Prison mail samples, suspected to be infused with NPS, were studied by means of LA‐APCI‐MS. The used instrumental setup was developed by Schmeinck and Karst [15]. In their study, they modified a commercially available APCI source to allow the ionization of a dry sample aerosol generated by LA instead of liquid samples and used it for molecular MSI. In MSI, the laser beam is focused on multiple spots across a single sample, sequentially scanning the whole sample in order to obtain spatial resolution. In the present study, this exact setup is also used, although not because of its imaging capabilities, but rather because of the minimal sample preparation needed and the high‐throughput analysis it allows. Because the system was optimized for fast imaging of single samples, rapid analysis of multiple samples can be achieved by focusing the laser beam on only one spot per sample across a set of multiple samples.

Traditionally, LC‐ or GC‐MS methods are used to test suspicious prison mail for NPS, which require laborious extraction steps and are generally time‐consuming. Here, chromatographic separation alone typically takes 10–20 min per sample [1, 2, 3, 8]. Using LA‐APCI‐MS, sample preparation was reduced to sampling each letter using a hole puncher and fixing the resulting cutouts to microscopy slides using double‐sided tape. Because the laser beam only penetrates the sample surface in the low micrometer range during LA and because the NPS are primarily located on the sample surface, mainly NPS are transferred to the resulting sample aerosol. This allows a direct analysis of mail samples in contrast to common liquid extraction, where major parts of a sample's constituents are extracted, making chromatographic separation mandatory.

Figure 2 shows a photograph of all 31 investigated samples being mounted onto two microscopy slides. Before providing the samples, GC‐MS analysis was carried out by the state criminal police office of Rhineland‐Palatinate to determine whether the letters were in fact infused with NPS and if so, which exact compounds were present. To ensure an unbiased approach, these results were not shared with us prior to our analysis, with the exception of one sample, which was selected randomly to facilitate method development. Therefore, the analyses were performed as a blinded study.

The randomly selected sample is labeled 18 in Figure 2. It contained two distinct substances, namely ADMB‐BINACA (N‐[(2S)‐1‐amino‐3,3‐dimethyl‐1‐oxobutan‐2‐yl]‐1‐butylindazole‐3‐carboxamide, Figure 3A) and JWH‐210 ((4‐ethylnaphthalen‐1‐yl)‐(1‐pentylindol‐3‐yl)methanone, Figure 3B). ADMB‐BINACA is a potent full agonist of both cannabinoid type 1 (CB₁) and type 2 (CB₂) receptors. It causes strong psychotropic effects similar to those caused by the partial CB₁ and CB₂ receptor agonist Δ⁹‐tetrahydrocannabinol (Δ⁹‐THC), the main active ingredient in natural cannabis [19, 20]. Unlike cannabis, ADMB‐BINACA is also known to cause many adverse side effects, for example severe hepatotoxicity likely caused by induction of oxidative stress [21]. ADMB‐BINACA was first detected by government authorities in Sweden back in 2019 [19, 22]. JWH‐210, on the other hand, was first synthesized by the research group of J.W. Huffman in 2005 as a tool to study the human endocannabinoid system and cannabinoid receptors, but was then also found in illicit products obtained via the internet in 2011 in Japan [23, 24]. Similar to ADMB‐BINACA, JWH‐210 is also a full agonist of the CB₁ receptor as well as a potent partial agonist of the CB₂ receptor. It is known to cause many adverse side effects with tachycardia, nausea and hypertension being reported most frequently [25].

