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Analytical methods > Mass spectrometry (MS) > Flashcards

Mass spectrometry (MS) Flashcards

1
Q

What is MS?

A

Weighing of ions

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2
Q

What are some clinical applicaitons of MS in biochemistry?

A
  • Immunosuppressant drug monitoring post-transplant
  • Steroid analysis
  • Metabolic disease
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3
Q

How is MS applied in toxicology?

A
  • drugs of abuse
  • legal highs
  • post mortem work
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4
Q

How is MS applied in haematology?

A
  • Haemoglobin variant analysis

- Homocystine and methylmalonic acid analysis

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5
Q

How is MS applied in microbiology?

A
  • Antibiotic drug monitoring

- Micro-organism identificaiton (MALDI-ToF)

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6
Q

How is MS applied in immunology?

A

Peptide measurements of MHC classes

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7
Q

How is MS applied in histology?

A

Intra-operative deteremination of tumour margins
- Knife used in operating a tumour can be hooked up with a MS which sucks up tissue to compare fingerprint of peptide of cancer tissue and normal tissue

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8
Q

Types of clinical MS

A

Gas chromatography MS
Time of flight (ToF)
Orbitrap
Liquid chromatography MS/MS

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9
Q

What are some advantages of MS?

A
  • Specificity is high
  • Small sample volumes
  • Multiple analytes per analysis
  • Cheap one time buy
  • Not limited to commercial assays
  • Flexibility of assay
  • Rapid run times compared to other HPLC detectors
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10
Q

Disadvantages of MS

A
  • Batch analysis
  • Manual preparation
  • High capital costs
  • Associated equipment: fume cupboard, N2 generator, plate sealer
  • Environmental requirements
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11
Q

Give an overview of LC-MS/MS

A

Inlet-> ionise -> mass analyse (seperate the analyte we are interested in) -> fragment -> mass analyse -> detect

All after ionise occur in a vacuum

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12
Q

Describe the function of inlet and source

A
  • Eluate from LC enters the source
  • Involves a method of removing solvent and creating ions
  • Uses heating element and heated nitrogen gas for desolvation
  • Charge applied to stainless steel capillary to aid ion formation (ESI)
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13
Q

State some ionisation techniques

A
  • ESI+, APCI
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14
Q

Describe ESI+

A

Analyte leaves via positively charged capillary tips. Assisted by nitrogen gas running either side. This creates multiply charged droplet where analyte is contained within a positive shield.
Solvent evaporates from the droplet producing analyte ions which are attracted to the cone (negative charge)

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15
Q

Give an overview of atmospheric- pressure chemical ionisation (APCI)

A
  • Sample solution flow through heated tube where it is volatilised and sprayed into a corona discharge with the aid of nitrogen nebulisation
  • Ions produced in discharge and extracted into the MS
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16
Q

What is APCI best suitable for?

A

Relatively polar, semi-volatile, low molecular weight samples
- An APCI mass spec usually contains the quasi-molecular ion, (M+H+)

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17
Q

What is the stepwise APCI ionisation process?

A

3 steps

  1. EI-ionisation of gas molecules: N2+, O2+
  2. Gas molecules ionise solvent molecules: HCO3+, CH3OH2+
  3. CH3OH2+ and/or H3O+ transfer proton to analyte molecule: (M+H)+
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18
Q

What are some advantages and disadvantages of ESI?

A

Adv

  • Most versatile
  • Ionises minor polarity and mid-polarity molecules to large proteins
  • Soft ionization, very little fragmentation

Dis

  • Can be dependent on the mobile phase used
  • Matrix effect
  • Analyte must occur in solution
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19
Q

What are some advantages and disadvantages of APCI?

A

Adv

  • Better for less polar compounds
  • Compatible with higher flow rates
  • Good sensitivity
  • Fewer matrix effects than ESI

Dis

  • Thermal degradation can occur
  • Not good for high or low masses
  • Manufacturer dependent
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20
Q

What are quadropole mass filters and what purpose di they serve?

A

Following ion focussing the quadrupole mass filters seperate the analytes according to mass

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21
Q

Describe how mass seperation occurs

A

Quadropoles are four rods arranged precisely with DC and RF alternating voltages applied to pairs

  • Due to the offset of RF voltage and DC the polarity of each pair of rods continually changes
  • Ions oscillate with quadrupoles
  • Amplitude of oscillation = m/z
  • Length of quadrupole determines the mass scale of the instrument

Only the analyte with the right mass to charge will have a stable trajectory

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22
Q

What occurs in the collision cells?

