GC–MS/MS analysis of synthetic cannabinoids 5F-MDMB-PICA and 5F-CUMYL-PICA in forensic cases

Abstract

Aim: Validate a method to quantify 1-(5-fluoropentyl)-N-(2-phenylpropan-2-yl)-1H-indole-3-carboxamide (5F-CUMYL-PICA) and methyl 2-[[1-(5-fluoropentyl) indole-3-carbonyl] amino]-3,3-dimethyl-butanoate (5F-MDMB-PICA) in blood samples using GC–MS/MS. Materials & methods: A solid-phase extraction (SPE) method has been developed to quantify 5F-MDMB-PICA and 5F-CUMYL-PICA in authentic human blood samples. Results & conclusion: The limit of detection (LOD) was 0.1 and 0.11 ng/ml for 5F-CUMYL-PICA and 5F-MDMB-PICA, respectively, while the limit of quantification (LOQ) was 0.50 ng/ml for both two compounds. Recovery was 91.40, 82.54 and 85.10% for SPE, supported liquid extraction (SLE) and ISOLUTE C18; matrix effects 15, 24 and 22.5% for SPE, SLE and ISOLUTE C18; accuracy was 2.4-5.5 and 3.9-7.3% for SPE, SLE and ISOLUTE C18, while precision was 4.6-7.7 and 6.4-8.3% for SPE, SLE and ISOLUTE C18, respectively. The concentrations of 5F-CUMYL-PICA and 5F-MDMB-PICA in the authentic human blood samples were 2.18 and 3.07 ng/ml, respectively. The validated method was successfully used in supporting the quantification of analytes in blood.

Plain language summary

Summary points.

• For clinical and forensic laboratory applications, it is necessary to develop analytical methods for the qualitative and quantitative analysis of synthetic cannabinoids (SCs) in various biological samples because they can seriously harm health and cause significant toxicity.

• Low detection of some SCs in some cases demonstrates the problem of illegal substances remaining undetected in current screening procedures for biological fluids as well as the significance of developing a solution for new and existing SCs.

• The challenges in developing analytical methodologies for SCs in biological samples are increased by a lack of reference materials, low body concentrations of SCs because of their potent nature and potential interference-causing structural similarities.

• The method has been validated according to international guidelines and was found to be selective for all tested compounds.

• The method was successfully applied to two individuals suspected of the use of a SC in which 1-(5-fluoropentyl)-N-(2-phenylpropan-2-yl)-1H-indole-3-carboxamide and methyl 2-[[1-(5-fluoropentyl) indole-3-carbonyl] amino]-3,3-dimethyl-butanoate were detected.

Synthetic cannabinoids (SCs) have recently increased alarmingly around the world, which has had a serious effect on world health. Contrary to traditional drugs of abuse, SCs contain a variety of structures, and most of them have never had their physiological and pharmacokinetic characteristics investigated in controlled studies, making it extremely difficult to identify and interpret them [1]. The European Monitoring Centre for Drugs and Drug Addiction was monitoring 224 SCs by the end of 2021, with an additional 13 emerging between January and July 2022 [2]. 24 new cannabinoids were reported to the EU Early Warning System in 2022, increasing the total number under observation to 245 (according to the latest report of the year 2023 published in June 2023: New psychoactive substances – the current situation in Europe, European Drug Report 2023) [3]. The structural variability of SCs is used by illegal drug dealers and clandestine laboratories to avoid analysis and detection and to deceive international law's prohibition [4].

