Short- and long-term stability of synthetic cathinones and dihydro-metabolites in human urine samples

Synthetic cathinones (SCt) are one of the largest groups of new psychoactive substances (NPS) in illicit drug markets [1]. Their inexpensive cost and ease of availability via online vendors are assumed to contribute to their expansion and popularity among drug users [2].

An extensive period of time before analysis may be needed when laboratories are encountered with backlogs or when samples must be retained over a long period of time to allow retesting (if requested). Hence, knowledge of their stability is of great importance to assist the interpretation of the results as some analytes may degrade during sample storage. Stability may be influenced by the storage conditions, chemical structure, and/or type of biological samples. These factors must be taken into account when comparing initial results and results after days, weeks, and months of storage.

A number of studies indicate instability of some SCt in biological samples, with the storage temperature being an important factor [3]. Al-Saffar et al. [4] studied the stability of methcathinone, buphedrone, mephedrone, 3-fluoromethcathinone (3-FMC), 4-fluoromethcathinone (4-FMC), methedrone, methylone, butylone, pentylone, methylenedioxypyrovalerone (MDPV), and naphyrone in urine samples maintained at − 20 °C (freezer), 6 °C (refrigerator), and 22 °C (room temperature (RT)) for 3 months. Degradation of analytes was higher at 22 °C than at 6 °C but less pronounced at –20 °C.

In addition to temperature, mechanisms of degradation can also be influenced by chemical structure and urinary pH. Glicksberg and Kerrigan [5] investigated the stability of 22 SCt in acidic (pH 4) and alkaline (pH 8) urine samples at –20 °C, 4 °C, 20 °C, and 32 °C for a period of 6 months. They found improved stability of tertiary amines SCt incorporating methylenedioxy (MD) groups over other tertiary amines-containing SCt, followed by secondary amines. Among secondary amines, flephedrone and its isomer 3-fluoromethcathinone (3-FMC) exhibited the worst stability at pH 8 even when maintained at –20 °C and preserved (1% sodium fluoride), while stability improved in those analytes at pH 4. Adamowicz and Malczyk [6] corroborated these findings when they reported that instability of SCt increase with the increasing pH.

Parent SCt share a carbonyl functional group in their chemical structure. This carbonyl is readily reduced to a hydroxyl group, leading to the so-called dihydro-metabolites [7]. It has been recommended that stability studies should involve both parent and major metabolites, as this may be helpful to establish a clear image of changes in drug concentrations and degradation [8]. While urine samples are typically characterized by the presence of metabolites, which can aid in determining the intake of the parent drug, most published reports on the stability of SCt mainly focused on the stability of parent drug [3]. A literature search only identified two published studies on the stability of SCt metabolites in urine. In a study performed by Concheiro et al. [9] dihydro-4-methylethcathinone (4-MEC), dihydro-buphedrone, and dihydro-mephedrone were found to be stable at RT for 24 h and at 4 °C for 72 h. In contrast, other authors have reported dihydro-4-MEC as being unstable in urine samples after 24 h of storage at 4 °C, whereby degradation of 22% from the initial SCt level was found [10]. With respect to other biological matrices, dihydro-mephedrone and dihydro-nor-mephedrone (one of the main phase I mephedrone metabolites) were consistently stable in whole blood for 10 days at either 4 and –20 °C, whereas a decrease up to 23.4% was observed in the corresponding parent cathinone at 4 °C [11]. Soh and Elliott [12] investigated the stability of 4-MEC in blood and plasma and found that dihydro-4-MEC was the prominent metabolite in the absence of 4-MEC (for instance via in vitro and ex vivo sample instability). In our previous work, dihydro-metabolites were more stable than their respective parent drugs in whole blood over 6 months regardless of storage temperatures [13]. Similar stabilities might be expected in urine samples, but this information appears to be scarce in the literature. Therefore, it is important to assess the stability of metabolites as they may be considered more appropriate biomarkers if the parent drugs are extensively degraded.

