you know gives you the right answer to a new thing)
in a bland altman plot what is bias and do you get this at low concentrations?
anything above 0 and yes
How is testosterone mesured?
Mass spec
Will you have to change the measurements of the assays dependant on gender, age and disease?
yes
what was the issues in a immunoassay of testosterone
other androgens where interfereing
what is the process of mass spec?
1) Separate out compound of interest from matrix
2) Vaporise the compound of interest to produce ions of interest
3) Separate ions based on their mass to charge ratio, m/z, where m = mass, z = charge
what is the purpose of seperation?
isolate it from other similar compounds
what are the main ways to seperate in mass spec?
1) Gas Chromatography-MS
2) Liquid Chromatography-MS
separation technique - chromatography
Chromatography is a physical method of separation in which the components to be separatedare distributed between two phases, one of which is stationary while the other moves in adefinite direction (mobile phase).
By what grounds are compounds seperated on chromatography?
molecular characteristics -size, volatility, ionisation, solubility
Gas chromatography Mass spec does splits things on what?
boiling point
How does gas chromatography mass spec work?
Inject substances into an injection port and as they boil they will flow through a column before going through a detector and a gas chromatogram - peaks show how much molecules are there
What does liquid chromatography mass spec separate on?
Achieved according to the partition coefficient of an analyte between solvents.
How do you calculate the partition coefficient?
Concentration of analyte in oily phase/concentration of analyte in water phase
How do you do liquid chromatography mass spec?
Put substance on a column and pour liquid through, this will pull the substances through in a certain order and they are then put through a detector and this creates peaks.
What is the backbone of the column?
Silica which can be modified to stick different compounds on top for more complicated mass spec.
What are the types of ionisation?
1) volatile low molecular weight compounds which are thermally stable - electron impact or chemical ionisation
2) non-volatile, thermally label compounds - Electrospray (LC), atmospheric pressure chemical ionisation (LC)
Electron impact - this is used in gas - how is this done?
You move ions from the filament to the anode and whilst they are travelling they get hit by electrons which knocks electrons and break bonds - this makes positive ions.
You can occasionally form negative ions.
What is seen on the peaks after electron impact and why?
The ionised compounds must provide energy and thus the ions formed fragments to smaller ions - so lots of peaks
What is the most abundant ion called and the molecular ion?
The most abundant ion is the base peak
Ion with mass of whole molecule = molecular ion
Non-volatile thermally labilie compounds - electrospray - on what type of mass spec would you use this on?
Liquid
How does an electrospray work?
Put the liquid into a charged capillary so the substance in the capillary is transferred charged. The liquid is evaporated and grows smaller which causes the molecules within the liquid to be unstable and explode - this then leads the molecule to have the positive or negative charge transferred onto it.
Groups which charge more readily to form ions? Yes or no
Yes
The electrospray peak?
These are simple, less fingerprints
Does gas chromatography or liquid chromatography take longer
Yes
What are the two main ways to detect the ions?
1) Beam - Magnetic sector, Time of flight, Quadropole
2) Traps - cyclotron
How do ions get into the detector?
Charged plates - beams them into the analyser
Quadrupole Mass analysers - how do these work?
The poles have radio frequency and charges meaning that when ions enter only one will have a stable trajectory - you can select for the drug or hormone you want.
Pros of quadrupoles?
Low cost,
Bench top, multi user, robust
Ideal for quantition
Cons of quadrupoles?
Limited mass range - can’t do proteins
Unit resolution
sensitivity drops and number of ions scanned increases
Triple quadropole - the first one was previously described, what does the second one do?
Adds a collision gas
Triple quadropole - what does the third one do?
Select for product
Why would you use the triple quadropole? This can be called ms-ms
Helps with selectivity as some ions will give you the same number - you therefore have to put them through transitions.
What are pros of ms-ms
Less back ground noise
Screens out interferences
Reduced sample prep times
Improved selectivity
Cons of ms-ms?
Expensive
Staff training time
What are reference methods?
Check amino assays or samples to see how much drugs there is
What is an internal standard?
A molecule you put into every analysis to make sure you are doing it correctly
How would you then quantify a drug with an internal standard?
Take the ratio
What should your internal standard be?
A chemically similar to your sample as possible e.g. isotopomer, isomer, same chemical class
Ideally isotopomer
What do you make to do you analyse - see if you are doing everything okay?
Make a standard curve by comparing internal standard and peak area and make it into a ratio (PLOT the RATIO).
Case study vitamin D - why did we have issues with an immunoassay
There is lots of different types of vitamin Ds and its hard to tell them apart
Quantitative analysis of concentration - what is therapeutic drug monitoring?
A way to measure drugs which have little therapeutic windows (they have to be monitored closely as they will go toxic with little prompt)
Qualitative analysis is used to do what?
See what isn’t meant to be there
Qualitative analysis - toxicology - how do they identify people taking drugs of abuse during sports?
Do a mass spec fingerprint (all the different peaks during gas chromatography mass spectrometry is this)
How would you know if someone is abusing testosterone?
Measure the ratio of testosterone to epitestosterone if the ratio is higher than 6 = you’ve taken testosterone. But people would just take epitestosterone to keep this ratio correct.
People usually take testosterone from plants and you can compare plant and animal derived testosterone.
What type of mass spectrometer do you use to compare plant and animal derived testosterone?
Isotope ratio mass spectrometer
This is boiled into carbon dioxide and you can measure the natural Animal testosterone and the plant testosterone carbon dioxide and compare it
Qualitative analysis - inborn errors of metabolism - paediatric- what are the things which could be wrong and need qualitatively analysed?
steroid biosynthesis
Bile acid biosynthesis
Disorders of mitochondrial oxidation
Purines, pyrimidines, porphyria, catecholamine
Why would you use mass spec on inborn errors of metabolism?
You need to know exactly what steroid they cannot metabolise in order to treat - you can look at peaks and see what’s being overproduced or underproduced.
