Liquid chromatography triple quadrupole mass spectrometry (LC-QqQ-MS)

Liquid chromatography mass spectrometry (LC-MS) is a widespread analytical technique for the identification of compounds from complex mixtures. The liquid chromatography is a first dimension that allows to separate compounds of a mixture on their distribution coefficient between the stationary and mobile phase. There are several combinations of chemical solid state materials and the selection of mobile phases that determine the chemical separation principle of LC. However, there is no chromatographic method that is ideal for all compound classes.⁵¹⁵ A powerful and widespread (estimated 80% of all HPLC applications⁵¹⁶) high resolution chromatographic mode is reversed phase (RP) LC. The stationary phase in RP chromatography is typically hydrocarbonaceous with a variety of functional groups with strongly hydrophobic surfaces.⁵¹⁶ Two retention mechanisms are discussed for RP. One states that the solute adsorbs to the liquid-solid interface due to hydrophobic interactions between the nonpolar analyte and the hydrophobic ligates on the surface of the stationary phase. Another claims the partitioning of solute into the bonded stationary phase chains and the transfer of analyte from the bulk solution into a immiscible solvent.^{516, 517} Therefore, the mobile phases used for RP chromatography are rarely pure water but rather hydroorganic mixtures or polar organic solvents that cause the

“solvophobic” effect. The high volatility of solvents typically used in RP chromatography and their ability to donate protons make it well compatible to atmospheric pressure ionization with electrospray ionization technology. A multitude of methods have been developed and several approaches for method development exist.⁵¹⁸ However, a major drawback of classic RP chromatography is that polar, weakly acidic or basic samples are typically not sufficiently retained or separated.^{519, 520} An approach to improve retention of compounds with too polar properties on RP stationary phase is the addition of an ion-pair reagent to the mobile phase.^{521, 522} This reagent is typically a lipohilic ion.⁵²³ When the ion-pair reagent is allowed to equilibrate with the column the non-polar end of the compound is strongly interacting with the stationary phase leading to a strong retention while the typically charged end of the molecule is sticking into the mobile phase thus attracting and retaining inversely polar or charged analytes.^{524, 525} Since it has been shown

that the simple formation of ion-pairs in the eluent decreases analyte retention the mode should be named ion interaction chromatography. However, the term ion-pairing chromatography meaning the combination of ion-paring reagent with RP chromatography (IP RP HPLC) is most commonly used. A typical indicator for the utility of ion-paring reagents can be if some compounds start to be retained at low pH on RP while others elute at the column dead time. Those non-retained compounds are most likely ionized bases.⁵²⁵ A major drawback of IP RP HPLC is that it is problematic for robust handling. Two of the multiple examples for problems in handling shall be mentioned. One is that ion-pair concentration in the stationary phase is difficult to control (content in mobile phase, mobile phase organic content, column temperature) and that gradient elution is nearly impossible.⁵²⁵ Furthermore, ion-pairing reagents are known to be non-removable from the analytical column and the same is true for the LC-MS system. Thus, alternatives to IP RP mode should be considered first and most laboratories would decide for it only if inevitable. An attractive alternative is the combination of several separation modes in one stationary phase.

This provides multimodal interactions of the analyte with the solid phase and therefore improves separation of complex analyte mixtures. The last decade has shown that the mixed-mode chromatographic stationary phases have become very popular.^526-528 The column material developed by Imtakt Corp. combines an octadecylsilica (ODS) stationary phase with anion and cation exchange properties. Figure 4 A and B depicts the schematic composition of column material with weak ionic ligands that are adequately bonded to the C18 material. The cartoon symbolizes the ODS ligands in a collapsed state near to the silica gel surface that is caused by strong hydrophobic repulsion by nearly pure water mobile phase.⁵²⁹ Because of the special composition of the stationary phase polar and non-polar retention can be achieved under RP conditions. However, due to the multimodal properties of the material these mixed-mode columns can be operated not only in RP mode but also in anion and cation exchange and normal phase mode all on the same column. Furthermore, chromatographic methods developed with this stationary phase material can be simultaneously optimized on organic, buffer and pH gradients.

