This study aimed to investigate the stability of the five synthetic cathinones in both liquid and DUS in acidic and mildly basic pH conditions across 14 days to determine which factor can aid in improving analyte stability. Stability assessments below 4 °C were deliberately omitted, as prior data from Aldubayyan et al. (2024) confirm that the analytes remain stable at −20 °C for up to 30 days even at basic pH (Aldubayyan et al. 2024), making further investigations into improved stability at colder temperatures within 14 days redundant. The time points were selected based on the complete instability of the analytes within 14 days in liquid urine; extending the stability assessment beyond this window was therefore unnecessary. Conversely, the absence of short-term time points in Aldubayyan’s study prompted the inclusion of hourly assessments in this investigation to capture early degradation kinetics and provide additional insight into the stability dynamics of synthetic cathinones. Similarly, assessments in basic urine were limited to three days as the experiment design focused on a target comparison to clarify the influence of pH, not replicate the full 14 day time course; furthermore, prior results had already demonstrated complete instability after three days under basic urinary conditions (Aldubayyan et al. 2024). The analytes selected were based on the degradation profiles of highly unstable analytes reported by Aldubayyan et al. (2024) (Aldubayyan et al. 2024), enabling a thorough investigation into improved stability profiles in DUS. An exception was MDPV, which generally exhibited exceptional stability in liquid urine, but was included in the current study as a positive control and to confirm previously reported stability data for this analogue (Adamowicz and Malczyk 2019; Ciallella et al. 2020; Glicksberg and Kerrigan 2018; Aldubayyan et al. 2024).

To ensure consistency across all conditions, both analytes and ISTDs were spiked at T0 for every sample, including liquid urine and DUS. This approach avoided introducing a secondary variable related to the timing of ISTD addition, which would have hindered direct comparison between matrices. As mephedrone-d3 was used as a surrogate deuterated synthetic cathinone, it may also undergo degradation. As a result, the stability values reported here reflect relative stability using analyte to ISTD ratios, rather than absolute signal loss. This does not affect the comparative outcomes of the study, as all conditions were treated identically, but it should be recognised as an inherent feature of the study design.

The most significant finding of this study was the confirmation that urinary pH is the dominant factor in controlling analyte stability, with almost all analytes performing better in acidic urine regardless of the storage temperature, while matrix format has varying effects. The influence of urinary pH on synthetic cathinone degradation pathways is well-documented. Extensive studies have shown that samples with an acidic pH (~ 4) exhibit considerably improved stability of various synthetic cathinone analogues compared with samples with a basic pH (~ 8) (Glicksberg and Kerrigan 2018; Adamowicz and Malczyk 2019; Tsujikawa et al. 2012; Glicksberg et al. 2018), emphasising a somewhat protective effect of an acidic matrix, and enabling urinary pH to be a strong predictor of synthetic cathinone decay. Many synthetic cathinones are primary or secondary amines; therefore, at basic pH, synthetic cathinones are more likely to undergo base-catalysed hydrolysis or oxidative deamination. Urinary acidification could suppress these pathways by limiting the formation of deprotonated, reactive species and microorganism growth that can produce basic by-products (Zaitsu et al. 2008). However, the mildly acidic (pH 5.98) urine in this study is unlikely to have fully suppressed microbial proliferation, as growth has persisted at pH 5–8 (Erdogan-Yildirim et al. 2011). The improved stability observed here therefore likely reflects suppression of base-catalysed degradation rather than total microbial inhibition.

Notably, 4-Cl-α-PPP, 4-CEC and NEH exhibited distinct degradation kinetics in basic urine. 4-CEC showed remarkable instability in basic urine in both storage conditions, potentially due to its simple secondary amine and halogenated ring substitution, which offer minimal steric protection. These structural features make 4-CEC susceptible to base-catalysed degradation as mentioned previously, while the activated ring further facilitates instability. Similarly, the extreme over-recovery (> 700%) of 4-Cl-α-PPP is likely an artefact arising from hydrolysis or deamination, which in turn may promote the formation of adducts, thereby enhancing signal intensity. Regardless, in both cases, DUS did offer some improved degradation kinetics, aligning with previous reports suggesting that DMS can attenuate degradation processes through matrix desiccation and reduced enzymatic activity (Jacques et al. 2022). By contrast, varied sample recoveries of NEH point to unique analyte-paper interactions. For this analyte, DUS in fact reduces stability in basic urine, which could be due to the use of a hydrophilic, cellulose-based paper substrate. Despite the long alkyl chain of NEH increasing its overall lipophilicity, cellulose-based paper may instead simply retain the polar functional groups leaving them exposed to structural degradation pathways.

