The Field Of Mass Spectrometry Biology Essay

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The field of mass spectrometry began more than a century ago with the work of Eugen Goldstein who found that cathode rays could be deflected in a magnetic field this led Arthur Schuster and Joseph John Thompson to the discovery that these rays were made up of electrons. Thompson developed a method for separating different kinds of atoms and molecules by the use of positive rays and found evidence that neon exists in two forms with different atomic weights which is an indication that isotopes exist among stable elements. This was confirmed by the work of Aston who received the Nobel Prize in Chemistry in 1922 ''for his discovery, by means of his mass spectrograph, of isotopes, in a large number of non-radioactive elements, and for his enunciation of the ''whole number rule''. In 1929 Dempster and Bartky were the first to propose the construction of a mass spectrograph that combine both velocity and direction ,however , it was Alfred Nier who perfected this technique leading to a higher resolution mass spectrometer and the ability to study more accurately the composition of the heavy elements.

In 1976, Sano et al coupled the principle of GC to a combustion MS (GC/C/MS) to study the metabolic pathways of aspirin labelled with 13C in humans. In 1978, Matthews and Hayes reported measuring C and N ratios at natural levels on computer controlled beam switching IRMS (Schierbeek et al., 2011). In 1980s, Barrie et al conducted the first demonstration of utilizing IRMS coupled with GC. There was a dramatic increase in research to improve the methods and instrumentation in IRMS for the determination of isotope compositions in volatile elements which lead to the first commercial IRMS being available in the 1990s (Griffiths et al., 1998).

One of the uses of IRMS is for the detection of Helicobacter Pylori. The spiral bacterium was discovered in 1979 by Robin Warren and was linked to the development of stomach ulcers by Barry Marshall in 1982. The H.Pylori presence in the gut arises in the development of duodenal and or gastric ulcers in 20% of infected individuals, 2% of these will develop stomach cancer while less than 1% may develop gastric MALT lymphoma. The first Urea Breath Test using urea labelled with 13C to detect H.Pylori was carried out in 1987 by Graham et al.

Basis of the technique

The principle of the test is based on the production of large amounts of the enzyme urease by Helicobacter pylori, discovered by Landenberg et al, when 13C-urea is orally administered. The urease causes hydrolysis of urea into ammonia and 13C which diffuses into the bloodstream, is expelled by the lungs and can be measured in the exhaled breath. The UBT is conducted by taking a breath sample prior to and 30 minutes after the patient consumes the isotopically labelled urea, 125mg in U.S and 100mg in Europe, dissolved in a liquid solution.

A test meal is given prior to the test to prolong the contact time the urease enzyme has with 13C-urea. Meals are chosen to delay gastric emptying such as food with high fat content or citric acid due to its ability decrease antral motility by reducing duodenal pH (Savarino et al., 1999).

The quantity of 13C in breath samples is measured using an Isotope Ratio Mass Spectrometry (IRMS). IRMS is based on the technique to separate and measure the ratios of the stable isotopes that compose the light elements (H, C, N, O). For the 13C UBT, the sample is introduced into the IRMS by gas chromatography (GC-IRMS). The GC separates CO2 from the other gases in the breath sample such as N2, O2 so that only purified CO2 and stream of Helium enter the mass spectrometer through an interface system (Muccio and Jackson, 2009).

A MS is an analytical instrument that can calculate the weight of chemical compounds by separating ions on the basis of mass-to-charge ratio (m/z) which can be used to determine isotope abundance ratios (Siuzdak, 1996).

There are 3 main components of the IRMS:

Ion Source - The purified gaseous molecules from GC enter the IRMS by an inlet system. During ionization, a beam of electrons interacts with molecules and removes an electron to form positively charged ions.

Mass Analyzer- The ions are accelerated through a magnetic field which separates the ions by mass-to-charge ratio. The deflection of the ions is inversely proportional to the ionic mass.

Ion Detector- An assortment of Faraday Cups collect the ions as they are bent by the magnetic field. The electric current is amplified and the voltage detected is converted to frequency. The signal is graphically expressed by a mass spectrum as mass-to-charge ratio versus isotopic abundance (Dass, 2001).

A vacuum is used between the components of the IRMS to ensure that the molecular ions reach the ion detector without collisions (Siuzdak, 1996).

