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A method using high-performance liquid chromatography (HPLC) and atmospheric pressure chemical ionization (APCI) mass spectrometry (MS) was established for the detection of 6-(methylsulfinyl)hexyl isothiocyanate (6-MITC) and its conjugate with N-acetyl-L-cysteine (NAC). The optimal chromatographic conditions were obtained on an ODS column (150×4.6mm, 3μm) with the column temperature at 37℃. The mobile phase consisted of a methanol-0.1% trifluoroacetic acid (TFA) mixture (50:50, v/v), and the flow rate was 0.3 ml/min. The detection wavelength was set at 220 nm. The identities of the peaks were accomplished by comparing retention times (t_R), UV and mass data. All calibration curves showed good linear regression (correlation coefficients for 6-MITC and NAC>0.999) within test ranges. The developed method provided satisfactory precision calculated as percent coefficient of variation with overall intra-day and inter-day variations of less than 5% (4.1 and 4.9% for 6-MITC; 4.2 and 4.9% for NAC). Both 6-MITC and NAC had good responses in the positive APCI and formed strong [M+H]^+ ions in the full scan spectra at an m/z of 206 and 164, respectively. The presence of the [M+H]^+ ion for the 6-MITC/NAC conjugate was also observed at an m/z of 369. To our best knowledge, this is the first report that describes the formation of the 6-MITC/NAC conjugate and its detection method by HPLC-MS.