Pressure sensitive adhesives and paper spray‐mass spectrometry for the collection and analysis of fentanyl‐related compounds from shipping materials

The rise of fentanyl and fentanyl analogs in the drug supply pose serious threats to public health. Much of these compounds enter the United States through shipping routes. Here we provide a method for fentanyl screening and analysis that utilizes pressure‐sensitive adhesive (PSA) lined paper to recover drug residues from parcel‐related surfaces. The paper used is commercially available repositionable notes (also called post‐it or sticky notes). From this paper, mass spectra were obtained by paper spray‐mass spectrometry (PS‐MS), where PSA paper served as both a sampling and analysis substrate. Seven fentanyl‐related compounds were analyzed: fentanyl, 4‐anilino‐N‐phenethylpiperidine (4‐ANPP), N,1‐diphenethyl‐N‐phenylpiperidin‐4‐amine (phenethyl‐4‐ANPP), valerylfentanyl, 4‐fluoroisobutyrylfentanyl (4‐FIBF), carfentanil, and p‐fluorofentanyl. These compounds were recovered by PSA paper and identified by PS‐MS from packaging tape and plastic at 50 ng and from cardboard and shipping labels at 100 ng. The impact of cutting agents on PS‐MS analysis of fentanyl analogs was explored. No trends of analyte suppression were found at high concentrations of the cutting agents caffeine, diphenhydramine, and lidocaine when recovered from surfaces. A cartridge that required no precise cutting of PSA paper prior to sampling or analysis was evaluated for use in PS‐MS for fentanyl screening. Recovery and detection of fentanyl from plastic sheeting was demonstrated with this cut‐free cartridge. The cut‐free cartridge showed somewhat less consistency and lower analyte signal than the standard cartridge, but performance was suitable for potential screening applications. In combining PSA surface sampling with PS‐MS for drug screening, both sampling and detection of fentanyl‐related compounds is simple, rapid, and low‐cost.


| INTRODUC TI ON
The rise of synthetic opioids since the early 2010s have led many to call these compounds the "third wave" of the US opioid epidemic While fentanyl and a few of its analogs are legally manufactured and diverted for abuse, clandestinely manufactured fentanyl is responsible for most of the illicit fentanyl supply [3,6]. The DEA has identified two major routes by which fentanyl enters the United States, both of which rely on shipping [7]. The primary route for fully synthesized fentanyl and fentanyl analogs is through direct shipment of fentanyl compounds in small quantities from China to the United States [7][8][9]. Synthetic precursors to fentanyl are also shipped to Mexico, as well as Canada and the United States for synthesis in clandestine laboratories [7]. These synthesized fentanyl analogs are trafficked into the United States across land borders [7]. In addition to these main routes, the shipping channels for fentanyl are expected to diversify in coming years, with fentanyl now originating in India and other nations [7,8,10]. Attempts to stem the flow of illicit fentanyl in the United States have resulted in increases in international shipping regulations; however, they have not resulted in a decrease in fentanyl in the US drug market [11,12].
While fentanyl and its analogs remain pervasive in the United States, developing methods to sample and detect these compounds, especially before they reach the consumer, is of utmost importance.
Due to their high potency and the perceived risk for accidental exposure by first responders, the safe sampling of these compounds is important [13][14][15]. One demonstrated method for low-cost collection of drugs is the use of pressure sensitive adhesive (PSA) lined papers [16]. Pressure-sensitive adhesives are a class of commercially available adhesives that form non-reactive bonds with a surface when pressure is applied [17]. There are a variety of commercially available applications of PSAs including tape, stickers, and stationary, including the popular stationary product, sticky notes [18].
These adhesives, in the form of commercially available sticky notes, have been demonstrated to recover drug residues from a variety of surfaces and serve a substrate for analysis by paper spray-mass spectrometry (PS-MS) [16]. In addition to offering an affordable, allin-one collection and analysis substrate, PSA paper also allows for sampling without direct human contact with the surface in question. In this work, the use of PSA papers combined with PS-MS was expanded to fentanyl-related compounds for their recovery from parcel-associated surfaces. The application to recovery of fentanyl compounds from parcel-related surfaces PSA was developed as a potential screening mechanism for fentanyl entering the United States by mail.
Paper spray-mass spectrometry has been previously applied to the detection of fentanyl and fentanyl analogs including detection in biofluids and bulk samples [19][20][21][22][23][24][25][26][27][28]. The combination of PS-MS with surface enhanced Raman (SERS) paper for fentanyl detection has been demonstrated, including on portable instrumentation [23,24,29]. Paper spray-mass spectrometry has also been applied to the detection of fentanyl analogs in casework-type samples including suspected illicit pharmaceutical tablets [25,26]. Paper spray has also been used in a pilot drug checking program, specifically for the detection of fentanyl compounds, with the goal of harm reduction [27,28]. Applications of PS-MS have largely been focused on the limited sample prep and quick analysis times offered by the technique. While sample preparation for PS-MS is simplified compared to techniques such as LC-MS, most published methods require the spotting of a prepared solution onto paper for analysis. With the use of PSA paper, these preparations steps are eliminated, streamlining the screening process.
The combination of PSA paper with PS-MS offers a low-cost sampling and analysis substrate for the screening of fentanylrelated compounds. While PS-MS has been utilized for fentanyl detection, many of these methods require additional sample preparation steps such as mixing and spotting a solution on the paper.
In utilizing PSA paper for sampling, these preparation steps are eliminated, simplifying the sample workflow, and minimizing risks for accidental exposure. Compared to using wet swabs for sample collection, PSA paper does not require solvent for collection,