(Sahra, 2005)

In IRMS analysis, the ratio of the abundance of the minor, heavier isotope relative to the major, lighter isotope (13C:12C) is the R-sample and is used to establish isotope enrichment. This isotopic abundance ratio of the sample is then compared to that of a reference gas, R-standard. The international standard for Carbon is VPDB (Brand, 1996).The variations of isotopic ratio of sample relative to standard are very minor and therefore as expressed using delta notation in per mille (°) and are used to measure accuracy and account for systematic error (Schierbeek et al., 2011).

The sample chosen for the R-standard values generally have high levels of the heavy isotope and therefore most of the substances studied will have negative delta values as they will have depleted levels of the heavier isotope compared to the standard (Muccio and Jackson, 2009).

δ13Csample = [(Rs/Rst) - 1] -1000

Rs = abundance ratio of 13C in the sample

Rst = is the ratio of an international standard

The difference between the δ values of the samples before and after 13C urea administration is called the Delta over Baseline Value (DOB), a cut off value of 5° is the recognized standard to distinguish between an uninfected and an infected patient

(Goddard and Logan, 2003).


The contact of 13C-urea with the urease from bacteria residing in the oesophagus or oral cavity may lead to false positive results for H.pylori detection (Savarino et al., 1999).

Prior to UBT, Proton Pump Inhibitors, anti-secretory drugs, H2 Blockers and antibiotics are required to be withdrawn in advance of the test as urease suppression could yield false negative results. (Savarino et al., 1999). Adverse side effects of standard of the anti-secretory drugs, ranitidine and omeprazole reported within 7 days of drug cessation suggests that withdrawal of H2 antagonists 7 days prior to UBT would be sufficient to avoid false negative findings (Savarino et al., 2000).

Another limitation that exists is the need for a skilled technician to operate the IRMS, as it involves a very complicated procedure. As well as this, experienced personnel are needed to read the integrated bar-codes.

















Urease test



























Stool Antigen





Figure 1: Comparisons between the methods for H.pylori detection (Logan and Walker, 2001)


There are many clinical applications of 13-C Breath Tests including 13C-octanoic acid breath test to measure gastric emptying of solids (Delbende et al., 2000), 13C acetate BT to measure gastric emptying of liquids (Braden et al., 1995) and cholesteryl-1-13C-octanoate BT for assessing fat malabsorption and exocrine pancreatic dysfunction (Ventrucci et al., 1998). The hepatic function of Hepatitis C chronic liver disease patients can be assessed using the isotope tracers 13C-aminopyrine, 13C-methacetin (Afolabi et al., 2012) (Oliveria et al., 2006), 12C-phenylalanine (Lara et al., 2000) and 13C-galactose to detect the presence of cirrhosis (Giannini et al., 2005). The validation of [1-13C] glucose), [U-13C6] glucose,(Schierbeek et al., 2011) and 13C-Glucose breath test (GBT) for insulin resistance and to measure glucose turnover for early diabetes diagnosis are being determined (Mizrahi et al., 2010).

The protocols for the UBT are constantly being researched to counteract limitations while reducing cost. The citric acid is the optimal test meal for the UBT, which gave a superior diagnostic accuracy, reduced time and cost compared to semi-liquid fatty meals (Dominquez-Munoz et al., 1997) with an administration of 75mg 13-C urea with a 2.5g citric acid meal proved to be sufficient (Graham et al., 2001). The administration of 13C-urea and citric acid in a tablet retains the specificity and sensitivity of the UBT while reducing the test duration to 10 minutes, eliminating the need for a test meal and while allowing patients to remain on acid-suppression treatments (Hamlet et al., 1999).

New cost effective techniques such as non-dispersive, isotope selective, infrared spectroscopy (NDIRS) and laser assisted ratio analysis (LARA) have been developed and both hold potential to serve as a replacement for IRMS in the 13C analysis of breath samples, although they cannot process the same amount of samples simultaneously as IRMS.

While there are many limitations associated with the 13-C UBT in association with GC/IRMS, the accuracy and sensitivity to measure very low isotopic enrichments and a precision of 0.0002% of the IRMS make it the optimal technique for the identification of H.pylori colonization in the gastric mucosa. (Schierbeek et al., 2011).