Figure 4A shows the mass spectrum obtained by LA‐APCI‐MS for the above‐mentioned mail sample. It was acquired by ablating a line over the sample for 1 min and averaging the recorded mass spectra in data processing afterwards. Parameters for both LA and MS were optimized to yield maximized signal intensities with minimal fragmentation. Three distinct signals can be observed at different mass‐to‐charge ratios (m/z), namely, m/z 286.1932, m/z 314.1878, and m/z 370.2170. Figure 4B shows the extracted ion currents (EIC) for all three signals. Line ablation started at around 0.6 min and lasted for 1 min. It can be observed that the EICs for all three signals greatly increase during laser ablation and decrease again afterwards when the laser is turned off. This indicates that the signals in fact originate from substances that are removed from the mail sample during laser ablation. Furthermore, it is observed that the EICs for all three signals greatly fluctuate during the continuous laser ablation, suggesting a heterogenous distribution of NPS across the mail sample. Here, single spot ablation could result in the analysis of only a sample region containing no to minimal analyte while line ablation samples, a greater surface area assures the analysis of sample regions containing large amounts of analyte. Hence, line ablation was selected for analysis of all following samples.

The signal at m/z 370.2170 corresponds to the protonated molecular ion of JWH‐210, which would be observed at an exact m/z of 370.2165. The remaining two signals correspond to fragments originating from ADMB‐BINACA. The ion at m/z 314.1878 is most likely formed by cleavage of the C–N bond of its terminal amide group resulting in an exact m/z of 314.1863. The ion at m/z 286.1932 is probably formed by further loss of carbon monoxide resulting in an exact m/z of 286.1914. Since both fragmentation pathways are not commonly observed in traditional APCI or electrospray ionization modes, it can be proposed that fragmentation does not occur during the ionization process but rather during laser ablation. The exposure of the sample to the high‐energy laser beam likely induces cleavage of the analytes resulting in the fragments observed in Figure 4. The proposed fragmentation pattern is shown in Figure 3A. Accurate m/z together with proposed ion formulae, exact m/z and mass deviations are summarized in Table 1.

Following method optimization, all remaining samples were analyzed by this LA‐APCI‐MS approach. Each sample was ablated for 1 min, leading to a total analysis time of just over 30 min for all 31 samples, demonstrating a stark contrast to the 5 to 10 h traditional LC–MS or GC‐MS analysis would take. Signals for both JWH‐210 and ADMB‐BINACA were observed in 9 further samples resulting in a total of 10 samples that were presumably infused with a mixture of both substances. In 15 of the remaining 21 samples, only the two fragment signals originating from ADMB‐BINACA were observed, implying that those samples were infused with only this one SCRA. No signals that differ from the background signals, which are observed when the laser ablation system was turned off, were detected for a total of 4 samples. These samples were therefore regarded as negative samples. It could be possible for regular prison mail to be confiscated and sent in for analysis if it looked suspicious, for example by featuring visible stains that are not originating from illicit substances. Finally, even though signals were observed for the two remaining samples, they neither matched the signals observed for JWH‐210 nor those for ADMB‐BINACA. In Figure 2, these samples are labeled 14 and 16 respectively.

Figure 5 shows the mass spectrum obtained by LA‐APCI‐MS for prison mail sample 16. Because the obtained mass spectra for samples 14 and 16 are almost identical, only one mass spectrum is shown here exemplarily. Seven distinct signals can be observed in this mass spectrum. Because the compounds corresponding to the signals were unknown this time, the signals were matched against the NPS Data Hub database. This database is a web‐based data repository for analytical data specifically related to NPS [26]. Using this approach, the signals at m/z 378.2200, m/z 358.2140, and m/z 290.1510 were matched to the protonated molecular ions of three distinct SCRAs.

The most intense signal in the spectrum at m/z 378.2200 corresponds to the protonated molecular ion for MDMB‐5F‐PINACA (methyl (S)‐2‐[1‐(5‐fluoropentyl)‐1H‐indazole‐3‐carboxamido]‐3,3‐dimethylbutanoate, Figure 6A), which would be observed at an exact m/z of 378.2187. Similar to the other SCRAs mentioned above, MDMB‐5F‐PINACA is also a potent agonist of the CB₁ receptor and therefore causes the cannabis‐like effects expected by most users [27, 28]. It also causes unexpected and unwanted adverse side effects like tachycardia, psychomotor agitation, confusion and anxiety [29]. MDMB‐5F‐PINACA was first identified in white powder seized in Hungary in 2014 [28]. Based on this database match, the signals at m/z 346.1924 and m/z 318.1984 can be explained as fragments originating from MDMB‐5F‐PINACA. The ion at m/z 346.1924 is most likely formed by loss of methanol from the methyl butanoate moiety, which would result in an exact m/z of 346.1925, while the ion at m/z 318.1984 is probably formed by loss of the whole methyl ester group altogether, resulting in an exact m/z of 318.1976. The proposed fragmentation pattern is shown in Figure 6A.

