Each year bee specialists report detection of larger numbers of bee colonies suffering from colony collapse disorder. One of the suggested causes is the increasing use of neonicotinoid pesticides in farming agriculture. To study the effects these pesticides have on the environment, it is necessary to establish a method to quantify the amount of imidacloprid in an environmental sample. Previously, we found that off-line C-18 cartridges were suitable for enriching imidacloprid from complex matrices and for preparing samples for analysis in GC-MS (gas chromatography-mass spectrometry). Our previous work primarily focused on achieving a reliable calibration curve by enhancing signal-to-noise in the GC-MS. To further evaluate our findings, a spiked environmental sample was subjected to C-18 extraction and analyzed. This method resulted in a limit of detection as low as 160 micromolar, which is similar to the 120 to 320 micromolar range for limits of detection achieved by other research groups using more complex extractions. This is important to the future study of imidacloprid because it allows us to progress towards the analysis of environmentally relevant concentrations of imidacloprid in soil, plant and honey samples.