Interference Study
Positive fentanyl solutions were prepared at 1 mg/mL and diluted down to 5 ng/mL. The FTS limit of detection (LOD) was determined by eye to be 25 ng/mL due to the presence of a dark testing band (Fig. 1, Left). Quantification of the testing bands correlates 25 ng/mL to 1152 counts (n = 5, SD = 72) (Fig. 1, Right) and is consistent with the FTS packaging stating a 20 ng/mL detection limit. The LOD value will be referenced as the fentanyl threshold line.
The FTS were assessed to determine prevalence of false positives when tested with stimulants such as cocaine, methamphetamine, and MDMA as well as over-the-counter medicines such as alprazolam, gabapentin, naloxone buprenorphine, and diphenhydramine. Solutions were prepared at 20 mg/mL in DI water and diluted down to 0.2 mg/mL. By eye, FTS with cocaine, alprazolam, gabapentin, and naloxone buprenorphine were negative even at the highest concentration, with the appearance of dark bands in both the control and test regions (Supplemental 1). However, the FTS testing band with methamphetamine, MDMA, and both diphenhydramine capsules and tablets did not appear when the analyte was in high concentrations. These samples could be read as false positives (Fig. 2). Figure 3 shows the integrated intensities of the test bands; the error bars are the standard deviation from replicate measurements (sub-graphs shown for each substance close to the fentanyl detection limit). The integrated intensities of test bands from solutions of cocaine, DI water, and tap water (which should all be negative for fentanyl) were well above the fentanyl threshold. However, the integrated intensities of the test bands from moderately concentrated samples of methamphetamine, MDMA, diphenhydramine capsules, and diphenhydramine tablets were below the fentanyl threshold, consistent with the visual false positive results. The critical concentration level for diphenhydramine capsules was 1 mg/mL, for diphenhydramine tablets 2.5 mg/mL, methamphetamine was 1.5 mg/mL, and MDMA was 2 mg/mL. At or above these concentrations, the FTS is likely to produce a false positive result.
While both the diphenhydramine tablets and capsules contain 25 mg of diphenhydramine HCl active ingredient, these are over-the-counter formulations. The diphenhydramine capsule’s average weight is 240 mg (n = 3, SD = 3) whereas the tablet’s average weight is 250 mg (n = 3, SD = 4). In this case, the tablets contain more cutting agents so the 20 mg of solid used for the test would have less diphenhydramine for the tablet measurement than the capsule and is supported by the lower false positive critical concentration.
FTS were ran with diphenhydramine tablets and capsules in three different temperature conditions (4oC, 25oC (Room Temperature), and 40oC) at three different concentrations (4 mg/mL, 2 mg/mL, and 1 mg/mL). FTS were ran and allowed 5 minutes to process in these temperature conditions. These concentrations were selected to cover the critical concentration of false positives for both diphenhydramine capsules and tablets. In the room temperature and warm conditions with the diphenhydramine tablet, the FTS behaved as expected with a false positive result at 4 mg/mL and negative results for 2 mg/mL and 1 mg/mL (critical concentration of 2.5 mg/mL). In the room temperature and warm conditions with the diphenhydramine capsule, the also FTS behaved as expected with false positive results at 4 mg/mL and 2 mg/mL with a negative result for 1 mg/mL (critical concentration of 1 mg/mL). In the cold condition, all FTS gave a false positive result (only appearance of control band). The FTS test band does not react properly at temperatures at or below 4oC. Images were taken 5 minutes after running and can be found in Supplemental 2.
The concentrations of diphenhydramine, methamphetamine, and MDMA are at levels that current drug detection protocols could generate false positives. For example, a drug user that is concerned about fentanyl in their sample, might use a FTS to test the residue in their cooker or the powder residue in a baggie prior to use of the drug. If we assume there is 5 mg of residue in the container, the sample composition is 100% methamphetamine, and the user adds 1 mL (many cookers and baggies can hold about 0.75-1 mL of water) of water to the container, there is 5 mg/mL of methamphetamine in the sample. As we know from our FTS bar quantification, this concentration of methamphetamine would likely trigger a false positive with the FTS. If the user instead dilutes with 50 mL of water, the methamphetamine concentration would be 0.1 mg/mL which is well below the critical concentration and would render a true negative on the FTS. If we take another methamphetamine sample that was 95% methamphetamine and 5% fentanyl, there would be 4.75 mg of methamphetamine and 0.25 mg of fentanyl present. If the user diluted again with only 1 mL of water, the methamphetamine would be at a concentration that would provide a false positive. However, if the user dilutes with 50 mL of water, the methamphetamine concentration is low enough to not interfere with the FTS (0.095 mg/mL) and the true positive would come only from the fentanyl present in the sample. Even if the user diluted the sample with 1000 mL of water, the FTS are still sensitive enough to detect the fentanyl present without the interference of methamphetamine.
Time Study
FTS were analyzed to determine how long a user should wait prior to reading the results. Panels A-J in Fig. 4 show images of a water control FTS. Panels a-j in Fig. 4 show the FTS images for a 1 mg/mL fentanyl standard solution. These photos were quantitatively analyzed using ImageJ and plotted in Fig. 5. The BTNX Inc. FTS product insert states to read the FTS between 5–10 minutes after sampling. (21, 22)
There is significant color change of the water control, water test, and fentanyl control bars over the first 120 seconds after running. Fentanyl test bar values never leave the x-axis due to the bar never developing in a positive fentanyl sample. The water control, water test, and fentanyl control bars develop at a rate of 143, 104, and 97 counts per second respectively over the first 120 seconds. From 120 seconds to the final measurement at 420 seconds, the water control, water test, and fentanyl control bars develop at a rate of 12, 10, and 16 counts per second indicating approximately a 10x color development rate over the first 2 minutes (120 seconds). After this point, both the testing and control bands become saturated and the color development rate levels out. From this data, a user should wait 2 minutes (120 seconds) at minimum when interpreting FTS.
The fentanyl threshold line was included in Fig. 5 to also show how quickly the test bars develop in comparison to the LOD of fentanyl detection. By approximately 15 seconds, the user should be able to determine if a test band will appear by eye; however, the manufacturer’s instructions should be followed when possible.