A simple, versatile, and automated pulse-diffusion-focusing strategy for sensitive milliliter-level-injection HILIC-MS/MS analysis of hydrophilic toxins.

The measurement of trace hydrophilic toxins in complex aqueous-rich matrices is a daunting challenge. To address this analytical bottleneck, pulse diffusion focusing (PDF), a novel sample injection technique for hydrophilic interaction chromatography-tandem mass spectrometry (HILIC-MS/MS), was developed. Theoretical and experimental investigations of the mechanism and key parameters revealed that the pulse-injection approach, assisted by solvent diffusion, efficiently solved the volume overload problem. This milliliter-level-injection HILIC-MS/MS technique was reported for the first time herein, and provided a significant enhancement in sensitivity compared to the conventional injection method, in addition to being an efficient approach to address the solvent incompatibility of off-line sample preparation and HILIC. The automated PDF-HILIC-MS/MS system was obtained by slightly modifying a commercial LC-MS/MS instrument in an easy and economical manner. The efficiency of the system was demonstrated through the detection of trace tetrodotoxin contents in plasma and urine samples. Low limits of detection (i.e., 0.65 and 2.2 ng·mL-1) were obtained using the simplified sample preparation method. The recoveries were in the range 91-113.3 % with intra-day and inter-day precisions of ≤9.6 %. Further experimental results proved the method to be versatile for various hydrophilic toxins.