The combination of these properties makes the mixed-mode chromatography ideal for a simultaneous analysis of non-polar and polar compounds. For the sake of completeness it shall be mentioned that normal phase chromatography, also known as HILIC (hydrophilic interaction chromatography), offers high potential to separate mixtures of small and polar compounds in combination with MS detector.^{519, 520}

Figure 4: Mixed mode liquid chromatography coupled to an Agilent 6410 triple quadrupole mass spectrometer with an electrospray ionization source.

A) Shows the magnification of the solid-state material of Imtakt Corp. Scherzo SM-C18 mixed mode column. The left side represents the stationary phase of the weak cation exchanger beads with octadecyl residues (black zigzag line) and weak cation residues (green circles) bound on the silica particles. Similarly, the right side shows the surface of the weak anion exchanger beads with octadecyl residues and weak cation (red circles) residues bound on the silica particles (Adapted from a product publication of Imtact Corp.).

B) Illustrated assembly of the bead material in the column forming the stationary phase of the analytical mixed mode column.

C) Schematic illustration of an Agilent 6400 triple quadrupole mass spectrometer with an ESI ionization source. The most important elements along the ion path are shown.

A schematic illustration of a typical QqQ-MS detector for the identification of molecules is given in Figure 4 C. It combines high sensitivity and high specificity with mass resolution and mass accuracy. One of the key elements of MS is the production of gas phase ions. An extensively used soft ionization principle is the electrospray ionization (ESI).⁵³⁰ Soft in this context means an ionization process that does not break down analyte molecules into fragments because little residual energy is retained by it. Introduced by Fenn et al. in 1989, ESI was the first technology to overcome the in-source fragmentation problem. A detailed presentation of the ESI is given in Figure 5 A. Briefly described, the LC eluent gets sprayed into the source by a nebulizer and is accompanied by heated drying gas. The electric potential applied to the capillary tip will cause the eluent to form a Taylor cone that emits a fine mist of droplets.^{531, 532} The initial ESI droplets have diameters of several micrometers. Evaporation causes the droplets to shrink and therefore the charge density increases until the surface tension is balanced by Coulomb repulsion. If the Rayleigh limit is reached a fission causes the formation of highly charged daughter droplets.⁵³¹ The shrinking of the droplets due to evaporation and fission proceeds until droplet diameters in the range of several nanometers are reached. These ultimately formed tiny and highly charged droplets release molecule ions into the gas phase. As the majority of analytes present in CDM are low molecular weight species they will probably be released by the ion evaporation model.⁵³¹ However, ESI is strongly matrix dependent as has been confirmed by many examples described in literature.⁵³³ This is not only caused by co-eluting molecules as described in Figure 5 A but also by for example mobile phase pH.⁵³⁴ However, matrix effect in MS is not a hindrance for quantitative methods and can be addressed with matrix evaluation experiments.⁵³³

The history of mass spectrometry can be traced back to the beginning of the 20iest century.⁵³⁵ The valuable contributions to triple quadrupole development by Enke and Yost in the late 1970ties have helped to turn mass spectrometry to the high sensitivity and selectivity tool with a wide dynamic range in analytical chemistry.⁵³⁶ The high sensitivity and selectivity, achieved by its ability to select ions from a sample and select fragments of those in a second stage, made it to the workhorse in quantitative mass spectrometry.^{537, 538} Before the invention of triple quadrupole MS

the mass spectrometry technology has been mainly used by physical and organic chemists for molecule identification only. An overview of a triple quadrupole MS and the main processes happening in the ion beam are depicted in Figure 5 B and C. Briefly described, the triple quadrupole consists of three quadrupoles with each having a distinct function in multiple reaction monitoring (MRM) mode. The first quadrupole is set to filter for targeted precursor ions.

