Mass spectrometry (MS) is a physico-chemical spectral technique used for the structural elucidation or confirmation of organic, bioorganic, organometallic and inorganic compounds. It is frequently used as a highly selective chromatographic detector for the quantitative analysis of environmental, pharmaceutical, forensic, and food/feed samples, to name a few. From a theoretical point of view, MS is not actually a “ spectral” technique since it does not deal with the absorption or emission of quantum of energy according to Planck´s law. However, due to the similarity of experimental approaches, the spectra’s appearance, and the range of applications, MS is frequently included in the family of spectral techniques.

Actual mass spectra are records of relative ion abundances (expressed as percent) vs. mass to charge ratio (x-axis). The most abundant ion in the spectrum is called the base peak and is assigned a relative intensity of 100%. The relative abundances of other ions in the spectrum are then normalized to the base peak.

Electron Ionisation mass spectrum of benzophenone (Click to enlarge)

Most of the ions carry just one charge, so the m/z values correspond directly to the masses of the particular ions. In case of some ionization techniques (especially electrospray ionization), ions may be produced with multiple charges. As a result, the observed m/z values are diminished by the factor 1/z.

The current mass spectrometric nomenclature recommends the use of the Thomson (Th) as a unit for m/z values, in honor of J. J. Thomson, who was awarded the Nobel Prize for physics in 1906 for the discovery and experimental proof of the existence of electrons and charged particles. The field of mass spectrometry has garnered four Nobel Prizes for chemistry and physics so far. Nowadays, mass spectrometry influences and supports research in many fields of chemistry, biology, medicine and physics.

Generally speaking, the mass spectrometer consists of 3 main parts:

1. Ion source. The role of the ion source is the conversion (ionization) of neutral species into charged particles (ions).
2. Mass analyzer. The mass analyzer then separates the ions according to their m/z values
3. Detector. The detector records the relative abundances of individual m/z values.

Scheme of the mass spectrometerIn addition to these basic parts, several other parts are essential to the function of the mass spectrometer. The sample introduction system introduces the solid, liquid or gas sample into the high vacuum required for ion analysis and detection, while ion optics transfer, focus and accelerate ions once in vacuum. And no mass spectrometer would be complete without a noisy vacuum system and a computer to control the instrument and provide data handling and reporting.

Mass spectrometry alone is best suited for the analysis of pure compounds, because the whole sample is introduced to the ion source at once. In practice, the analysis of more complex mixtures is often required, so we need to couple the mass spectrometer to some separation technique.

In the past, the target compound had to be isolated first and then analyzed with a direct insertion probe, a time-consuming process that also made the analysis of trace impurities difficult. During the last several decades, the coupling of gas chromatography to electron ionization (EI) sources their cousins, chemical ionization (CI) sources has made MS a routine technique. GC/MS (gas chromatography MS) is used for the analysis of complex mixtures of gas phase compounds, which limits the range of analytes to relatively volatile, non-polar molecules.

To prevent extensive fragmentation, soft ionization techniques are used. Initially, HPLC-MS also relied on EI or CI, but due to the limited sensitivity and robustness of such devices, intensive research brought new soft ionization techniques, which are ideally suited for HPLC/MS coupling since they combine multiple functions into one step, including:

1. Interface between the column and MS system (Sample transfer into gas phase,
2. Sample ionization

Nowadays, the coupling of HPLC and MS in analytical laboratories is very common. MS is compatible with the whole range of analytical flow rates (from nl/min to 2 ml/min) and the mobile phase composition (except for normal phase eluents without a polar modifier), thanks to the innovative ionizers developed in the last few decades. The main remaining limitation lies in the choice of chromatographic conditions, especially the selection of buffers and additives. Non-volatile additives (e.g. phosphate buffers, tetraalkylammonium ion-pairing agents, etc.) should be replaced by more volatile analogues (e.g. ammonium acetate or formate, formic or acetic acid, ammonia, tri- or dialkylammonium acetate, etc.), which should be used at the lowest possible concentration (usually 5-10 mmol/l at maximum) in order to avoid contamination of the mass spectrometer or ion suppression effects. Contamination and suppression effects can also be reduced by using orthogonal ion source geometry, the standard for current HPLC/MS systems.

How can ion suppression influence your spectrum?
The analyte may give a different MS response in a mobile phase without ionic additives in comparison to the identical system containing ionic and/or non-volatile additives, such as phosphate buffer. The suppression occurs in the ionizer when ionic species in the mobile phase successfully compete with the analyte for charges at the droplet surface, thus reducing the ionization of target analytes. In addition to ionic additives in the mobile phase, sample matrix components can also result in the suppression, or sometimes enhancement of response when they co-elute with target analytes.