Highlights
• Collection of fentanyl analogs by pressure-sensitive adhesive (PSA) paper (sticky notes) is reported.
• PSA paper is used as a substrate for paper spray-mass spectrometry analysis of seven common fentanyl analogs.
• Fentanyl analog signal explored in the presence of cutting agents.
• Performance of cartridge accommodating full sized sticky note reported.
reduces the risk of damage to fragile wet paper, and picks up residues by particle adhesion rather than dissolution. In this work, the recovery of a subset of common fentanyl-related compounds from four different shipping materials was determined. In addition to screening for fentanyl analogs, the impact of common cutting agents on fentanyl analog signal was evaluated. The use of a cartridge that did not require cutting of the paper tip for analysis by PS-MS was assessed and the performance compared to that of a previously developed cartridge that required cutting the paper to a particular shape [16].
Individual 10 ppm solutions of each fentanyl precursor and analog were prepared for method development. Three cutting agents, caffeine, lidocaine, and diphenhydramine were prepared at 10, 30, and 30 ppm, respectively. When appropriate, 50-100 ng of standard solution, spotted on a surface in 5 μL aliquots. All standard solutions were prepared in a 75:25 water to methanol solvent ratio to reduce dispersal of the analytes when deposited on a surface.

| Pressure-sensitive adhesive and paper spray-mass spectrometry
Commercially available PSA paper was prepared in two ways. The standard PSA tips were prepared by cutting five-layers of PSA paper into 0.5 cm by 1 cm rectangles with adhesive present on 75% of the tip area. A triangular tip with an angle of 45° was cut into the portion of the paper lacking adhesive (Figure 2A). The tip was reduced to three layers by discarding the top and bottom layer prior to sampling to lower the potential for contamination during storage or handling.
Sampling was conducted by dabbing the surface three times with the PSA-lined portion of the tip, applying firm pressure to the tip while in contact with the surface. The tip was then placed, adhesive side up, in a 3-D printed polypropylene cartridge containing a solvent well and high voltage wire channel ( Figure 2B).
A comparison between the standard cartridge used for all previous PSA and PS-MS experiments and a cartridge requiring no paper cutting (referred to as cut-free cartridge) was conducted. For experiments involving the cut-free cartridge a single, full-sized, PSA ticket was used for sampling. Sampling with full size PSA ticket was conducted in the same manner as the cut tickets. The adhesive portion of the ticket was placed in the cut-free cartridge and excess paper torn from the ticket and discarded ( Figure 2C). The cartridge was comprised of 3-D printed polypropylene containing three solvent wells, an opening for high voltage application via a ball bearing and an open corner to achieve paper spray ionization ( Figure 2D).