The purpose of this study was to investigate the stability of a panel of SCt and dihydro-metabolites in human urine samples under the typical conditions used in forensic toxicology. Storage conditions included RT for 3 days, 4 °C for 14 days, and –40 °C over 12 month. A recently reported fully validated liquid chromatography–tandem mass spectrometry (LC–MS/MS) method was used to assess the stability of a total of 26 analytes (Fig. 1) at each storage condition [14].

The stability data expressed as the percentage remaining of each tested analyte after 3 days at RT and 14 days in refrigerated (4 °C) (Fig. 2) and over 12 month in frozen (–40 °C) storage conditions are summarized in Fig. 3.

Contrary to parent drugs, most dihydro-metabolites, except for dihydro-4-CEC, dihydro-4-Cl-α-PPP, and dihydro-4-F-PHP (which had more than 60% remaining), were stable after 3 days and 14 days at RT and 4 °C, respectively.

Concerning long-term stability (Fig. 3), freezing the urine samples maintained good stability across all parent drugs for up to 1 month of storage. On day 90, parent analytes demonstrated minimal variation within the stability criteria where the overall loss was less than 25% of the original contents, except for halogenated analytes that experienced significant degradation, especially 4-CEC and 4-Cl-α-PPP, in which only about 50% of their initial values remained detectable. With the exception of halogenated analytes (recoveries of about 40–55%), the concentration remaining for all parent drugs did not decrease by more than 65% after 6 months of storage and was relatively maintained to these levels until the end of the study period.

Given both α-PVP and 4-Cl-α-PVP were part of the tested panel, each analyte was fortified separately and stored at RT for 3 days before separately run following the procedure described in “stability study design” section. Visual inspection of the MRM chromatogram of α-PVP following analysis of 4-Cl-α-PVP revealed a peak at the same RT (6.72 min) for the MRM transitions m/z 232 > 91 and m/z 232 > 105 (Fig. 4c). Of note, the reverse was not observed (Fig. 4d).

Information on extended stability is of paramount importance since it may address the maximum storage time of a drug in a particular biological matrix. In addition to parent analytes, this study incorporated a selection of dihydro-metabolites that have been recently detected in toxicological casework – either with or without their respective parent drug [14, 17] – but where stability considerations are lacking in the literature. This study presents, for the first time, the stability data of SCt and dihydro-metabolites over a much longer period of time in human urine samples.

The influence of storage temperature appears to be more significant for halogenated analytes, which are typically the most unstable SCt in different matrices [3]. This observation has been previously reported and has been attributed to the presence of a halogen atom (e.g., chlorine or fluorine) on the benzene ring that adversely affects SCt stability [6, 13]. In parallel, mephedrone, one of the most commonly detected SCt in toxicology casework [18] had also significant loss at RT and 4 °C storage conditions, which accounted for approximately 60% of the initial mephedrone concentration. The stability of mephedrone and MDPV in urine at RT and 4 °C for up to 14 days has also been studied elsewhere [19]. Both mephedrone and MDPV were found to be fairly stable when stored refrigerated throughout the study period, but instability in mephedrone was observed at RT (about 65% remaining) following 7 days of storage [19]. While these results agree with those seen in this study for mephedrone and MDPV stored at RT, our results were contradictory with the previous work in that mephedrone concentration dropped to approximately 60% remaining after 14 days at 4 °C. These results indicate that while it is evident that the degree of degradation for most SCt stored at RT was larger than those kept at 4 °C, degradation rate was dependent on the chemical structure. Thus, these conclusions agree with those of Glicksberg and Kerrigan [5], who found that N-pyrrolidine derivatives are more stable than those of primary and secondary amine groups.

By demonstrating the relatively rapid degradation of certain SCt in urine samples, our analysis of dihydro-metabolites in this matrix has shown that long-term use can be monitored not only due to extensive metabolism but also because they are more stable. The different stabilities between parent and dihydro-metabolites affirm findings in Concheiro et al. [9] in which degradation of dihydro-metabolites in urine was substantially less than that of parent drugs. These results are also in line with Al-Saffar et al. [4] for the stability of SCt. That study showed most SCt were relatively stable in urine when stored at –20 °C for up to 3 months, while a higher decrease (up to 40% remaining) in concentration was observed for analytes substituted with halogen atom (i.e., 4-fluoromethcathinone and 3-fluoromethcathinone) [4].

