Mass Spectroscopy

MS measures MASS OF INDIVIDUAL IONS produced by fragmentation of the analyte molecule.

Different from other forms of spectrometry

• Does not involve absorption or emission of radiation.
• Sample is destroyed in the analysis - use only a small aliquot.

Original and most widely used form is ELECTRON IONIZATION MS. (Figure below)
[CHEMICAL IONIZATION gives simpler ionization patterns]

Electron Ionization Mass Spectrometer

Theory of Electron Ionization MS

Electron bombardment displaces orbital electrons - molecules are fragmented to +ve ions.
Ions accelerated by electric field.
Magnetic field deflects ion stream along curved path

Measure ion current at detector as magnetic field is varied (electromagnet)
---> MASS SPECTRUM

Segment of Mass Spectrum

Qualitative Analysis by Mass Spectrometry

Molecular mass of the analyte
Peak at highest magnetic field strength = the MOLECULAR ION
(but ignore small peaks from ¹³C)
Mass spectrum highly individualised - complicated fragmentation patterns

Very characteristic for a particular compound ----> IDENTIFIER of compounds. (Search database of mass spectra)
ID using (conventional) MS requires ...

1. PURE analyte for fragmentation
2. VOLATILE analyte
   derivativisation of polar groups: ESTERIFICATION or SILYLALATION
3. MOLECULAR MASS of analyte less than or equal to 1000

But modern adaptations have fewer limitations.

Gas Chromatography - Mass Spectrometry
(GC-MS)

In biochemical analysis sample not usually pure compound.
Frequent need to identify and/or quantify one component in a complex biological matrix.

Apply powerful separation technique to resolve the mixture into its separate components, then present to mass spectrometer.

GAS CHROMATOGRAPH (GC) ideally suited for coupling to the MASS SPECTROMETER, because it provides HIGH RESOLUTION SEPARATION OF COMPONENTS IN THE VAPOUR STATE - can be passed directly into the MS for analysis.

Flow Chart of GC-MS

Applications of GC-MS

GC-MS = very powerful combined technique
---> both qualitative and quantitative analysis of complex mixtures.

• RESEARCH : eg protein chemistry - take peptide fragments from protein hydrolysate - separate and identify as step in protein structure determination.
  
• MEDICAL/FORENSIC: screen metabolic fluids to detect and quantify abnormal metabolites, toxins, drugs.
  
• ENVIRONMENTAL: identify and quantify toxic pollutants in plant and animal tissues, water supplies etc.
  

Quantitative Analysis by Mass Spectrometry

VERY ACCURATE quantitative analyses of complex metabolites, drugs, toxins etc in biological fluids by MS using a STABLE ISOTOPE DILUTION METHOD.

"STABLE" here means non-radioactive.
(Well-established isotope dilution method uses radioactive isotopes -see various texts.)

MS provides an isotope dilution assay of higher accuracy while avoiding the hazards of using and disposing of radioactivity.

5.4.1 MS Stable Isotope Dilution Assay

1. Synthesise sample of analyte species with one or more atoms replaced by heavier or lighter isotope of the same element,
   eg codeine with a -CH₃ group modified to -CD₃ (D = deuterium).
   
2. Add identical amount of isotopically modified species to samples and analyte standards.
   
3. Carry out GC-MS. Measure in mass spectrum the size (area) of peak for a fragmentation ion that displays peaks for both ANALYTE and its ISOTOPICALLY MODIFIED FORM - will give separate peaks in mass spectrum because different masses.

(if mass of isotopic form is constant)

Calibrate Area ratio vs Mass for standards - hence evaluate mass of analyte in samples.

HIGH ACCURACY and generally FREE FROM INTERFERENCE - "gold standard" method for clinical and environmental work.