| Data acquisition
Prior to data acquisition, a spray solvent was applied to the PSA tips before analysis. A spray solvent of 90:10 MeOH:H 2 O with 0.1% FA was used for all experiments. The spray solvent was selected based on its common use in PS-MS and the solubilities of fentanyl and fentanyl analogs [30]. For the standard cartridge, a spray solvent volume of 70 μL was applied to the solvent well over 5-10 s. For the "cut-free" cartridge a total of 200 μL of spray solvent was used, with 150 μL applied in the three solvent wells prior to analysis and the final 50 μL applied to the tip during data collection to sustain the spray.
All spectral data was collected on a Thermo LTQ-XL linear ion trap mass spectrometer (Thermo Scientific Inc.). For all experiments, data acquisition was carried out for 0.7 min with voltage application of +4.5 kV (45° paper angle) or +5.0 kV (90° paper angle) for 30 s.
A higher voltage is required as the angle of the paper spray tip increases [31]. Both full MS and MS/MS spectra were collected during analysis. Instrument parameters for MS/MS analysis are detailed in Table S1.

| Data analysis
The impact of cutting agents on analyte signal was examined. The two cartridges were compared by averaging the AUC:TIC for fentanyl. The relative standard deviation (RSD) (s/x × 100%) was calculated. A student t-test (p-value = 0.05) was used determination of statistical significance between data sets when applicable. A total of five replicates were collected for each experiment unless otherwise noted.

| Surface recovery experiments
To demonstrate the ability of PSA paper to recover fentanylrelated compounds from packaging materials, 100 ng of the fentanyl analogs both individually, and in a mixture, were deposited on packaging tape, cardboard, shipping labels, and a plastic sheet. Due to absorption into the shipping label and cardboard surfaces, 100 ng was first dried on the plastic sheet and trans-

| Cutting agent experiments
To assess differences in analyte signal caused by cutting agents, three unregulated cutting agents were assessed (caffeine, lidocaine, and diphenhydramine). Cutting agents and fentanyl analogs were combined by mixing a 5 ppm fentanyl analog solution 1:1 with solutions of 5 ppm lidocaine, 15 ppm caffeine, or 15 ppm diphenhydramine. The cutting agents and fentanyl analogs were recovered from each of the four surfaces.

| Cartridge comparison
The performance of the standard and cut-free cartridges were assessed. A total of 50 ng of fentanyl was added directly to the different PSA tips and recovered from a plastic sheet. For the full-sized PSA tip, recovery of the fentanyl residue was focused on the corner of the tip used for paper spray ionization. A total of eight replicates were carried out for each condition and average fentanyl signal, failure rate, and relative standard deviation were compared. Differences in cartridge performance were evaluated for statistical significance.
In addition to regulated compounds, phenethyl-4-ANPP, a synthesis byproduct, was selected because it can serve as indicator of fentanyl synthetic route [33]. The four surfaces, cardboard, packaging tape, shipping labels, and plastic were selected as they are commonly used in shipping.
PSA-lined paper was selected as a sampling mechanism and substrate based on a previous study showing a marked increase in drug residue recovery by PSA paper compared to standard paper [16].
To establish the use of PSA paper for the recovery of fentanyl analogs, full MS and MS/MS spectra were collected for individual fentanyl compounds spotted and recovered from each of the four surfaces  Figure 3G). The lack of a peak at 188 m/z is consistent with fentanyl analogs that have an additional, stabilizing, substituent group at position 4 of the piperidine ring [34]. The recovery of a mixture of all fentanyl-related compounds from each surface was also demonstrated ( Figure 4, Figure S1). For the plastic sheet and packaging tape, 50 ng on the surface was a sufficient amount for identification. For shipping labels and cardboard, 100 ng was required for identification, perhaps owing to the greater porosity and roughness of these materials.

| Cutting agents
Three cutting agents, caffeine, lidocaine, and diphenhydramine were examined for their impact on fentanyl-related analyte signal.
Fentanyl analog purity was from 50% for lidocaine and 25% for caffeine and diphenhydramine. These purities were selected based on casework samples where fentanyl or fentanyl analogs were the primary controlled substance [35]. Each cutting agent was examined for impacts on analyte signal when recovered from each surface.
The cutting agent data for fentanyl is represented in Figure 5. As indicated by Figure 5  F I G U R E 5 Normalized fentanyl signal in the presence of caffeine, lidocaine, and diphenhydramine for recovery from cardboard, shipping label, packaging tape, and plastic by PSA paper.