Studies are required to improve the identification of new SCs because the parent structure of SCs in biological matrices is frequently challenging to identify. Forensic toxicology researchers now face a great challenge in the identification and quantitative analysis of SCs in various biometrics, primarily because sensitive, reliable and concentrated analytical methods are required [4]. The SCs were originally synthesized to be able to act as agonists or antagonists on and canonical cannabinoid (CB₁) and/or CB₂ receptors by bearing some structural similarity to anandamide (AEA), 2-Arachidonoylglycerol (2-AG), or different phytocannabinoids. Compared with delta-9-tetrahydrocannabinol (THC), some SCs have higher potency for cannabinoid receptors CB₁. However, THC is a partial agonist of the CB₁ receptor, and some SCs work as full agonists of the receptors. According to reports, SCs have psychoactive effects like those of THC that exhibit a variety of effects due to their different mechanisms of action. Every case of intoxication with SCs should therefore be evaluated separately by offering important details, including the concentration that was found. Low doses of SCs can produce hallucinations and paranoia. Repeated use of SCs has been linked to hallucinations and paranoia. In some cases, it has also been linked to the syndrome known as ‘excited delirium’, which appears as severe psychomotor agitation and aggressive behavior toward oneself and the environment [5,6]. Additional signs of intoxication include dehydration, renal failure and muscle damage, perhaps leading to multiple organ failure and eventual death [7].

The compound 1-(5-fluoropentyl)-N-(2-phenylpropan-2-yl)-1H-indole-3-carboxamide (5F-CUMYL-PICA) is categorized in Supplementary Figure S1; it is relatively common in Middle Eastern countries and mainly sold on the internet or in retail stores in the form of shredded tobacco, tobacco leaves or e-liquid [4], and is considered an active and potent SC [8]. The compound 5F-CUMYL-PICA is a brand-new SC, which shares structural features with several other carboxamide-derived SCs, including N-[(1-pentyl-1H-indazol-3-yl)carbonyl]-L-valine, methyl ester (AMB) and 5F-AMB. The year 2014 saw the first detection of 5F-CUMYL-PICA in SC products from Europe [9] and it was recently detected in toxicological analyses of blood samples in Germany [10]. The compound 5F-CUMYL-PICA was listed as SGT67 and SGT25, respectively, in a 2014 patent [11], which is a Schedule I substance in the USA [12].

The compound methyl 2-[[1-(5-fluoropentyl) indole-3-carbonyl] amino]-3,3-dimethyl-butanoate (5F-MDMB-PICA) is classified as an SC (Supplementary Figure S1) and has a structure like methyl (S)-2-[1-(5-fluorophenyl)-1H-indazole-3-carboxamido]-3,3-dimethyl butanoate (5F-ADB), and it was first synthesized and described for pharmacological research characterized by valinate or tert-leucinate moieties by Banister et al. [15]. The compound 5F-ADB is a popular SC, which was associated with numerous intoxications and death cases worldwide, and has the indole analogous 5F-MDMB-PICA [13,14]. In the USA, 5F-ADB is classified as a Schedule I substance and has been identified in nine cases since January 2018 [15]. Banister et al. synthesized several compounds with valine amide or tert-leucine amide substituents to study the structure–activity relationships of this subclass of SCs. The most effective substance in this group was 5F-MDMB-PICA, which acted via the human CB1 with an activity 380-times higher than that of 9-THC [15], which led to severe outcomes/risks. For clinical and forensic applications, the analysis of SCs in biological samples is an analytical challenge due to the changing structures, which present at a very low concentration, and the lack of reference materials, particularly regarding the detection of novel substances in biological matrices [16,17].

Recently, there have been several published methods for the identification and quantification of SCs in biological specimens using GC–MS [18–23], LC–high-resolution MS or LC–MS/MS [24–28], ultra-HPLC–MS/MS [29–31], and LC–MS-TOF [32,33]. GC–MS/MS with the two ionization modes ‘chemical ionization and electron ionization’ is one of the most applied techniques for the analysis of drugs in forensic toxicology laboratories and has been described in some literature for the analysis of SCs in blood and urine samples [18–23]. Mass spectral data for SCs using electron ionization shows extensive fragment ions with the absence of molecular ions for most compounds.

This study aims to develop a qualitative and quantitative analysis of 5F-CUMYL-PICA and 5F-MDMB-PICA in authentic human blood samples using the GC–MS/MS method discussing chromatographic and mass spectrometric results. Additionally, these forensic case samples are used to evaluate the effectiveness of three commonly used SPE methods.