Generally, samples containing dihydro-metabolites had an overall smaller degree of loss as opposed to parent analytes under all conditions for similar time periods. While this is the first long-term study concerning the degradation of dihydro-metabolites in urine samples, they are consistent with our previous findings on these analytes in whole blood [13], although reflecting greater stability in whole blood than in this study when stored frozen at –40 °C for 6 months. This might be explained by pH differences of both matrices (pH 7.41 in whole blood in the previous study vs. pH 7.63 in our study), which is a typically less stable medium for these analytes. In another study in urine, no degradation was found at pH 4, but when pH increased to 8, most of SCt concentrations significantly decreased after 6 months of storage at –20 °C [5]. Cook et al. [20] found a slight increase in urine pH (+ 0.7) over 2 weeks even when stored frozen. However, stability at RT and 4 °C were much higher than those in whole blood [13], suggesting the impact of other factors (e.g., enzymatic activity). Nevertheless, as stated earlier, the goal of this work was to simulate casework samples since urine pH is not commonly adjusted. As such, this stability study is probably more representative of what may be experienced in real toxicological cases.

Another crucial aspect in stability studies is the investigation of potential degradation products of target analytes, more specifically, the halogenated analytes that exerted significant degradation effects under all storage conditions, suggesting a distinct reaction may be involved in the degradation of halogenated analytes. Following analysis of 4-Cl-α-PVP, the degradation products corresponding with the m/z 232 might be attributed to α-PVP. A tentative explanation might be due to the dehalogenation of 4-Cl-α-PVP to generate α-PVP via instability.

Unfortunately, non-halogenated reference materials for 4-CEC, 4-Cl-α-PPP, and 4-F-PHP were not available at the time of the test to provide more insight into this reaction. Nevertheless, LC–QTOF-MS in full scan and auto MS/MS modes were used to attempt to identify potential 4-CEC, 4-Cl-α-PVP, 4-Cl-α-PPP, and 4-F-PHP breakdown products along with metabolites. In addition to untargeted analysis, targeted analysis was performed based on the results of previous in vivo and in vitro experiments in human biological samples, namely, carbonyl group reduction, hydroxylation, N-deethylation, and demethylenation [21, 22], and a list of predicted degradation products was used as potential targets (Supplementary material; Table S1). No potentially postulated degradation products were detected in any of urine samples with the present method. While it can be assumed that breakdown products were missed due to instability, the formation of undetected breakdown products in these samples, leading to analytes degradation, cannot be ignored. This hypothesis is supported by earlier suggestions that only trace amounts of dihydro-metabolites were detected via in vitro instability in whole blood and plasma [12].

This study reported herein evaluated the stability of selected SCt in human urine in a more comprehensive manner due to the inclusion of metabolites and longer timeframe than previously published work. From the obtained results, the stability of SCt was highly influenced by the storage temperature, whereby storage at high temperatures accelerated the rate of degradation. Although slower when stored frozen, degradation of these analytes increased over time. Moreover, parent analytes containing halogen such as 4-CEC, 4-Cl-α-PPP, 4-Cl-α-PVP, and 4-F-PHP were characterized by extreme instability in urine samples, but marked stability differences were observed for their respective dihydro-metabolites in all storage conditions, making them potential better biomarkers of SCt use.

While –20 °C is routinely used as a frozen condition in toxicology laboratories, it is also important to test the hypothesis that deep freezing (–40 °C or lower) could be more suitable condition for urine samples suspected to contain SCt, which might help more efficiently preserve the analytes for a longer period of time. Thus, urine samples potentially containing SCt should be stored frozen and analyzed in a timely manner to avoid any significant losses.