What to do with non-volatile compounds e.g. buffers?
HPLC/MS coupling has only one serious limitation concerning the choice of mobile phase composition, which is the use of non-volatile inorganic buffers and additives, such as phosphate buffers, inorganic acids, non-volatile ion-pairing agents, cyclodextrins, etc. When the HPLC method containing such reagents is converted to HPLC/MS, non-volatile additives should be substituted by more volatile additives.

How can we convert an HPLC method containing non-volatile mobile phase additives to MS-friendly conditions without the loss of chromatographic performance?
HPLC/MS coupling places only one serious constraint on HPLC mobile phase composition, ruling out the use of non-volatile inorganic buffers and additives, including phosphate buffers, inorganic acids, non-volatile ion-pairing agents, cyclodextrins, etc.

When an HPLC method containing such reagents is adapted for HPLC/MS coupling, non-volatile additives should be substituted by more volatile additives at the lowest possible concentration, for example:

• Ammonium acetate or formate (usually up to 5 mmol/l), formic or acetic acid (up to 0.1%, in some special cases a little bit more)
• Ammonium hydroxide for basic pH values (up to 0.1%).
• For analyses employing ion-pairing reagents, HPLC/MS is compatible with di- or trialkylammonium acetates or formates (for cationic analytes) at or perfluorocarboxylic acids (for anionic analytes), all of which can be used at concentrations up to 3mmol/L.

The advantage of so called soft ionization techniques arises the fact that they primarily produce protonated or deprotonated molecules and relatively few fragment ions (thus the term “soft”), which makes the molecular weight (MW) determination relatively simple. At the same time, the absence of fragment ions may be considered a disadvantage for structure elucidation since MW information alone is not sufficient to tease out molecular structure. This drawback may be overcome by using tandem mass spectrometry (MS/MS), whereby the first mass analyzer is used for the isolation of a selected precursor ion (previously called a "parent ion"), which is then fragmented to give product ions (originally knows as "daughter ions") for subsequent MS analysis.

Tandem mass spectrometry principle

In this manner, we can obtain information the sub-structure of each precursor ion, which should represent a portion of the molecule. This process may be repeated for several precursor ions, typically using triple quadrupole or ion trap analyzers.

Tandem MS allows to repeat isolation and fragmentationThe ion trap analyzer allows repeated isolation and fragmentation steps, producing fragments of fragments of fragments, a technique known as multistage tandem mass spectrometry (MSⁿ). This is valuable for studying fragmentation paths, as well as confirming molecular structures.

What is the difference between in-source collision induced dissociation (CID) and collision induced dissociation in tandem mass spectrometry?

In-source CID does not enable precursor ion isolation, so all the ions present in the ion source at a given time are fragmented as a group without prior isolation, which is feasible with true tandem mass spectrometry. Another difference is that in-source CID occurs during the ionization process in the ion source, while CID in MS/MS occurs in the ion trap or in the collision cell, in case of QqQ (triple quadrupole) or QqTOF instruments. The absence of the isolation step for in-source CID may not be of concern if chromatographic resolution is adequate.