| Cartridge comparison
A cartridge was designed to eliminate the need to cut PSA paper to a sharp tip prior to sampling. Instead, the existing corner of the PSA tips (~90° angle) was used for ionization. This is an increase in the bond angle of the tip by a factor of approximately 2. The cut-free cartridge required only a single sheet of PSA paper as opposed to the three layers used in the standard tip. A reduction in layers was possible because of the support built in to the cartridge, which increased the stability of the tip during analysis ( Figure 2D). While no cutting of tips was required prior to sampling, excess paper not lined with PSA was torn off and discarded to reduce solvent dispersal away from the tip. With the increase in surface area between the standard tip and the full-sized PSA ticket, solvent volume was increased to 200 μL.
Comparison between the standard cartridge focused on failure rate and fentanyl signal when fentanyl was added directly to paper and recovered from plastic. Failure rate was defined as a sample that did not ionize or produce consistent ionization. Paper spray chronograms and mass spectra obtained for both cartridges are shown in Figure 6.
The standard cartridge had a lower failure rate than that of the cut-free cartridge with only 1 of the 16 samples failing to produce consistent ionization. The cut-free cartridge has a failure rate four times greater with 3 out of 16 samples producing inconsistent ionization. Overall, ionization with the cut-free cartridge had more variability than the standard cartridge. The spray instability of the new cartridge as it compared to the standard cartridge is illustrated in Figure 6A,B. Reduced spray stability in the cut-free cartridge was attributed to the much larger paper angle. A previous study showed that larger paper angles required higher spray voltages and also required more precise positioning of the paper tip relative to the inlet [31]. There was a statistically significant difference in the fentanyl signal between the standard and cut-free cartridges for the neat addition of fentanyl and recovery from plastic (p = 0.0100 and 0.0131, respectively). The average fentanyl signal for the standard cartridge was approximately two times greater with the standard cartridge than the new cut-free cartridge for the neat addition of fentanyl directly to the PSA paper ( Figure 7). For the recovery of fentanyl from plastic sheeting, fentanyl signal remained significantly greater F I G U R E 6 Spectral comparison between standard cartridge and cut-free cartridge for the recovery of 100 ng fentanyl from plastic surface. (A) chronogram (TIC) for standard cartridge (B) chronogram (TIC) for cut-free cartridge, (C) Full MS spectrum for standard cartridge, (D) Full MS spectrum for cut-free cartridge.
for the standard cartridge but only by a factor of approximately 1.4 times (Figure 7). These results, taken together, suggest that the paper spray MS step is somewhat less effective when performed from the 90° corner, but that there may be improved recovery for the larger paper that partially offsets this loss. Despite reduced spray stability and fentanyl signal, the molecular ion for fentanyl was still identifiable with the cut-free cartridge ( Figure 6C,D).
While the cut-free cartridge generally had poorer performance than that of the standard cartridge, it may still be suitable for qualitative analyses and screening. The cut-free cartridge with a full-sized PSA ticket was able to recover and identify fentanyl at the 50 ng level. Increasing analysis time to accommodate the larger paper may allow for more of the analyte to ionize, making the cartridge more comparable to the standard cartridge. Moreover, future work will involve design of an apparatus to position the paper tip more exactly in front of the mass spectrometer, which should reduce failure rate due to slight mispositioning of the 90° paper tip.

| CON CLUS ION
This work expands the use of paper spray-mass spectrometry as a screening tool for fentanyl-related compounds by applying pressuresensitive adhesive paper for drug recovery. In combining the sampling mechanism with the paper spray substrate, this method offers a rapid and highly affordable method for drug identification that

CO N FLI C T O F I NTE R E S T S TATE M E NT
The authors have no conflicts of interest to declare. F I G U R E 7 Average signal (AUC:TIC) for 50 ng of fentanyl neat and recovered from a plastic surface using the standard and cut-free cartridges.