Materials & methods

Chemicals & materials

Certified reference standards of 5F-CUMYL-PICA, 5F-MDMB-PICA and 1-methyl-N-[(1R,5S)-9-methyl-9-azabicyclo[3.3.1]nonan-3-yl] indazole-3-carboxamide monohydrochloride (Granisetron hydrochloride) 1.0 mg/ml free base in methanol were purchased from Cayman Chemicals (MI, USA) (Supplementary Figure S1). Methanol (HPLC grade), ethyl acetate and acetonitrile (HPLC grade) were obtained from Fisher Scientific Co. (PA, USA). (Strata-X Solid 60 mg/ml (SPE) was purchased from (Phenomenex Inc. part no. 8B-S128-UBJ, Torrance, CA, USA), supported liquid extraction (SLE) from (Thermo, Hemel Hempstead, UK), and ISOLUTE C18 (100 mg/3ml; Biotage, Sweden).

Instrumental

The analysis of compounds was carried out using an Agilent GC 7890 gas chromatograph coupled with an Agilent 7000A triple quadrupole mass spectrometer and an Agilent 7693 Autosampler (Agilent Technologies, CA, USA), with HP-5MS 15 m × 0.25 mm × 0.25 μm (part no. 19091S-433) (Agilent Technologies, Palo Alto, USA). Typical autosampler, GC and MS method parameters used for the octafluoronaphthalene detection limit studies are shown in Supplementary Table S1. The multiple reaction monitoring (MRM) mode was used to detect and quantify the compounds.

Sample collection

The drug-free blood samples were obtained from staff at the College of Criminal Justice, Naif Arab University for Security Sciences. Fifteen authentic human blood samples suspected of 5F-CUMYL-PICA and 5F-MDMB-PICA were received from the forensic laboratory. The samples were stored at -20°C until analysis. The study protocol was given the approval no. Nauss-Rec-22-07 from the research and ethical committee of the Naif Arab University for Security Sciences, Riyadh, Saudi Arabia.

Working & calibration standards

Standard stock solutions of 5F-CUMYL-PICA and 5F-MDMB-PICA 1 mg/ml were prepared by transferring 1 ml of standard stock solutions of 10 mg/ml into a 10 ml volumetric flask and made up to 10 ml with methanol. These solutions were stored at -20°C. To prepare a range of concentrations necessary for the experiment, working standard solutions were prepared by dilution of the prepared stock standard solution 1 mg/ml with methanol. Drug-free blood samples were fortified with the appropriate concentrations of 5F-CUMYL-PICA and 5F-MDMB-PICA working solutions. The intra-day and inter-day precision and accuracy were established using three quality controls (QCs) at concentrations of 25, 75 and 450 ng/ml. The three QCs were prepared by mixing an adequate volume of the intermediate solution mixture with a blank blood sample.

Sample pretreatment

Briefly, calibrators were prepared by the addition of 0.5, 1.0, 5, 10, 50, 100 and 500 ng of SCs into 0.5-ml samples of drug-free whole blood. Then, 50 μl of 5 μg/ml granisetron hydrochloride as internal standard (IS) was added to these samples. A negative control sample was prepared by adding only the IS 5 μg/ml to a sample of drug-free whole blood 0.5 ml. A negative control samples (drug-free blood) were prepared by adding only 50 ul of (5 ug/ml) IS. Then, 5 ml of 100 mM sodium acetate buffer (pH 5) was added to the negative control, calibrators and test samples. These were then well mixed on a vortex mixer for 1 min and centrifuged at 3000 r.p.m. for 10 min before application on individual SPE columns. All determinations were performed in duplicate. After being decanted into a clean tube, the clear supernatant was immediately subjected to SPE, SLE and C18 cartridges.

Extraction with Strata-X polymeric SPE

The SPE cartridge was conditioned with methanol, water and 1 ml of 100 mM sodium acetate buffer (pH 5). The blood samples were loaded onto the cartridge. Washing was carried out by the addition of water, and 2 ml of acetonitrile: water (30:70). Elution was performed by 2 ml ethyl acetate: isopropanol (85:15).

Extraction with SLE

The blood samples were loaded onto the cartridge with a silica gel vacuum and then at gravity. A total of 2.5 ml ethyl acetate was added and eluted under gravity. The low vacuum was applied to complete elution.

