Sample Preparation: Two sets of samples (acetonitrile and apple juice matrix) were prepared by spiking 10 ppm level of toxin compounds into 10 ml of the matrix and then adding 10 ml of acetonitrile. Spiked toxins in the acetonitrile sample were Colchicine, Strychnine and Aconitine; Lobeline and Solanine were spiked in the apple juice sample. The mixtures were shaken for 30 minutes and stored at 4℃ until further analysis. Solvent and matrix blanks were prepared in the same way without spiking any compounds. Samples were analyzed by ultrahigh pressure liquid chromatographymass spectrometry (UHPLC-MS) with the Thermo Scientific Accela 1250 pump and the Q Exactive™ benchtop mass spectrometer with full scan and data dependent MS/MS with a 6.5-minute gradient. The identity of the spiked compounds was not known by the analyst prior to the analysis. The spiked compounds were thus “unknown” to the analyst.

Liquid Chromatography:

Mass Spectrometry:

FIGURE 1. Q Exactive Benchtop Orbitrap Mass Spectrometer

Data Analysis: Data acquisition was performed using Thermo Scientific Xcalibur 2.1 software. Differential analysis in Thermo Scientific SIEVE 1.3 software was used to analyze spiked and control samples with principal component analysis(PCA).

Figure 2 shows the overall SIEVE workflow for the unknown screening. Briefly, acquired LC chromatograms are first aligned based on the selected reference raw file to correct the retention time variance from LC runs. Then all peaks found above given intensity threshold will be ordered based on intensity. Frames of 0.30 min x 0.010 amu (see Figure 3) are defined based on the orders from the most intense to second highest peak and so on, without overlapping with previous frames. The capacity of 2,700,000 frames in 9-minute gradient are used to define analytes that may exist in the sample by PCA analysis. The ions found from differential analysis is identified through ChemSpider™ online database search and confirmed with MS/MS spectra.

FIGURE 2. SIEVE Workflow

FIGURE 3. Frame Parameters