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A technique is described where an atmospheric pressure-thermal desorption (AP-TD) device and electrospray ionization (ESI)-mass spectrometry (MS) are coupled and used for the rapid analysis of Bacillus subtilis spores in complex matrices. The resulting AP-TD/ESI-MS technique combines the generation of volatile compounds and/or pyrolysis products with soft-ionization MS detection. In the AP-TD/ESI-MS approach, an electrospray solvent plume was used as the ionization vehicle of thermally desorbed neutrals at atmospheric pressure prior to mass spectrometric analysis using a quadrupole ion trap mass spectrometer. The approach is quantitative with the volatile standard dimethyl methylphosphonate (DMMP) and with the use of an internal standard (diethyl methylphosphonate, DEMP). A linear response was obtained as tested in the 1–50 ppm range (R2 = 0.991) with a standard error of the estimate of 0.193 (0.9% RSD, n = 5). Bacterial spores were detected by performing pyrolysisin situmethylation with the reagent tetramethylammonium hydroxide (TMAH) for the detection of the bacterial sporebiomarkerdipicolinic acid (DPA) as the dimethylated derivative (2Me-DPA). This approach allowed spore detection even in the presence of growth media in crude lyophilized samples. Repetitive analyses could be performed with a duty cycle of less than 5 min total analysis time (including sample loading, heating and data acquisition). This strategy proved successful over other direct ambient MS approaches like DESI-MS and AP-TD/ESI-MS without the in situderivatization step to detect the dipicolinic acidbiomarker from spores. A detection limit for the dimethylated DPAbiomarker was estimated at 1 ppm (equivalent to 0.01 μg of DPA deposited in the thermal desorption tube), which corresponded to a calculated detection limit of 105 spores deposited or 0.1% by weight spore composition in solid samples (assuming a 1 mg sample size). The AP-TD/ESI source used in conjunction with the in situmethylation step allowed the differentiation of bacterial spores from other ‘suspicious white powders’ using a single stage for mass analysis and with minimum sample preparation, making this approach suitable for simple field-portable MS instrumentation and pattern recognition data analysis.