Extraction with ISOLUTE C18

The cartridge was conditioned with 3 ml methanol. The blood samples were loaded with 0.5 ml blood sample and 0.5 ml of 100 mM sodium acetate buffer (pH 5). Washing was carried out with 2 ml of 5% acetonitrile in water. The cartridge was dried for 1 h and eluted with 2.5 ml ethyl acetate (or dichloromethane: isopropanol [80:20]), under gravity.

All the three extraction's elution were dried at 45°C with a gentle nitrogen stream, and the residue was then reconstituted with 50 μl of ethyl acetate and transferred to the GC–MS vial with micro-insert and 2 μl was injected into the GC–MS/MS.

Results & discussion

GC–MS/MS optimization

For the target SCs, a highly sensitive and selective GC–MS/MS method was developed using multiple parameters, including reagent gas selection, collision energy and MRM transition ions. The major compounds were identified by initial analysis using GC–chemical ionization/MS/MS. The most intense peak in the GC spectrum appeared at 10.947 and 9.796 min, which corresponded to a molecular ion at 377 and 367 m/z (Figure 1) and product ions (232, 204, 144 and 249, 206, 119 m/z) (Supplementary Figure S2 & Supplementary Table S2) for 5F-MDMB-PICA and 5F-CUMYL-PICA, respectively. The mass spectra and the proposed fragmentation of 5F-MDMB-PICA and 5F-CUMYL-PICA are given in Figure 2. The reagent gas source pressure was adjusted to produce the base peak ion's maximum intensity for each reagent condition. The MRM mode was used to operate and monitor the transitions from precursor ions to dominant product ions. Each compound was determined using two specific transitions, the most intense transition being used for quantification (quantifier transition) and the second being used for confirmation (qualifier transition) (Supplementary Figure S3) [34]. This method optimizes the appropriate collision energy for each transition using Agilent software. The list of analytes together with the corresponding precursor ions, and product ions of 5F-CUMYL-PICA, and 5F-MDMB-PICA are displayed in Table 1.

Figure 1.

Total ion chromatogram of methyl 2-[[1-(5-fluoropentyl) indole-3-carbonyl] amino]-3,3-dimethyl-butanoate (377) and 1-(5-fluoropentyl)-N-(2-phenylpropan-2-yl)-1H-indole-3-carboxamide (367).

EIC: Extracted ion chromatogram; SC: Synthetic cannabinoid.

Figure 2.

Mass spectra of 5F-CUMYL-PICA and 5F-MDMB-PICA at optimized CE, by NH₃-CI.

5F-CUMYL-PICA: 1-(5-Fluoropentyl)-N-(2-phenylpropan-2-yl)-1H-indole-3-carboxamide; 5F-MDMB-PICA: Methyl 2-[[1-(5-fluoropentyl) indole-3-carbonyl] amino]-3,3-dimethyl-butanoate; CI: Chemical ionization.

Table 1.

Recovery of synthetic cannabinoids from blood by GC–MS/MS.

Analyte	ISOLUTE C18	(% CV)	Supported liquid extraction	(% CV)	Strata-X polymeric SPE	(% CV)
	Recovery (%)		Recovery (%)		Recovery (%)
1-(5-Fluoropentyl)-N-(2-phenylpropan-2-yl)-1H-indole-3-carboxamide	86.30	6.5	80.13	4.5	93.70	5.6
Methyl 2-[[1-(5-fluoropentyl) indole-3-carbonyl] amino]-3,3-dimethyl-butanoate	83.90	4.5	84.94	5.5	89.10	3.5
Average	85.10		82.54		91.40

CV: Coefficient of variation; SPE: Solid-phase extraction.