A

Filled with inert gas. Collision energy is applied to inert gas to give it kinetic energy. The collision with ions of interest causes fragmentation.

Collision energy and collision flow gas can be optimised

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23
Q

Is there a difference in function between first and second quadropole?

A

Only difference is the first one selects the ion and the second one select fragments of interest

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24
Q

What are some common detectors?

A

Electron mutlipliers, photomultiplier detector

25
Q

What is a different way of producing ions

A

Adduct formation

- attaching something else that is charged to analyte of interest, e.g. ammonium in immunosuppressant analysis

26
Q

What are some data acquisition modes?

A

For quantitative analysis we use selected reaction monitoring

  • The analyte is the only thing with enough amplitude to be able to pass through the Q1 and into the collision cells
  • The collision cells will bash the analyte to fragment it and then propel it into Q2, which is specific to a certain fragment – all others will have the wrong amplitude

MRM

  • Same mechanism as SRM
  • MS/MS will do one SRM then switch to do another SRM for different m/z
  • Time spent countig for any particular m/z is the dwell time
  • The MS/MS can switch between m/z in milliseconds
  • Allows simultaneous determination of multiple analytes and their internal standard
27
Q

Name some other acquisition modes.

A

Product ion scan-drug screening

Neutral loss scan - amino acid analysis

Precursor ion scan - Acyl carnitine analysis

28
Q

What does chosen method of sample preperation depend on?

A
  • Sample type
  • Conc of analyte of interest
  • Interfering substances
  • Ion suppression
  • Equipment availability
29
Q

What is sample dilution?

A
  • Dilute any interferance with an acqeous matrix
  • Suitable to aqueous samples where matrix effects wont be an issue
  • Example - Urine 5-HIAA: works because urine is a simple matrix with 5-HIAA appearing in larger quantities
30
Q

Describe protein precipitation as a sample preperation method

A
  • Zinc sulphate is used to precipitate large proteins e.g. Igs. In a whole blood assay, it also lyses the cell to release contents
  • Acetonitrile or methanol is then added to precipitate smaller proteins and also zinc sulphate
  • Samples are centrifuged before analysis and only supernatant is injected
    E.g. includes immunosuppressant drug monitoring
31
Q

Describe liquid liquid extraction as a sample preperation method

A
  • Seperate compounds based on their relative solubilities in two immiscible liquids
  • Solvent commonly used incl. hexane, CDM and MTBE
  • Solvent added to sample plus internal standard, then mixed to extract
  • Solvent layer is seperated from the matrix, and can be dried down to add a conc step
32
Q

What is the function of zinc sulphate in protein precipitation?

A

Precipitate larger proteins.

- In whole blood assays it also lyses the cell to release the contents

33
Q

Describe supported liquid extraction

A
  • Uses a modified diatomaceous earth as solid support for liquid liquid extraction
  • Sample is diluted with water (make it less viscous) and internal standard, then applied to the support (to adsorp onto the diatomaceous earth)
  • After adsorption onto the diatomaceous earth, the analyte is eluted, then the solvent evaporated and sample reconstituted
34
Q

What is good alternative to liquid liquid extraction?

A

Solid phase extraction

- Uses the affinity of solutes dissolved in the liquid mobile phase for the stationary solid phase

35
Q

Describe solid phase extraction process

A
  1. Condition the solid phase to ensure all the particles are wetted. Maximum activation of all the particles
  2. Equilibrate with a similar matrix to what the sample is in to ensure maximum analyte retention on the solid phase
  3. Load sample
  4. Wash - get rid of interferances
  5. Elute with the solvent
36
Q

What is the purpose of solid phase extraction?

A

Isolate one or more analyte from liquid sample by extracting, partitioning, and/or adsorbing onto a solid stationary phase.

Changes the original matrix to a simpler matrix environment to ensure the least amount of interference

37
Q

When is derivitisation appropriate?

A

Used for molecules that are difficult to ionise. Can make compound that will only ionise in negative mode able to ionise in posititve mode.

38
Q

What is the clinical significance of derivitisation?