Structural features of synthetic cathinone analogues play a central role in determining their stability. Analogues containing 3,4-methylenedioxy ring substitutions and N-pyrrolidine substitutions, such as MDPV, have been shown to exhibit significantly less degradation than synthetic cathinone compounds without these features, complementing the findings in this study. It has been speculated that the combination of these two structural features has a dual-stabilising effect (Ellefsen et al. 2016; Glicksberg and Kerrigan 2018). Analogues containing tertiary amines, such as NEH, have been found to exhibit greater stability than secondary amines (Glicksberg et al. 2018), such as 4-EMC, followed by structures containing secondary amines with or without halogenated ring substitutions, such as 4-Cl-α-PPP and 4-CEC, being the most unstable (Adamowicz and Malczyk 2019; Aldubayyan et al. 2021; Glicksberg and Kerrigan 2017). These observations are largely reflected in the findings in this study, whereby 4-CEC was found to be unstable in acidic urine within seven days at room temperature; however, the instability of NEH is contradictory to the previously documented findings relating to its structure, exhibiting the most pronounced degradation kinetics across the study at room temperature in both pH conditions and matrix forms except acidic liquid urine, and a sudden loss of stability in basic DUS at 4 ºC only. As previously highlighted, this may be due to specific interactions between the analyte, DUS, and the paper substrate.

From a forensic toxicology standpoint, these results have several implications. Firstly, the use of urine in a forensic context is not obsolete, despite whole blood often being the matrix of choice, with case reports of fatal and non-fatal concentrations of synthetic cathinones in urine routinely reported (Froberg et al. 2015; Wright et al. 2013; Pieprzyca et al. 2023, 2022; Kuropka et al. 2023). Secondly, the use of DUS still presents considerable toxicological impact. The approach demonstrated robustness across variable urine matrices and reduced-dependence on cold-chain storage. Although spot homogeneity was not separately assessed, potential spatial variation within DMS is unlikely to influence results, as PSI utilises the entire wetted area for ionisation rather than discrete punches, reducing position-related bias. This suggests that one could deposit urine on to a paper strip for transport, storage, or in the case of sample backload, for up to 48 h to maintain analyte integrity, whether for subsequent PSI analyses or traditional LC–MS techniques.

Future considerations in synthetic cathinone stability studies include investigations into their photostability, and pH monitoring over the course of a study. Although light exposure was controlled in the present study, with samples stored in the absence of direct light, future studies could examine photodegradation as an independent variable. Cathinone within the Catha edulis plant has been found to be converted into dimers or inactive metabolites under natural sunlight (Katz et al. 2014), likely due to its β-keto-phenethylamine structure, which is conserved across many synthetic cathinones. Therefore, there is a mechanistic basis to hypothesise that synthetic cathinone analogues possess features conducive to similar degradation pathways. Given that these compounds share photochemically active functional groups, such as the β-keto group and aromatic ring, it is reasonable to infer that synthetic cathinones may also be prone to light-induced degradation under environmental or analytical conditions. This possibility is further supported by the general instability of synthetic cathinones and their tendency to degrade under various stressors, though photodegradation has yet to be specifically isolated and quantified in a controlled experimental framework. Additionally, dynamic changes in urinary pH can arise from the bacterial decomposition of amino acids and urea, leading to the formation of amines and ammonia, and therefore the alkalisation of urine (Zaitsu et al. 2008). Such fluctuations in pH may influence the stability of some analytes over time, confounding interpretation of the results. Due to the nature of DMS, there is an inability to monitor pH throughout associated studies, however, it is important to be cognisant of potential pH fluctuations when assessing liquid urine. Another avenue of future work could incorporate external calibration or delayed ISTD addition to aid in distinguishing absolute from relative degradation kinetics.