Multiple heart-cutting two dimensional liquid chromatography quadrupole time-of-flight mass spectrometry of pyrrolizidine alkaloids.

Pyrrolizidine alkaloids (PAs) and their and the corresponding N-oxides (PAs-ox) are genotoxic plant metabolites which can be present as unwanted contaminants in food products of herbal origin like tea and food supplements. PAs and PAs-ox come in a wide variety of molecular structures including many structural isomers. For toxicity assessment it is important to determine the composition of a sample and to resolve all isomeric PAs and PAs-ox, which is currently not possible in one liquid or gas chromatographic (LC or GC) run. In this study an online two dimensional liquid chromatography quadrupole time-of-flight mass spectrometry (2D-LC QToF-MS) method was developed to resolve isomeric PAs and PAs-ox. After comprehensive column and mobile phase selection a polar endcapped C18 column was used at pH 3 in the first dimension, and a cross-linked C18 column at pH 10 in the second dimension. Injection solvents, column IDs, flow rates and temperatures were carefully optimized. The method with column selection valve switching described in this study was able to resolve and visualize 20 individual PAs/PAs-ox (6 sets of isomers) in one 2D-LC QToF-MS run. Moreover, it was shown that all isomeric PAs/PAs-ox could be unambiguously annotated. The method was shown to be applicable for the determination and quantification of isomeric PAs/PAs-ox in plant extracts and could be easily extended to include other PAs and PAs-ox.

A chemical profiling solution for Chinese medicine formulas using comprehensive and loop-based multiple heart-cutting two-dimensional liquid chromatography coupled with quadrupole time-of-flight mass spectrometry.

Chinese medicine formulas represent an excellent illustration for "complex matrix". The complexity lies in a big array of small molecules with high chemical diversity. The present paper describes a novel chemical profiling solution for complex matrix by combining comprehensive and multiple heart-cutting two-dimensional liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (2DLC/qTOF-MS). This solution includes method optimization, combinational separation, and structural characterization. It was exemplified by fully profiling the chemical constituents of a four-herb traditional Chinese medicine formula, Gegen-Qinlian Decoction (GQD). GQD was separated by a RP × RP (C18 × Phenyl-Hexyl) 2DLC system, and eluted with acidic × alkaline mobile phases with an optimized shift gradient elution program. In the comprehensive 2DLC mode, 280 peaks were detected, and 125 compounds were characterized within 42 minutes. The multiple heart-cutting (MHC) mode extended the (2)D modulation time to 3.0 min to effectively separate the minor compounds. The (1)D eluate within 4.4 min was loaded into eleven 40-µL loops. These fractions were successively separated by (2)D to resolve 13 additional compounds. The combination of comprehensive and MHC 2DLC/qTOF-MS provides a powerful technique for global chemical profiling of Chinese medicine formulas and other complex systems.