Technical Report On-line LC-GC×GC-MS/MS: A Powerful Unified Separation-science Tool In-depth Analysis of a Very Complex Mixture Mariosimone Zoccali1, Peter Q. Tranchida1, Paola Dugo1, 2, Luigi Mondello1, 2 A b s tra c t: This Technical Report demonstrates the potential of the on-line combination of high performance liquid chromatography (HPLC), cryogenically modulated comprehensive two-dimensional gas chromatography (GC×GC), and triple quadrupole mass spectrometry (MS/MS). The selectivity of the HPLC dimension enabled the separation of chemical classes; each fraction, transferred to the GC×GC instrument via a syringe-based interface, was subjected to a specific programmed temperature vaporizer (PTV) GC×GC-MS/MS untargeted or targeted analysis. Keywords: liquid-gas chromatography, comprehensive gas chromatography, triple quadrupole mass spectrometry 1 . I n tro d u ctio n The detailed characterization, or untargeted analysis, of very complex mixtures of organic volatiles (and semi-volatiles) represents one of the main challenges in the field of analytical chemistry. Sample complexity, as such, is not only related to a potentially high number of constituents (e.g., ≥ 1000), but also to the range of different chemical classes (from apolar to highly polar) and concentration levels (from trace amounts to highly concentrated). In terms of mass spectrometry (MS) unit-mass devices such as single quadrupole (quad MS) or low-resolution time-of-flight (LR ToF MS) are sufficient for the scopes of an untargeted experiment. In a targeted experiment, the analyst has an a priori knowledge of the name and number of solutes requiring investigation. In targeted GC-MS experiments, the use of quad MS, LR ToF MS, high-resolution (HR) ToF MS and triple quadrupoles (MS/MS) is popular. Consequently, the satisfactory profiling of a very complex mixture would require: 1) enhanced separation power; 2) high selectivity; 3) increased sensitivity; 4) wide dynamic range. High-resolution gas chromatography (GC) methods, such as comprehensive 2D GC (GC×GC), are advisable for the untargeted analysis of a complex sample. The combination of GC×GC, with quad MS, LR ToF MS, HR ToF MS, and MS/MS systems generates very powerful analytical tools that can be employed in both untargeted and targeted applications. However, as will be seen, the separation power of comprehensive 2D GC is often unsufficient, and many co-elutions still occur at the end of the analytical process. Fig. 2 TIC-GC×GC-MS chromatogram of coal tar 1 Department of Pharmaceutial Sciences and Health Products, University of Messina 2 Chromaleont S. r

Probably, the best choice to improve selectivity, due to the high number of stationary phases, is the exploitation of an HPLC preseparation step. HPLC is very useful for the performance of polaritybased separations (e.g., hydrocarbons, aromatic constituents); after, simplified fractions can be subjected to a GC-based analysis, injecting large sample volumes, enabling the detailed analysis of complex samples. The potential of the novel LC-GC×GC-MS/MS combination was evaluated in the analysis of a highly challenging sample, namely coal tar (Figs. 1-2). The normal-phase LC step was successful in...

Full scan mode I) non aromatic hydrocarbons were transferred from 1.35 to 1.85 min (175 |jL) II) aromatic compounds (with and without S) were transferred from 1.85 to 7.00 min (1802 jL) III) oxygenated compounds were transferred from 8.95 to 14.10 min (1802 jL) Scan speed : 20,000 u/sec Acquisition frequency : 33 Hz Ion source temperature : 250°C Interface temperature : 280°C MRM mode Argon (200 kPa) was employed as collision gas For MRM transitions and collision energies see Fig. 5 3-3. GCxGC analysis I) non aromatic hydrocarbons (Fig. 3) Split ratio 200:1 for 1 min, then splitless for...

4 . R e s u l ts a n d D iscu ssio n As can be observed in the figures, the pre-separation step was very useful in generating simplified sub-samples of coal tar, which is an extremely complex sample. The purification step made quali/quantitative analysis easier. All the fractions were analyzed in the full-scan mode; moreover, the second fraction was analyzed in the MRM mode for the determination of the sulphur compounds. Fig. 8 MRM LC-GC×GC-MS/MS chromatogram of DBT isomers Finally the third fraction containing the oxygenated compounds was analyzed in the full scan mode. A positive off-set of...