Extraction

In this study, SPE, SLE and C18 cartridges were assessed to extract the target analyte from blood samples. The recovery was studied to compare and choose the best SPE cartridges for extracting 5F-CUMYL-PICA and 5F-MDMB-PICA in blood. The extraction protocol using three cartridges results in nearly complete recovery and minimizes matrix effects for all analytes. With an overall average recovery of 91.40, 82.54 and 85.10% for SPE, SLE and C18, respectively, the two analytes exhibit recoveries of 80% or greater within the acceptable limits ranging in CV less than 6.5% (Table 1 & Figure 3). In contrast to other SPE sorbents, SLE skips the condition/equilibration stage without affecting recovery. Our results indicate that using SPE led to cleaner chromatograms and very high extraction efficiencies for all analytes. QC testing using drugs-of-abuse probes from blood samples [34].

Figure 3.

Recoveries for synthetic cannabinoids in blood at reporting limit using ISOLUTE C18, SLE and Strata-X polymeric SPE columns.

SLE: Supported liquid extraction.

Method validation

The method validation was performed using linearity, specificity (selectivity), matrix effect, recovery, LOD, LOQ, accuracy and precision [35]. Seven calibration standards with a concentration range of 0.5–500 ng/ml were prepared using pooled negative blood from volunteers and then analysed on three different days. The criteria for acceptance of linearity were an R² value of ≥0.998 and a 15% difference in percentage for calibration standards. Additionally, no more than two points were excluded from each set of standards [35]. The ratio of the analyte's measured concentration to its anticipated concentration following extraction and preparation is known as recovery. The influence of the co-extracted components on the analyte's ionization and detection is known as the matrix effect. Studying recovery and matrix effect helps to understand their contribution to obtained process efficiency. The equation below was used to calculate the analyte recovery:

$$\% \text{Recovery}=\left(\frac{\text{Area} \text{A}}{\text{Area} \text{B}}\right)\times 100\%$$

where A is the extracted sample peak area and B is the extracted matrix sample peak area where the compounds were added after extraction. The equation below was used to calculate the matrix effects:

$$\text{Matrix} \text{effect}=\left(\frac{\text{Peak} \text{area} \text{in} \text{the} \text{presence} \text{of} \text{matrix}}{\text{Peak} \text{area} \text{in} \text{the} \text{absence} \text{of} \text{matrix}}\right)\times 100\%$$

The peak area of an extracted matrix sample to which the compounds were added after extraction is referred to as the peak area in the presence of a matrix. In the absence of a matrix, analytes in a clean solvent solution are referred to as the peak area [35]. The lowest calibration standard with a value ≥3*S/N was used as LOD, whereas the lowest calibration standard with a value ≥10*S/N was used as LOQ. Specificity was estimated through seven distinct zero-control blood samples (IS added exclusively), and blank blood samples (no analyte or IS) were evaluated [35].

The precision and accuracy of the analysis method were assessed in six replicates over three days. Three QCs (QC₁, QC₂ and QC₃) with concentrations of 25, 75 and 450 ng/ml were used. Precision was expressed as the percent relative SD of the replicates while accuracy was expressed as bias. Precision was acceptable at ≤15% at any concentration level and accuracy was within ±15% of the actual value [35].

The characteristic calibration results for all analytes provide a dynamic range from the LOQs to 500 ng/ml and are summarized in Table 2. The LODs for 5F-CUMYL-PICA and 5F-MDMB-PICA were 0.1 and 0.11 ng/ml, while LOQ was 0.5 ng/ml for both compounds (Table 2). The matrix effects across the panel were less than 20% with an overall average of 15% for SPE, and more than 20%, namely 24 and 22.5% for SLE and C18, respectively (Table 3) [36]. The intra- and inter-assay precisions were found to be in the range of 4.6–7.7% and 6.4–8.3%, and (2.4-5.5 and 3.9-7.3%), respectively showing good precision and accuracy were achieved. The accuracy values were in the ranges of 2.4–5.5% and 3.9–7.3%, indicating the acceptable accuracy of the proposed extraction method for 5F-CUMYL-PICA and 5 F-MDMB-PICA. The precision and accuracy values are presented in Table 4. The results revealed that two out of 15 authentic humane blood samples contained 5F-MDMB-PICA and 5F-CUMYL-PICA with concentrations of 3.07 and 2.18 ng/ml, respectively, and the remaining samples were free of the analytes. Figure 4 represents the extracted ion chromatogram of 5F-CUMYL-PICA and 5F-MDMB-PICA.

