Chapter 1
LC/MS Coupling

1.1 State of the Art in LC/MS

Oliver Schmitz

1.1.1 Introduction

The dramatically increased demands on the qualitative and quantitative analysis of more complex samples are a huge challenge for modern instrumental analysis. For complex organic samples (e.g., body fluids, natural products, or environmental samples), only chromatographic or electrophoretic separations followed by mass spectrometric detection meet these requirements. However, at certain moments, a tendency can be observed in which a complex sample preparation and pre-separation is replaced by high-resolution mass spectrometer with atmospheric pressure (AP) ion sources. However, numerous ion-molecule reactions in the ion source - especially in complex samples due to incomplete separation - are possible because the ionization in typical AP ion sources is nonspecific [1]. Thus, this approach often leads to ion suppression and artifact formation in the ion source, particularly in electrospray ionization (ESI) [2].

Nevertheless, sources such as atmospheric-pressure solids-analysis probe (ASAP), direct analysis in real time (DART), and desorption electrospray ionization (DESI) can often be successfully used. In ASAP, a hot nitrogen flow from an ESI or AP chemical ionization (APCI) source is used as a source of energy for evaporation, and the only change to an APCI source is the installation of an insertion option to place the sample in the hot gas stream within the ion source [3]. This ion source allows a rapid analysis of volatile and semi-volatile compounds, and, for example, was used to analyze biological tissue [3], polymer additives [3], fungi and cells [4], and steroids [3, 5]. ASAP has much in common with DART [6] and DESI [7]. The DART ion source produces a gas stream containing long-lived electronically excited atoms that can interact with the sample and thus desorption and subsequent ionization of the sample by Penning ionization [8] or proton transfer from protonated water clusters [6] is realized. The DART source is used for the direct analysis of solid and liquid samples. A great advantage of this source is the possibility to analyze compounds on surfaces such as illegal substances on dollar bills or fungicides on wheat [9]. Unlike ASAP and DART, the great advantage of DESI is that the volatility of the analyte is not a prerequisite for a successful analysis (same as in the classic ESI). DESI is most sensitive for polar and basic compounds and less sensitive for analytes with a low polarity [10]. These useful ion sources have a common drawback. All or almost all substances in the sample are present at the same time in the gas phase during the ionization in the ion source. The analysis of complex samples can, therefore, lead to ion suppression and artifact formation in the AP ion source due to ion-molecule reactions on the way to the mass spectrometry (MS) inlet. For this reason, some ASAP applications are described in the literature with increasing temperature of the nitrogen gas [5, 11, 12]. DART analyses with different helium temperatures [13] or with a helium temperature gradient [14] have been described in order to achieve a partial separation of the sample due to the different vapor pressures of the analyte. Related with DART and ASAP, the direct-inlet sample APCI (DIP APCI) from Scientific Instruments Manufacturer GmbH (SIM) was described 2012, which uses a temperature-push rod for direct intake of solid and liquid samples with subsequent chemical ionization at AP [15]. Figure 1.1 shows a DIP-APCI analysis of a saffron sample (solid, spice) without sample preparation with the saffron-specific biomarkers isophorone and safranal. As a detector, an Agilent Technologies 6538 UHD Accurate-Mass Q-TOF was used. In the upper part of the figure, the total ion chromatogram (TIC) of the total analysis and in the lower part the mass spectrum at the time of 2.7 min are shown. The analysis was started at 40 °C and the sample was heated at 1°s-1 to a final temperature of 400 °C.

Figure 1.1 Analysis of saffron using DIP-APCI with high-resolution QTOF-MS.

These ion sources may be useful and time-saving but for the quantitative and qualitative analysis of complex samples a chromatographic or electrophoretic pre-separation makes sense. In addition to the reduction of matrix effects, the comparison of the retention times allows also an analysis of isomers.