Furthermore, MDMB‐4en‐PINACA (methyl (S)‐3,3‐dimethyl‐2‐(1‐(pent‐4‐en‐1‐yl)‐1H‐indazole‐3‐carboxamido)butanoate, Figure 6B) was matched to the second most intense signal of the spectrum at m/z 358.2140, which corresponds to the expected m/z of 358.2125 for its protonated molecular ion. MDMB‐4en‐PINACA was first identified in seized material in Germany in 2017 [30]. Its molecular structure only differs slightly from MDMB‐5F‐PINACA, which features a 5‐fluoropentyl tail group while MDMB‐4en‐PINACA features a 4‐pentene tail group. It also is a potent full agonist of the CB₁ receptor [27, 31]. Thus, it is regularly consumed as a substitute for regular cannabis although many users self‐report adverse side effects like chest pains, irregular heartbeat, vomiting, confusion and auditory as well as visual hallucinations [30, 31]. The observed signals at m/z 326.1876 and m/z 298.1930 indicate that MDMB‐4en‐PINACA shows the same fragmentation pattern as MDMB‐5F‐PINACA. Here, the ion at m/z 326.1876 is probably the product from the loss of methanol while the ion at m/z 298.1930 is most likely formed by the loss of the whole methyl ester which theoretically would be observed at m/z 326.1863 and m/z 298.1914, respectively. The proposed fragmentation pattern is shown in Figure 6B.

Finally, the signal at m/z 290.1510 was assigned to MDMB‐INACA (methyl (S)‐2‐(1H‐indazole‐3‐carboxamido)‐3,3‐dimethylbutanoate, Figure 6C). Here, the protonated molecular ion would theoretically be observed at an exact m/z of 290.1499. In contrast to other SCRAs, MDMB‐INACA is a rather weak agonist of the CB₁ receptor and therefore causes less potent cannabis‐like effects. It is rather considered a precursor sold in “Do‐It‐Yourself (DIY)” kits for the synthesis of controlled SCRAs [32]. These kits became common after China, the suspected origin of most internationally sold SCRAs, introduced a class‐wide ban on SCRAs in 2021 [8, 9, 33]. Since then, MDMB‐INACA has been detected in seized material in many different European countries [32, 34]. Although MDMB‐INACA features the same methyl‐3,3‐dimethylbutanoate moiety for the linked group as MDMB‐5F‐PINACA and MDMB‐4en‐PINACA, no fragment ions were observed for MDMB‐INACA. This might be explained through the rather low abundance of MDMB‐INACA in the tested mail samples, where possible fragments are either below the detection limit or are affected by ion suppression effects. Accurate m/z together with proposed ion formulae, exact m/z and mass deviations are summarized in Table 2 for all seven signals observed in the two mail samples.

Following analysis of all 31 prison mail samples, the results were compared to those obtained by GC‐MS by the state criminal police office of Rhineland‐Palatinate for method validation. Comparison showed that all 27 samples infused with SCRAs were identified correctly by LA‐APCI‐MS as positive samples and that there were in fact four negative samples where no SCRAs were detected. Furthermore, identification of the specific SCRAs present in the positive samples matched fully with 10 samples containing a mixture of ADMB‐BINACA and JWH‐210, 15 samples being infused with solely ADMB‐BINACA and the two remaining samples being laced with MDMB‐5F‐PINACA, MDMB‐4en‐PINACA and MDMB‐INACA. A detailed overview of the qualitative results is provided in the Supporting Information (Section S2, Table S2.1).