A

Measurement for steroids

e.g. dansyl chloride for E2

39
Q

Match these statements to sample prepertation methods.

A. Dilute out any interferenence, when analyte occurs in large concentrations
B. This method is useful for analytes that are hard to ionise
C. This method is used for more challenging analytes

  1. Derivitisation
  2. Liquid or solid phase
  3. Sample dilution
A

A3
B1
C2

40
Q

When are internal standards used? and what purpose do they serve?

A
  • Added early in the sample prep to all calibrators, quality controls and samples
  • Used to calculate response
41
Q

What is response?

A

Amount of analyte detected/amount of internal standard detected

42
Q

What does internal standards help identify?

A

Whether any sample is lost during the extraction, so is the corresponding amount of internal standard

  • Matrix components may vary from sample to sample which may cause slight differences in ionisation
  • The response will stay the same as the internal standard is compensating for these effects
43
Q

Requirements of an internal standard?

A
  • Behave same as our analyte in matrix
  • Behave same as our analyte during sample preparation or extraction
  • Chromatograph the same
  • Behave the same in the MS
44
Q

What are some types of internal standards?

A

Isotopes and analogues

45
Q

How are isotopes used as internal standards? Give an example

A

Incorporation of a stable isotope into compound being measured
- Typically deuterium 2H, 13C

46
Q

How are analogues used as internal standards? Give an example

A

Usually used when no better option is available

  • Structurally similar to analyte
  • Non-endogenous
47
Q

What is ion suppression?

A

It is a matrix effect

- Competition for ionisation efficicent in the ionisation source, causing reduction in detector response/signal:noise

48
Q

How does ion suppression affect ionisation?

A

It causes a decrease in ionisation of the analyte of interest.
- It varies between patient samples, so it is important to identify ion suppression in an assay to remove it if possible or to compensate for it

49
Q

Name causes of ion suppression.

A

Co-elution with analyte of interest of:

  • Non-volatile components e.g. salts
  • Endogenous compounds e.g. peptides, amino acids, phospholipids
  • Plasticisers
  • Drugs/metabolites
  • Ion pair reagents
50
Q

Is there a concrete mechanisms of ion suppression in ESI?

A

No one really knows. Theories at the moment:

  • Competition for charge between analyte and suppressor
  • Large conc of non-volatile agents affecting the surface tension of the droplet & affecting evaporation efficiency
51
Q

How is ion suppression detected?

A
  • Qualitative - post column infusion of analyte or internal standard whilst simultaneously injecting prepared sample
  • Quantitative - analyte is spiked into matrixed samples post-preparation and the signal compared to a non-matrixed sample
52
Q

How is ion suppression overcome?

A
  • Stable isotope internal standard: compensate for ion suppression
  • Alter chromatography: move peaks of interest away from ion suppression
  • Change sample preperation method: exclude things causing ion suppression
  • Change ionisation method:
53
Q

What are some other challenges to MS?

A

Isobaric interference

  • Refers to another molecule with same mass
  • These may also fragment to give same daugther ions
54
Q

How is isobaric interferences dealt with?

A

Chromatography is required to seperate isobaric interferences

  • Can be challenging due to the similar structure
  • It is a particular problem for steroids: structural isomers, M+2 isotopes
55
Q

Why is an internal standard utilised in Mass Spec?

A

Added early in the separation stages to all calibrators to calculate the response
- Any sample lost during the extraction, so is the corresponding amount of internal standard

56
Q

What are some issues with calibrators for mass spec?

A

Kits are available, but anchoring them to reference material or comparison with reference laboratory is often not possible or too expensive.

Calibrator matrix matching is hard.
- eg. Serum/plasma is simple for drug assays but for steroids there will interferences from other steroids that exist endogenously.

Animal serum, charcoal stripped serum and aqeous based are all alternatives but they are not matching the matrix well enough

57
Q

Why might animal and charcoal stripped serum not be used?

A

The matrix matching is not perfect and is quite far from the normal human serum/plasma

58
Q

Describe the two transitions for compounds

A

2 transitions tuned per compound

  • Most sensitive is used for quantifiaciton
  • Second transition is for qualification
59
Q

What does differences in ion ratio (from two transitions) tell you?

A

That there is interferance present causing one transition to go up and not the other

Analytical methods (15 decks)

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