Techniques - Mass Spectrometry

Using the way that a charged sample component passes through an electromagnitic field to diagnose its mass and hence molecular structure.

Principle
Uses and Applications
Strengths
Watch-outs
Results Interpretation

In most applications, the sample is separated into individual compounds before entering the mass spectrometer (MS), either using gas (GC) or liquid chromatography (LC). Because the MS works at a high vacuum, there is an interface between the chromatographic equipment and the MS, to separate out the sample from the gaseous or liquid carrier medium.

Image copyright: Food Authenticity Network

Ionisation

There are many systems to ionise sample molecules, and each has its application, advantages, and disadvantages. There are three groups: gas phase, spray and desorption systems. Some of the more common systems are listed below:

Gas Phase

Electron Ionisation (EI)	A beam of electrons interacts with a solid or gaseous molecule, to produce a positively charged molecule or fragments. It is most commonly used for GC applications and with lower molecular weight molecules. It tends to fragment the molecular ion (break it into charged pieces, each piece giving diagnostic structural information)
Chemical Impact (CI)	An ionised reagent gas ionises the sample molecule. Both positive and negatively charges sample molecules can be produced depending on the reagent. CI occurs at lower energy than EI, and hence produces more unfragmented molecular ions (i.e. diagnostic information about the molecular mass) In this respect, it complements EI.
Direct Analysis in Real Time (DART)	The sample is held on a probe at atmospheric pressure. It is rapid, good for volatile and labile compounds and has a good potential for portable devices
Inductively Coupled Plasma (ICP)	Uses a plasma to ionise the sample and is the standard technique for ionising elements: metals and minerals.

Spray Methods

Electrospray (ESI)	A high voltage is applied to a liquid medium producing an aerosol, which then evaporates leaving a charged sample molecule. It is the standard ionisation system for LC-MS
Desorption Electrospray (DESI)	Employs a fast-moving charged solvent stream, at an angle relative to the sample surface, to propel the secondary ions towards the mass analyser. It has wide application for food samples and macromolecules.

Desorption Methods

Atmospheric Pressure Chemical (APCI)	An alternative to electrospray for LC-MS analysis. It ionises at lower energy and so it is good for diagnosing intact molecular ions rather than "fingerprints" of fragments..
Matrix Assisted Laser Desorption (MALDI)	The direct introduction of ionised samples spotted on a plate using laser energy with minimal fragmentation. It is used for analysing large biomolecules, proteins, and DNA fragments.
Rapid Evaporative (REIMS)	Used for the direct analysis of meats, fats (originally developed for pathology/surgery) using a highly charged metal edge (“ion knife”) to cut into the sample. Technology owned by Waters Corporation.

Ion Separation or Deflection

The ionised particles can be separated in various ways depending on requirements for scanning speed and resolution between different mass/charge (m/z) ratios.

Electromagnetic field ("Sector")	A magnetic field disperses or bends the path of the ionised molecules or fragments in trajectories according to their m/z ratios. Large, expensive, traditional technique capable of very high resolution. Tends to be used in research environments.
Time-of-flight (ToF)	Ions are separated according to their m/z ratio based on the length of time it takes them to travel through a flight tube of known length to reach a detector. Fast electronics, suitable for full spectral scanning at high resolution.
Quadrupole	Ions entering the quadrupole have their trajectory deflected by electrical potential in a manner that is proportional to their m/z value. Changing the potential allows only ions of specific m/z values to reach the chamber end and be detected. Relatively cheap, sensitive, low resolution, the workhorse mass separator for routine food analysis applications.
Ion trap	Similar to a quadrupole, but the electrodes are ring shaped and ions are separated and detected by discharging ions with unstable oscillations from the system and into the detector rather than detecting those with stable oscillations. Reputation for poor ruggedness but modern instruments are much better.
Orbitrap	Two electrically isolated cup-shaped outer electrodes face each other with a spindle-like central electrode around which ions of a specific m/z spread into orbiting rings. The conical shape of electrodes pushes ions toward the widest part of the trap and the outer electrodes are then used for current detection. It is the only method described here that uses an image current rather than some detection device to detect the ions. Has more current use in bioanalysis and sports antidoping than for food analysis.
Tandem mass spectrometry (MS-MS)	Joining one type of mass spectrometer to another (either same or different type) in sequence to increase resolution and/or mass accuracy capability. The ionised molecule or fragment emerges from the first MS, and is then broken into smaller fragments, each of which then passes down the second spectrometer. If a quadrupole is coupled with a fast-scanning spectrometer (Q-ToF or Q-Trap) this gives the ability to pre-select the molecular ion in the quadrupole, then identify it by the full scan of its fragments (optimum selectivity). Modern Q-ToFs can be operated either as a Q-Q system or as a Q-ToF, giving scope for dual-stage screening and confirmatory analysis.