1.1.2 Ionization Methods at Atmospheric Pressure

In the last 10 years, several new ionization methods for AP mass spectrometers were developed. Some of these are only available in some working groups. Therefore, only four commercially available ion sources are presented in detail here. The most common atmospheric pressure ionization (API) is ESI, followed by APCI and atmospheric pressure photo ionization (APPI). A significantly lower significance shows the atmospheric pressure laser ionization (APLI). However, this ion source is well suited for the analysis of aromatic compounds, and, for example, the gold standard for polyaromatic hydrocarbon (PAH) analysis. This ranking reflects more or less the chemical properties of the analytes, which are determined with API MS:

Most analytes from the pharmaceutical and life sciences are polar or even ionic, and thus efficiently ionized by ESI (Figure 1.2). However, there is also a considerable interest in API techniques for efficient ionization of less or nonpolar compounds. For the ionization of such substances, ESI is less suitable.

Figure 1.2 Polarity range of analytes for ionization with various API techniques. Note: the extended mass range of APLI against APPI and APCI results from the ionization of nonpolar aromatic analytes in an electrospray.

Dieses Bild haben wir in O. J. Schmitz, T. Benter in: Achille Cappiello (Editor), Advances in LC-MS Instrumentation, AP laser ionization, Journal of Chromatography Library, Vol. 72 (2007), Kapitel 6, S. 89-113 publiziert

1.1.2.1 Overview about API Methods

Ionization methods that operate at AP, such as the APCI and the ESI, have greatly expanded the scope of mass spectrometry [16-19]. These API techniques allow an easy coupling of chromatographic separation systems, such as liquid chromatography (LC), to a mass spectrometer.

A fundamental difference exists between APCI and ESI ionization mechanisms. In APCI, ionization of the analyte takes place in the gas phase after evaporation of the solvent. In ESI, the ionization takes place already in the liquid phase. In ESI process, protonated or deprotonated molecular ions are usually formed from highly polar analytes. Fragmentation is rarely observed. However, for the ionization of less polar substances, APCI is preferably used. APCI is based on the reaction of analytes with primary ions, which are generated by corona discharge. But the ionization of nonpolar analytes is very low with both techniques.

For these classes of substances, other methods have been developed, such as the coupling of ESI with an electrochemical cell [20-31], the "coordination ion-spray" [31-46], or the "dissociative electron-capture ionization" [37-41]. The APPI or the dopant-assisted (DA) APPI presented by Syage et al. [42, 43] and Robb et al. [44, 45], respectively, are relatively new methods for photoionization (PI) of nonpolar substances by means of vacuum ultraviolet (VUV) radiation. Both techniques are based on photoionization, which is also used in ion mobility mass spectrometry [46-49] and in the photoionization detector (PID) [50-52].

1.1.2.2 ESI

In the past, one of the main problems of mass spectrometric analysis of proteins or other macromolecules was that their mass was outside the mass range of most mass spectrometers. For the analysis of larger molecules, such as proteins, a hydrolysis and the analysis of the resulting peptide mixture had to be carried out. With ESI, it is now possible to ionize large biomolecules without prior hydrolysis and analyze them by using MS.

Based on previous works from Zeleny [53], and Wilson and Taylor [54, 55], Dole and co-workers produced high molecular weight polystyrene ions in the gas phase from a benzene/acetone mixture of the polymer by electrospray [56]. This ionization method was finally established through the work of Yamashita and Fenn [57] and rewarded in 2002 with the Nobel Prize for Chemistry.

The whole process of ion formation in ESI can be subdivided into three sections:

• formation of charged droplets
• reduction of the droplet
• formation of gaseous ions.

To generate positive ions, a voltage of 2-3 kV between the narrow capillary tip (10-4 m outer diameter) and the MS input (counter electrode) is applied. In the exiting eluate from the capillary, a charge separation occurs. Cations are enriched at the surface of the liquid and moved to the counter electrode. Anions migrate to the positively charged capillary, where they are discharged or oxidized. The accumulation of positive charge on the liquid surface is the cause of the formation of a liquid cone, as the cations are drawn to the negative pole, the cathode. This so-called Taylor cone resulted from the electric field and the surface tension of the solution. At certain distance from the capillary, there is a growing destabilization and a stable spray of drops with an excess of positive charges will be emitted.

The size of the droplets formed depends on the

• flow rate of the mobile phase and the auxiliary...