Table 2.

Method validation parameters and synthetic cannabinoids concentration in blood samples.

Analytes	Correlation coefficient†	Linear range (ng)	CV (%)	Concentration (ng)	Limit of detection (ng)	Limit of quantification (ng)
1-(5-Fluoropentyl)-N-(2-phenylpropan-2-yl)-1H-indole-3-carboxamide	0.997 ± 0.003	0.5–500	4.19	2.18	0.1	0.50
Methyl 2-[[1-(5-fluoropentyl) indole-3-carbonyl] amino]-3,3-dimethyl-butanoate	0.996 ± 0.001	0.5–500	4.23	3.07	0.11	0.50

^†Mean of three replicates of calibration curves.

CV was calculated from the mean value of the response factor (ratio between peak area and analyte quantity) (n = 2).

Table 3.

Matrix effects of synthetic cannabinoids from blood by GC–MS/MS.

Analyte	C18	(% CV)	Supported liquid extraction	(% CV)	Strata-X polymeric SPE	(% CV)
	Matrix effects (%)		Matrix effects (%)		Matrix effects (%)
1-(5-Fluoropentyl)-N-(2-phenylpropan-2-yl)-1H-indole-3-carboxamide	22	5.5	23	5.4	14	7.6
Methyl 2-[[1-(5-fluoropentyl) indole-3-carbonyl] amino]-3,3-dimethyl-butanoate	23	5.6	25	6.4	16	4.7
Average	22.5		24.0		15.0

Table 4.

Method validation data: precision and accuracy.

Quality control (ng/ml)	Precision (CV,%)		Accuracy (bias,%)
	Intra-assay	Inter-assay	Intra-assay	Inter-assay
25	5.8	6.4	2.4	3.9
75	7.7	8.3	4.3	7.3
450	4.6	7.7	5.5	6.2

^†Mean of five replicates (n = 15) along 3 subsequent working days.

Figure 4.

Chemical ionization MS/MS chromatograms (MRM) of 5F-CUMYL PICA and 5 F-MDMB PICA in authentic human blood samples.

5F-CUMYL PICA: 1-(5-fluoropentyl)-N-(2-phenylpropan-2-yl)-1H-indole-3-carboxamide; 5 F-MDMB PICA: Methyl 2-[[1-(5-fluoropentyl) indole-3-carbonyl] amino]-3,3-dimethyl-butanoate; MRM: Multiple reaction monitoring.

In the present study, the proposed method was optimized to develop an analytical method for the determination of 5F-MDMB-PICA and 5F-CUMYL-PICA in authentic human blood samples. The analytes were identified in the samples by the molecular ions at 376.47 and 366.50 m/z, respectively. The confirmation of 5F-MDMB-PICA and 5F-CUMYL-PICA was achieved through comparison with the retention time and fragment abundance of the reference material. To optimize MRM conditions, the triple quadrupole was separately merged with each component. The dominating adducts were then determined while running the MS instrument in full scan mode. Fragment ions were then detected using the product ion scan mode, and the three strongest ions were automatically selected. The fragment and the collision energy were optimized for each transition by applying a 150eV and a 5–15 eV ramp, respectively. The positive chemical ionization mechanism was initially examined in this work using NH₃ as a reagent gas, which is considered the optimal soft ionization of the target analytes [37].

As shown in Supplementary Figure S2 & Figure 2, using NH₃ provides a superior ionization of all the target analytes with less fragmentation, whereas the protonated molecules are the dominant, as shown in Supplementary Table S2. Such results can be explained by the term ‘proton affinity’ (PA). PA is a thermochemical reaction between reagent gas ion (NH₄⁺) and the analytes to transfer protons. According to the literature, to generate a protonated molecule, the PA of the analyte should be greater than that of the reagent gas ion. This is because the difference between the PA of the reagent gas ion and the PA of the analyte increases the amount of fragmentation of the analyte increases, due to more internal energy being transferred to the analyte the by reagent gas ion. Referring to the literature, NH₃ has a high PA value of 205 kcal/mol compared with the PA of most organic molecules, therefore less energy transfers to the analyte. The selected transitions used for each compound are shown in Supplementary Table S2. The results indicated that the abundance of product ions was higher for the analytes using NH₃-positive chemical ionization (NH₃-PCI) (Figure 2).