After that, the precursor ions are fragmented by collision induced dissociation in the second quadrupole into product ions. More strictly spoken the second quadrupole is a hexapole in the shown Agilent 6400 triple quadrupole but generally scientists speak of quadrupoles in these instruments. Subsequently, the third quadrupole filters the formed fragment ions of interest with distinct mass to charge ratios and guides them to the detector. The fragmentation of a precursor to a product ion is referred to as an ion transition and is highly specific for each analyte. The advantage of MRM scan mode is its capability to apply selected reaction monitoring to multiple product ions from one or more analyte/s eluting from the first dimension (LC). Thus, a high number of compounds can be monitored in a single run. Though the MS is a powerful detector it is not universal and can, just as others, not cover the entire chemical complexity of CDM. The applicability of the MS detector has a high dependence on the successful formation of gas phase ions. Metal ions are analytes that have principally be shown to be detectably by ESI-MS but only with special means, as charge reduction on solvation sphere or ion pairing.⁵³⁹ More recent work has shown that ESI-MS is capable to reveal information about analyte elemental composition by measurement of metal ligand complexes.^{540, 541} But these approaches are highly specialized and complex and thus the key strength of ESI triple quadrupole MS should be used to first develop quantitative methods for as many organic CDM compounds as possible.

Figure 5: Mechanistic description of Agilent 6410 triple quadrupole mass spectrometer with an electrospray ionization source.

A) Detailed schematic depiction of an ESI ionization source operated in the positive ion mode. The analyte flow is coming from the left. For the purpose of simplification, the heated nitrogen drying gas is not shown. The capillary in the nebulizer is held at an electric potential of several kV with positive potential versus ground. The form of the liquid at the surface of the nozzle is determined by two main forces. A surface tension derived force that pulls the liquid back and an electrostatic Coulomb attraction that pulls the liquid to the counter electrode.⁵³² From the very tip of the formed Taylor cone a spray is emitted. The magnification after the bracket shows four exemplary depictions of type of droplets that can be formed.

Example 1: Ion and counter-ion equal charge and thus the strong ion pairs are kept in the interior neutral phase of the droplet and prevent the analyte from capturing charge in the surface phase of the charged droplet. Example 2: Shows a droplet very crowded with analyte ions that compete for the limited excess surface charge thus decreasing the likelihood for each individual one to get ionized. Furthermore, the black dots symbolize that gas phase reactions (charge neutralization, charge stripping and charge transfer) can happen impacting the ESI response. Example 3: Three different co-eluting analytes in one droplet get all transferred from liquid to gas phase by Coulomb explosions when the Rayleigh limit is reached in the evaporating droplet. Example 4: Incomplete evaporation, caused by for example polar matrices or the presence of nonvolatile salts, can cause ion-suppression as well. Parts of the figure were derived from Mei Hong et al..⁵³³

B) Schematic illustration of an Agilent 6400 triple quadrupole mass spectrometer with an ESI ionization source.

C) Zoom into schematic mechanisms in the ion beam passing through the MS. The skimmer aperture removes neutral and lighter molecules (e.g. drying gas) that have not enough momentum to pass the aperture. Thus, they are deflected and removed from the ion beam. The octopole ion guide focuses the ions with radio-frequency voltage applied to the octopole rods. The ions focused by the octopole pass into the quadrupole. The opposing metal rods in the quadrupole are electrically connected. Alternating positive and negative radio frequency (RF) voltage with a direct current (DC) voltage offset is capable to selectively filter a distinct population of ions. The chance of ions hitting the rods, and therefore being filtered out, depends on the mass to charge ratio of the ions and the strength of the field and the oscillation frequency. The filtered ions that pass the first quadrupole filter are directed through a collision cell. This second element of the triple quadrupole mass spectrometer is typically called the second quadrupole. But in the case of the instrument used in this thesis the collision cell is hexapole filled with nitrogen (same as the drying gas). The collision of analyte ions with the nitrogen molecules causes them to fragment into product ions. These fragment ions are then sent to the third quadrupole which works with the same principle as in the first quadrupole. It is set to filter for distinct fragment ions that pass into the detector. The conversion dynode is a kind of deflector electrode that is put in front of the detector (electron multiplier). It is held at high voltage with reverse polarity to the analyte ions. The physical principle behind the conversion dynode is an effect called secondary electron emission. If charged particles like electrons or ions strike the surface of a dynode secondary electrons are released from the atoms in the surface. The number of emitted electrons and thus the signal intensity depends on the type of incident

1.3.3 Inductively coupled plasma mass spectrometry (ICP-MS) and inductively coupled plasma