The still developing and rapidly growing drug market implies the need to develop new and sophisticated methods of identification of drugs in biological matrices. Recently, new psychotropic substances and especially SCs have been important targets of interest for both forensic and clinical laboratories. There are also more and more reports of poisonings with SCs, analytical confirmation of which is essential for professionals. The ever-increasing numbers, variability of chemical groups, many structural modifications, and low concentrations in biological materials as well as numerous and fast metabolic pathways of SCs hinder the complex analysis. Thus, the screening methods should be sensitive and discriminate a large number of different (but sometimes also with a very similar structure) substances. One of the better solutions to this problem is the use of the LC–MS/MS technique as a screening method that can be easily and quickly modified and expanded for new compounds. To be fully adequate for routine laboratory work, new procedures must also be characterised by short sample preparation and analysis. Unfortunately, there is a lack of comprehensive screening methods for simultaneous detection of a large group of SCs.

Taking into account the above issues, an attempt was made to develop an LC–MS/MS screening approach for the detection and identification of SCs in whole blood. The developed procedure allowed the simultaneous qualitative screening, detection, and identification of 72 compounds from different chemical families in whole blood: naphthoylindoles, naphthoylindazoles, benzoylindoles, phenylacetylindoles, tetramethylcyclopropylindoles, indole-3-carboxylic acid esters, indole-3-carboxylic acid amides, indazole-3-carboxylic acid amides, and others.

Coverage of many compounds with different physicochemical properties by one preparation procedure is a difficult task. Most researchers have used liquid-liquid extraction (LLE) for SCs isolation from whole blood, serum, and plasma [9]. SCs were also isolated from blood using solid-phase extraction [19]. Urine preparation included simple dilution, protein precipitation, LLE, supported liquid extraction, or salting-out LLE, and required also a hydrolysis step often with β-glucuronidase [9]. The necessity for the high-throughput and universal method of the isolation of analytes from whole blood compelled us to propose simple protein precipitation. It provided sufficient extraction efficiency, together with simplifying the extraction and significantly shortening the total time of sample preparation, which is very important in screening methods to be used for routine laboratory work. It should be emphasized that high lipophilicity of SCs facilitates their isolation from aqueous medium. For precipitation, we have used frozen acetonitrile that is one of the most efficient protein precipitants [20]. The use of ice-cold solvent improved the isolation process, which is a well-known phenomenon [21, 22].

MRM was applied in the developed method. This is a very sensitive and selective detection mode, but as the number of monitored analytes increases, the performance of the mass spectrometer decreases. The total 217 pairs of MRM were monitored in the method; for each analyte, three pairs with the exception of ADB-FUBINACA and AM-1220, for which only two transitions were observed (the fragmentation of these compounds was poor and only two transitions of high intensity were obtained). It must be noted that, for identification, it is sufficient to monitor two pairs of MRM, while in our method for most compounds, we monitored one precursor and three product ions for increased specificity. The application of dynamic MRM mode increased sensitivity by utilising the retention time window (± 0.5 min) of each analyte.

The core-shell particles in the used Kinetex C18 column provided very good resolution and high sensitivity and ensured the stability of retention times. The chromatographic separation and detection were significantly better when compared to columns with porous particles. The use of the gradient of the mobile-phase composition and the application of variable flow conditions shortened the analysis time, improved the separation of analysed compounds, and made all of the compounds well-separated by their retention times and/or transitions. These conditions allowed the chromatographic analysis and the stabilization of the phase composition in only 16 min. Most of the tested compounds were eluted in less than 7 min, in times from 2.53 to 9.15 min (Fig. 1).