According to previous studies, the total times needed to prepare the blood samples for SPE, SLE and C18 are 20 min, 45 min and 65 min, respectively. In addition, SLE and C18 need method development with different sample pretreatment or extraction solvents for different classes of analytes, while SPE uses a simple, generic three-step extraction technique to remove salt, proteins and phospholipids [38]. After loading, SLE needs 5 min to wait for the sample to completely absorb into the support matrix. The analytes must be given a further 5 min to interact with the extraction solvent after the extraction solvent has been administered. Additionally, the flow starts in the SLE sample loading step, which is accomplished by introducing a very gentle vacuum of 20684.3 Pa for 2–5 s. This step is extremely subtle and difficult to master without patience and practice. The aqueous sample will not be able to successfully immobilize the sorbent if the initiation time is too short, less than 2–5 s or the pressure is too low. The blood sample will directly elute if the duration is too long or the pressure is too high, producing a murky elution solution and higher matrix factors. According to the results and the previous studies cited above, SPE demonstrates excellent and consistent recoveries (for acidic analytes) across all the investigated analytes, with an average recovery of 91.4%, leading to clear chromatograms and extremely high extraction efficiency for all analytes (Table 1) [38]. The target analytes in the SLE and C18 recovered well, with average recoveries of 82.54 and 85.10%, respectively. However, the extraction efficiency was affected by these recovery levels [39]. The GC–MS/MS method and determination of coefficient (R²) >0.99 demonstrated excellent linearity for the two analytes over the entire concentration range of 0.5–500 ng (Table 2). The LODs, LOQs, accuracy and precision for the back-calculated concentrations of each calibrator were within acceptable limits [33,34].

The total matrix effect of the SPE was less significant compared with SLE or C18. For SPE, all matrix effects were below 20%; for SLE and C18, they were all above 20% (Table 3). This goes back to removing salts, proteins and phospholipids during the three-step SPE process, producing very clean final extracts with little to no matrix effects for all different analytes. The higher matrix effects in SLE may be the result of contaminants recovered from the SLE sorbent, although SLE extracts used the same material and extraction solvent as SPE extracts. Also, SPE might be able to remove additional impurities that could reduce ions, but it appears to be more effective in removing analytes from blood. When the sample matrix contains a wide variety of compounds (including acidic SCs compounds of varying polarities), SPE showed excellent recovery and minimal matrix effects (Figure 3). On the other hand, SLE produced acceptable recoveries, with much higher matrix effects for acidic analytes. Due to its low extraction efficiency, C18 exhibited acceptable recoveries (84.66% lower in recoveries compared with SPE). Additionally, C18 showed higher variability in matrix effects, especially for acidic substances like SCs. Accordingly, acidic SCs analytes cannot be efficiently recovered with an SLE or C18 extraction cartridges, and additional technique development would be necessary to enhance recoveries.

Our validated method was successfully applied to detect and quantify 5F-MDMB-PICA and 5F-CUMYL-PICA from authentic human blood samples. The chromatograms for 5F-CUMYL-PICA and 5F-MDMB-PICA are represented in Figure 4. The results obtained agree with Sykutera et al., who reported that two cases of fatal intoxications with 5F-MDMB-PICA were presented in Poland [40]. The determined blood concentrations of this compound in these cases were 3.7 and 1.8 ng/ml, respectively [40]. Twelve examples of documented 5F-MDMB-PICA ingestion were reported by Kleis et al., including three fatalities, four instances of drugged driving and five other criminal acts [41]. Concentrations in the blood or serum ranged from 0.1 to 16 ng/ml. Nine out of the 12 cases, including all fatal ones, involved co-consumption of additional drugs. In a few cases, the only exogenous substance was 5F-MDMB-PICA. However, in two cases, the determined concentrations in femoral blood were 0.28 and 0.32 ng/ml. Ketoacidosis was assumed to be the cause of death, probably with some drug usage contributing [42]. In previous studies by Krotulski et al., authentic blood case samples (more than 3487) were analysed [43]. A total of 2.6% of samples sent to NMS Labs (Horsham, PA, USA) from March 2018 to March 2019 included 5F-MDMB-PICA.