Detection and Mass/Charge Determination (Resolution and Mass Accuracy)

The resolution is how well a spectrometer can differentiate between different m/z ions. This is related to its mass accuracy. Mass accuracy is important because molecules do not have integer masses; their precise mass runs to many decimal places. There might be 150 hypothetical fragments that would give a measured m/z of 201.1 (the typical accuracy of a quadrupole), but only a single explanation for a measured m/z of 201.09587 (a typical high-resolution accuracy).

There is a trade-off. High resolution instruments tend to be more expensive, less sensitive, and require a tightly controlled operating environment and specialist operator.

Examples of food authenticity applications		Critical aspects
Volatile aroma profiles		Is the database comprehensive with authentic varieties, species, location where grown or reared?
Adulterants such as artificial colours, melamine		Limit of detection, certainty in identification
Non-targeted analysis for standardised profiles of authentic samples combined with chemometrics- e.g. metabolites (metabolomics),		Is the reference database fully representative of an "authentic" test sample in terms of all agricultural production inputs, processing steps, seasonality etc. How robust is any machine learning protocol or discrimination algorithm.
Peptide analysis of proteins (proteomics) – where specific proteins are isolated and hydrolysed by trypsin enzyme. The peptides produced are compared to a bioinformatics database, which will normally give one or more specific peptides that are unique for the speciation of meat, fish, gelatine, plant varieties etc. (see example below).		Although, intuitively, proteomics should be suitable for quantitation this has not proven to be the case. It has been validated as a qualitative technique. Lack of quantification can lead to difficult interpreting the difference between cross-contamination and adulteration. Other critical factors includ the suitability of bioinformatics database, and whether the peptides are unique to the species /varieties?
Stable Isotope Ratio Analysis		see separate entry

Example: Unique Bovine and Porcine Peptides from Derived from Beef and Pork Collagen/Gelatin (crown Copyright)

Mass spectrometry is a widely adopted, and well used in food laboratories. Mass spectrometers can be connected to either gas or liquid chromatography with the appropriate interface to act as a detector or identify complex mixtures. It has a wide scope of application, gives confidence in identification, and can be used quantitatively at very low detection levels. With full scan techniques, there is scope for retrospective data mining; looking at historical data for signals that were not sought or considered significant at the time.

The equipment is expensive, and generally unsuitable for point-of-use testing. High resolution in particular, needs skilled operators and a well-controlled environment. Test methods must be developed, optimised and validated for each specific application, and in many cases need extensive sample preparation and clean-up.

MS signals can easily be enhanced, suppressed or missed, if interfering compounds enter the detector at the same time as the analyte. Electrospray LC-MS, in particular, is succeptible to these "matrix effects" and quantitation can be uncertain if stable-isotope are not used during calibration to compensate for matrix effects.

Mass spectrometry is capable, in principle, of results which meet the evidencial confidence criteria of a court of law. But this confidence is highly dependent on the specific type of MS, it's configuration (e.g. resolution, number and nature of mass ions or transitions monitored) and the sample extraction and clean-up. There are international guidelines for the appropriate use of mass spectrometry for "confirmatory" confidence in results in other fields of analysis such as sports anti-doping or residues/contaminants analysis in food. These can equally be applied to many food authenticity applications.

It is important that the configuration, and hence confidence in results, are communicated by the laboratory to the customer. It should no be assumed that all mass spectrometry results share the high confidence typical in forensic applications.