• Atmospheric pressure chemical ionization (APCI) - soft ionization technique typically used for HPLC/MS coupling and the analysis of small organic molecules with low to medium polarity.
• Atmospheric pressure photoionization (APPI) - soft ionization technique, nearly identical applications as for APCI, but it extends the polarity range slightly towards non-polar or very labile molecules.
• Base peak - peak with the highest abundance in the spectrum. Its relative abundance is set to 100%, relative abundances of other peaks in the spectrum are related to the base peak and fall in the range 0-100%.
• Chemical ionization (CI) - first soft ionization technique, used mainly in GC/MS.
• Dalton – a unit of molecular weight frequently used in mass spectrometry.
• Deprotonated molecule - even-electron ion [M-H]^- with an m/z ratio one mass unit lower than the molecular weight. This is typically the base peak in negative-ion mass spectra taken with soft ionization techniques.
• Electron ionization (EI) - first ionization technique generally used for GC/MS coupling. It is sometimes referred to as a "hard" ionization technique because ionized species can obtain large amounts of internal energy, which leads to extensive fragmentation and the complete absence of the molecular ion for about 10% of all the volatile compounds amenable to EI.
• Electrospray ionization (ESI) - the softest ionization technique, especially useful for polar to ionic compounds, biopolymers, non-covalent complexes or any extremely labile compounds.
• Elemental composition - sum of individual atoms present in particular ion or molecule.
• Fragmentation - process whereby the ion is cleaved into smaller parts called fragments.
• Exact mass - precise mass of particular a particular ion calculated to at least four decimal places, taking into account the number of electrons, used to extract molecular formula information from highly accurate and precise mass spectra.
• Gas chromatography - mass spectrometry (GC/MS) -coupling of gas-phase separation technique and mass spectrometry, typically with electron ionization. This is a very powerful tool for the identification of volatile compounds because of the availability of large EI spectral libraries and well defined spectral interpretation rules.
• High-performance liquid chromatography - mass spectrometry (HPLC/MS) - coupling of these two techniques is useful for the qualitative and quantitative analysis of complex mixtures.
• Ion cyclotron resonance (ICR) – very precise mass analyzer requiring Fourier Transformation of its detector signal to provide the highest available resolution and mass accuracy among mass analyzers, but at the expense of the most rigorous vacuum requirements and high cost.
• Ion trap (IT) – a relatively recent type of mass analyzer capable of iterative tandem mass spectrometry.
• Ionization - the process of converting a neutral molecules into a charged species (ion).
• - Magnetic sector analyzer - the oldest type of mass analyzer. Ions are separated because different m/z values result in different trajectories through the magnetic field. Typically used in series with an electrostatic analyzer to increases resolution.
• Mass accuracy – the difference between theoretical and measured m/z values, reported as ppm. Mass accuracy better than 5 ppm is generally considered the minimum necessary for exact mass determination, which can allow determination of the elemental mass composition.
• Mass spectrometry (MS) – A spectral technique useful for structural elucidation and sensitive chromatographic detection.
• Mass-to-charge (m/z) – the quantity graphed on the x-axis of a mass spectrum. Determines the interaction of the ion with magnetic and electrical fields, a fact that is exploited in ion analyzers.
• Matrix-assisted laser desorption/ionization (MALDI) - soft desorption ionization technique not frequently coupled to separations. Very useful for biopolymers and synthetic polymers with high molecular weights. Sometimes coupled to HPLC off-line via fraction collection.
• Molecular ion - odd-electron ion, it may be M^+. in positive-ion or ^—. in negative-ion mode.
• Molecular weight (MW) - sum of masses of the most abundant isotopes for each atom in the molecule. Note that MW determination using the most abundant isotopes (used in MS) differs from MW determination on the basis of averaged isotopic masses (used in all fields of chemistry except for MS. For example, a mass spectrometrist should count bromine as 79 (since ⁷⁹Br is the most abundant isotope) rather than 80 (average of ⁷⁹Br and ⁸¹Br isotopes in the ratio approximately 1:1)
• Nominal mass - the integer value of particular ion calculated from most abundant natural isotopes.
• Orbitrap - the newest type of FT mass analyzer introduced in 2005, it provides high resolution and high mass accuracy by detecting the oscillation of ions in an electric field.
• Protonated molecule - even-electron ion with m/z value higher than the molecular weight by one mass unit, the [M+H]⁺ ion is typically the base peak in positive-ion mass spectra generated with soft ionization techniques.
• Quadrupole analyzer (Q) - low resolution mass analyzer commonly coupled to chromatography.
• Resolution – A measure of the mass spectrometer’s ability to distinguish (separate) two adjacent spectral peaks. There are two basic definitions: 1) the mass of the target peak divided by the difference between two neighboring peaks with the same heights and 10% valley overlap (R_{10% valley}), 2) the mass of target peak is divided by the peak width at the half height of this peak (R_FWHM). The second definition is more widespread and is generally accepted nowadays, although the 10% valley definition is still common for magnetic sector instruments. Roughly, R_FWHM is approximately half of R_{10% valley}.
• Soft ionization techniques - a group of ionization techniques with the common feature that the molecular ion or deprotonated molecule usually correspond to the base peak of mass spectra with the lack or low abundances of fragment ions.
• Tandem mass spectrometry (MS/MS) - coupling of two or more analyzers (both ion traps and ion cyclotrons can actually achieve MS/MS with at single analyzer) used for the isolation of precursor ion, its subsequent fragmentation, and the detection of their product ions.
• Thomson (Th) - unit for m/z recently proposed by mass spectrometric nomenclature.
• Time-of-flight (TOF) - mass analyzer based on the precise measurement of the flight times of ions accelerated by an electric field.
• Triple quadrupole (QqQ) - tandem mass analyzer consisting of three quadrupole rods. The first quadrupole is used for precursor ion selection, the second one serves as a collision cell for fragmenting precursors into product ions, which are then analyzed by the third quadrupole.