Gas Chromatography with Soft Ionization Mass Spectrometry for the Characterization of Natural Products

The application of gas chromatography (GC) combined with atmospheric pressure chemical ionization mass spectrometry (GC–APCI-MS) and with supersonic molecular beam ionization mass spectrometry (GC–SMB-MS) is described in this article. These ionization modes offer complementary information that helps to unravel the complexity of extracts from natural products. They are also compatible with high-temperature GC, which extends the GC application range to higher-molecular-weight apolar solutes. Additionally, soft ionization can be very useful for the characterization of polar fractions analyzed by GC after derivatization (silylation). Derivatized sugars, organic acids, and amino acids show strong fragmentation in electron ionization–mass spectrometry (EI–MS) with non-characteristic fragment ions, whereas GC–APCI-MS and GC–SMB-MS offer easier feature extraction and compound identification.

Figure 1: Schematic overview of the four classes of natural product constituents.
Natural products contain a broad range of organic molecules, including low- to high-molecular-weight compounds and apolar to polar solutes. Several analytical methods are therefore required to unravel the complexity, characterize the product, and identify important constituents of natural products. In a very schematic way, natural product constituents can be divided into four classes based on their molecular weight and polarity (Figure 1). Classical gas chromatography–mass spectrometry (GC–MS) with electron ionization (EI) is typically applied for the analysis of low-molecular-weight, apolar (and thus relatively volatile) solutes, resulting in high sensitivity and library searchable spectra. For apolar high boiling solutes, such as lipids, high-temperature GC or liquid chromatography (LC) are used. Polar constituents, such as sugars and amino acids can also be analyzed using LC or GC but require derivatization. Finally, the polar high-molecular-weight constituents such as proteins and oligosaccharides are typically analyzed by LC (including size exclusion chromatography [SEC]) or by electrophoretic techniques.

The borders between the four quadrants in Figure 1 should not be considered as "solid," but rather as "transition zones." Since GC is characterized by high resolving power, research in GC–MS is continuously exploring ways to­ extend the applicability from the low-molecular-weight apolar zone — the "classical" GC–MS application area — into the higher molecular weight zone and the more polar fraction. Three main obstacles are encountered:

Firstly, in the apolar fraction, compounds such as waxes and alcohols show strong fragmentation, and no molecular ion is detected in GC–EI-MS making unequivocal identification difficult. Secondly, for the elution of high-molecular-weight compounds, high-temperature GC conditions, such as the selection of a column with a high phase ratio (wide bore + thin film), high column flows (> 5 mL/min), and very high temperatures, are needed. These conditions are not compatible with classical benchtop MS systems. Finally, the polar fraction of natural products is important. While this fraction can be analyzed by GC after derivatization (for example, using the metabolomic method developed by Fiehn [1,2], involving oximation and silylation), identification of derivatized sugars, organic acids, and amino acids is difficult because of strong fragmentation in electron ionization.

For these reasons, soft ionization techniques combined with MS can offer complementary information. The best known are (positive ion or negative ion) chemical ionization (3) and, more recently, single photon ionization (4). In this article two other alternatives for GC soft ionization MS were evaluated: GC hyphenated to atmospheric pressure chemical ionization (APCI) on a high resolution time-of-flight (TOF) MS system (5) and GC combined via a supersonic molecular beam (SMB) interface and a single-quadrupole MS system (GC–SMB-MS) (6). These ionization modes are also compatible with higher capillary column flows that are often used in high temperature GC, and are therefore quite interesting to extend the range of "GC-amenable" solutes to higher-molecular-weight solutes, such as long chain alcohols and lipids. The aim of this article was not to provide a detailed technical description of these ionization modes, but rather to illustrate their application in natural product research. Both hyphenated methods were evaluated using tobacco leaf extracts (both apolar and polar extracts) as an example, but the GC–APCI–TOF-MS and GC–SMB–MS methodology can be extended to a wide range of applications in natural products, including tea, herbal medicines, plant extracts for cosmetics, and more.