There are several analogues with 5F-CUMYL-PICA, such as 1-(4-cyanobutyl)-N-(2-phenylpropan-2-yl)-1H-indazole-3-carbxamide, 2,5-Dihydro-2-(1-methyl-1-phenylethyl)-5-pentyl-1-pyrido[4,3-b]indol-1-one, that have cumyl groups and heterocyclic cores substituted with cyanobutyl, pentyl or fuoropentyl functional groups. Some of these chemicals have been found in cases that have tragic results. Eleven cases of CUMYL-4CN-BINACA intoxications in which the blood concentrations of the victims ranged from 0.4 to 34.3 ng/ml were reported by Yeter [44]. Of the total cases, three involved falling from a height, and two involved drowning after ingesting; however in other cases, CUMYL-4CN-BINACA poisoning alone was the cause of death. Blood concentrations for seven post-mortem cases involving CUMYL-PEGACLONE were reported by Nash et al. to be in the range of 0.73–5.5 ng/ml [45]. One clinical toxicological case was presented by Abouchedid et al. with a blood concentration of 5F-CUMYL-PINACA of 0.8 ng/ml [46]. Contrary to the previously mentioned, no fatal intake of 5F-CUMYL-PINACA, 1-(5-Fluoropentyl)-N-(2-phenylpropan-2-yl) pyrrolo[2,3-b]pyridine-3-carboxamide and 5F-CUMYL-PICA have yet to be documented. Although the concentration of the blood level of 5F-CUMYL-PICA in the present cases 3.07 ng/ml is the highest recorded to date, it may have been significantly higher at the time of the accident.

The pharmacology and toxicology of 5F-CUMYL-PICA and 5F-MDMB-PICA in the human body are little known. Common side effects after using SCs include seizures, hypothermia, acute respiratory distress syndrome, acute renal injury, myocardial infarction, pulmonary oedema, ischemic stroke and multiple organ failure [47].

Conclusion

The most common synthetic cannabinoid compounds in Middle Eastern are 5F-CUMYL-PICA and 5F-MDMB-PICA. The method described in this work has been validated over a wide concentration range which provides good linearity, accuracy, and intra- and inter-day precision, and approaches one of the highest recorded sensitivities to date. Also, this study has demonstrated the effect of the validated extraction method on the extraction of 5F-CUMYL-PICA and 5F-MDMB-PICA from 15 authentic human blood samples through short, low-cost and eco friendly procedures with a high recovery rate and good sensitivity. The validated method was a simple, fast and reliable analytical tool, so it must be offered for SCs quantification in forensic laboratories. To our knowledge, the quantification of authentic human blood samples associated with 5F-MDMB-PICA and 5F-CUMYL-PICA is the first to fully validate and quantification of SCs in blood. Finally, the appearance of the new SCs 5F-MDMB-PICA and 5F-CUMYL-PICA will increase the morbidity and death of drug users.

Funding Statement

The work was funded by the Naif Arab University for Security Sciences, Riyadh, Saudi Arabia, under grant no. PR2-06.

Author contributions

H Ahmed: the idea of the manuscript, the practical part of the manuscript and the discussion of the results. S Mujeebuddin: the practical part of the manuscript.

Financial disclosure

The work was funded by the Naif Arab University for Security Sciences, Riyadh, Saudi Arabia, under grant no. PR2-06. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.

Competing interests disclosure

The authors have no competing interests or relevant affiliations with any organization or entity with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, stock ownership or options and expert testimony.

Writing disclosure

No writing assistance was utilized in the production of this manuscript.

Ethical conduct of research

The study protocol was given the approval no. Nauss-Rec-22-07 from the research and ethical committee of the Naif Arab University for Security Sciences, Riyadh, Saudi Arabia.

Data availability statement

All relevant data are within the manuscript.