The validation parameters were determined according to the recommendations of the SWGTOX. The validation was performed for 50 out of 72 compounds due to fact that the method initially covered 50 compounds, but during several months of development, a further 22 compounds were included without validation. Detection limits ranged from 0.01 to 0.48 ng/mL. It is worth emphasizing that LODs were calculated for the transition with the lowest intensity of the three monitored, and the peaks for the most intense transition can be observed even at significantly lower concentrations. LODs in other previously published methods involving extraction of a large group of SCs from blood/serum/plasma were in the range of 0.01–2.0 ng/mL [9, 23]. Comparing estimated LODs with the limits determined by the authors of other methods for SCs, it can be concluded that they were in similar ranges. Such values of LODs also demonstrated that the method was suitable for detection of SCs in authentic whole-blood samples, because the expected concentrations of these compounds in the blood specimens are low. Maximum concentrations of SCs in blood are reached quickly; drugs are metabolized smoothly and the concentrations decrease rapidly. Gurney et al. [24] described over 60 cases of SCs where the concentrations were in the range of approximately 0.1 up to 230 ng/mL. The concentrations of SCs reported in serum specimens were in the range of 0.1–190 ng/mL in poisoning cases [6]. In fatal cases, the concentrations were similarly in the range of 0.1–199 ng/mL. However, the concentrations of SCs in blood in non-fatal cases, e.g., driving under the influence of SCs, were significantly lower, and have rarely exceeded several nanograms per millilitre [5, 25, 26].

The matrix effect was also determined and the most astonishing results were those obtained for some compounds reaching a few thousand percent. We have the possible explanation for such results. Many lipids are present in whole blood, and cannabinoids are lipophilic [27, 28]. Cannabinoids are adsorbed on plastic and glass vials, meaning that during matrix effect experiments, SCs are lost in vials without matrix, while they are kept in vials with matrix due to binding with lipids [29, 30]. Such processes can greatly affect and falsify the results, and significantly increase the matrix effect values. Therefore, special attention should be paid when conducting matrix effect experiments according to the Matuszewski method, especially for compounds from the SCs group [18]. In subsequent studies, we intend to confirm (or disprove) our thesis. At this stage, the obtained results of the matrix effect appeared to be unreliable. It is also worth noting that for different blood samples (both antemortem and postmortem), no significant variation was observed for matrix effects for several tested compounds, which means that even such results are not a significant disadvantage of the developed method, but rather an advantage in view of detection sensitivity.

The developed procedure allows performing rapid qualitative screening analysis for SCs. It is being used in our laboratory in routine work for analyses of authentic blood samples collected from drug abusers, drivers, and other individuals in cases where there was a need to prove or exclude the presence of SCs. MDMB-CHMICA, ADB-FUBINACA, 5F-AMB, AB-CHMINACA, and MAB-CHMINACA were detected in 14 cases. These results correspond to the most popular SCs currently present in the Polish drug market. The application to real samples has confirmed the suitability of the proposed procedure for the toxicological screening of SCs in biological samples.

Upon comparing the developed procedure with a few published methods for SCs determination, it can be concluded that it covers a large group of compounds. Kneisel et al. [12, 14] presented LC–MS/MS methods for the analysis of 30 SCs in serum and oral fluid. Hess et al. [10] described simultaneous detection of 93 SCs in plasma using LC–MS/MS technique; however, there is a lack of screening procedures for identification of SCs in whole blood and there are great needs to analyse haemolysed whole-blood samples in forensic toxicological laboratories. The method described by Tynon et al. [11] is capable of determining 34 SCs in whole blood. Our procedure was intended for detecting 72 SCs in whole blood. The number of compounds is somewhat less than in the method developed by Hess et al. [10], but both of these methods seem complementary. As many as 39 compounds in our procedure were not covered by the Hess et al. method (many of them belong to JWH and AM SCs). Both methods are designed to analyse different materials, plasma and whole blood, and different procedures for SCs isolation (LLE and protein precipitation) were adopted. In addition, different instruments were also used for the analyses. Hess et al. [10] used a sensitive chromatograph coupled to a quadrupole linear ion trap mass spectrometer, while we applied a popular triple-quadrupole MS detector. Therefore, the parameters presented in Table 2 will be useful in laboratories using such instruments. The most important advantage of our method is that the procedure can be easily modified for more SCs. The LC–MS/MS equipment is becoming more and more popular, which makes the method more easily adaptable in other laboratories.